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Novel luminescentimmunoassays
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LOUGHBOROUGH UNIVERSITY OF TECHNOLOGY
LIBRARY
AUTHOR/FILING TITLE
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3 JUL 1990 1 1 JUN 1992
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NOVEL LUMINESCENT IMMUNOASSAYS
by
VIJAY KUMAR MAHANT
A Doctoral Thesis J
submitted in partial fulfilment of the requirements for the award of
DOCTOR OF PHILOSOPHY of the Loughborough University of Technology
Supervisor: Professor J N Miller Department of Chemistry
Loughborough University of Technology
June 1983
© V K MAHANT (1983)
i
: flf T~cr..r,4: ~! L:':-i.ry ~
;~::--V%i--~ ~-~ ~-.. -- .----! l C!,., i .. --------~ j ::.~. OCl'l.<t-"'¥ !-01, I
CERTIFICATE OF ORIGINALITY
This is to certify that I am responsible for the work submitted in this thesis, that the original work is my own, except as specified in acknowledgements or in footnotes, and that neither the thesis nor the original work contained therei~ has been submitted to this or any other institution for a higher degree.
i i
To my first and most devoted teachers - my parents
i i i
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to Professor J N
Miller for his guidance, constructive criticism and invaluable
assistance throughout the research period.
The assistance received from the following members of
the technical staff is thankfully acknowledged: Messrs J N
Swithenbank, M L Patel, A F Bower and M R Coupe.
The encouraging support received from all my friends, in
particular Mr J S Hunjan and Dr D K Bhanot is greatly apprecia
ted.
My thanks go to all my laboratory colleagues and other
research students for their friendly cooperation in every
possible way.
Thanks are also due to Mrs J Smith for her patience and
competence in transforming the pencil-written draft into this
typed manuscript.
Finally, my wholehearted thanks go to my parents and all
the members of my family for their words of wisdom, inspiration
and financial assistance.
iv
SUMMARY: NOVEL LUMINESCENT IMMUNOASSAYS
Chemiluminescence and fluorescence immunoassays are increa
singly being employed in analytical sciences in order to circumvent
problems associated with radioimmunoassays. Fluorescence immuno
assays in practice suffer from background interferences, and have
inferior limits of detection. Chemical excitation of conventional
fluorescent labels via peroxyoxa1ate offers an attractive approach
for improving the detection limits of fluorescence immunoassays.
Initial studies were primarily concerned with (a) development
of a peroxyoxa1ate-f10w injection analysis 1uminometer, because
reproducibility was poor in the static system; (b) solubility of
bis-(2,4,6-trich10ropheny1)oxa1ate in aqueous environments; and
(c) analytical parameters affecting peroxyoxa1ate chemiluminescence.
Following these studies, simple methods to remove serum fluores
cence and the peroxyoxa1ate chemiluminescence quenching problems
were examined. Calcium hydroxy1apatite, Blue Sepharose CL-6B,
etc were employed to reduce serum fluorescence. The non-specific
quenching problem, on the other hand, was eliminated using
imidazo1e-HC1 buffer.
Fluorescence immunoassays utilising fluorescamine- and
fluorescein-thyroxine were investigated for developing a polari
sation immunoassay and a chemiluminescence immunoassay based on
peroxyoxalate excitation. The preliminary work included: (a) studies
into poor reversibility of the fluorescence enhancement immunoassay
when fluorescamine-thyroxine was utilised, (b) possibility of
v
recycling immobilised antibodies, and (c) a simple approach for
enhancing assay sensitivity, namely the two-phase sequential
addition technique. Further work with thyroxine involved the
development of chemi- and photoluminescence assays based on the
enzyme-linked immunosorbent assay technique. The performance
of these assays against the standard colorimetric procedure is
discussed.
Finally, a viable chemiluminescence enhancement immunofssay
for human serum albumin based on peroxyoxalate excitation was
developed. In this study a wide variety of fluorescent labels,
for example, fluorescein isothiocyanate, dansyl chloride,
fluorescamine, etc and the effect of fluorophor:protein ratio
on the chemiluminescence signal were examined. The kinetics of
the enhancement effect were briefly investigated to provide
an insight into the mechanism. The results obtained using the
chemiluminescence enhancement immunoassay correlated well with
a standard human serum sample and the colorimetric procedure
based on bromocresol green. The potential of this technique in
the development of a generally-applicable chemiluminescence enhance
ment immunoassay is discussed.
vi
CONTENTS
Frontispiece Certificate of Originality Dedication Acknowledgements Summary: Novel Luminescent Immunoassays
CHAPTER ONE: 1.1
I NTRODUCTI ON General Background: Antibodies
the Dual Role of
Page No
i
i i
iii iv v
1
1
1.2 Classification of Ligand Assays 3 1.2.1 cZassification of Immunoassay
Techniques 3
1.3 Principles of Immunoassay 4 1.3.1 Non-Gorrrpetitive Binding Assays 7
1.4 Non-isotopic Immunoassays 9 1.5 Luminescence Spectroscopy: Fundamental
Concepts 9 1 .5. 1 Chemie~ectronic Reactions .,. 15 1 .5.2 Peroxyoxa~ate CL: Genera~
Background 18 1.6 Fluoroimmunoassays: Basic Principles 20
1.6.1 Fluorescent Labels
1.6.2 Heterogeneous FIAs
1 .6 .3 Homogeneous F IAs
· ..
1.6.4 Major Advances in FIA Techniques
1.7 Bio- and Chemiluminescence Immunoassays 1.7.1 General Background
1.8
1. 7.2 Indicator Reactions: Bio- and
1. 7.3 Enzyme 1.8.1
Chemi~uminescence .••
Luminescence Immunoassays
Immunoassays General Background
1.8.2 Heterogeneous Assays
1.8.3 Homogeneous Assays
· .. · ..
· ..
· .. 1.8.4 Some Recent Advances in EIAs
22 22
24
29
30 , 30,
3l 33 35
35
36
38
39
Page No
1.9 Novel Luminescent Immunoassays: Research Programme 40
CHAPTER TWO: MATERIALS AND METHODS 41 2.1 Materials 41
2.1.1 Synthesis of TCPO • • . 44 2.2 Labelling Techniques: Some Fundamental
Aspects 44 2.2.1 Conjugation Procedures Employed
in LIA Studies 48
2.3 Instrumentation 50 2.3.1 2.3.2
Fluorimetry ... Lwninometry
2.3.2.1 Design of the Peroxyoxalate-fia Lwninometer
2.3.2.2 General Analytical Procedure .,.
50 50
52
56
2.3.2.3 Analytical Parameters Affecting PeroxyoxaZate CL 56
2.3.2.4 Performance of the Peroxy-oxaZate-fia Lwninometer 60
CHAPTER THREE: STRATEGIES FOR REDUCING 8ACKGROUND INTERFERENCES IN PHOTO- AND CHEMILUMINESCENCE ANAL YSIS 64
3.1 Introduction: the Background Interference Problem 64
3.2 Experimental 71 3. 2. 1 Genera l : photo- and ChemiZwnines-
cenae Measurements 71 3 . 2 . 1 • 1 Procedures: Background
Photolwninescenae Redun-tion 71
3.2.1.2 TCPO-ExCited Lwninescenae: Non-specific Quenahing and Elimination Procedures 74
3.3 Results and Discussion 75 3.3.1.1 Background Photolwninescenae
Reduntion: Evaluation of Adsorbents 75
3. 3 . t 2 Elimination of Peroxyoxa late CL Quenahing: Physico-Chemical Approaches 85
3.4 Conclusion 90
Page No
CHAPTER FOUR: LUMINESCENCE-LINKED IMMUNOASSAYS FOR THYROXINE 93
4.1 General Introduction: Biological Role
4.2
4.3
and Assay Techniques 93 Experimental: Luminescence and Radioimmunological Procedures 4.2.1 General-
4.2.1 .1 Fl-uorimrmowassays uti-
97
97
Using FL-T 4 • • • 98
4.2.1.2 Reayal-ing of Antibodies 99
4.2.1.3 Luminesaenae Immuno-assays Util-ising FITC-T4 100
4.2.1.4 Luminesaenae Immunoassays Based on ELISA 10]
Results and Discussion 4.3.1.1 Fl-uoroimrmowassays Util-ising
FL-T4 4.3.1.2 Reayal-ing of Antibodies ...
4.3.1.3 Luminesaenae Immunoassays Util-ising FITC-T 4
4.3.1.4 Luminesaenae Immunoassays Based on ELISA
103
103 111
113
122
4.4 Conclusion 125
CHAPTER FIVE: ENHANCEMENT CLIA FOR HUMAN SERUr~ ALBUMIN ... 129
5.1 Introduction: Physiological Role and Assay Techniques 129
5.2 Experimental 132
5.3
5.4
5.2.1 General- 132
5.2.1.1 Comparative Studies of Anal-ytiaal- Parameters 132
5.2.1.2 Immunoassay Studies
Results and Discussion 5.3.1 .1 Comparative Studies of Anal-y
tiaal- Parameters
5.3.1.2 Immunoassay Studies
Conclusion
133 134
134 138
152
CHAPTER SIX: GENERAL CONCLUSION REFERENCES
Page No
154
160
CHAPTER ONE: INTRODUCTION
1.1 General Background: the Dual Role of Antibodies
An elegant feature of the immune defence system is its
abil i ty to "tailor" an anti body in response to exposure to an
immunogent (bacteria, virus, etc) and its subsequent inactivation
to abolish the harmful effects. The hallmark of adaptive immunity
is the specific non-covalent interaction between an antigen*{Ag)
and an antibody (Ab). namely immunoglobulin G (IgG). Immuno
globulin G is a glycoprotein of molecular weight of 160,000 and
cons i sts of two long "heavy" cha ins and two shorter or "1 i ght"
chains [1]. The chains are linked together by disulphide bonds
to form a V-shaped structure. The ends of the heavy and the light
chains of IgG form two identical antigen binding sites, which are
highly antigenic specific. The high specificity and sensitivity
of antibodies was realised by Thomson [3] in 1941; he stated:
"conceivably. the endocrinologist may utilise the action of
specific anti-hormones in the study of the interactions of the
endocri ne gl ands" . Immunoassays, thus exploit the two outstandi ng
properties of antibodies: structural specificity. representing
the capacity of Abs to differentiate between Ags of similar
structure [4]; and high intrinsic sensivitity, referring to the
utilisation of antibodies for the assay of subnanomolar quantities
t Immunogen: a substance which provokes an immune response [2].
* Antigen: a substance which combines with a specific antibody [2].
1
of an antigen. Thus, the greater the difference in affinity of
the antibody for the two closely related structures, the more
specific the assay.
All immunochemical techniques are based on the use of anti
bodies as specific binding proteins, but these techniques differ
in the mechanism by which the Ag-Ab interaction is monitored.
Thus the classical methodologies [5], as exemplified by precipita
tion methods, rely upon the interaction of an antibody (at least
divalent) with its corresponding antigen (multivalent) to register
a visible or instrumentally-detected response. These techniques
are hard to use for the quantitation of low molecular weight
species, for example haptens1", which do not form a precipitate
upon reaction with antibody. The other shortcomings [5] of these
methodologies include: lack of sensitivity, a requirement for a
relatively large amount of monospecific antibody, and a relatively
long incubation period. However,these shortcomings are circum-
vented through the use of labelled immunochemical reactant
(labelled Ag or labelled Ab) techniques. These techniques exploit
the physico-chemical properties of a label (a luminescent group)
to serve as a qualitative index of Ag-Ab interaction. The distribu
tion (or a change in the signal) of the labelled reactant in an
assay system permits quantitation of the sample (unknown) from a
standard dose-response curve.
The explosive impact of the labelled immunoassay techniques
1" Hapten: a low-molecular weight (~ 4000) substance that is an antigen but not an immunogen, unless otherwise coupled to an immunogenic carrier such as albumin [1].
2
is quite evident from its plethoric applications. for example
clinical diagnosis. pharmacology. taxonomy. quality control.
forensic. and environmental sciences.
1.2 Classification of Ligand Assays
Ligand assays make up a group of assay techniques [6] that
depend upon specific non-covalent interactions between ligands
(substance to be bound) and binding proteins and which obey the
law of mass action. They are usually subdivided [5,6,7] into
three categories: (a) immunoassays. employing antibody and anti
gen; (b) receptor assays, employing receptor proteins. for
example steroid receptors for the assay of steroids; and (c)
protein binding assays, for example thyroxine binding globulin
for the assay of thyroxine. The assays are either heterogeneous
or homogeneous. Heterogeneous assays, for example, radioimmuno
assays always require a physical separation of Ag and Ab-Ag
complex prior to measurements. On the other hand, homogeneous
assays exploit a change in the physico-chemical properties of a
label. for example, a fluorescent group in fluorescence enhancement
immunoassays and hence avoid the need for a separation procedure.
Immunoassays currently dominate the area of ligand assays.
1.2.1 Classifioation of Immunoassay Techniques
This is based [8] on the reaction mechanisms in which labelled
immune reactants are employed. namely
3
a) By the nature of the reaction - this reaction can either be
competitive or non-competitive.
Competitive binding assays are basically of two types: the
simultaneous addition assay technique in which all reagents
are incubated together, and the sequential addition assay
technique in which an unlabelled antigen is incubated with a
specific antibody prior to the addition of a labelled anti
gen. Non-competitive binding assays are exemplified by the
immunoradiometric and their variations (see Table 1);
b) On the basis of whether an antigen or an antibody is labelled;
c) On the basis of whether an antigen or an antibody is to be
detected;
d) On the basis of any separation step employed.
1.3 Principles of Immunoassay
Radioimmunoassay (RIA) techniques pioneered by Yalow and
Berson [9] represent a class of competitive binding assays. The
mathematics [6,10] of the Ag-Ab reaction are based on two assump
tions: the reactants obey the law of mass action; and that
the antibody is incapable of discriminating between a labelled
antigen and an unlabelled antigen. The equilibrium between an
antibody and its corresponding antigen can be represented (equa-
tion 1) as:
kl [Ag] + [Ag*] + [Ab]<=, =:kr==. =' [Ab Ag*] + [Ab Ag]
2 standard
or known
Limited
4
(1 )
where: [Ag] = the molar concentration of unlabelled Ag,
[Ag*]= the molar concentration of labelled Ag,
[Ab] = the molar concentration of unlabelled Ab,
[Ab Ag*] = the molar concentration of Ab Ag* complex
[Ab Ag] = the molar concentration:ofAb Ag complex
k1 = the rate association constant for the forward
reaction
k2 = the rate dissociation constant for the backward
reaction
k1/k2 = Ka' the affinity constant [11] of the Ab
The proportion of *Ag bound to the Ab is thus inversely
proportional to the amount of unlabelled Ag in the sample and
is determined from a standard dose-response curve (Figure 1).
Since the labelled antigen produces no significant change upon
binding to its corresponding antibody, a separation step is
therefore an integral part of R1A, thus making the assay rather
difficult to automate. The separation procedures [7,12] usually
employed are: (a) use of second precipitating antibodies,
(b) the use of solid-phase antibodies, (c) fractional precipita
tion of antibodies, for example with ammonium sulphate, po1yethy-
1ene glycol, alcohol, etc, and (d) adsorption of the free antigen
on dextran-coated charcoal, silicates, etc. One of the principal
advantages of utilising radioactive (y-emitter such as 125 1) labels
is the sensitivity with which the radioactive emission can be
detected to determine extremely low analyte concentrations, often
picomolar to femtomo1ar quantities. However, R1As being hetero-
geneous are (a) difficult to automate, (b) time-consuming, and
5
5000
\ \ \
Counts miri- 1
o
\
\ \ , , ,
..... ...... ---1 Concentration of Ag (ng.m1 )
FIGURE 1: Example of a standard dose-response curve showing both the decrease of the antibody-bound labelled antigen and the increase of the free labelled antigen resulting from the addition of increasing concentrations of unlabelled antigen in an assay system
6
Ag*
(c) error-prone due to complexities in sample manipulation and
due to non-specific adsorption of sample. In addition, radio
isotopes present health hazard problems. Consequently, there has
been an upsurge in non-isotopic label techniques (Section 1.4) since
the last decade.
1.3.1 Non-Competitive Binding Assays
These assays were developed by Miles and Ha1es [13] in order
to circumvent a number of disadvantages associated with the compe
titive binding assays. The main criticisms [14] of competitive
binding assays are: (a) all the unknown antigen does not react
with the antibody, (b) there is a substantial background activity
(ca. 50~) at zero antigen dose, and (c) labelling an antigen may
alter the immunoreactivity. The use of labelled antibodies in immuno
radiometric techniques was therefore suggested for circumventing
these disadvantages. Furthermore, antibodies are easier to label
than most antigens, and also, they can be labelled with higher
specific activity than most antigens, thus resulting in enhanced
assay sensitivity. A severe limitation of the immunoradiometric dS~YS
and the i r va ri ants (sandwi ch, double sandvli ch etc, see Table 1),
however, is the dissociation of hapten-Ab complexes in the presence
of solid-phase Ags. Moreover, sandwiches cannot be prepared with
hapten. As a consequence, immunoradiometric techniques and their
variants are confined to analysis of macromo1ecu1ar antigens for
example IgG, albumin etc. The principles of the major types of
non-competitive binding assays are outlined in Table 1.
7
Principles (bound or unbound Assay format fraction measured after appro
priate incubation)
Immunoradiometric Ab* + Ag ... Ab*Ag + Ag-l
Sandwich (two-s He)
Doub 1 e sa ndwi ch
Indirect sandwich (e.g. radio-all ergosorbe nt test)
+ separa te AgAb* + Ab*Ag-l
(I- = sol id-phase)
f-Ab + Ag ... AgAb-l
+ Ab* Ab*-Ag-Ab-I
Ab t-Abl Ag· S Ab2AgAb~
+ Ab*
Ab*Ab2AgAbj1
(Abl and Ab2 represent first and second Ab)
i-Ag + Ab (IgE)
+ bAgAb + Ab* (anti-IgE)
+ rAg - Ab - Ab*
TABLE 1: Major types of non-competitive binding assays
8
1.4 Non-isot opic Immunoassays
The non-isotopic immunoassays (NIIAs) are of special interest,
because they overcome the problems of health hazards and special
disposal problems, the non-isotopic labels have longer shelf-life
than the radioactive labels and they avoid the use of specialist
radioactive counting equipment. Furthermore, the assays may be
homogeneous and hence easily automated. The various types of NIIAs
are summarised in Table 1.1. Currently, immunoassay techniques
employing fluorescence, chemiluminescence and enzyme labels are
the methods of choice, because of (a) high intrinsic sensitivity, -1 ng.m1 in many cases; (b) economical advantages, fluorescent
and chemi1uminescent labels are inexpensive, and simple equipment
such as filter fluorimeter can be used for detection; (c) rela
tively simple labelling procedure; and (d) assays may be homo
geneous. On the other hand, NIIAs,for example, meta11oimmuno-
assays, are heterogeneous and require the use of complicated
equipment. However, the decisive factors upon which choice of
assay is likely to be based will be the sensitive determination
and practicality (ease of performance, low cost, etc). Non-
isotopic immunoassays have been reviewed in a number of pub1ica-
tions [15,16,17,18,19].
1.5 luminescence Spectroscopy: Fundamental Concepts
luminescence is the light emitted by an atom or a molecule
after it has absorbed energy to go to an excited state [20]. The
term chemiluminescence (Cl), bioluminescence (Bl), and fluorescence
9
~
o
Immunoassay Technique
Enzyme Immunoassay
Particle Counting Immunoassay
Viroimmunoassay
Electroimmunoassay (Voltammetric immunoassay)
Fluoroimmunoassay
Bio- and Chemiluminescence Immunoassays
Label
(a) Enzymes (b) Enzyme cofactors (c) Enzyme inhibitors (d) Enzyme substrates
Particles: (a) La texes (b)L iposomes (c) 501s (d) Microspheres (e) Erythrocytes
Virus (bacteriophage)
Electroactive groups
(a) Fluorochromes (b) Fluorogens (c) Fluorescence
quenchers
Bio- and Chemiluminescent
TABLE 1.1: Major types of NIIA techniques
Comments
Homogeneous assays possible, simple labelling procedure, long shelf-life and simple detection system required, for example spectrophotometer
Homogeneous assays possible, simple labelling procedure, long shelf-life (except liposomes) and simple detection system required (spectrophotometer, particle counting etc)
Homogeneous assays possible, limited shelf-life and difficult labelling procedure
Homogeneous assays possible, difficult labelling procedure, and susceptible to background interferences
Homogeneous assays possible, simple labelling procedure, long shelf-life, simple equipment required, but susceptible to background interferences (serum fluorescence and scattering)
Homogeneous assays possible, long shelf-life, high intrinsic sensitivity (pico- to attomole levels), simple detection system, but have poor precision due to time-dependence of signal
describe a particular example of the general phenomenon of lumines
cence. These different forms are concerned with the excitation
mechanisms (see below), which occur prior to the emission of
luminescence. Aristotle (384-322 BC) described the BL of prutefying
fish (bacteria) and fungi in "De Anima". The term fluorescence
was applied by Sir G Stokes in 1852, to the blue-white observed
in the mineral f1uorospar(Latin f1uo, to flow). The present
discussion deals with molecular luminescence only.
The fundamental step of any photoluminescence process is the
excitation of a molecule from its ground state to a higher elec
tronic state (Sl' S2 etc) by absorption of a quantum of electro
magnetic radiation of an appropriate wavelength [20,21]. The
excited states are transient and quickly collapse to the ground
state with a concomitant release of some of the absorbed energy.
In most molecules all the energy is released as thermal energy to
the surroundings, but in some cases some of the energy is emitted
in the form of fluorescence or phosphorescence, depending on the
pathway followed when returning from the excited state (Figure 1.1).
Alternatively, the excited states (singlet or triplet) formed
initially as a result of photon excitation (or by chemical excita
tion) in certain circumstances may transfer their excitation energy
to an energy deficient acceptor mo1ecu1e.-The principal mechanisms
[21,22,23] of energy transfer (ET) are:
a) Radiative ET. This process has been termed [24] "trivial",
not because it is unimportant, but because it is easy to conceive
and explain: life on earth depends upon radiative ET from the
11
Se ----.~-,I---
Absorption (1015 sec- l )
I
Internal conversion (1013 sec- l )
Fluorescence 8 -1 (10 sec )
Intersystem crossing 8 -1 (10 sec )
......... ~
T e
I
I I
Phos phorescence (100- 3 sec- l )
S o
I I V
FIGURE 1.1: Electronic energy state diagram showing the photophysical events of absorption, fluorescence, and phosphorescence. Typical unimolecular rate constants in sec- l are given in brackets. Radiative and non-radiative transitions are represented by continuous and broken lines, respecti vely.
12
/
sun [25]. The mechanism of ET depends on the capture of a photon
by an acceptor, A, emitted by ,a donor, D (equation 1.1):
D* -----"""""- D + hv (l. 1 )
A + hv - """ A* (excited state)
Radiative ET is characterised by (a) change in the donor emission
spectrum, (b) invariance of the donor emission lifetime, and (c)
lack of dependence upon viscosity of medium. The process is
considered to be the dominant mechanism of ET in dilute solutions
and can occur if the donor and acceptor chromophores are more
than 1000A apart. The probability of reabsorption by a given
acceptor varies as r-2, r being the donor-acceptor separation.
b) Non-radiative ET: This process entails the transfer of non
radiative energy from an excited fluorophor (donor) to the ground
state of a neighbouring chromophore (acceptor). The possible types
of ET (equations 1.20-1.23) between donor and acceptor molecules
are:
i ) Singlet-singlet ET
'D* + 'A ... 'D + 'A* (1.20)
ii) Triplet-singlet ET
3D* + 'A ... 'D + 'A* (l. 21 )
i i i ) Singlet-triplet ET
'D* + 'A ... 'D + 3A* (l. 22)
13
iv) Triplet-triplet ET
(1.23 )
These transfers are considered under the resonance ET and the
exchange ET process. The resonance ET is a long-range process
and can occur over considerable distances (SOoA or more). The
efficiency of ET varies as the inverse sixth power of the distance
between the donor and acceptor chromophores. The singlet-singl,et
ET is fully permitted, because the process does not entail a change
in spin, in either component (equation 1.20). Triplet-singlet ET
(equation 1.21), however, is less common due to spin-forbidden
transition. Singlet-triplet ET (equation 1.22) and triplet-triplet
ET (equation 1.23) resulting in triplet-sensitised phosphorescence
are also not generally observed due to spin-forbidden transitions.
The electron exchange process depends on the interchange of
electrons between D* and A (equation 1.3):
D* + A ~ (D---A)* ~ D + A* (1. 3)
The exchange mechanism of ET is a short-range (lO-lSOA) phenomenon
compared with resonance ET. The permitted transfers in this mecha
nism are singlet-singlet, triplet-singlet, triplet-triplet
(commonly) and singlet-triplet (see equations 1.20-1.23). The
exchange mechanism of ET is a diffusion limited process and con
sequently, its rate depends upon the viscosity of the medium.
The rate of ET is also subject to an exponential decrease with
14
decreasing acceptor concentration. On the other hand, the
resonance ET process is not diffusion limited, it does not depend
upon solvent viscosity and can be observed at lower concentrations.
1.5.1 ChemieZectronic Reactions
These are reactions [26] in which at least one elementary
step involves the conversion of chemical energy into electronic
excitation energy. The relationship [27] between photo- and chemi
luminescence is depicted in Figure 1.2. It is apparent that the
emitted light should have the same energy distribution in photo
and chemiluminescence. If, however, the luminescent species is not
a reaction-product molecule formed directly as a result of an
exergonic reaction (e.g. luminol) then sensitised (ET) CL can
occur (equation 1.4)
A + B + - -> D* (electronically excited)
D* + Acceptor -> D + Acceptor*
*Acceptor -> Acceptor + Light
The requirements [28] for an electron transfer reaction
(1.4 )
are that the electron must approach the acceptor at an energy
level equal to or greater than the first excited state, and
secondly, the excited system must be free from a strong quenching
environment. The criteria [29] for the occurrence of CL reaction
include:
a) sufficient excitation energy
15
2
-hv
c
---------
:3
+hv
(Ground state)
FIGURE 1.2: A diagrammatic repres~ntation of the relationships between photo- and chemiluminescence process. Path (l) and (2) = chemical excitation and CL emission, respectively; path (3) and (2) = photon
.excitation and photoluminescence, respectively.
16
b) species capable of transfer into an electronically excited
s ta te,
c) a chemical reaction proceeding at a relatively high rate to
provide the excitation energy, and
d) a system of reaction coordinates favouring excited state
production.
Chemiluminescence efficiency, QCl' is the number of photons
emitted per molecule reacting [30]. The overall QCl (equation 1.5)
is a product of two separate efficiencies: excitation efficiency
(Qex) of the chemical reaction, that is, the number of electronically
excited product molecules formed per molecule reacting; and
fluorescence efficiency (Qf)' representing the number of photons
emitted per electronically excited molecule.
The Cl intensity (Cll; equation 1.6) is a product of reaction
ra te and QCl.
dc CLl = QCl x d£
(1,. 5)
(1 .6)
where: Cll = number of photons emitted per second;
dc d£ = reaction rate (number of molecules reacted per second).
Generally, Cll decreases with time because of reactant consumption.
The principal advantages of Cl methods over conventional fluorimetry
17
are lower limits of detection and simple equipment requirements.
1.5.2 Peroxyo:rnZate CL: GeneraL Background
Peroxyoxalate CL is a generic name representing a class of
reactions involving peroxyoxalate intermediates [31]. It is
reported [32] to be the most effective (Qex = 60%) non-biological
CL reaction. Peroxyoxalate CL has been reviewed in a number of
publications [30,32,33,34]. The oxalate esters such as bis(2,4,6-
trichlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate
(DNPO) react with hydrogen peroxide in the presence of a suitable
fluorophor (anthracene) to produce CL (see below) via an energy
transfer process involving a high energy intermediate.
The electron transfer in the excitation step proceeds via a
charge-transfer complexation process. In general, charge-transfer
complexation (equation 1 . .]) occurs [35,36] when one partner is an
electron donor, D and the other is an electron acceptor.
(1 .7)
The formation of a charge-transfer complex is interpreted in terms
of interactions between the w electrons of the donor-acceptor pair.
McCapra [32] has proposed a pathway (scheme 1) which permits
electron transfer as the excitation step in the peroxyoxalate CL.
The most commonly employed oxalate ester in peroxyoxalate CL
studies is TCPO, because (a) DNPO is not as shelf-stable as TCPO,
(b) DNPO is difficult to synthesise, and (c) CL of DNPO is of shorter
18
TCPO
+
+ Anthracene
Radical cation
ca e -2
+ CO2
Radical anion
l,2-dioxetanedione
a a
a-a
Charge-tra nsfer comp1 ex
El ectron~ transfer
L
OCX)'" '" * I I ~G ~ ....-:: ....-:: H
T
+ Excited state
2c oe
SCHEME 1: Mechanism of TePO-excited luminescence, using anthracene as an example
19
duration [37] than CL of TCPO. On the other hand, DNPO gives a
brighter emission than TCPO, because QCL increases with increa
sing electronegative substitution [38]. The most attractive
feature of peroxyoxalate CL is versatility:emission wavelength
can be varied and is characteristic of the excited fluorophor.
Furthermore, the kinetics of the emission can be modified by
alterations in solvent [39]. Evidently (Table 1.2), the most
efficiently excited fluorophors are those with high fluorescence
efficiency and lO~1 excitation energy [31,40]. The ma,jor limita
tion of the peroxyoxalate system, however, is that the oxalate
esters (TCPO, DNPO, etc) yielding efficient CL are not soluble
in water.
1.6 Fluoroimmunoassays: Basic Principles
The use of fluorescent labels in immunoassay techniques
provides an elegant approach, because such labels lend themselves
to simple conjugation, high intrinsic sensitivity and excellent
selectivity. The sensitivity advantage of the technique arises
from the direct emission of the excited fluorescent label. The
selectivity advantage of the technique is derived from the depen
dence of fluorescence on the excitation and the emission wave
lengths of the fluorescent label. The utilisation of fluorescence
lifetimes and fluorescence polarisation enables further assay
selectivity. Moreover, the use of photophysical parameters such
as polarisation enables the development of homogeneous assays.
20
Fl uorophor Relative CLl Qf t.xcltatlon t.!Jfrgy
(KCal.mol) Emission Wavelength (nm)
Peryl ene 900 0.94 66 470
Anthracene 42.3 0.36 76 400
Pyrene 1.6 0.32 77 385
Phenanthrene 1.4 0.13 83 365
Fluorene 1.35 . 0.80 95 310
TA8LE 1.2: ·Comparison of data [31] for TCPO-excited luminescence of various fluorophors
1 .6.1 . Fluorescent wbe Ls
Coons and co-workers [41] in 1941 utilised f1uorescein
labelled antibodies as topographical markers for the localisation
and identification of antigens in tissue cells. It is only
recently that an extensive use of fluorescent labels in immunoassay
techniques has been made. The choice of a label is critical [42,43]
in developing FIAs. The label should, therefore, be (a) sensitive
to the environment (b) soluble in water, (c) stable during storage
and experimental conditions, (d) not influenced by matrix effects
including light scattering, (e) highly fluorescent (high Qf) and
(f) easy to conjugate. t10reover, the label should not produce
any adverse immunological effects. The most widely employed
labels in FIA techniques are shown in Table 1.3.
1.6.2 Heterogeneous FIAs
The main types of heterogeneous assays are (a) competitive,
(b) f1uoroimmunometric (immunof1uorimetric), (c) sandwich, and
(d) indirect sandwich. The solid-phase procedures have received
most of the attention thus far, because of simplicity of separa
ting the bound and the free fractions. The commercially available
FlAX system developed by International Diagnostic Technology
(Santa C1ara, California, USA) is based on the use of plastic
dipsticks called ".STIQ". In this system, the antigen or antibody
is immobilised on the surface of the "STIQ" sampler. The sampler
is incubated with the sample containing a fixed amount of the
labelled antigen and washed prior to fluorescence measurements
22
N W
Fluorescent Label Sens it i v ity to Excitation/Emission Environment Maxima. (nm)
Fl uram Sensitive 394/475
DNS-Cl Sens itive 340/480-520
MDPF Sens itive 395/485
RBITC Stabl e 550/585
FITC Stab 1 e 492/518
TABLE 1.3: Physico-chemical properties of fluorescent lables.
Abbreviations: Fluram = 4-phenylspiro-(furan-2(3H)-1-phthalan)-3,3-dione DNS-Cl = Dimethylaminonapthalene-5-sulphonyl chloride MDPF = 2-methoxy-2,4-diphenyl-3(2H)-furanone RBITC = rhodamine-B 200-isothiocyanate FITC = fluorescein-isothiocyanate
Remarks
Prone to inferference by serum fluorescence
Prone to interference by scattered 1 i gh t and serum fluorescence
to remove the unbound labelled antigen and the sample materials.
The Immuno-Fluor assays developed by Bio-Rad Labs (Richmond,
California, USA) employs transparent polyacrylamide immunobeads
of 5-10 microns in diameter. The FlAX and the Immuno-Fluor systems
can both be adapted to perform competitive binding assays, sand
wich assays, etc.
The principal advantage of the heterogeneous assays tech
nique is enhanced assay sensitivity due to the removal of back
ground interference (ChapterThree). The detection limits of the
technique thus-depends on the intrinsic fluorescence of the
solid-phase (immunobeads) materials and light scattering
(mainly Tyndall and Rayleigh) interferences. On the other hand,
the shortcomings of the technique include (a) high-cost of the solid
phase immobilised antibodies or antigens, (b) poor precision mainly
due to non-specific adsorption of sample materials and sample
losses, and (c) difficulties in automation.
1.6.3 Homogeneous FIAs
Table 1.4 shows the various types of homogeneous FIA tech
niques. Some of these are described below:
a) Enhancement and quenching FIAs: The interaction of a labelled
antigen upon binding to its appropriate antibody may induce a
change in the polarity of the microenvironment of the fluorophor
and hence affect its signal. The first enhancement FIA for
thyroxine utilising a fluorescein-thyroxine conjugate was des-
24
Technique Reference(s)
Enhancement FIA 44,45,46,47 Quenchi ng FIA 42,48 Energy transfer FIA 42,47,48,49 Polarisation FIA 42,47,50,51 Double antibody binding FIA 42,52 (mixed binding reagent FIA) Protection FIA 42,48 "D; fferentia 1 heavy-atom" quenchi ng FIA 53 (solvent quenching FIA) Internal reflection FIA 54 Release FIA 55 Autocorrelat;on FIA 56 Cytometric FIA 57
TABLE 1.4: Homogeneous FIA techniques
25
cri bed by Smith [44]. The fluorescence enhancement phenomenon
was ascribed to intramolecular quenching of the fluorescence
of fluorescein-thyroxine by the iodine atoms of thyroxine, possibly
via the heavy-atom effect, the effect being relieved or abolished
upon binding to anti-thyroxine antibodies. Lim [45] recently
developed enhancement FIAs for haptens and macromolecular anti
gens utilising the fluram enhancement effect. The quenching FIA,
on the other hand, is based on quenching of the fluorescence of
a labelled antigen when an antibody complex is formed [47].
Thus, for example, binding of fluorescein-gentamicin to anti
gentamicin antibodies induces quenching, the effect being reversed
in the presence of unlabelled gentamicin.
The attractive feature of these techniques is that they are
simple to perform. However,the techniques suffer from background
serum fluorescence (Chapter Three) and therefore have inferior
limits of detection.
b) Energy transfer FIAs: The process of energy transfer entails
the transfer of non-radiative energy, ' .. (Section 1.5) from an
excited fl uorophor (donor) to the ground state of a nei ghbolJri ng
choromophorR(acceptor). The requirements for an efficient energy
transfer process are that the fluorescence spectrum of the donor
should overlap the excitation spectrum of the acceptor, and secondly,
the donor-acceptor pair should be in close proximity (~ 5 nm).
The technique [48,49] is based on the use of an antigen and an
antibody labelled with a suitable donor (e.g. fluorescein) and
26
an acceptor (e.g. rhodamine) lapel, respectively. The interaction
of the labelled antigen 9nth the labelled antibody results in
fluorescence quenching of the donor label and possibly fluorescence
enhancement of the acceptor label. The presence of unlabelled
antigen causes the effects to reverse. The advantage of this
technique is that it is applicable to both haptens and macro
molecular antigens. Furthermore, the use of labelled molecules
cancels experimental errors in some cases. However, the technique
requires the use of moderately purified labelled antigens and it
is susceptible to background interferences. In addition, the
need to label two molecules is a disadvantageous feature of the
assay, because labelling may involve tedious purification proce-
dures, for example the use of affinity chromatography.
c) Polarisation FIA: When a fluorescent molecule is excited
with polarised light, the fluorescence is polarised to a degree
that is inversely proportional to the Brownian motion that occurs
between absorption and emission of light. Thus, for example, the
smaller the fluorophor the faster will be its Brownian motion
and hence the degree of polarisation will be low. On the other
hand, macromolecular fluorophors have relatively high polarisa
tion values due to restriction in the Brownian motion. The degree
of polarisation (equation 18) is defined [50] as:
p = (18)
27
where: 11\ = intensity of the emitted light polarised parallel
to the excitation polarisation.
11 = intensity of the emitted light polarised perpen
dicular to the excitation polarisation.
Polarisation FIA thus exploits an increase in the polarisation
value when a small labelled antigen (usually a hapten) binds
to its antibody. The concentration of the unknown antigen is .
calculated from a standard dose-response curve, which relates the
degree of polarisation to the standard concentration. Recently,
Jolley et al [58] have described an automated system for the
assay of gentamicin, theophylline, phenytoin and phenobarbital.
Polarisation FIA offers the advantages of being rapid, precise,
and intrinsically sensitive. It is limited in practice to
determination of antigens with molecular weight of "" 20,000
because the technique depends on measurement of increase in the
polarisation value following antigen-antibody interaction. Further
more, rotation of the antigen-antibody complex and incomplete
immobilisation of the antibody-bound labelled antigen reduces
the theoretical maximum polarisation value of 0.5 for fully
oriented immobilised molecules [47]. The other shortcomings of
this technique include the requirement for polarisation equipment
and the susceptibility of the technique to background inter-
ferences.
d) Autocorrelation FIA: This technique [56] is based on the
antigen-antibody reaction occurring at the surface of carrier
28
microparticles (acrylamide or polystyrene). Determination of
the number fluctuation and diffusion of these particles in a
microlitre volume of the solution enables discrimination of
the slowly-diffusing particles bound to the labelled antigen and
the relatively fast-diffusing unbound labelled antigen, including
endogeneous (serum) fluorophors.
1.6.4 Major Advances in FIA Techniques
a) High Performance Liquid Chromatography (HPLC) FIA: This
approach enables enhanced assay sensitivity due to the excellent
separation capabilities of HPLC combined with the high intensity
and high monochromaticity of laser excitation [59). The HPLC
fluoroimmunoassay described by Lidofsky et al [60) for insulin
has detection limits of 0.4 ng.ml- l . The relatively high-cost
of the equipment, however, is a disadvantageous feature of the
technique.
b) Time-resolved FIA: This technique [43,61) provides an
elegant approach of enhancing assay sensitivity by enabling the
removal of background fluorescence interferences (Chapter Three).
Time-resolved fluorimetry is based on discrimination of fluoro
phors with different decay-time. The rare earth metals (europium,
terbium, etc) and the polycyclic hydrocarbons (pyrene derivatives),
are particularly useful labels in FIA techniques. The fluorescence
decay-time of the metal chelates is usually in the range of 10-
1000 ~s compared with 1-20 ns of endogeneous serum fluorophors.
29
Thus, using pulsed excitation and time-gated detection system,
the background emission is allowed to decay to negligible levels
and the fluorescence of the label is measured. Furthermore,
with pulsed excitation photobleaching and photolysis of the
sample are minimised. The principal advantages of fluorescent
metal labels are (a) high intrinsic fluorescence, (b) large
Stokes' shift (270 nm for europium), (c) narrow emission maxima,
and (d) emission wavelength may be selected outside the range
of serum fluorescence, for example 590 nm and 613 nm for the
europium che1ates. Time-resolved FIAs have recently been
described by Pettersson et a 1 [62] and Hemi 11 a et a 1 [63] ,
using the che1ates of europium.
1.7 Bio- and Chemiluminescence Imunnoassays
1 .7 .1 GeneraZ Background
Bio- and chemiluminescence immunoassays (CLIAs) are analo
gous to FIAs, differing only in the mechanism of sample excita
tion. Thus, for example, FIAs are based on photon excitation
and bio- or chemiluminescence immunoassays are based on chemical
excitation. Bioluminescence (BL) is an enzyme-mediated chemi
luminescence phenomenon, but from the analytical point of view the
two phenomena are identical [30]. Bio- and chemiluminescence
immunoassays are thus generally classified [64] as luminescence
immunoassays (UAs). The major advantages [6'4] of the procedures
based on BL systems accrue from their high quantum efficiency
and high specificity of the reaction compared with the techniques
30
based on CL procedures. Consequently, BL procedures exhibit
the potential of detecting attomo1e (10-18 moles) quantities
of an antigen [64]. The other attractive feature of these pro
cedures is that conjugation of an antigen with a BL/CL label
can influence [30,39] the reaction (BL/CL) by modifying (a) the
reaction rate, (b) the fluorescence efficiency, and (c) the
excitation efficiency. Furthermore, these three parameters
are also sensitive [30] to the changes in the environment, for
example binding of a labelled antigen to its antibody. Conse
quently, a number of possible effects can be exploited. For
example, a change in the kinetics of a BL/CL reaction upon
antibody-antigen formation may permit quantitatio'n of a sample
antigen from a single intensity-time curve.
1 .7.2 Indicator Reaations: Bio- and Chemi Zwrrinesaenae
A severe practical limitation of chemi- and bioluminescence
labels is that at the present the ,number of available reactions
yielding efficient luminescence is relatively small [30].
Furthermore, most conjugated labels (including fluorescent labels
exhibit diminished luminescence compared with the unconjugated
forms [30,65]. Consequently, this imposes a severe constraint on
the development of sensitive luminescence immunoassays (LIAs).
However, coupled reactions involving CL and BL extend the
range of analyses. For example, any reaction that produces or
consumes adenosine triphosphate (ATP), nicotinamide adenine
dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate
(NADP), flavin mononucleotide (FMN), can be coupled to luminescent
31
reactions. The most widely employed BL reactions [30,66] are:
a) Firefly (photinius) reaction):
ATP + luciferin + O2
Luciferase 2+ > Mg
AMP = adenosine monophosphate
PP = pyrophosphate.
b) Bacterial reaction:
AMP + PP + oxyl uciferi n +
+ Oxidoreductase NADH(NADPH) + FMN + H ) NAD+(NADP+) + FMNH2
Luciferase FMNH2 + RCHO + O2 Mg2+ ~ FMN + RC0 2H + light (Amax =
The limits of detection and luciferase stability depend on
490 nm)
purity of the sample. The purified enzymes enable low detection
limits and are stable for at least a day at room temperature
[30,67]. Recently, bioluminescent labels such as ATP, NAD, luci
ferase, peroxidase and glucose oxidase have also been employed in
immunoassay techniques [30,68,69].
-The most widely employed labels in CLIA techniques are
luminol and isoluminol. Luminal and its analogues (see below)
produce light in the presence of suitable oxidising systems, for
example H202/peroxidase, H202/transition metal ions (Cu2+,
2+ CO ) etc. The reaction of luminal CL is:
~"""N/'10 '1 ....... N/'l lIf
rAlO l'rH OXidiS;g ~O 1 ...... 09 + Ne
VL--..('NH system ~ 4fP0 b . t \9
(1 ) 32 LlghC (). = 430 nm) max
When, Rl = Hand R2 = H, then (1) is luminol [70].
When, Rl = Hand R2 = -~-Cl, then (1) is luminol-carbamyl o ,
chloride [70]. When, Rl = Hand R2 = -C-C-SH, then (1) is mercapto-" I
acetyl-l umi no 1 [70]. 0 For coupling [30] luminol to an antigen, luminol is usually
transformed into ei ther a diazoni urn salt or an azo carboxyl
methyl phenyl derivative. The potentials of l,2-dioxetanes
(containing adamantyl groups) labels in immunoassay techiques
has recently been suggested by Wynberg et al [71]. An attractive
feature of these labels is that thermal activation is required
to induce CL.
1.7.3 Luminescence Immunoassays
The four major types of LIAs are summarised in Table 1.5.
Some of the recently developed LIA techniques i ncl ude:
a) Solid-phase Electrochemiluminescence Immunoassay
This technique [72] is based on the use of a pair of electrodes
to oxidise the labelled antigen bound to the surface of
an anode immobilised with the antibodies. The light generated by
oxidation of the label is transmitted through a transparent cathode
and measured using a photomul tiplier tube. Alternatively, the
light can be measured directly by making the cathode an integral
part of the photomultiplier tube.
33
Immunoassay technique Label(s) Typica 1 Indicator Reaction
(a) Luminescence immunoassay Lumi no 1, i so 1 umi no 1 H202/peroxidase, H202/microperoxidase
(b) Luminescence enzyme multiplied Dehydrogenase NADH/bacterial luciferase system immunoassay technique
(c) Luminescence cofactor immuno- ATP derivatives Luciferin/firefly 1 uci ferase assay
(d) Luminescence enzyme immuno- Peroxidase Lu rn; n01 . /H202 assay
TABLE 1.5: The four major types of LIA techniques
b) Energy transfer CLIA
This technique [73] as exemplified for the assay of cyclic
adenosine monophosphate (AMP) involves the use of anti -At·IP
anti bodi es 1 abe 11 ed with 1 u minol • Peroxidase and H202 are
added to the assay sample and luminescence (sensitised) derived
from the antibody-bound fluorescein-AMP is measured at 540 nm.
c) ONPO - excited LIA
Boguslaski and Carrico [74] have described a DNPO-excited
LIA for sisomicin. In the described assay, sisomicin was labelled
with rhodamine-B and DNPO was used to excite the labelled antigen.
Increasing amounts of the unlabelled antigen in the assay caused
an increase in the luminescence of the labelled antigen.
The principal advantages of LIAs are (a) high sampling rate,
because most BL/CL reactions are fast; (b) high specificity; and
(c) low detection limits, picomolar quantities in many cases.
On the other hand, LIAs suffer from poor precision due to time
dependence of the signal. This problem, however, can be minimised
using flow injection analysis (Section 2.3.2). In addition,
homogeneous LIAs suffer from environmental influences (Chapter Three)
for example quenching.
1.8 Enzyme Immunoassays
1 .8. 1 Genera Z BaakgroW'ld
Initially, enzyme labelled antibodies were utilised [75,76] in
immunohistochemical procedures and later the enzyme labels were shown
35
to be qui te suitabl e for use in immunoassay techniques [77,78].
The high specificity combined with the catalytic properties of
an enzyme enables enhanced assay sensitivity and specificity.
Enzyme immunoassays (EIAs) have been reviewed in a number of
publications [8,79,80,81]. Automation of EIAs has been des
cribed by Saunders et al [81]. Heterogeneous EIAs are basically
of two types:
a) Competi ti ve
i) wi th enzyme-l ab ell ed anti gen
ii) with enzyme-labelled antibody.
b) Non-competitive
i) with enzyme-labelled anti-immunoglobulin for the assay
of specific antibodies
ii) with enzyme-labelled antibodies for the assay of antigens.
Homogeneous assays are exemplified by procedures such as enzyme
multiplied immunoassay technique (EMIT) and enzyme-channelling
immunoassay (see below). An example of heterogeneous assay is the
enzyme-linked immunosorbent assay (ELISA). The most widely used
labels in EIA techniques are given in Table 1.6.
1.8.2 Heterogeneous Assays
ELISA is the most popular analytical procedure, because the
use of solid-phase (polystyrene tubes, glass beads, etc) immobilised
antibodies or antigens facil itates the separation of the bound and
the free fracti on. Moreover, ELISA has sensiti vity comparable to
radioimmvnoassay techniques, because of the removal of matrix
36
Label (source)
Malate dehydrogenase (Pig heart mitochondria)
Lysozyme (Egg white)
Glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides)
Peroxidase (Horseradish)
Alkaline phosphatase
(Calf intestinal mucosa)
B-galactosidase
(Escherichia Coli)
Typical Indicator
NAD
Micrococcus luteus
NAD
P-creso 1 /H202 O-dlnisidine/H202 P-nitrophenylphosphate
o-nitrophenylB-galactoside
Technique
EMIT
EMIT
EMIT
EUSA
ELISA
EUSA/ EMIT
TABLE 1.6: Enzyme immunoassay techniques and labels
37
effects. The catalytic activity of the labelled antigen bound
to the solid-phase is inversely proportional to the amount of
sample antigen. The detection [81,82,83] of the label may be
performed (a) co10rimetrica11y using chromogenic substrates
(Table 1.6); (b) f1uorometrically using f1uorogenic substrates
such as tyramine; (c) chemi1uminometrica11y using 1umino1 CL;
(d) thermometrica11y based on heat measurements, for example
using catalase and hydrogen peroxide; and (e) e1ectrochemica11y
(see below).
Some of the problems [84,85] inherent in the ELISA procedure
are steric hindrance and diffusion limitations at the solid-phase
affecting the enzyme reaction, resulting in poor assay performance.
1.8.3 Homogeneous Assays
There are basically two types of homogeneous assays:
a) EMIT
These are of two main types:
i) Enhancement assay. This technique [86,87] is based on the
principle that binding of a labelled antigen to its antibody results
in enhanced activity of the label. Enhancement assays have been
described for haptens [88] (e.g. thyroxine) and proteins [86,87],
using labels such as malate dehydrogenase and a-galactosidase,
respecti ve1y.
38
ii) Steric hindrance or quenching assay: This technique [89]
is based on the principle that binding ofa labelled antigen to
its antibody results in inhibition of the activity of the label;
Rubenstein [87] has suggested that inhibition of the enzyme
activity is either due to steric exclusion of the enzyme substrate
or due to conformational changes in the label. Steric hindrance
assays have been developed for a number of analytes, for example
morphine using lysozyme as the label [90].
b) Enzyme-channelling immunoassay. This technique [91,92] is
based on rate acceleration generated by introducing two enzymes
that act sequentially into a microenvironment. In this technique
the antigen and the label (e.g. glucose-6-phosphate dehydrogenase)
are co-immobilised on agarose particles. Binding by antibody
labelled with a second enzyme (e.g. hexokinase) in the presence + of a suitable substrate (e.g. glucose/ATP/NAO ) results in an
increase in the rate of product (6-phosphogluconolactone/ADP/
NADH) forma ti on.
1.8.4 Some Recent Advances in EIAs
a) Electrode-based EIAs
Meyerhoff and Rechnitz [93] have described solid-phase assays
for cyclic AMP and bovine serum albumin using urease as the label
and urea as the enzyme substrate. The amount of the labelled antigen
bound to the solid-phase was measured using an ammonia gas sensing
el ectrode.
39
b) Simultaneous Dual-enzyme Immunoassay
B1ake et a1 [94] have developed a simultaneous dual-enzyme
immunoassay for thyroxine and triiodothyronine. In the described
assay thyroxine and triiodothyronine were labelled with alkaline
phosphatase and a-D-ga1adDsidase, respectively, and the antibody
bound fraction was measured ca1orimetrica11y using phenolphthalein
monophosphate and O-nitropheny1-a-ga1acoside as the substrates for
the enzymes. The advantages of this technique over the single
assay procedure include: (a) better diagnostic efficiency, for
example of thyroid disorders, (b) shorter overall assay, (c)
low-cost, and (d) smaller sample volume requirements.
1.9 Novel Luminescent Immunoassays: Research Programme
This research programme is primarily concerned with the
following developments:
a) methods for removing background interferences inherent in
photo- and chemiluminescence procedures;
b) luminescence-linked immunoassays using thyroxine (T4) as a.mode1
system;
c) TePO-excited LIA for human serum albumin (HSA) with flow
injection analysis.
40
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials
Materials/Description
Human proteins/serum
Albumin, lyophilised, electrophoretic purity 100%
Standard human serum
Sterile pooled serum (normal)
Thyrogl obul i n
Protein standard solution
Animal proteins/polypeptides
Bovine albumin (fraction 5 -purity 96-99%)
Bovine y-globulin (electrophoretic purity 99% + <1% NaCl)
Bovi ne i nsul in
Anti bodi es
Purified and monospecific rabbit immunoglobulins to human albumin and a-2-macroglobulin
Sheep-phenytoin antiserum
41
Source
Hoechst (UK) Ltd, Hounslow, Middlesex
Hoechst (UK) Ltd.
Miles Laboratories Ltd Stoke Poges, Slough
Sigma Chemical Co Ltd. Poole, Dorset
Si gma (Poole)
Sigma (Poole)
Sigma (Poole)
Sigma (Poole)
DAKO-immunoglobulins Ltd, Mercia Brocade L td, 14eyb ri dge , Surrey
Guildhay Antisera, Surrey.
Materials/Description
Monoclonal anti-T4 T 4 -anti serum
Bi ndi ng reagent
Insulin-binding reagent
Ch roma tog ra ph i c
Sepharose CL-5B
Blue Sepharose CL-5B
Column PD-10 (containing Sephadex G-25M)
DEAE-Sephadex A50
Sephadex G-25 (medium grade)
Calcium hydroxylapatite (Bio-Gel HTP)
Chemicals. Reagents. etc
Fl uorescami ne
MDPF
l-dimethyl aminonapthalene-5-sulphonyl chloride
Fluorescein isothiocyanate isomer I
Fluorescein isothiocyanate isomer I (10% on celite)
42
Source
Miles Labs (Slough)
Advanced Labs. Kent.
Wellcome Reagents Ltd. Beckenham. Kent.
Pha rmaci a L td. Hounslow. Middlesex
Pharmacia (Hounslow)
Pharmacia (Hounslow)
Pharmacia (Hounslow)
Pharmacia (Hounslow)
Bi-Rad Labs. Watford. Hertfordshi re
Roche Diagnostics Ltd. Welwyn Garden Ci ty.
Roche Diagnostics Ltd.
BDH Chemicals Ltd. Poole. Dorset
BDH (Dorset)
Calbiochem Ltd. Bishops Stortford.
Materials/description
L -thyroxi ne
Thiomeroso1 (sodium salt)
Tyrami ne hydroch 1 ori de
Hydrogen peroxide. 20 volumes (AR grade)
Titani urn di oxi de
Titanium (11) oxide granular 'Patinal' (0.8 - 2 mm)
Imidazole (SLR grade)
2.4.6-trich10ropheno1 (98%)
Oxalyl chloride (98%)
Source
Sigma (London)
Si gma (London)
Sigma (London)
Fisons Scientific Ltd. Loughborough
Fisons (Loughborough)
BDH (Dorset)
Fisons (Loughborough)
A1drich Chemical Co Ltd. Gi11ingham. Dorset
A1drich Chemical Co Ltd ( Dorset)
'TABLE 2.1: A List of the Materials Utilised
Protein Molecular Weight Reference
Human serum albumin 66.241 95 Human thyrog1 obu1 i n 660.000 96 Bovin albumin 69.000 95 Bovine insulin 5.734 97 Bovine y-g10bu1in 160.000 98 Rabbit IgG 140.000 99
TABLE 2.2: Molecular Weights of Proteins
43
2.1.1 Synthesis ofTCPO
TCPO was synthesised according to the methodology of Mohan
and Turro [37). A solution of O.lM 2,4,6-trichlorophenol (TCP)
in 250 ml of benzene was dried (50ml) azeotropically by distilla
tion. The TCP solution was cooled to lOoC and 0.H4 of freshly
distilled triethylamine was added dropwise. This was followed
by the addition of 0.055 mole of oxalyl chloride. The reaction
mixture was allowed to warm to room temperature and stand over
night. Benzene was removed from this mixture using a rotary
evaporator and to the residue 50 ml of petroleum ether was added.
This mixture was stirred for 15 minutes, filtered and further
washed with petroleum ether. The white solid was dried under
vacuum for one hour and recrystallised using chloroform [31).
The white product had a melting point of 189-191 0 C. This product
was stored (dark-brown bottle) at 40C in a desiccator.
2.2 Labelling Techniques: Some Fundamental Aspects
A prerequisite [100) in labelling a protein or other molecule
is that it should be as pure as possible, and secondly, it should
not be liable to denaturation during the labelling reaction. The
fluorescent label should possess [101) chemical groups capable of
reacting directly with proteins to form stable linkages such as
azo, carbamido or sulphonamido bonds. The chemical groups,
isocyanate, azide, diazonium chloride, and acid chloride are
examples of such groups. The fluorescent label should also be
chemically pure, because the presence of contaminants (e.g. HC1,
degradation products and isomer 11 in FITC preparation) often
44
results [100, 102] in poor protein 1 abell i ng and fl uorescence
deterioration under long storage conditions.
A variety of techniques have been employed [102] in labelling
procedures, for example the use of organic solvents (e.g. acetone
dioxane for dissolving labels), impregnated filter paper, celite
powder, dialysis labelling and the direct addition of a label.
The variables affecting a particular labelling procedure include:
concentration of buffer, pH, light, temperature, the mode of
addition (label), and the extent of the reaction between a label
and a protein. Thus, it is important to control precisely these
reaction conditions in order to optimise a labelling procedure,
and to obtain reproducibility of labelling.
The mechanism of conjugation of proteins with fluorescent
labels such as dansyl chloride can be quite complex involving
[102] :
i) reaction with cr-amino groups,
ii) reaction with E-amino groups of lysine,
iii) bonding with cysteine resulting in the formation of S-sulphonyl
cys tei ne,
iv) formation of ring-labelled compounds with the imidazole group
(histidine), and
v) O-dansyl a ted deri va ti<ves with the groups of tyros i ne.
A principal disadvantage associated with a number of fluorescent
labels such as dansyl chloride and FITe is the need to separate the
fluorescent conjugates from the unreacted fluorochromes. As a
result, Wigele et al [103] introduced fluorescamine and its analogue
45
MDPF as suitable immunofluorescent reagents. Fluorescamine(FL) and
MDPF react with primary amines to yield fluorescent derivatives. Fur-
thermore, fluorescamine is non-fluorescent and excess reagent
is hydrolysed to form non-fluorescent, water-soluble products.
thus obviating the need for additional purification steps. The
reaction (Scheme 2.1) proceeds as:
Hydrolysate (non-fl uorescent)
Fluorogenic reagent (non-fluorescent)
o Fl uorescami ne
SCHEME 2.1: Reaction of fluorescamine with a primary amine and in the presence of water
46
Conj uga te (fl uorescent)
When it is necessary to separate the labelled proteins
from the unreacted fluorochromes the purification may also
(depending on the method and label used) remove organic solvents
and hydrolyti c products from a 1 abe 11 ed product. Thes e impuri ti es
can be removed from a labelled conjugate by dialysis or by gel
filtration techniques. One of the principal advantages associated
with gel filtration is that it is less time-consuming than dialysis.
The purified labelled conjugates can be further fractionated with
respect to the degree of labelling by ion-exchange or by electro
phoretic techniques to yield fractions containing a more homoge
neous population of labelled conjugates.
The purified labelled conjugates are characterised with
respect to fluorophor:protein (F:P) ratios in a number of ways
[100). For example, the protein can be assayed spectrophoto
metrically, by the micro-Kjeldahl method or by using colorimetric
methods such as the bi uret or Lowry methods. Methods based on
absorbance measurements, however, must be corrected for absorption
by the fluorescent label at the wavelength of measurement (280 nm).
The Lowry method [97) for the assay of proteins is based on the
reaction between Folin-Ciocalteau reagent and proteins. This
reaction is mediated by copper ions and the resulting coloured
solution is measured at ",700 nm; the method is thus not usually
subject to interference by the fl uorophor. The fl uorescent dye is
assayed spectrophotometrically using its molar extinction coeffi
cient. The F:P ratios can be calculated from the use of formulae
[100) and nomographs [104).
47
2.2.1 Conjugation Procedures EmpLoyed in LIA Studies
F1uorescamine and MDPF-T4 conjugates: The conjugation
procedure described by Hand1ey et a1 [46] was employed.
This method involved rapid addition of D.5 m1 of 0.03% solution
of the label in acetone to 2.0 m1 solution containing 10 ~g m1- 1
of T4 in 0.1 sodium phosphate buffer, pH 8.0, which was being
vortex-mixed.
FITC-T4 conjugate: The method described by Smith [44] was
employed for conjugating FITC to T4. This method involved reac
ting 1 volume of 51.4 mM FITC (isomer I) with 2 volumes of
25.7 mM of T4 in pyridine/H20/triethy1amine medium of volume
composition 9:1.5:0.1. The reaction was incubated at room temp
erature in the dark for a period of 1 hour. The crude product was
precipitated with 20 volumes of 0.2M ammonium acetate buffer,
pH 4.0. The mixture was centrifuged (10 minutes) and the super
natant was decanted. The precipitate was washed by suspending it
in twenty volumes of distilled water and centrifuged (10 minutes).
The product was re-dissolved (with the aid of minimal amounts of
ammonia solution) in eight volumes of 0.05M NH4 HC03 solution.
The labelled -T4 product was chromatographed on a Sephadex G-25
column (10 m1) pre-equi1ibrated with 0.05M NH4 HC03 solution. The
impurities were removed by washing the column with twelve column
volumes 0.05M NH4 HC03 solution. The purified FITC-T4 conjugate
(bound) was eluted from the column using distilled water. The
product (FITC-T4) was lyophilised and stored at 40 C in a desiccator.
48
Fluorescein-HSA (FITC-HSA) conjugates: The methodology of
Rinderknecht [105] was employed to prepare the FITC-HSA conjugates.
FITC (10% on celite) was added to HSA (2.5 - 10 mg ml- l ) in
sodium carbonate-bicarbonate buffer, pH 9.0. Celite was removed
from the labelled solution mixture by centrifugation. This solu
tion was then chromatographed on a Sephadex G25 PO-10 column
(pre-equilibrated with 10 mM phosphate buffer, pH 7.2) to
separate the unreacted dye. The FITC-HSA conjugates were eluted
using the above buffer. The Lowry method was applied to determine
the protein concentration and a value of 7.2 x 104 M-I cm- l at
493 nm [106] was utilised for the molar absorption coefficient of
the fluorescein thiocarbamide group.
Fluorescamine - and MOPF-HSA conjugates: These were prepared
essentially according to the methodology of Weigele et al [107].
HSA (25-35 mg) was dissolved in 2.0 ml of 0.1 sodium phosphate
buffer, pH 9.5. Then, to a constantly stirred (magnetically) HSA
solution, 50 )11 or more (depending on the molar F:P ratio
required) the label solution (3-4 mg ml- l ) in acetone was added.
The labelled-HSA conjugates were chromatographed on a Sephadex
G25 PO-10 column (pre-equilibrated with 10 mM phosphate buffer,
pH 7.2) to remove the hydrolytic products of the label and to
remove the acetone. The concentration of HSA was determined by the
Lowry method. The bound-fl uorophors were assayed spectrophotomet
rically using the absorption coefficient value of 6400 ~ 60 Mrl cm- l
and 6300 ~ 100 W l cm- l at 385 nm for the MOPF- and FL-HSA conju
gates, respectively [104].
49
Dansyl-HSA (D-HSA) conjuga tes : The procedure descri bed by
Chen [108] was employed. Dansyl chloride (3-5 mg ml- I in acetone)
\~as added gently to 2.0 ml of a magnetically stirred HSA solution
(16-25 mg in O.lM NaHC0 3 maintained at ice-bath temperature).
The reaction was then allowed to proceed at room temperature for a
further period of ninety minutes. The undissolved material was
removed by centrifugation and the labelled solution mixture was
chromatographed on a Sephadex G25 PD-10 column pre-equilibrated
with 10 mM sodium phosphate buffer, pH 7.0. The concentration of
HSA was determined using the Lowry method. The bound fluoro
phores were assayed spectrophotometrically using an extinction
coefficient value of 3.4 x 10 3 M-I cm- I at 340 nm [108].
2.3 Instrumentation
2.3.1 Fluorimetry
The following fluorescence spectrophotometers were used:
Perkin-Elmer MPF 44B (Perkin-Elmer Ltd, Beaconsfield, Bucks)
Perkin-Elmer Model 2000 (Beaconsfield) and its associated polarisation accessories
Fluorescence measurements (in arbitrary fluorescent intensity
(FI) units) were performed at room temperature and the background
signal due to buffer, protein etc was substracted in FlA studies.
2.3.2 Luminometry
A prinCipal advantage of CL and BL methods is that these
methods require simple instrumentation. The required components
50
for a typical luminometer [30,109] are a light detector, a light
tight reaction chamber, provision for mixing the reactants, and
electronics to read out and process data. However, CL measurements
can also be performed using detection systems designed for other
purposes, for example fluorimeters, liquid scintillation counters,
and spectrophotometers. A major drawback of these instruments
is that these lack provision for in situ mixing of the reactants.
This is important, in particular for good repeatability, and in
the generation of a complete intensity-time curve. On the other
hand, most luminometers have provision for in situ mixing and
thus these permit recording of a complete intensity-time curve,
and also improve precision. However, luminometers employing
conventional syringe-injection methods to initiate CL reactions
are subject to poor precision. This is a consequence of force of
injection and the exact positioning of the syringe tip, which may
vary from measurement-to-measurement. Other factors affecting
precision include bubble formation upon injection (increasing
scattered light) and incomplete mixing of reagents, in particular
mixed aqueous-organic solvent systems which may be required for
dissolving certain reagents e.g. TCPO. As a result, most of the
analytical work to date involving peroxyoxalate CL has made use
of flow systems [110]. since such a system provides better control
of mixing.
Flow-injection analysis t (fia) was introduced by Ruzicka
and Hansen [111] in 1975 as a simple and convenient approach to
continuous-flow analysis. Flow injection analysis is based on three
t Designated fia to avoid confusion with FIA (fluoroimmunoassay)
51
principles: (i) sample injection into an unsegmented carrier
stream, (ii) controlled sample dispersion, and (iii) reproducible
timing of injection. The reaction products are monitored before
"steady-state" conditions are established and as a result, a
high sampling rate is possible. The other features of this
technique include low sample consumption, and good precision.
Furthermore, it offers flexibility, since by varying experimental
conditions (sample volume, flow rates and tube dimensions) a wide
range of sample zone concentrations can be' manipulated and sample
dispersion can be tailored to suit a particular analytical proce
dure.
A number of reviews [112,113,114] have been published on Ha
techniques and these techniques have been applied [30,115,116,117,118]
to monitor CL reactions.
2.3.2,1 Design of the Pe1'O:r:yoxaZate - fia Lwrrinometer
The following preliminary design studies were undertaken using
a Model 1250 luminometer (LKB Ltd, Croydon UK):
a) The use of a 'T'-shaped flow-cell (glass), in order to
i niti ate the CL reacti on in front of the detector. Thi s
arrangement was found to be unsuitable as it required synchro
nisation of flow rates of two streams, containing H202, and
sample plus TCPO, respectively.
b) The use of a helical flow-cell constructed from glass. This
suffered from leakage problems at the joints due to high
back-pressure.
52
c) The use of an injection-port (flap valve type). This also
suffered from leakage problems due to high back-pressure and
poor repeatability, probably due to variations in the force
of injection and/or poor precision of sampling.
d) The effect of tubing internal diameter on analytical para
meters. Reliable results were not achievable with tubing of
internal diameter less than 0.8 mm, probably due to poor
mixing of the reactants.
e) Determination of the length of tubing between the valve and
the point of winding on the sample tube (Figure 2.1). The
shortes t, techni ca lly feasi b 1 e 1 ength of tubi ng was employed
in order to detect maximum CL intensity.
f) The use of 0.6 ml, 0.3 ml, and 0.1 ml sample loops. The 0.6 ml
loop was found to be unsuitable, its use 1 eadi ng to producti on
of multiple sample peaks. This suggests that the CL reaction
proceeded at different rates, probably due to limited dispersion
of the sample. The use of a 0.1 ml sample loop was preferred
to a 0.3 ml loop, the former giving better repeatability and
economy of injected sample.
g) The use of a gelatin filter between the helical flow-cell and
the detector reduced the CL signal of blank buffer (phosphate,
Tris etc) solutions by about 23.5%. As a result, this filter
was employed in all measurements in which the transmission
was at least 80%. The emission maxima of TCPO-H 202 has
recently been identified (119) at 440 nm and at 550 nm.
53
si gna1 The removal of the 440 nmtmay partially explain the resul ts,
because the transmission of the filter at 450 nm was 0.04%.
The use of a pressure-bottle eliminated the air-bubble
problem which occurred with a peristaltic pump. The rate of air
bubble formation in pump-tubing often increased during its
operative period (after ten minutes wi th an aged tubing and
twenty-five minutes with new tubing): an increase in the rate ,
of H202 decomposition may explain this effect. The decomposition
process was probably catalysed by contaminants (metal ions)
present in the pump-tubing material.
The final design of the peroxyoxalate-fia luminometer was
based on these preliminary studies. The apparatus is depicted
in Figure 2.1. A helical flow-cell was constructed by winding
polyethylene flow-injection tubing (ID ~ 0.8 mm) around a cylin-
drical luminometer sample tube. The length of flow-injection
tubing around such a tube was 37 cm, corresponding to ten helical
turns (other dimensions are shown in Figure 2.1). The exposed
portions of the flow-injection tubing (near the valve and the
walls) were wrapped with black insulating tape to prevent a "fibre
optic" effect introducing ambient light into the detector. A gelatin
filter (Lee Filters Ltd, Andover, Hants) with 85% transmission at
500 nm was placed between the flow-cell and the detector. The
sample was injected into a flowing stream of H202 using a 4 way
Rheodyne valve (HPLC Technology, Macclesfield), with a sample loop
volume of 100 111. The H202 stream was propelled using either
a miniature peristaltic pump (Model 840 Ismatec, Forest Instruments
54
'" '"
H202
r-----------
T 10 turns
~ Rheodyne
valva
f .. waste
17. SCr---- ... ~ r,"' -~ -------------, ~-+-II n i I - , -:-
-', , -,, , -~
--:-
-,-I
-:-, -'-, ,
__ 1_" , ...
K2 ~I
Detector
l' Filter
Light-tight chamber , L _______________________________ J
FIGURE 2.1: Design of the Peroxyoxa1ate - fia Luminometer
Wokingham) or using a pressure-bottle (EA 1101; Roth Scientific
Co Ltd, Hampshire) connected to a nitrogen cylinder.
2.3.2.2 GeneraZ AnaZytiaaZ Proaedure
TCPO was dissolved with the aid of an ultrasonic bath at an
initial concentration of 4 mg ml- 1 in dry and distilled acetone.
This solvent minimised the rate of TCPO hydrolysis and reduced the
background CL signal. Preliminary experiments showed that the TCPO
solution (10 ~l) was soluble only in imidazole-HCl buffer (400 ~l)
As a result, this buffer was utilised (see Table 2.2 and below)
in most analyses. Hydrogen peroxide was utilised in all fia at an
initial concentration of 1.02 g ml- 1 •
2.3.2.3 AnaZytiaaZ Pa~eters Affeating PeroxyoxaZate CL
Figure 2.2 shows that increasing the concentration of KCl to
lM (in 0.22M imidazole - HCl buffer, pH 7.0) increased the CL
intensity to a maximum level. The CL intensity was measured either
in millivolt (mV) or in arbitrary units (Figures 2.3, 2.4 and all
subsequent measurements). However, decreasing the concentration
of imidazole increased the CL! (Table 2.3). A similar effect was
observed when the concentration of 1ris was reduced (Table 2.3).
However, pH exerted a considerable effect on the CL! (Table 2.3)
and an optimum response was obtained at pH 7.0. These results
(reducti on in CLl) compare favourab ly with those reported by
Williams et al [116] using mixed solvent systems.
56
Initial Concentration Observation upon Addition of Imidazole buffer, pH 7.0 of 10 vl of TCPO*/400 vl of
Sample Solution
0.02SM Turbid solution
O.OSM Turbi d sol uti on
o .125M Turbidity cleared after ten seconds of addition of TCPO sol uti on
O.20M Turbidity cleared within ten seconds of addition of TCPO solution
0.22M Clear solution
TABLE 2.2: Solubility of TCPO Solution as a Function of Buffer Concentra tion
* TCPO solution was measured using a Hamilton microlitre syri nge
57
2·8 ->
5 2.7 ....... ......J U
QJ
> 2·6 .-...... c.n re 0> ~
QJ
0:: 2.5
2·4lj,~: ~---r----.:'";:-"~1~----:M~11--0·6 0-7 0·8 0·9 lO 1·1 1-2
[ K Cl ] FIGURE 2.2: ClI as a function of KCl concentration in 0.22M imidazole-HCl buffer, pH 7.0.
The concentration of FL-T4 was 50 x 10-9m. Flow rate of H202
: 5 ml min- 1
Final imidazole concentration containing lM KCl (pH 7.0)
o .125M
0.22M
Final Tris concentration in 0.125M imidazole containing lM KCl (pH 7.0)
OM
0.05M
0.25M
0.5M
pHt of 0.125M imidazole containing 1 M KCl
5.5
7.0
9.5
CLl (mV)
6.2
2.8
CLl (mV)
6.5
6.3
5.5
5.5
CLl (mV)
0.7
6.2
0.7
TABLE 2.3: Analytical Parameters Affecting Peroxyoxalate CL. The concentra ti on of FL -T 4 was 50 x 10-9 M and the flow rate of H202 was 5 ml min- 1 (propelled using the pump)
t pH values were adjusted using concentrated HCl
59
The system based on imidazole-HCl buffer exhibited poor
reproducibility at pH 5.5 and 9.5. This may be a consequence
of an increase in the rate of TCpa hydrolysis (catalysed by acid
and base respectively). Preliminary experiments supporting this
effect showed that:
a) analytes containing TCPO solution (pH 5.5 or 9.5) if left
longer than five minutes before analysis yielded a signal
indistinguishable from the background, and
_ b) TCPO solutions prepared in dry acetone were stable for at
least thirty hours.
Consequently, a period of about eight seconds between the pre
mixing of TCPO and sample and injection was deemed necessary to
ensure reproducibility. The 0.22M imidazole-HCl buffer (pH 7.0)
was employed in most of the subsequent workt. This is because the
TCPO solution dissolved instantaneously in this buffer and hence
the rate of TCPO hydrolysis was minimised. Moreover, because
the sample solutions were not turbid and the non-specific quen
ching effect described in Section 3.3l2 was not encountered with
this buffer.
2.3.2.4 Performance of the Pe~oxaZate-fia Luminometer
Evidently (Figure 2.3). the optimised system is highly
reproducible; the coefficient of variation (Table 2.4) being
~3.5%. The relationship between CLl and FL-T4 concentration is shown
in Figure 2.4. Recently. Sigvardson and Birks [119] using peroxy
oxalate CL-HPLC system reported the CL signal to be linear with
t Exceptions will be specified wherever appropriate
60
~ 30s ~
L ~ -
FIGURE 2.3: An example of the recorder traces showing reproduGibility of the technique. The concentration of FL-T4 was 6 8 x 1O-9t~ (0.22M imidazole/HCl + 1.OM KCl buffer, pH 7.0) and the flow rate of H202 was 8 ml min- 1
(propelled using Ismatec pump)
Imidazole concentration (concentration of FL-T4 = 68 x 10-9M)
0.20M + lM KC1, pH 7.0
0.22M + Hl KC1, pH 7.0
cv (%)
3.5(n=7)
3.4(n=5)
TABLE 2.4: Precision of the peroxyalate-FIA system
61
CT> N
20
16
8
4
O~--~--~--~----~~----.-~----~~ 1x10-12 1x10 -11 1x10-10 1x10-9 1x10-8
FIGURE 2.4:
[FL -T 4] x M
Relationship between CLl and FL-T4 concentration. (propelled using the pressure bottle)
-1 Flow rate of H20
2: 5.6 ml min
fluorophor (anthracene, pyrene, etc) concentration over three
orders of magnitude. The CL intensity (equation 2) for a first
order reaction [301 is:
(2)
where: K = the first order rate constant for the light generating
reacti on
[A(t)l = concentration of an analyte as a function of
time
The non-linear relationship (Figure 2.4) between CLl and
FL-T4 concentration may thus be due to two major factors:
variations in the reaction rate and possible changes in QCL'
because efficiency Po] of an energy transfer is a concentration
dependent process.
63
"
CHAPTER THREE: STRATEGIE~ FOR REDUCING INTERFERENCES IN PHOTOAND CHEMILUrUNESCENCE ANALYSIS
3.1 Introduction: the Background Interference Problem
Luminescence spectrometry is well-known for sensitivity
(nanomolar to attomolar quantities), and selectivity. The detec
ti on 1 imits, however, in practi ce depend [120] on the performance
of the instrument; intrinsic properties of the luminescent
analyte; and various other factors. For example, various endo
geneous/exogeneous factors present in biological fluids (serum,
urine, etc) may cause an apparent enhancement or reduction in
the signal of a luminescent analyte [42]. Background inter
ferences due to the presence of exogeneous factors ari se mai nly
from poor experimental techniques, such as fingerprints on sample
cells, and from impurities (luminescent metal ions) in solvent
or sample cells. These can be minimised [120] by good experimental
techniques. Examples of background interferences due to endo
geneous factors are (a) inner filter effects, (b) quenching,
(c) luminescent materials, and light scattering, for example
Rayleigh in photoluminescence.
The inner filter effects are due to sample matrix which
absorb part of either the excitation or the emission light, and
hence cause attenuation in the luminescence signal. The quenching
effect, on the other hand, is a consequence of reduction in the
64
luminescence quantum efficiency. There are three principal
mechanisms [20, 21, 22, 120] by which the excitation energy
of a luminescent species, for example, a fluorophor may be
quenched by a quencher (Q). These are:
i) non-radiative energy transfer (energy transfer quenching)
due to resonance interaction between F* and Q (see Section
l. 5 ) ;
ii) metastable complex formation (static quenching) as exempli
fied by the formation of a charge-transfer complex between
F and Q;
iii) by collision (dynamic or collisional quenching) between
F* and Q.
The excitation of the (FQ) complex formed by process (ii),
or the formation of the transient (FQ)* complex formed by process
(iii) may result in quenching of the excitation energy in the
fo 11 ow.i ng ways:
a) by the heavy atom/ion (ethyl iodide, Ag+, etc) or paramagnetic
(oxygen, nitric oxide, etc) effects, which enhance intersystem
crossing in F*;
b) by radiationless or radiative transitions in Q*;
c) by radiationless transitions in (FQ)*, resulting in the forma
tion of a stable FQ complex;
d) by electron transfer as shown below:
Q +3F + -~
Q + F* ') Q • F ~ Q + F pol ar 1 solvent Q+ + F- (solvated ion pair)
65
An electron is transferred from Q to F*, resulting in the + -formation of the ion pair Q. F. This ion pair can dissociate
(i) to give either a triplet state, 4F, and Q, or (ii) fluorophor,
F in the ground state and Q. Processes (i) and (ii) dissipate
the excitation energy in the form of heat.
One of the most serious interferences encountered in homo-
geneous FIAs (Section 1.6.3) arises mainly from the use of serum
samples. This fluorescence interference has excitation and emission
maxima of 340 nm and 450-470 nm, respectively [43]. Unfortunately,
many fluorescent labels used in FIAs exhibit emission maxima in
the same spectral region as those of serum and antiserum. As a
result, the detection limits of most fluorescent labels in serum
samples are reported [43] to be 50 to 1000-fold worse than for
those in a pure buffer solution.
Background interference problems inherent in homogeneous
FIAs are also encountered in homogeneous CLIAs. Luminescence
(BL/CL) quenching has thus far been a major problem in analysis,
and as a result, hindered the development of viable homogeneous
LIAs. However, in energy transfer LIAs, effects due to environ-
mental interferences may be more complex: luminescence Signals
may be enhanced or quenched. For example, the homogeneous assay
of the drug sisomicin.using peroxyoxalate CL is susceptible to
quenching [74]. Hence, it follows that any technique (instrumental,
chemical etc) that is capable of obviating these problems will
enhance the detection limits of luminescence analysis.
66
Photo- and CL methods differ in the mechanisms of sample
excitation (Section 1.5). As a consequence, some of the methods
used to eliminate/remove background interferences in photo- and
CL analysis are different. For example, problems such as Rayleigh
scattering do not arise in CL measurements. This is because of
chemical excitation as opposed to photon excitation, and secondly,
endogeneous chemilumigenic substances do not occur in serum
samples. Some of the instrumental approaches used in eliminating
background fl uorescence are:
a) Bleaching 1 ifetime measurement - thi s technique [42] exploits
the difference between the bleaching lifetime of fluorescent
background and fluorescent label using an intense pulsed
exci tation and an appropriate time-gated detection system.
b) Phase-sensitive detection of fluorescence [121] - this tech
nique involves exciting the sample with sinusoidally modulated
light using a phase fluorimeter having a phase-sensitive
detector. If the lifetime of the fluorophor (proteins,
label, etc) are different, then their emissions exhibit
different characteristic phase angles. The detector phase
angle can be selected to be out of phase with the background
fluorescence, and as a result background fluorescence is
reduced.
cl Time-resolved fluorimetry (Section 1.6.4).
Some of the methods available to minimise light scattering
problems include: (i) interference filters, (ii) physical removal
of macromolecules, lipids etc, (iii) laser excitation (Section 1.6.4).
67
(iv) derivative spectroscopy [122], (v) synchronous scanning
[47], and (vi) use of a horizontal polariser [123].
Techniques based on physical/chemical removal of interfering
substances (proteins); kinetic measurements; sample dilution;
interference filters; and automatic blank subtraction are
applicable in both photo- and CL techniques. One of the most
effective methods reported [47] thus far involves pretreatment of
the sample with peracids such as peracetic or persulphuric acid.
Thi s method el imi nates 90% of serum fl uorescence. However, it
suffers from one major drawback: "not all analytes can survi ve
this treatment" [47]. Oeproteinisation methods based on ultra
filtration and precipitation of proteins using ethanol or trich
loroacetic acid (10%) have also been employed [43,124]. The prin
cipal disadvantages [43,47] of these approaches are: denaturation,
(using organic solvents) of certain samples, in particular proteins,
and fluorescent material(s) with excitation and emission wavelength
of 340 nm and 450-470 nm, respectively are not wholly removed.
In addition, the use of organic solvents in luminescence analysis
may introduce luminescent impurities into the sample. These methods
are thus unlikely to be widely employed.
Matrix effects are also quite common, for example in luminol
CL: oxidising systems such as H202 in the presence of transition
metal ions (Fe 2+, Cu2
+, etc) are capable of catalysing the CL
reaction [30]. The most widely used method of minimising this type
of interference involves treating the sample with 1,2-diamino-
ethanetetracetic acid (EOTA). EOTA forms complexes with the
68
3+ 2+ interfering ions (endogeneous Fe , Cu , etc) and thus render them
i nacti ve as ca ta 1ysts,
Elimination of background interferences by instrumental
approaches are unlikely to be widely used at present, on account
of the high cost and complexity of the methods. Simpler and re1a-
tive1y inexpensive techniques are preferred. This chapter describes
the use of several inorganic adsorbents as possible methods of
eliminating background photoluminescence interferences from
biological samples, and also, the approaches adopted to eliminate
non-specific quenching of peroxyoxa1ate CL. The possibility of
exp1 oiti ng adsorbents to remove f1 uorescent materi a1 s from
biological samples became evident from the ability of certain
adsorbents to bind proteins. Some of the materials which display
such properties are:
a) Calcium hydroxy1apatite (CH) - this is a crystallised form
of calcium phosphate, [Ca3(P04)2]3 Ca(OH)2] [125]. CH exhibits
a negligible adsorption affinity for low-molecular weight
substances (steroids, amino acids, etc), but a higher binding
affinity for macromolecules. Macromolecules are bound to
the surface of CH by electrostatic interaction in a low ionic
strength buffer ~t neutral pH and are quantitatively displaced
by increasing the ionic strength of buffer (usually phosphate),
or salt (NaC1). CH has been employed for the fractionation of
serum proteins [126,127] and nucleic acids [128], and in
radioimmunoassays [129].
69
b) . Blue Sepharose CL-6B (BS) - this material [130,131] consists
of a sulphonated dye of the triazine class, Cibacron Blue
F3G-A, which has been immobilised by direct formation of
ether linkage between the triazine ring and the hydroxy
groups of agarose, Sepharose CL-6B. BS exhibits high
binding affinity for a number of proteins (dehydrogenases,
lipoproteins, human albumin, etc), and since the immobilised
ligand (dye) does not have a biological relationship to
the proteins it binds, its affinity has thus been termed
pseudo-l i gand [132]. BS interacts [133] wi th protei ns vi a
hydrophobic bonding; affinity binding; ion-exchange; and
exclusion-diffusion. BS exhibits high binding capacity
[130] for human serum albumin (~ 40 mg.ml- 1 ), but it does
not bind [134] bovine serum albumin (BSA), chicken albumin,
or rabbit albumin. BS-bound proteins (albumin) can be
eluted [130,135] with high salt concentrations (1.5M KC1),
or with chaotropic reagents such as 0.2M sodium thiocyanate
in 50 mm Tris-HCl buffer, pH 8.0.
Pseudo-l i gand affi nity chromatography of enzymes and the
selective removal of human serum albumin from serum/plasma
has been described in a number of publications [136,137,138].
c) Titanium dioxide (Ti02) - this adsorbent, like CH, exhibits
high binding affinity for ptoteins. Laboratory grade Ti02 has been modified [139] into porous spheroidal Ti02 particles
of size 50-600 ~m. The principal advantage of utilising this
material is that molecular exclusion can be utilised to enhance
70
the fractionating properties of the adsorbent. The Ti0 2-
bound material(s) can be eluted (depending on the protein)
with citrate, phosphate, or pyrophosphate buffers.
3_2 Experimental
3.2.1 General: Photo- and Chemiluminescence Measurements
Photo- and CL measurements were performed at room temperature
us i ng the MPF-44B Fl uorescence Spectrophotometer and the peroxy
oxalate-fia luminometer, respectively (see Section 2.3). Sample
and reagents for CL measurements were prepared as described in
Section 2.3.2.2. The hydrogen peroxide stream was propelled using
the peristaltic pump in all experiments, except those involving
imidazole-HCl buffer: these experiments were performed using
the pressure-bottle. Proteins, serum samples, anti-T4, FL-T4,
FITC-T4, and H202 were stored at 40 C and equilibrated to room
temperature prior to analysis. Corrections were made where appro-
priate for the background luminescence signal contributed by
buffer, and added proteins, including human serum and anti-T4 anti serum.
3.2.1.1 Procedures: Background Photoluminescence Reduction
a) Calcium hydroxylapatite
Human serum (250 ~l) was chromatographed on a CH column
(5.0 ml), equilibrated with 2 mM sodium phosphate buffer, pH 7.0.
The column was eluted with the same buffer in a total volume of
15.25 ml. The CH-bound material was eluted with 0.2M sodium phosphate
71
buffer containing 0.2M NaCl, pH 7.0. The fluorescence intensities
of the eluate were measured at a number of wavelengths and compared
with those of control samples which had been untreated, but simi
larly prepared and diluted.
Chromatography of urine was performed using a fresh auto
urine sample. The sample was filtered (Gelman filter, 0.45 ~m)
to remove the materials in suspension. 400 ~l of this sample
was applied to a 10.0 ml CH column, equilibrated with 5.0 mM
sodium phosphate, pH 7.0. In a similar experiment, a 400 ~l
of the urine sample containing 37.5 x 1O-5M bovine serum albumin
(BSA) was applied to an identical CH column. The columns were
eluted with 15.0 ml of the same buffer. Fluorescence intensities
of the el uate were measured and compared with those of control
sampl es.
b) Blue Sepharose CL-6B and Sepharose CL-6B
BS and Sepharose CL-6B columns (5.0 ml each) were equilibrated
with 50 mM Tris-HCl buffer containing O.lM KC1, pH 7.0, and
250 ~l of human serum (HS) sample was applied to each column.
The columns were eluted with the same buffer in a total volume of
15.25 ml. The material bound to the matrix was desorbed with 50 mM
Tris-HCl containing 0.2M NaSCN, pH B.O. Fluorescence intensities
of the eluate were measured at a number of wavelengths and compared
with those of control samples passed through identical columns.
72
c) Titanium dioxide
The method was performed by adding one gram of Ti02 to 5.0 ml
each of the sample solutions: (a) human serum (1 :200) in 10 mM
-phosphate buffer, pH 7.2, (b) 51 x 10-9M FL-T4 in 10 mM
phosphate containing 0.145M NaCl, pH 8.0, (c) 96 x 10-8M
anti-HSA in 10 mM sodium phosphate containing 0.145M NaCl, pH 8.0,
(d) 96 x 10-8M anti-HSA in 10 mM phosphate buffer, pH 7.2,
and (e) 5 x 10-GM human serum albumin in 1 mM phosphate buffer,
pH 7.2. Each sample was vortex-mixed for five seconds, incubated
for half-an-hour at room temperature, and centrifuged. Fluorescence
intensities of the Ti02-treated samples were compared with those
of control samples.
d) Titanium oxide
Small columns (1.5 ml) of TiO; particle size 0.125-0.25 mm
were equilibrated with 10 mM sodium phosphate buffer containing
O.145M NaCl buffer, pH 8.0. A 20 ~l aliquot of each of the samples:
(i) anti-a-2-macroglobulin, (ii) human serum, and (iii) human serum
albumin (6.62 x lO-GM) were applied to the columns. The samples
were eluted with 3.0 ml of the same buffer. Fluorescence intensi
ties of the total eluate were compared with those of the control
samples.
73
3.2.1.2 TePO-excited Luminescence: Non-specific Quenchin E um-natwn Proce ures
Quenching experiments were perfonned in SO mM sodium phosphate
buffer containing O.lM NaCl (pH 7.2), using Sl x 10-9M FL-T4 in
the presence of 34 x 1O-9r~ bovine insulin, 34 x 10-9M human serum
albumin, 34 x 10-9M anti-HSA, 34 x 10-9M human thyroglobulin, anti-T4
antiserum diluted 1 :60, and human serum diluted 1 :2000. In a sepa
rate experiment, tryptophan (30.S x 10-aM) and FL-T4 (Sl x 10-9M)
solutions were prepared in SO mM sodium phosphate buffer, pH 7.2,
containing O.lM NaCl" Other experiments were investigated for
their compatibility with the peroxyoxalate CL analysis
i) lS% polyethylene glycol (PEG) containing Sl x 10-9M FL-T4 in SO mM sodium phosphate, pH 7.0.
ii) 60% ammonium sulphate containing Sl x 10-9M FL-T4 in SO mM
sodium phosphate buffer, pH 7.0.
iii) Ethanol, diluted 1:1 with SO mM sodium phosphate buffer, pH 7.0,
containing Sl x 10-9M FL-T4"
iv) Calcium hydroxylapatite (O.S g ml- 1 ), containing Sl x 10-9M
FL-T4, in S mM sodium phosphate buffer, pH 7.0.
The solubility of TCPO (lOJll/0.4 ml) was tested in barbital
buffer (7S mM, pH 8.6), Tris-HCl (O.Ol-O.lM, pH 6-8), Tricine
(O.OS-O.lM, pH 6.8), N-2-hydroxy-ethylpiperazine-N'-ethanesulphonic
acid (O.OS-O.lM, pH 6.8), and imidazole-HCl (O.ol-O.SM, pH 6.0-9.S).
The effect of proteins on the CL signal was studied in 0.22M
imidazole-HCl buffer, pH 7.0. These experiments were conducted
74
using 51 x 10-9M FL-T4, containing 10 x 10- 8M human serum
albumin, or human serum diluted 1 :200. The CL intensities of
Fl-T4 were compared with those in the presence of human serum
albumin and human serum. A similar experiment was conducted
using 26.6 x 10-9M FITC-T4, and anti-T4 antiserum diluted 1 :240
and 1:60.
3.3 Results and Discussion
3.3.1.1 Baak~ound Photo~uminesaenae Reduation: of A orbents
Eva~uation
The results (Table 3.1) show that the human serum sample
exhibited three major fluorescent bands: excitation/emission
(Ex/Em), 290/335 nm; Ex/Em, 344/440 nm; and Ex/Em, 425/490 nm.
The fluorescent band, 290/335 nm is predominantly due to
tryptophan residues of proteins [120].
The phenylalanine and tyrosine residues (Ex/Em, 280/310 nm),
however, also contribute .. The fluorescent band detected at
Ex/Em, 344/440 nm is possibly due to a fluorescent ligand bound
to albumin [140]. Soini et a1 [43] have detected this fluorescent
band (Ex/Em, 340/440-470 nm) in human serum, but the fluorescent
material was not identified. Recent studies of Schwertner et al
[141] have revealed that the fluorescent ligand occurs in
renal-diseased sera, normal urine, hemofi1trates, etc and that
the fluorescent material is a low-molecular weight « 1000) species,
suspected to be either NADH, pyridoxine (vitamin 86) or its
metabo1ities. The fluorescent band observed at Ex/Em, 425/490 nm
75
is due to bilirubin-albumin complex [142]; a similar band
(Ex/Em, 422/S00 nm) has been reported by Soini et ·al [43], but
the fluorescent material was not identified.
Human serum treated with CH exhibited (Table 3.l{a) and
Figure 30 1) negligible (Ca.S% of the total) fluorescence, and
an overall reduction of Ca.9S% in the fluorescence signal was
obtained at the wavelengths studied. The fluorescence signal
(Ca.S%) observed from the CH treated serum sample suggests the
presence of low-molecular weight species, for example aromatic
amino acids. Desorption studies showed almost quantitative
recovery (Ca.80-9S%) of the fluorescent materials. These results
thus demonstrate that most (Ca.9S%) serum fluorescence is
associated with proteins or fluorescent ligands bound to proteins,
and furthermore, CH is an excellent material for reducing serum
fluorescence. However, some problems [139] posed by CH are
compressibility of the material resulting in low and unpredictable
flow rates; non-uniform particle size resulting in non-reproduci
ble results from batch to batch; presence of fines; and regenera
tion problems. CH prepared [139] in the form of porous spheroidal
particles (SO-600 ~m), and HA-Ultrogel (LKB) do not have these
drawbacks. In addition, molecular exclusion chromatography can be
performed on the spheroidal CH to enhance the fractionating
properties of the adsorbent. CH may thus have two important roles
in FIAs: reduction of serum fluorescence/inner filter effects in
homogeneous assays, and separation of haptens/macromolecular ligands
(proteins), provided the antibody-bound ligand and the free ligand
76
can be efficiently separated. Spheroidal CH offers a possible
role in heterogeneous imrnunoassays, because molecular exclusion
can be used to enhance the fractionating properties of the adsorbent.
The efficiency of CH in reducing serum fluorescence, however, will
depend on several factors, for example protein concentration,
presence of competitors (citrate, NaCl, phosphate, etc), diet,
drugs/vitamin content.
Photoluminescence studies performed on the urine sample showed
(Table 3.2) the presence of three fluorescent bands: (i) a very
(compared with the BSA) weak band at Ex/Em 290/330 nm, (ii) a
strong fluorescent band at Ex/Em, 330/420 nm, and (iii) a weak
band at Ex/Em, 390/510 nm. The fluorescent band detected at Ex/Em,
330/420 nm is similar to the band detected in serum samples
(cf Table 3.1); an Ex/Em maxima of 332 ! 4 nm/415 ! 5 nm has
been reported [141] for the fluorescent ligand present in urine.
Experiments performed with pure NADH solution suggested that the
330/420 nm fluorescent band is not due to the presence of NADH in
the urine sample, because the Ex/Em maxima of the pure NADH
solution was 345/450 nm. The fluorescent band detected at Ex/Em,
390/510 nm is due to the presence of urobilin in the sample.
The results show (Table 3.2) that the urinary fluorochromes
did not bind to the CH column. Similar results were obtained for
the urinary fluorochromes in the presence of bovine serum albumin.
The possibility of exploiting this protein to reduce the fluorescence
was considered because albumin binds a number of endogeneous
(bilirirubin) and exogeneous (drugs) substances and CH binds albumin
77
and fluorescent 1igands bound to albumin. This approach, however,
was unsuccessful, because BSA apparently did not bind the urinary
f1 uorochromes. Thi s may be due to two major factors: the 10w
molecular weights of the urinary f1uorochromes and hence their
negligible binding affinity for CH, and competition between CH
and the f1uorochromes for the binding sites on albumin.
The results of the experiment with BS showed (Table 3.1 (b))
appreciable reduction (Ca.40-60%) in the serum fluorescence at
Ex/Em, 290/335 nm; Ex/Em, 344/440 nm; and Ex/Em, 425/490 nm.
Studies performed withSepharose CL-6B (Table 3.1 (C)) under
conditions identical to BS demonstrated that the fonner material
did not bind the fluorescent materials present in the serum sample.
Sepharose CL-6B does not have the 1 i gand, F3GA, and consequently
1 acks in its abil ity to bi nd protei ns. The recovery of the BS-bound
material (mainly albumin) was quantitative (Ca.100%).
BS is inferior to CH, because BS reduced serum fluorescence
by only 40-60% (cf. Table 3.1(a) and Table 3.1(b)), depending on
the wavelength. Nevertheless, this approach is highly effective
for the se1 ecti ve removal of f1 uorescence associ a ted with human
serum albumin. In addition, unlike CH, desorption of the BS-bound
materials is not significantly affected by high buffer/salt concen
trations, and as a result, the need to desa1t (certain samples),
or the need to use low ioni c strength buffers at neutral pH are
obviated.
78
Experimental . F1 uorescence Intensi ty
(human serum Ex/Em Ex/Em Ex/Em di 1 uted to 290/335 344/440 425/490 1 :61) nm nm nm
a) Calcium hydroxy1-apatl te
Control (untreated) 71.0 40.5 20.0 prepared in 2 mM phosphate buffer Eluate in 2 mM 4.0 0.5 1.0 phosphate buffer
------------------------ ,.------------- ------------ ---------.----
Control prepared in 0.2 H phos pha te con- 71.5 38.5 18.5 taining 0.2 M NaC1 El uate 69.5 33.0 14.5
b) Blue Sepharose CL-6B Control prepared in 50 mM Tris-HC1 con- 83.0 60 17.5 taining O.lM KC1 El uate 48.0 22 9.5
------------------------1-------------- ------------ -------------Control prepared in 0.2 M NaSCN + 50 mM 70.0 37.0 13.5 Tris-HC1 El uate 38.0 36.5 8.0
c) Sepharose CL-68 Control prepared in 50 mM Tri s -HC1 con- 73.0 38.5 23.0 taining O.lM KC1 El uate 71.0 38.5 23.0
------------------------ -------------- ------------ -------------Control prepared in 0.2M NaSCN + 50 mM 62.5 23.0 15.5 Tri s -HC1 El uate 0.5 0.0 0.0
TABLE 3.1: Chromatography of human serum on (a) Calcium hydroxypatatite, (b) Blue Sepharose CL-6B, and (c) Sepharose CL-6B
Excitation slit: 12 nm Emission slit: 8 nm 79
CL.
Cl> > +-'
'" Cl>
'"
Contro 1
Eluate
310 370 nm
FIGURE 3.1: Chromatography of human serum sample on CH showing reduction in the fluorescence
80
Fluorescence Intensity Experimenta 1 (sample diluted Ex/Em Ex/Em Ex/Em 1:37.5) 290/330 330/420 390/510
nm nm nm
Control prepared in 5.0 43.0 16.0 5.0 mM phosphate buffer
El ua te 4.8 41.0 16.0
Control containing BSA, prepared in 5.0 mM 47.5 50.0 19.0 phosphate
Eluate 6.0 44.0 15.0
TABLE 3.2: Chromatography of human urine on CH
Excitation slit: 12 nm Emission slit: 8 nm
81
Fluorescent studies performed on Ti02-treated samples of
anti-HSA, human serum albumin, and human serum showed (Table 3.3)
substantial reduction (Ca. 75-98%) in the fluorescence band at
Ex/Em, 290/335 nm. Similar studies performed with FL-T4 at
Ex/Em, 390/475 nm, however, demonstrated low binding (Ca. 32%)
to Ti02. Chromatographic studies performed by Thomson and
Miles [139] using spheroidal Ti02 demonstrated that bovine serum
albumin can be eluted with 1 mM sodium phosphate, and that
elution of y-globulin can be effected with 20 mM sodium pyro
phosphate buffer, pH 8.0. Factors such as the physical form of
Ti02 (laboratory grade) and the batch separation technique employed
in this work may explain the discrepancies. Further work was not
pursued with this material. because the material was too fine to
be employed for satisfactory work. Nevertheless, this preliminary
work demonstrates that Ti02, like CH, exhibits high binding
affinity for macromolecules. Furthermore, binding of macromolecules
to Ti02 is not significantly affected by sodium chloride.
Chromatographic studies performed using TiO showed (Table
3.4): substantial binding (Ca. 76%) by anti-a-2 macroglobulin;
a relatively low binding (Ca. 32%) by human serum albumin and a
negligible binding (Ca. 16%) by the constituents (proteins) present
in the serum sample. These results thus de~onstrate that TiO
exhibited binding properties similar to those reported [139] for
spheroidal Ti02. For example, bovine serum albumin can be eluted
from spheroidal Ti02 [139] with 1 mM sodium phosphate buffer.
The TiO particles (0.125 - 0.25 mm) employed in this preliminary
82
Fl uorescence Intensi ty
Experimental Ex/Em Ex/Em 290/335 nm 390/475 nm
Anti -HSA in 10 mN phosphate 18.0 -containing 0.145 M NACl
~bove treated with Ti02 . 1.0 -
Anti-HSA in 10 mM phosphate buffer
24.5 -Above treated with Ti02 0.5 -
HSA in 1 mM phosphate buffer 46.5 -
Above treated with Ti O2 6.5 -
HS (1: 200) in 10 mM 82.5 -phosphate wffer
Above treated with Ti O2 19.5 -
FL-T in 10 mM phosphate containing 0.145M NACl - 72.0
Above treated with Ti02 - 49.0
TABLE 3.3: Effect of treating macromolecules, serum and a lowmolecular weight species with Ti0
2
Excitation slit: 8 nm Emission slit: 9 nm
83
Experimental (sample diluted 1 : 150)
Anti-~-2-macrog10bu1in in 10 mM phosphate containing 0.145M NAC1
Eluate
HSA in 10 mM phosphate containing 0.145M NACl
El uate
HS in 10 mM phosphate containing O. 145M NACl
El uate
Fluorescence Intensity
Ex/Em 290/330 nm
40.0
9.5
46.5
31.5
59.5
50.0
TABLE 3.4: Chromatography of macromolecules and serum on TiO
Excitation slit: 8 nm Emission slit: 9 nm
84
work were irregular, and less satisfactory for chromatographic
studies. However, modification of TiO may significantly improve
adsorption/molecular exclusion properties of the material. These
preliminary results demonstrate a potential role for TiO in
hetero- and homogeneous FIAs.
3.3.1.2 Elimination of PeroxyoxaZate CL Quenching: PhysicoChemical Approaches
The results of preliminary experiments demonstrated
(Figures 3.20 and 3.21) that TCPO-excited luminescence of Fl-T4 was subject to non-specific quenching. This phenomenon apparently
occurred in all protein-containing solutions and increased with
the relative molecular weight (M.Wt) of the protein, being maximal
(Ca. 85-90%) when the relative molecular weight exceeded about 105
and negligible in the case of bovine insulin (M.Wt. 5734). Similar
results were obtained in the presence of human serum (1:2000) and
anti-T4 antiserum (1:60). The addition of anti-T4 antiserum to
FITC- and Fl-T4 conjugates is well-known [46] (see Chapter Four)
to result in substantial enhancement of fluorescence. These results
demonstrated that using peroxyoxalate excitation of Fl-T4, the
fluorescence enhancement was replaced by the non-specific quenching
effect. This effect has been well documented for a number of
luminescence (Cl/8l) assays:
i) a ClIA based on peroxyoxalate excitation [74].
ii) in a ClIA for thyroxine based on an isoluminol-T4
conjugate
and catalysed by the H202-hematin system; a substantial
85
quenching (50-80%) of the CL signal by bovine serum
albumin has been reported [149]
iii) bioluminescence assays [30,143], for example based on
luciferase.
From these results, it thus became apparent that homogeneous
CLIAs utilising fluorescence enhancement effects cannot be
exploited. Preliminary experiments using FL-T4 to assess the
compatibility of peroxyoxalate-fia system with conventional
separation systems showed: (a) poor reproducibility using PEG,
possibly due to the high viscosity of the solution; (b) non
reproducibility using ammonium sulphate; (c) high background and
poor reproducibility using ethanol; and (d) reduced CL signal
using CH, due to binding (Ca.20%) of FL-T4 and low (5 mM) ionic
strength of phosphate buffer. These approaches were deemed
unsatisfactory, and consequently, attempts were made to minimise
the problems caused by the poor solubility of TCPO in aqueous
systems, and problems of non-specific quenching. The results of
these experiments showed that amongst the buffers (barbital, Tris
HC1, Tricine, imidazole-HC1, etc) examined for solubility of
TCPO solution, only imidazole-HCl (O.22M, pH 7.0)was effective,
because the TCPO solution dissolved instantaneously and as a
result,its rate of hydrolysis was minimised. Further experiments
performed with this buffer, using fluorescein- and FL-T4 conjugates
in the presence of human serum, human serum albumin and anti-T4 antiserum showed (Table 3.5) that the non-specific quenching
effect did not occur, and furthermore, the specific enhancement
86
HSA
_ .... u. ___ ,
FIGURE 3.20: Examples of recorder traces showing quenching of TePa-excited luminescence of 51 x 10-9 M FL-T4 by 34 x 1O-9M HSA, and anti-T4 antiserumdiluted 1 :60.
87
-1 2.2 m1 min
00 00
100 0
y--80
0"1 C .--5 60 c <U
c3 -.J 40 LJ
~ o 20
01 c ..., --.-o 100 200 300 400 500 600 700
M.Wt. x 103 FIGURE 3.21: The effect of protein's molecular \~eight on the TePO-excited luminescence
of FL-T4 " FloVl rate of H20f 2.2 ml min- l
Experimenta 1 Re 1 a ti ve CLl
51 -9 x 10 M FL-T4 2
51 x 10-9M FL-T4 + HS (1:200) 3
51 x 10-9M FL-T4 + 10 x 10-8M HSA 5
26.6 x 10-9M FITC-T4 2
26.6 x 10-9M FITC-T4 + Anti-T4 (1 :240) 11 .8
26.6 x 10-9M FITC-T4 + Anti-T4 (1 : 60) 16.5
TABLE 3.5: Effect of addingHS, HSA, and anti-Ta antiserumto TCPO-excited luminescence of f1uorestein-and . FL-T4 conjugates
89
I I I
observed in FIAs was restored. This effect was exploited in the
development of an enhancement CLIA for thyroxine (Chapter Four),
and an enhancement CLIA for human serum albumin (Chapter Five).
The mechanism of non-specific quenching is obscure, but is
unlikely to be a consequence of energy transfer (non-radiative)
to proteins, because experiments performed using peroxyoxa1ate
excitation of FL-T4 in the presence of 30.5 x 10-~M tryptophan
showed negligible (Ca.15%) reduction in the CL signal; and the
quenching effect did not occur in imidazo1e-HC1 buffer. These
results thus suggest that the interaction of certain protein
groups with the high energy intermediate, 1,2-dioxethane dione/
charge-transfer complex (Scheme 1) are responsible for the quenching
effect. The possible mechanism of the CL enhancement is described
in Chapter Five.
3.4 Conclusion
The studies performed in this work provided a valuable
insight into some of the interference problems in luminescence
spectrometry. The nature of the interference in the two different
mechanisms of sample excitation varied: photo-excitation of
serum samples at the excitation maxima of endogeneous f1uorophor
(tryptophan, bilirubin, etc) caused an apparent increase in the
fluorescence signal; on the other hand, TCPO-excited luminescence
of FL-T4 was subject to non-specific quenching by proteins.
Background fluorescence interferences arising at Ex/Em,
290/335 nm are of little consequence in homogeneous FIAs.
90
But, the fluorescent bands located at Ex/Em, 344/440 nm and
Ex/Em, 425/490 nm are likely to impose inferior detection limits
in homogeneous FIAs. For example, fluorescein isothiocyanate
(492/518 nm), dansyl chloride (340/480-520 nm), and fluorescamine/
MDPF (394/475 nm) will suffer interferences (see Table 1.3).
The fluorescent material, with Ex/Em, 344/440 nm has thus far
not been identified, but it has been well established (also
evident from this work) that the fluorescence comes at least
partly from ligands bound to albumin. Unquestionably, any tech
nique that is capable of minimising this interference will enhance
the detection limits of photoluminescence analysis. Calcium
hydroxyl apatite, because of its high binding affinity for pro
teins, was able to reduce approximately 95% of serum fluorescence.
However, it was ineffective in reducing urine fluorescence,
possibly because CH exhibits negligible binding affinity for low
molecular weight species. This finding confirms that a substan
tial amount of serum fluorescence is associated with proteins.
Blue Sepharose selectively adsorbed human serum albumin
(constitutes >50% of the serum proteins) and as a result reduced
fluorescence by Ca. 50% at all wavelengths. This again suggests
that human serum albumin is a predominant source of fluorescent
material in serum. The adsorption properties of Ti02 and TiO may
also be exploited to reduce background fluorescence; transforma
tion of these materials (laboratory grade) into porous spheroidal
particles may enhance their usefulness in achieving this.
The full impact of this simple, mild, economical and effective
91
(in particular CH) approach of background fluorescence reduction
in homogeneous FIAs has yet to be realised. In addition, this
approach can be employed to minimise inner filter effects
by removing light absorbing species, and to separate the free
antigen from the antibody-bound antigen. Evidently, the ultimate
success of this approach in FIAs, capable of providing sub
nanomo1ar detection range will depend to a great extent on the
efficiency of the adsorbent to remove fluorescent materials from
the ligand to be assayed, and on the vulnerability of the fluores
cent label to background interferences.
The principal disadvantages of peroxyoxa1ate CL are poor
solubility of TCPO in buffers (Tris-HCI, barbita1, etc) and
non-specific quenching (Ca. 85-90%) in protein-containing.solu
tions. The non-specific quenching effect is of major consequence,
because homogeneous CLIAs utilising the fluorescence enhancement
effect cannot be developed. However, the quenching effect, and
the solubility problem can be minimised using imidazo1e-HC1
buffer. One of the most elegant features of this approach is
simplicity: additional steps, special reagents, or instrumental
approaches are not required to eliminate the effect.
The mechanism of the non-specific quenching is inconclusive,
but it most likely occurs via interaction of specific protein groups
with the high energy intermediate, 1,2-dioxetanedione ana/or
the charge-transfer complex (Scheme 1). The replacement of the
quenching effect by the specific CL enhancement effect offers
great potential in the development of homogeneous CLIAs.
92
CHAPTER FOUR: LUMINESCENCE-LINKED IMMUNOASSAYS FOR THYROXINE
4.1 General Introduction: Biological Role and 'Assay Techniques
The hormones thyroxine (3,3' ,5,5'-tetraiodothyronine, T4),
and 3,3' ,5-triiodothyronine (T3) are secreted by the endocrine
system of the thyroid gland. Thyroxine (X=I) and T3 (X=H) have
the structure:
9tI
H H H 0 I I I 11 HO 0 D~O>-or-n-C-DH
I H H NHe
These hormones are regulated [144,145) via a sensitive feedback
system involving the hypothalamus and pituitary gland. Thyroxine
is transported [4,146) bound principally to thyroxine-binding
globulin (TBG), and secondarily to prealbumin and albumin.
However, it is the free (Ca. 0.03%) fraction which is biologically
active, being involved in metabolic controls (147) and physical/
mental development of an individual. The normal range of -1 physiological T4 in serum is 4-11.5 ~g d~. The measurement
of T4 levels is an important indicator of thyroid status.
The need for an assay combining specificity, sensitivity, and
practicality (ease of performance, low-cost, etc) has limited the
number of clinically useful T4 and T3 techniques. In addition,
endogeneous T4 binding proteins in serum (TBG, etc) interfere
severely with the measurements. This problem, however, can be
minimised by displacing protein-bound T4, for example using
sodium merthiolate, 8-anilino-l-na~halene sulphonic acid (ANS),
salicylates, etc [148,149]. Currently radioimmunoassays are most
commonly employed for routine T4 measurements. However, recent
developments in non-isotopic immunoassay technology have intro-
duced:
a) Fluorescent labels - the first fluorescence enhancement
immunoassay for T4 based on a FITC-T4 conjugate was described
by Smith [44]. The enhancement effect was ascribed to
quenching of the fluorescence of FITC-T4 by the iodine atoms
of T4, possibly via the heavy-atom effect, the effect being
relieved or abolished upon binding to anti-T4 antibodies.
Handley et al [46) likewise found that an enhancement FIA
for T4 could be developed using the more convenient (ease of
preparation) labels, fluram and MDPF. Miller [150) has sugges-
ted that the enhancement effect occurs as a consequence of a
change in the polarity of the environment of the fluorescer.
The principal advantage of enhancement FIAs is simplicity of
the technique, but due to serum protein (TBG, albumin, etc)
interferences and background serum fluorescence problems the
technique suffers from severe limitations. However, the
,problems inherent in the enhancement FIAs can be partially
circumvented using:
94
Fluorescence protection immunoassay - the technique [47]
involves incubation of sample with anti-T4 antibodies
followed by incubation with anti-FITC and measurement of
the resulting fluorescence signal. Unlike the enhancement
FIA, FITC-T4 binds either anti-T4 antibodies or anti-FITC
antibodies, and consequently, fluorescence enhancement due
to endogeneous T4 binding serum proteins (TBG, albumin etc)
is minimised. However, the technique suffers from background
interference due to serum fluorescence and light scattering
(see Table 1.3 and Chapter Three).
Differential heavy-atom quenching FIA (solvent quenching
[53]) - the technique is based on differential quenching
of the unbound rhodamine-T4 and background fluorescence;
the fluorescence of the rhodamine-T4 bound to the antibody
being unaffected. The procedure is prone to light scattering
interference due to small (26 nm) Stokes' shift of rhodamine
(Table 1.3).
b) Enzyme labels - these have been utilised in homogeneous
(EMIT) and heterogeneous (ELISA) immunoassays. EMIT is based
on the principle [88] that T4-malate dehydrogenase is inactive.
The addition of anti-T4 antibodies, however, reactivates the
enzyme. The active enzyme converts NAD+ to NADH, resulting
in an absorbance change at 340 nm and this change is correlated
with the sample T4 concentration. In ELISA, T4 ;s conjugated,
for example with horseradish peroxidase (HRP). The immunoassay
95
is conveniently performed using anti-T4 antibodies immobi
lised on solid-phase, for example plastic tubes. The peroxi
dase activity of the antibody-body HRP-T4 complex can be
measured (i) colorimetrically [151], using the chromogen
di-ammonium 2,2'-azino-bis(3-etnylbenzothioline-6 sulphonate,
ABTS); (ii) by photoluminescence [152] using the fluorescence
reaction of peroxidase-tyramine-H202; and (iii) by chemi
luminescence [152], using the luminol-H202 reaction. However,
the high intrinsic sensitivity of luminescence methods
permits lower (10 to lOO-fold) detection limits than the
colorimetric methods. Recently, Blake et al [94] have
described a dual-enzyme immunoassay (Section 1.B.4). This
assay is based on the use of T4 and T3, conjugated with
alkaline phosphatase and 'B-D-galactosidase, respectively.
The principal advantages of this technique over the single T4
immunoassays are: better assessment of thyroid abnormalities
and microvolume sample requirement.
This chapter is concerned with:
a) preliminary investigations into poor (7-10%) reversibility
of the fluorescence enhancement immunoassay utilising FL-T4;
b) the possibility of reusing a recycled solid-phase with
immobilised anti-T4 antibodies;
c) comparison of two-phase sequential addition FIA (two-phase FIA)
and simultaneous addition FIA utilising FITC-T4;
d) development of a polarisation FIA, based on a sequential
approach;
96
e) development of a TCPO-excited luminescence immunoassay
utilising the fluorescence enhancement effect of FITC-T4 ,
f) performance of the ELISA monitored with (i) the standard
co1orimetric method, (ii) 1umino1 CL, (iii) photoluminescence
method utilising the fluorescence reaction of peroxidase-
tyramine-H 202; and (iv) TCPO-excited luminescence of the
peroxidase-tyramine-H202 reaction.
4.2 Experimental: Luminescence and Radioimmuno1ogica1 Procedures
4.2.1 Genera~
F1uorescein- and FL-T4 conjugates were prepared as described
in Section 2.2.1. Serum samples, protein solutions, anti-T4 antiserum, H202, FITC-T4 and FL-T4 solutions were stored at 40 C
in the dark and equilibrated to room temperature prior to
measurements. Sample and reagents for peroxyoxa1ate CL measure-
ments were prepared as described in Section 2.3.2.2.
Radioimmunoassays were performed at room temperature using
the Panax Auto-Bioscint gamma counting system (Panax Equipment
Ltd, Willow Lane, Mitcham, Surrey, UK). TCPO-excited luminescence
of f1uorophors was conducted using the peroxyoxa1ate-fia 'lumino
meter (Section 2.3.2.1). Experiments involving 1umino1 CL were
conducted at room temperature in a non-flow system using the
1uminometer, coupled to its display/printer unit (LKB Model
1250-101). The MPF-448 Fluorescence Spectrophotometer (Section
2.3.1) was employed in photoluminescence measurements, except
polarisation fluorimetry. These latter measurements were
97
performed using the Model 2000 Spectrofluorimeter fitted with
polariser control unit and sample holder equipped with an excita
tion polariser (fixed) and an emission polariser (rotatable)~
see Sect ion 1.6.3.
The background (radioactivity and luminescent) signal was
subtracted in all measurements.
4.2.1.1 Fluoroimmunoassays Utilising FL-T4
The assay was performed using 47.5 x 10-gM FL-T4 prepared in
50 mM sodium phosphate containing O.lM NaCl (pH 7.4); incubation
was for 30 minutes in the dark at room temperature. In a similar
experiment, monoclonal anti-T4 anti-serum was added to 42.5 x 10-9M
FL - T 4 prepa red in 75 ml1 ba rbita 1 buffer conta i ni ng 2.5 mM sodi urn
merthiolate (pH 8.6). The experiments involving the specific •
and the non-specific fluorescence enhancement effects were
performed by incubating 13.6 x 10-8M FL-T4 for 30 minutes at
room temperature with each of the solutions: anti-T4 antiserum
(1:225), anti-phenytoin antiserum (1:150), human serum albumin
(6.7 x 10-614), human serum (1:600), anti-a-2-macroglobulin (1:450),
and insulin-binding protein (1 :600). These solutions, including
FL-T4, were prepared in 50 mM sodium phosphate containing O.lM
phosphate saline buffered, pH 7.4. In a similar experiment,
the above reagents (FL-T4, anti-T4 , etc) were prepared in the
barbital-merthiolate buffer, pH 8.6. The competitive binding
fluorescence assay based on FL-T4 and insulin-binding protein -9 were performed at room temperature using 47.5 x 10 M FL-T4
98
made up in the phosphate-saline buffer, pH 7.4.
The solid-phase studies involving FL-T4 and anti-T4 anti
bodies immobilised on stick-tube system (Ventre/Sep Solid
phase T4; Seward Lab. London) were performed by incubating
FL-T4 solutions for thirty minutes at room temperature. The
FL-T4 solutions were made up in the barbita1-merthio1ate buffer,
pH 8.6. Fluorescence measurements were performed by removing
the supernatant from each of the solid-phase tubes; the
fluorescence signal was compared with the fluorescence signal
of an equal concentration of FL-T4, which were similarly pre
pared and incubated, but added to tubes without the immobilised
anti-T4 antibodies.
The radioimmunoassay studies were conducted using the solid
phase system. 1.0 ml of the l25 1_T4 was added to 25 ~l of each -1 -1 of the solutions: 19.5 ~g dt FL-T4 and 19.5 ~g dt unlabelled
T4. The bound counts per minute (CPM) were compared in the two
solid-phase systems.
4.2.1.2 Recycling of Antibodies
These experiments were performed by washing the used (first
R1A) stick-tube system five times with 2M KSCN solution, followed
by washing twice with 1 mM sodium phosphate containing 0.15M
NaCl, pH 7.2 and 15 minutes incubation at room temeprature with
2.0 ml of the above buffer, pH 7.2. The sticks and the tubes
were dried using tissue paper and reused in a second radioimmuno
assay, a correction being made for the unremoved l25 1_T4 radio
activity.
99
4.2.1.3 Luminescence Immunoassays Utilising FITC-T4
Experiments comparing the two-phase FIA and the simultaneous
addition FIAs were performed as described below. Anti-T4 -1 antiserum diluted 1 to 54, 12 ng.ml FITC-T4, and T4 solutions
were prepared in 75 mM barbital buffer, pH 8.6. The simultaneous
addition FIA was performed by adding anti-T4 to solutions contai
ning FITC-T4 and T4. The immunoassay was incubated at 250 C for
30 minutes, followed by fluorescence measurements. This assay
was further incubated for 2.5 hours at 250C, followed by fluores-
cence measurements. The two-phase sequential FIA was performed
as follows:
Phase One - T4 was added to anti-T4 antiserum, followed by incub
bation for 30 minutes at 250C.
Phase Two - the sample solutions (phase one) were incubated
+ 0 for 2.5 hours at 4 - 0.5 C. Subsequently, the pre-incubated
FITC-T4 solution (4 ! 0.50C for 2.5 hours) was dispensed into
each of the sample solutions and incubated for 40 minutes.
, Experiments involving FITC-T4 and human serum were performed
as described above for the simultaneous addition assay, except -1 that 6 ng. ml FITC-T4 and T4-depleted human serum (Ventre/Sep
T4 kit) were utilised.
The polarisation FIA was performed by the sequential addition
technique: anti-T4 antiserum (1:500) was added to T4 solution
in 10 mM sodium phosphate containing 0.145M NaCl, pH 7.4,
followed by incubation for 45 minutes at room temperature.
100
Subsequently, FITC-T4 at a final concentration of 10 ng.ml- l was
added to each of the sample solutions, followed by 45 minutes
incubation at room temperature.
The peroxyoxalate CL experiments were conducted using -9 33.3 x 10 M FITC-T4, T4 and anti-T4 antiserum, made up in 0.22M
imidazole-HCl buffer, pH 7.0. The immunoassay was performed as
described above for the two-phase assay. The flow rate of H202 was 48 ml mlnute- l .
4.2.1.4 Luminescence Immunoassays Based on ELISA
These assays were monitored with photo- and chemiluminescence
detection using the Enzymun-Test T4 kit (Immunodiagnostics,
Boehringer Mannheim, London). Serum thyroxine was determined
in four ways:
a) Colorimetrically - the standard procedure described in the
technical bulletin for the kit was employed. The method
involved (i) adding 20 ~l of the standard serum samples to
Ab-coated plastic tubes, (ii) adding 1.0 ml of T4-HRP to each
of the above tubes, followed by 2 hour incubation at room
temperature, (iii) decantation of the contents of each of the
tubes, followed by rinsing each of the tubes with cold
(4oC) tap water, (iv) addition of 1.0 ml of the substrate
(ABTS, supplied with the kit) solution, followed by 1 hour
incubation at room temperature and measurement of the absor-
bance of the sample solutions at 405 nm against the blank
(substrate solution).
101
Experiments (b), (c) and (d) were conducted after completion
of step (iii) above.
b) Luminol CL - the procedure [152,153] consisted of adding
0.5 ml of O.lM NaOH to solubilise the heme content. Then,
0.5 ml of 1.5 mM luminol solution (in O.lM NaOH) containing
0.1% EOTA was added, followed by 30 minutes incubation at
room temperature. Finally 0.2 ml of 3% H202 solution con
taining 0.1% EOTA was added to each of the above solutions.
Each of the sample tubes was vortex-mixed for 5 seconds and
the CL signal was measured 5 seconds after the mixing step.
c) Photoluminescence - the method [152] involved adding 0.8 ml
of 50 ~1 sodium phosphate containing 0.1% BSA (pH 7.4) to
each of the sample tubes. This was followed by the addition
of 0.05 ml of 1% tyramine-HCl solution containing 0.007%
H202. Each of the tubes was vortex-mixed for 5 seconds and
incubated for 1 hour at 370 C. The solutions were removed
from each of the sample tubes at a constant interval of
1 hour.
d) TCPO-excited luminescence - this was performed as described
above for the photoluminescence method, except that O.22M
imidazole-HCl containing 0.1% BSA (pH 7.0) was employed.
102
4.3 Results and Discussion
4.3.1.1 FluoroimmunoassaysUtilising FL-T4
The fluorescence enhancement immunoassay (Table 4.1(b))
exhibited a negligible (ca. 7.5%) reversal effect in the presence
-9 ) of 214 x 10 M unlabelled T4. The experiments (Table 4.2(a)
involving FL-T4 and anti-phenytoin antiserum, insulin-binding
protein, human serum, etc suggested that non-specific-binding
of FL-T4
to endogeneous T4 binding proteins (TBG, albumin, etc)
might be responsible for the poor reversal effect. The possibility
of expl oHi ng the fl uorescence enhancement effect in a competHi ve
binding assay based on insulin-binding protein was therefore
briefly investigated. The results (Table 4.3(b)) from this
experiment showed a negligible (Ca. 13%) reversal effect in -9 the presence of 214 x 10 M unlabelled T4. Lim [45] likewise
found that addition of immunoglobulin M (IgM) to desmethylnor
triptyline resulted in substantial (355%) fluorescence enhancement.
However, comparison of the magnitude of the fluorescence enhance-
ment in phosphate-saline buffer, pH 7.4 (Table 4.2(a)) and
barbital-merthiolate buffer, pH 8.6 (Table 4.2(b)) show:
a) anti-T4 antiserum produced Ca. 192% and 32% fluorescence
enhancement in the phosphate-saline buffer and the barbital-
merthiolate buffer, respectively;
b) human serum albumin produced Ca. 138.5% and no fluorescence
enhancement in the phosphate-saline buffer and the barbital-
merthiolate buffer, respectively;
103
Anti-T4 antiserum FI (Ex/Em, 390/470 nm) (dilution)
FL-T4 22 FL-T4 + 1:800 29
FL- T4 + 1 :400 43 FL-T4 + 1 :200 60 FL- T 4 + 1: 100 59
TABLE 4.l(a): Effect of adding anti-T4 antiserum to 47.5 x 10-9M FL-T4 Excitation slit: 8 nm Emission slit: 12 nm
Experimental FI (Ex/Em, 390/470 nm)
-9 + Anti-T4 47.0 o + 47.5 x 10 M FL-T4 10.7xlO-9MT4+ " " + " 47.5 2l.4X10-9MT4+ " " + " 45.5
107xlO-9M T4+ " " + " 44.0
2l4xlO-9M T4+ " " + " 43.4
TABLE 4.l(b): Effect of increasing T4 concentration on the fluorescence signal, in the presence of 47.5 x 10-9M FL-T4 and anti-T4 antiserum (1:400)
Excitation slit: 8 nm Emission ·slit: 12 nm
104
FI % Experimental (Ex/Em 390/470 nm) Enhancement
FL-T4 13.0 -FL-T 4 + Anti-T4 antiserum 38.0 192.31 FL-T 4 + Human serum albumin 31. 0 138.46
FL-T4 + Anti-phenytoin antiseruIT 47.0 261.54
FL-T 4 + Human serum 28.0 115.38 FL-T4 + Insulin-binding protein 36.0 176.92 FL-T4 + Anti-cr-2-macroglobulin 15.5 19.23
TABLE 4.2(a): Effect of addi ng 13.6 x 10-811 FL -T to each of the above in phosphate-saline buff~r. pH 7.4.
Excitation slit: 8 nm Emission slit: 12 nm
Experimental FI % (Ex/Em. 390/470 nm) Enhancement
FL-T4 25.0 -FL-T4 + Anti-T4 antiserum 33.0 32
FL-T4 + Human serum albumin 24.0 -FL-T4 + Anti-phenytoin antise~m 46.0 84
FL-T4 + Human serum 34.0 36 FL-T4 + Insulin-binding protein 31.0 24
TABLE 4.2(b): -8 Effect of adding 13.6 x 10 M FL-T to each of the above in barbital-merthiolate ~uffer. pH 8.6
Excitation slit: 8 nm Emission slit: 14 nm
105
c) anti-phenytoin antiserum produced Ca. 261.5% and 84%
fluorescence enhancement in the phosphate-saline and the
barbital-merthiolate, respectively;
d) insulin-binding protein produced Ca. 177% and 24% fluores
cence enhancement in the phosphate-saline and the barbit~l-
merthiolate, respectively;
e) human serum produced Ca. 115.4% and 36% fluorescence
enhancement in the phosphate-saline and the barbital-merthio-
late, respectively.
Apparently, the relatively low (32%) fluorescence enhancement
effect observed with the anti-T4 antiserum is a consequence of
inhibition of FL-T4 binding to the endogeneous T4 binding
proteins (TBG, etc) in the antiserum, because the barbital-mer-
thiolate buffer (pH 8.6) does not [149] significantly affect
the binding properties of anti-T4 antibodies. It was therefore
decided to purify the anti-T4 antiserum on DEAE-Sephadex A-50
column [154]. The FIA results of a preliminary investigation showed
that the reversal effect was comparable in magnitude to that
achieved using the non-purified antiserum (above). Further FIA
studies (Figure 4.1) involving monoclonal anti-T4 antiserum
also showed a negligible (Ca. 10%) reversal effect in the presence -6 of 2.8 x 10 M unlabelled T4.
experiment (Table 4.4) showed
However, the solid-phase FIA
that FL-T4 (3.4 - 101 x 10-BM)
did not bind the immobilised anti-T4 antibodies as evident
from comparison of the fluorescence signals: solid-phase
106
Insulin-binding protein FI (Ex/Em, 390/470 nm) (dilution)
FL-T4 + 0 27.0 FL-T4 + 1:800 37.0 FL-T4 + 1:400 55.0
FL - T 4 + 1: 200 64.0
FL - T 4 + 1: 1 00 73.0
TABLE 4.3(a): Effect of adding insulin-binding ~rotein on the fluorescence signal of 47.5 x 10- M FL-T4
Experi menta 1 tl
(Ex/Em,390/470 nm)
FL-T4+Insu1in binding protein 45.0 -9 . " + " " " 45.5 21.4x10 M T4+
107x10-9M T4+ " + " " " 40.0
214x10-9M T4+ " + " " " 39.0
TABLE 4.3(b): Competitive binding fluorescence assay utilising 47.5 x 10-9M FL-T4 and insulin-binding protein (1:600)
Excitation slit: 8 nm Emission slit: 12 nm
107
B
E c
C) en 00
x LW A .-. CL
~ > .~
+' ~
~ C<
500 nm
FIGURE 4.1: Examples of recorder traces showing poor reversibility of the fluoresence enhancement effect.
-9 -9 A ; 42.5 x 10 M FL-T4; B ; 42.5 x 10 M FL-T4 +
monocloncal anti-T4 antiserum (1:60); and -9 -6 C ; 42.5 x 10 M FL-T4 + 2.B x 10 M unlabelled
T4 + monoclonal anti-T4 antiserum (1 :60)
Excitation slit: B nm Emission slit: 12 nm
lOB
FI of Supernatant Ex/Em, 370/470· nm
Concentration of FL-T4 TuDe+Immobilised Tube without immo-
Ab bilised Ab
33.6 x 10-9M 1.0 2.0
168 x 10 -9M 10.0 12.0
335 x 10 -9M 26.0 28.0
670 x 10-9M 59.0 59.0
1010 x 10-9M 91.0 90.0
TABLE 4.4: Effect of adding.equal concentrations of FL-T solution to each of the tubesimmobilised with4 and without the antibody.
Excitation slit: 8 nm Emission slit: 14 nm
109
immobilised with the antibodies and the tubes without the
immobilised antibodies. The results from this experiment compare
favourably with those reported by Handley [155] using a similar
solid-phase system and FL-T4. The solid-phase (Ventre/Sep)
employed in this work was for the measurement of T4 by the
RIA method; thus a relatively high molar ratio of FL-T4:
immobilised anti-T4 antibodies and also, steric hindrance due 125 to larger size of FL-T4 than I-T4 may explain the results.
However, in a radioimmunoassay experiment the counts per minute
obta i ned -1 -1 using 19.5 pg d~ FL-T4 and 19.5 pg d~ unlabelled
T4 in the presence of 125I_T4 buffered (Ventre/Sep) solution
were 5511 and 1561, respectively. The solid-phase FIA and the
RIA results thus suggest that either the FL-T4 solution contains
unlabelled T4 or FL-T4 due to steric hindrance is unable to
bind as many sites on the immobilised antibody as the unlabelled
T4. It has been well established [156] that the reaction (Sec
tion 2.2) of fluorescamine with primary amine occurs with a half
time of milliseconds and proceeds with 80-95% of the theoretical
yield. Fu rth"ermo re , the labelling studies performed by Handley
[155] have shown that the molar ratio of fluorescamine conjugated
to T4 is 0.91. As a result, preliminary experiments were conduc
ted to label T4 using 0.03-1% fluorescamine and MDPF solutions.
The FIA investigations performed using FL-T4 and MDPF-T4 conjuga
tes showed that the reversal effects were comparable in magnitude
to those described above. As a result, further investigations
with FL-T4 and MDPF-T4 were not pursued for the following
110
reasons: FL-T4 and MDPF-T4 would not be suitable for the
studies of TCPO-excited LIA util ising the fluQrescence enhance
ment effect, because of the poor (7-10%) reversible effect
observed in the enhancement F1A, and moreover, because F1TC-T4 might be more efficiently excited with TCPO than FL-T4 or MDPF-T4 due to high (0.85) fluorescence quantum yield and high Ex/Em
(492/518 nm) maxima of f1uorescein.
4.3.1.2 RecycZing of Antibodies
The investigations showed that 11.25% of the original
(first R1A, Figure 4.2) radioactivity was not removed from the
recycled stick-tube system using 2M KSCN solution. The unremoved
(11.25%) radioactivity results compare favourably with the 12%
value reported by Akker et a1 [157]. The non-specific binding
of 1251-T4 to the stick-tube system and hence its unremova1
with 2M KSCN solution might explain the results. The second
(corrected for the non-specifically bound 1251_T4 ) radioimmuno
assay (Figure 4.1), however, demonstrated that the recycled
immobilised anti-T4 antibodies exhibited 39% of its original
binding. The 61% loss in the activity of the immobilised anti
bodies compared with the 37% value reported by Akker et a1 [157]
might principally be due to the differences in the conditions
employed for renaturing the immobilised antibodies: Akker et
a1 [157] renatured the antibodies by resuspension in 1 mM phos
phate containing 0.15M NaCI at 40C for three days. On the other
hand, the anti-T4 antibodies in this work were renatured by adding
2.0 m1 of the above buffer to the stick-tube system, followed
111
100 ----, Recycled immobilised antibodies
90
80
70
40
30
r---I I I I
20 0 S 10 1S 20 2S [14] pg/ dl.
FIGURE 4.2: Radioimmunoassay of Ta and reuse of the recycled antibodies in a secona radioimmunoassay
-1 % B/Bo; Average CPM of standard T4 (0-20 ~g d£ ) x 100
11 "11 11 "( 0 IJg dR. -1 )
112
by incubation for 15 minutes at room temperature. This instant
recycling procedure is ineffective (61% loss). However,
optimisation with other (2M KI, 10% dioxane, etc) ligand
desorbing agents and longer antibody renaturing time at 40C
might improve the procedure. This technique would thus have
economic advantages.
4.3.1.3 Luminescence Immunoassays Utilising FITC-T4
The FIAs (Figure 4.3) based on FITC-T4 show reversibility
of the enhancement phenomenon by unlabelled T4, and thus demon
strate suitability (cf. FL-T4) of FITC-T4 in LIAs. Furthermore,
it is evident (Figure 4.3) that the two-phase FIA exhibits lower
limits of detection (T4) than the corresponding simultaneous
addition FIAs. The maximum binding of unlabelled T4 by the anti
body in phase-one and the minimal [158] dissociation of the
antibody bound unlabelled T4 in phase-two may explain the results.
It is also evident (Figure 4.3) that the 30 minutes incubated
simultaneous addition FIA exhibits lower limits of detection
than the same assay incubated for 3 hours. These results thus
suggest that incubation of the assay for 3 hours results in the
reaction (see below) proceeding to equilibration.
Free Complex
The principal advantage of the two-phase technique is that the
sensitivity of an assay can be enhanced in a very simple way.
113
26
22
-l.1...
QJ 1B > .--I'D
...J
QJ
a:::
14
0
•
[J
0 Simultaneous addition FIA • (3 hours)
• Two- phase FIA ~
[J Simultaneous addition FIA ~ (30 mi nutes)
10~--~--~~--~--~--~~ o 4 8 12 16 20 [T4] n Q/ ml.
FIGURE 4.3: Comparison of two-phase FIA and simultaneous addition FIA techniques
114
On the other hand, the dependence of the technique on low (40C)
temperature and longer assay time than the simultaneous
technique are disadvantageous features.
Experiments (Table 4.5(a)) involving FITC-T4 and T4-depleted
human serum (Ventre/Sep) demonstrates that the non-specific
fluorescence enhancement phenomenon is of serious consequence
in homogeneous FIAs. Furthermore, the competitive binding
assay (Table 4.5(b)) based on T4-depleted human serum exhibited
negligible (Ca. 7%) reversal effect in the presence of
49.7 x 10-7M unlabelled T4. This is possibly due to an over
whelming competition by TBG and to a lesser extent, competition
by a 1 bumi nand prea 1 bumi n, because these studi es were' conducted
in 75 mM barbital buffer (pH 8.6) and moreover, because T4 binds
predominantly to TBG. Smith [44] likewise found that non-specific
binding of FITC-T4 to endogeneous T4 binding proteins in sheep
serum produced the fluorescence enhancement effect. These results
thus demonstrate the problems associated with the fluorescence
enhancement immunoassay. In addition, serum fluorescence (Chapter
Three) due to the albumin-bilirubin complex and light scattering due
to the small (26 nm) Stokes' shift of fluorescein will further
worsen the limits of detection in homogeneous FIAs. It is there-
fore not surprising that the development of viable homogeneous
(enhancement and polarisation) FIAs for serum T4 has thus far
been unsuccessful. However, alternative techniques which are
less prone to interferences (serum fluorescence, non-specific
binding, etc) have been developed, for example heterogeneous
FIA [42] based on FITC-T4, heterogeneous CLIA [159] utilising
115
Dilution of T4-dep1eted human serum in FI -1 the presence of 6 ng.m1 FITC-T4 Ex/Em. 492/535
TABLE 4.5(a):
0 1: 96000 1:12000 1 :4800 1:1600 1 :800
Effect of adding T4-dep1eted human FITC-T4 solution in 75 mM barbita1 pH 8.6
T4 concentration in the presence of
9.0 9.5
9.5 11. 5 21.0 23.5
serum to buffer .•.
FI 6 ng.m1- 1 FITC-T4 and T4-dep1eted Ex/Em. 492/535
human serum diluted 1 to 1600
0 21.0 -9 1. 7 x 10 M 20.5 -9 5.3 x 10 M 20.5 -9 9.9 x 10 M 20.5
20 x 10-9M 20.5 49.7 x 10-9M 19.5
TABLE 4.5(b)): Competitive binding fluorescence assay showing the effect of adding unlabelled T4
Excitation slit: 12 nm Emission slit: 14 nm
116
nm
nm
-
isoluminol-T4, fluorescence protection immunoassay (Section
4.1), and differential heavy-atom quenching FIA (Section 4.1).
The anti-T4 antiserum dilution curve (Figure 4.4(a)) construc
ted using the polarisation technique involving FITC-T4 demonstra
tes that the polarisation value is increased on binding with
the anti-T4 antibodies. The standard dose-response curve
(Figure 4.4(b)) generated using the sequential addition tech
nique shows that unlabelled T4 caused a decrease in the observed
polarisation value with increasing concentration of added
unlabelled T4. Unfortunately, determination of serum T4 levels
using the polarisation technique were unsuccessful when the
immunoassay was performed in the presence of barbital-merthiolate
buffer (pH 8.6) to inhibit the binding of FITC-T4 to the endo
geneous T4 binding proteins in serum and to displace T4 bound
to the serum proteins. The possible explanations are:(a) incom
plete immobilisation [47] of antibody-T4-FITC complex due to
flexing of the T4 moiety and the fluorescein mOiety; (b) back
ground interferences due to serum fluorescence (albumin-bilirubin
comp 1 ex) and 1 i ght scatteri ng due to the sma 11 (26 nm) Stokes'
shift of fluorescein, and (c) binding of FITC-T4 to the serum
proteins as found in the non-specific fluorescence enhancement
studies (above) involving FITC-T4 and T4-depleted human serum.
These results thus imply that pre-extraction of serum T4 will
be essential prior to determination. Unquestionably, this is an
undesirable feature, because the principal advantage of homoge
neous immunoassays is their avoidance of a separation procedure.
117
~
~
(Xl
0·14 c 0 -Cl) 0·12 VI .-. c-m -J
d? 0·10
0·08
006~--~--~--~~--~--~--~--~--~ 1:100 1:300 1:500 1:700 1:900 1:1100 1:1300 1:1500
Final anti-T4 serum dilution FIGURE 4.4(a): Antibody dilution curve showing binding of 10 ng.ml- l FITC-T
4 to serial
dilutions of anti-T4
antiserum
c 0 '--
0'13
0·11
Sequential addition polarisation FIA
~ '0·09 .-c.. to -J
0 a..
0·07
I i i
0'05 6 5 110 115 20 . 25 [T4J ng/ml
FIGURE 4.4(b): Standard dose-response curve for T4
119
I
The anti-T4 antiserum dilution curve (Figure 4.5(a)) construc
ted, based on TCPO-excited luminescence of FITC-T4 shows that
the CL signal is enhanced on binding with the anti-T4 anti
bodies. Figure 4.5(b) demonstrates the use of the two-phase
chemiluminescence enhancement immunoassay of pure T4 solution.
These results thus confirm that TCPO excitation of the antibody
bound FITC-T4 is a consequence of the fluorescence enhancement
effect (cf. Figure 4.3). The possible mechanism of the CL
enhancement effect is described in Section 5.3J.2. Experiments
involving measurement of serum T4 using 0.22M imidazole containing
barbita1-merthio1ate (pH 8.6) were, however, unsuccessful, because
of CL quenching. The heavy-atom effect of sodium merthio1ate
(Hg-containing compound) and an increase in the rate of TCPO
hydrolysis at pH 8.6 may explain the results. These results
further demonstrate the problems (binding to endogeneous serum
proteins, quenching, etc) of T4 determination in serum samples.
Schroeder et a1 [159] likewise found that the CLIA for serum T4,
based on iso1umino1-T4 suffered from non-specific (human serum)
quenching problems, in addition to vulnerability of the assay
to interference by the endogeneous T4 binding proteins in
serum. Consequently, Schroeder et a1 Q59] adopted a hetero
geneous approach to remove this interference. Further work
was not pursued with this technique because the barDita1-
merthio1ate (pH 8.6) and the baribta1-ANS buffer (pH 8.6)
are not compatible (hydrolysis, quenching with merthio1ate)
with the TCPO-imidazo1e system. Nevertheless, the principal
advantage of this technique is that excitation of f1uorescein
120
FIGURE 4.5(a): Anti-T4 antiserum dilution curve
14 •
~ 12 -...l u
~ 10 4-re
--' QJ
0::8
6~ ________ ~ ______ ~~ ____ ~ 1:850 1:1250 1:50
18
17
14
1:450 Anti -T4 dilution
Two-phase CL enhancement immunoassay
13~--~~--~--~--~--~--~ o 4 8 12 16 20 24
Final [T4] ng/ml . FIGURE 4.5(b): Standard dose-response curve for T4.
Flow rate of HO: 4.8 ml min-l 2 2 121
via TCPO is not subject to light scattering (Rayleigh).
4.3.1.4 Luminescence Immunoassays Based on ELISA
The performance of the ELISA based on the label HRP-T4 was
briefly investigated by monitoring the immunoassay with photo
and chemiluminescence (luminol and TCPO-excitation) detection.
The results (Figure 4.6) show that the immunoassay monitored
with luminol CL compare favourably with the standard colorimetric
procedure based on the chromogen, ABTS. On the other hand, the
immunoassays (Figure 4.6) monitored with photo- and chemi
excitation (TCPO) of the fluorescent product of the peroxidase-
tyramine-H202 reaction are relatively insensitive in the range,
0-5 ~g d~-l T4. These discrepancies might be a consequence of
several factors; (a) lack of optimisation (concentration of
the indicator reaction, incubation time, pH, etc) of the
experimental immunoassays; (b) sterical hindrance and diffusion
problems [85,151] at the solid-phase affecting peroxidase activity
and hence the indicator reaction; (c) product inhibition [85]
as evident from the assays utilising the fluorescence reaction
of peroxidase-tyramine-H202; (d) time-dependent [153] release
of hematin from peroxidase affecting the assay monitored with
luminol CL; and (e) low overall CL efficiency of the TCPO-
excited luminescence of the peroxidase-tyramine-H202 reaction,
because of its fluorescence band at 320/405 nm, thus requiring
a relatively high energy to excite the fluorescent product
(tyramine reaction) more efficiently. In addition, peroxyoxalate
122
90 0 o , Luminol CL
• . , TCPO-excited Cl Cl , Photoluminometric
o c 0, Colorimetric
70
-III -C ~
..0 50 '-« --10 C Cl'
Vl
30
10
o 5 10 15 20 25 lT4] pg/ dl
FIGURE 4.6: Performance of the ELISA, monitored with colorimetric, photo- and chemiluminescence detection
123
CL and luminol CL are subject to poor reproducibility, because
the reactions are fast and TCPO is vulnerable to hydrolysis.
These problems will, however, be minimal in automated systems,
for example fia with merging zone principles. The principal
areas of further work would thus be optimisation of the indica-
tor reaction, for example concentration of reagents, pH,
incubation time, etc and preparation of fluorogenic substrates
with emission maxima of 500-550 nm, in particular for excitation
via TCPO. Nevertheless, the experimental immunoassays described
in this study demonstrate versatility and potentiality. Further-
more, there is a possibility of performing simultaneous dual
enzyme immunoassay according to the scheme:
Solid-phase
Colorimetrically
(a) O-Nitrophenyl-S-galactoside
~Ab-T3 -/J - 0
~Ab-T4 - HRP
- galactosidase
(b) Luminol
Measure CL
124
4.4 Conclusion
The fluorescence enhancement immunoassay based on FL-T4 was subject to poor (7-10%) reversibility effect in the presence
of competing unlabelled T4. Furthermore, the fluorescence
enhancement effect was also produced on binding of FL-T4 to
serum proteins (TBG) and non-specific antiserum, for example
anti-phenytoin antiserum. The radioimmunoassay experiment
involving FL-T4 and unlabelled T4, however, suggested that
the presence of unlabelled T4 in the FL-T4 solution might be
responsible for the poor reversible effect observed in the
enhancement FIA. It has been well established that the reac-
tion of fluorescamine with primary amines occurs with a half
time of milliseconds and proceeds with 80-95% of the theoretical
yield; thus the presence of 5-20% of unlabelled T4 in the FL-T4 solution might explain the results. On the other hand, the
fluorescence enhancement immunoassay based on FITC-T4 did not
suffer from the poor reversibility problem as evident from the
standard dose-response curves (two-phase FIA and sequential
,addition FIA). These results thus further indicate that labelling
T4 with fluorescamine might not be efficiently achievable under
the conditions employed.
The investigations involving the reuse of recycled immobilised
antibodies in a second radioimmunoassay suggested that the
recycling procedure employed in this work was ineffective,
because of the substantial (61%) loss in binding of l25 I_T4 by
the recycled antibodies. However, optimisation (desorption,
renaturing conditions, etc) might improve the recycling procedure
125
and obviously, the reuse of antibodies in immunoassays would
then have economic advantages.
Evidently, the two-phase FIA has lower limits of detection
than the corresponding simultaneous addition FIAs, incubated for
thirty minutes and three hours. This is due to two major factors:
maximum binding of unlabelled T4 in phase-one of the immunoassay
and minimal dissociation of the antibody-T4 complex in phase-two
of the immunoassay. The principal advantage of the two-phase
technique is thus high intrinsic assay sensitivity. The fluor
escence enhancement effect produced as a result of FITC-T4 binding to the endogeneous T4 binding proteins in serum, however,
limits the technique to assays in pure solutions. As with the
enhancement FIA, the sequential polarisation FIA also suffered
from serum protein (TBG, etc) interferences. Unquestionably,
serum fluorescence due to albumin-bilirubin complex and light
scattering due to the small (26 nm) Stokes' shift of fluorescein
will further worsen the limits of detection in the polarisation
and the fluorescence enhancement immunoassay. The problems of
background interference and serum fluorescence, however, can
be eliminated by TCPO-excited luminescence of FITC-T4. The TCPO
excited LIA utilising the fluorescence enhancement effect is
vulnerable to two major interferences: CL enhancement due to
FITC-T4 binding to the endogeneous T4 binding proteins in serum,
prepared in 0.22M imidazole-HCl buffer (pH 7.0); quenching
when the immunoassay is performed in 0.22M imidazole containing
barbital-merthiolate (pH 8.6). The polarisation and the lumines
cence enhancement immunoassays are indeed simple to perform and
126
hence they can be easily automated, for example using fia with
merging zone principles. However, the present results suggests
that pre-extraction of T4 from serum samples will be essential
prior to measurements using the homogeneous immunoassay tech
niques. The pre-extraction step will undoubtedly be an undesirable
feature of the assay, because the principal advantage of homo
geneous immunoassay is their avoidance of separation. Alterna
tive approaches of circumventing the interference problems
would be (a) development of fluorescent labels incapable of
binding the endogeneous T4 binding serum proteins; (b) the use
of fluorescent labels with Ex/Em maxima outside the serum
fluorescence range, and (c) the use of heterogeneous immuno-
assays.
Finally, ELISA monitored with luminol CL compared favourably
with the standard colorimetric procedure based on the chromogen,
ABTS. On the other hand, the photo-excited and the TCPO-excited
luminescence assays utilising the fluorescence reaction of
peroxidase-tyramine-H202 were insensitive in the range, 0-5 ~g d~-l
T4. These discrepancies are partly due to lack of optimisation
of the indicator reaction (luminol, tyramine, etc) and partly
due to several other factors, for example steric hindrance and
diffusion problems at the solid-phase affecting the assays based
on measurement of the peroxidase activity, but not the luminol
monitored assay, because this assay is based on measurement of
hematin content of peroxidase. The experimental immunoassays
based on ELISA demonstrate versatility and potentiality. Further
more, there is a possibility of performing a simultaneous dual-
127
enzyme immunoassay for T4 and T3. The ELISA technique for the
simultaneous determination of T4 and T3 would involve labelling
T4 and T3 with two different enzymes, whose catalysed reactions
can be distinguished from each other by CL or co1orimetrica11y,
for example HRP-T4 could be measured using lumino1 CL. However,
at the present, determination of serum T4 with the homogeneous
(enhancement, polarisation, etc) immunoassay techniques is
still a challenge for an analyst.
128
CHAPTER FIVE: ENHANCEMENT CLIA FOR HUMAN SERUM ALBUMIN
5.1 Introduction: Physiological Role and Assay Techniques
Human serum albumin is synthesised almost exclusively by
the liver and comprises of about 60% by weight of the proteins
circulating in plasma/serum [4,160]. The normal range of p1asma/
serum albumin is 35-45 gm ~-1. Some of the major functions of
albumin include: maintenance of homoestasis, transport of
physiological (hormones, 1ipids, etc) and non-physiological
(drugs) substances, nutritive role, and detoxification of unconju
gated b1\~Yu~'n,-~leasurement of albumin levels is thus an important
indicator of pathological conditions such as malignancies, hepa-
tic dysfunction, malnutrition, and nephrotic syndrome. One of
the most widely employed non-immunochemica1 approaches for
measuring albumin levels is based on the affinity of albumin for
dyes such as bromocresol green (BCG) and methyl orange. The dye
binding method lacks the specificity and sensitivity required for
the determination of albumin in urine or cerebrospinal fluid.
Immunoassay techniques, because of their high sensitivity and
excellent specificity are thus the methods of choice. Furthermore,
assays may be performed using micro1itre quantities of sample.
Obviously, this is advantageous in situations where the sample
is available in limited amounts, for example samples from a new1y
born child. Some of the recently described non-isotopic immunoassay
techn i ques a re:
129
a) So~id-phase FIAs
The FlAX system (Section 1.6.2) developed by International
Diagnostic Technology is available commercially and is based on
a "STIQ" sampler with immobilised antibodies. Nargessie et al
[161] have described the use of magnetic particles with immo
bilised anti-HSA antibodies to separate the bound and the free
fraction. The label utilised in each case was fluorescein
isothiocyanate.
b) Energy transfer FIA
Miller et al [162] have described an energy transfer immuno
assay utilising albumin and anti-HSA antibodies labelled with
fluorescein (donor) and rhodamine (acceptor). respectively.
The technique has been automated [45] using stopped flow injec
tion analysis with merging zone principles.
c) F~uoresaenae enhanaement immunoassay
Fluorescence enhancement immunoassays utilising the fluram
enhancemen t effect of fl uram and its ana 1 ogue HDPF have been
developed by Lim [45].
d) F~uoresaenae proteation immunoassay
This technique [163] is based on the use of albumin labelled
with fluorescein isothiocyanate. anti-HSA antibodies and anti
fluorescein antibodies. The sample containing FITC-HSA is
incubated first with anti-HSA antibodies and this is followed by
130
incubation with anti-fluorescein antibodies. The fluorescence
of the free FITC-HSA is quenched when the anti-fluorescein
antibodies bind the fluorescein moiety. The antibody-bound
albumin, however, is sterically protected, and therefore, not
quenched. The degree of quenching thus depends on the amount of
albumin present in the sample.
e) Thermometric ELISA
In the described [164] assay HSA was conjugated with cata
lase and the activity of the antibody-bound fraction was measured
using hydrogen peroxide as the enzyme substrate.
f) SoZid-phase CLIA based on peroxidase
In the described [84,165] assays HSA was labelled with
peroxidase. The antibody-HSA-peroxidase complex was measured
using luminol CL.
g) SoZid-phase CLIA based on hemin
This technique [166] utilised HSA labelled with hemin and
solid-phase with immobilised anti-HSA antibodies. The fraction
bound to the solid-phase was determined with luminol detection.
This chapter describes the development of a semi-automated
chemiluminescence enhancement immunoassay based on TCPO-excited
luminescence of HSA labelled with fluorophors such as fluorescein,
131
dansyl chloride, fluram, and MDPF. The performance of the assay
was evaluated against a standard human serum sample and the colori
metric method based on bromocresol green.
5.2 ~xperimental
5.2.1 Genera~
Human serum albumin was conjugated (Section 2.2.1) with each
of (a) fluorescein isothiocyanate, (b) dansyl chloride, (c) MDPF,
and (d) fluram. The labelled HSA solutions, H202, anti-HSA
antibodies, serum samples and protein solutions (anti-a-2-
macroglobulin, BSA, etc) were stored at 40 C and equilibrated at
room temperature prior to measurements. Sample and reagents for
peroxyoxalate CL were prepared as described in Section 2.3.2.2.
Experiments involving fluorescence studies were conducted using
the MPF-44B Fluorescence Spectrophotometer (Section 2.3.1).
The peroxyoxalate-fia luminometer (Section 2.3.2.1) was employed
in all experiments, except those involving kinetic studies. These
were performed in a non-flow system using the luminometer, coupled
to its display/printer unit (LKB Model 1250-101). The hydrogen
peroxide stream was propelled using the pressure-bottle in all
studies, except those involving titrations: these studies were
conducted using the peristaltic pump. The background CL signal
was subtracted in all measurements.
5.2.1.1 Comparative Studies of Ana~ytiaaZ Parameters
An experiment comparing the TCPO-excited luminescence of
labelled HSA conjugates was performed using D-HSA (F:P ratio, 2.1:1),
132
FITC-HSA (F:P ratio, 2.3:1), and MPDP-HSA (F:P ratio 2.4:1).
The effect of F:P ratio on the luminescence signal was examined
using 28.1 x 10-gM FITC-HSA and 28.1 x 10-gM D-HSA.
5.2.1.2 Immunoassay Studies -g
The titration curves were constructed using 28.1 x 10 M
labelled HSA conjugates. Incubation with the anti-HSA antibodies
was performed for one hour at 250 C. Experiments involving the
speci fi city of the assay were conducted us i ng 28.1 x 1 o-gl~ D-HSA
(F:P ratio, 1:1), 28.1 x 10-gM FITC-HSA (F:P ratio, 2.3:1),
28.1 x 10-gM MDPF-HSA (F:P ratio, 0.7:1), and 33.0 x 10-gM FL-HSA
(F:P ratio, 17.4:1). These experiments were performed by incuba-
ting the sample with non-specific antiserum, bovine serum albumin,
etc. The kinetic experiment was performed by incubating
28.1 x 10-gM D-HSA (F:P ratio, 1:1) with 50 x 10-gM anti-HSA
antibodies. The control (minus the antibody) was similarly
prepared and the samples were incubated in the dark for one hour
at 250C. The samples (0.1 ml) containing 0.1 ml hydrogen peroxide
(1.02g ml-l ) and 5 ~l TCPO solutions were vortex-mixed for five
seconds and analysed in a non-flow system.
The simultaneous addition assay technique was performed
using 0.6 ml of 28.1 x 10-gM (final concentration) D-HSA (F:P ratio,
1:1) containing 0.6 ml of the sample (known or unknown). The
samples were vortex-mixed and to each 0.6 ml of anti-HSA anti--9 body solution was added at a final concentration of 85 x 10 M.
The samples were vortex-mixed and incubated for one hour at 250C.
133
The two-phase sequential addition technique was performed as
follows:
Phase~one: To 0.6 m1 of HSA solution, 0.6 m1 of·anti-HSA anti-
body solution was added. The samples were incubated
for one hour at 250C.
Phase-two: The samples (phase-one) were incubated for 3 hours at
4 ! 0.50C. Subsequently, the pre-incubated D-HSA
(F:P ratio, 1:1) was added to each of the sample
solutions. The final concentration of anti-HSA anti--9 -9 bodies and D-HSA were 85 x 10 M and 28.1 x 10 M,
respectively. The samples were incubated for one hour
at this temperature.
The co10rimetric procedure based on bromocresol green was
performed using the Sigma (Poo1e, Dorset) diagnostic kit (No. 630).
5.3 Results and Discussion
5.3.1.1 Compa~tive Studies of AnaLyticaL Parameters
Apparently (Figure 5.1), the most efficiently excited
labelled HSA conjugate amongst D-HSA (F:P ratio 2.1:1), FITC-HSA
(F:P ratio, 2.3:1) and MDPF-HSA (F:P ratio, 2.4:1) is the D-HSA
conjugate. Consequently, the D-HSA conjugate can be measured with
relatively high sensitivity and with high precision. The high
excitation efficiency of the D-HSA conjugate compared with the
excitation efficiencies of the FITC-HSA and the MDPF-HSA conjugates
may partially explain the results. This is because the fluorescence
134
~
w (J1
100~-------------------------------
80
>-4
-I LJ 60 aJ >
4-
~ 40 aJ er:
20
- D 0-0, D-HSA (F:P ratio, 2.1:1)
-, FITC-HSA (F:P ratio, 2.3:1)
MDPF-HSA (F:P ratio, 2.4:1)
200 400 [ Labelled - HSA]
FIGURE 5.1: Comparison of labelled HSA conjugates.
800
------ ---- - ----
quantum yield of dansyl chloride is low (0.3) compared with the
value of 0.85 for fluorescein isothiocyanate. The discrepancy
in the results may be due to two other factors: differences in
the F:P ratio of the conjugates and heterogeneous population of
labelled conjugates in a single preparation. The results are
thus semiquantitative. Nevertheless, such a comparison is impor
tant in choosing a label in an immunoassay technique. It has
been well documented [42,106,161] that the fluorescence of
proteins (HSA, globulins, etc) labelled with fluorescein, dansyl
chloride, etc is dependent on the F:P ratio of the conjugate.
Experiments (Table 5.1) to investigate this effect were performed
with TCPO excitation of FITC-HSA and D-HSA conjugates. The effect
of F:P ratio on the luminescence of these conjugates is shown
in Table 5.1. As an example, the CL signal of a heavily (F:P
ratio, 13.6:1) labelled FITC-HSA and a lightly (F:P ratio, 2.3:1)
labelled FITC-HSA conjugate was 13 and 24 units,respectively.
Chen [106] attributed the decrease in the fluorescence of FITC-HSA
conjugates to concentration quenching, collisional interactions
and decrease in the fluorescence efficiency. The effect of these
on the CL signal may partially explain the results, for example,
a decrease in the fluorescence efficiency of the label and hence
a consequent reduction in the CL efficiency. This is because CL
efficiency (equation 1.5) is a product of fluorescence efficiency
and the excitation efficiency of a chemical reaction. Nargessi
et al [161] likewise found that the fluorescence of FITC-HSA
decreased when the F:P ratio of the conjugates exceeded 6:1.
136
Expe ri men ta 1 CLl
28.1 x 10-9M FITC-HSA (F:P ratio, 2.3: 1 ) 24.0
28.1 x 10-9M FITC-HSA (F:P ra ti 0, 7.4:1) 19.0
28.1 x 10-9M FITC-HSA (F:P ratio, 13.6:1 ) 13.0
28.1 x 10 -9 t1 D-HSA (F:P ratio, 1 : 1 ) 21.5
28.1 -9 x 10 M D-HSA (F:P ratio, 2.1 : 1 ) 25.0
28.1 -9 x 10 M D-HSA (F:P ra ti 0, 7.8:1) 20.0
TABLE 5.1: Effect of F:P ratio on the TCPO-excited luminescence' of labelled HSA conjugates
137
A valid comparison, however, cannot be made due to hetero
geneous labelling of HSA in a single preparation. Nevertheless,
such a preliminary study is important (see below) in developing a
luminescence immunoassay.
5.3.1.2 Immunoassay Studies
The titration curves (Figures 5.2-5.5) show the CL enhance
ment effect. The magnitude of this effect, however, depends on
F:P ratio of the conjugates and the concentration of the anti-HSA
antibodies. The effect (see above) of F:P ratio on the CL signal
and the formation of cros~linked aggregates (Ab-Ag~Ab, Ab-Ag-Ab
Ag, etc) may explain the results. For example, sterical protection
of the labelled sites in an aggregate may partially be responsible
for reduction in the CL enhancement effect. Attempts to reproduce
the enhancement effect utilising D-HSA conjugates with photon
excitation demonstrated (Table 5.2) that the effect was not
repl aced. L im [45] 1 i kewi se found that fl uorescence enhancement
effect did not occur with the D-HSA conjugates. This effect,
however, occurs with FL -HSA and ~1DPF -HSA conjugates. In additi on
to HSA, the fluram enhancement effect has been utilised [45] to
develop assays for tricyclic antidepressants, human IgG, trans
ferrin, etc.
The mechanism of the CL enhancement was investigated briefly
in a non-flow system using D-HSA (F:P ratio, 1:1) and anti-HSA
antibodies. The CL reaction was initiated externally due to two
factors: the luminometer was modified for flow injection analysis
138
---1 LJ
ClJ >
4-ro -ClJ
0:: ~
W <D
0 0 , F:P ratio, 17.4: 1
40 <>-<> , F:P ratio, 0.8: 1
.......... , F:P ratio, 2.2:1
30
20
10
OL-------~~------~--------~--------~--~ o 40 80 120 160 [Anti-HSA] x10-9M
FIGURE 5.2: Titration curves showing the effect of anti-HSA antibodies on the luminescence of FL-HSA conjugates.
-1 Flow rate of H202: 6 ml min
~
-J LJ
aJ > -~ ro .., --'
0 aJ a:::
60
45
30
15
o 0
, F:P ratio, 3.2:1
, F:P ratio, 2.4:1
, F:P ratio, 0.8:1
~-. - ___ a ----____ a
40 80 120 160 [Anti-HSA] x10-9 M
FIGURE 5.3: Titration curves showing the effect of anti -HSA antibodies on the luminescence of MDPF-HSA conjugates
-1 Flow rate of H
20
2: 6 ml min
....... -1 u QJ
> -.!9 QJ
~
'" CY ~
25r-------------~~~~----------~
20
15
10 • • • F:P ratio, 2.3: 1
5 G 0 • F:P ratio, 13.6: 1
<> 0 • F:P ratio, 7.4: 1
o o d
40 80 120 160
[Anti-HSA] X 10-9 M FIGURE 5.4: Titration curves showing the effect of anti-HSA antibodies on the luminescence
of FITC-HSA conjugates.
-1 Flow rate of H202: 6.4 ml min
..,. N
55 -, F:P ratio, 1 : 1
-0 0, F:P ratio, 2.1 : 1
.45 F:P ratio, 7.8: 1 --I LJ
QJ 35 > 4-ro ~
QJ 25 0:::
15
40 80 120 160 [Anti-HSAl x10-9M
FIGURE 5.5: Titration curves shO\~ing the effect of anti-HSA antibodies on the luminescence of D-HSA conjugates
-1 Flow rate of H
20
2: 6.4 ml min
Ex/Em, 325/525 nm Experimenta 1 FI of FI of
blank sample
D-HSA (F:P ra ti 0, 1 : 1 ) 49.2 53.5
" " " " -9
+ 10.6 x 10 M anti-HSA 49.2 53.2
" " " " + 21 x 10 -9M " 49.5 55.0
" " " " + 43 x 10-9~1 " 49.0 54.2
" " " " + 85 x 10-9M " 49.2 54.8
" " " " + 160 x 10-9M " 49.0 55.0
D-HSA (F:P rati 0, 7.8:1) 49.2 55.0
" " " " -9
+ 10.6 x 10 M anti-HSA 49.2 55.0
" " " " + 21 x 10 -9M " 49.5 56.0
" " " " -9
+ 43 x lOt·' " 49.0 52.0
" " " " + 85 x lO-9M " 49.2 5~.2
" " " " + 160 x 10-9M " 49.0 54.0
TABLE 5.2: Effect of adding anti-HSA antibodies to 28.1 x 10-9M D-HSA conjugates
Excitation slit: 8 nm
Emission slit: 12 nm
143
and therefore generation of a complete intensity-time curve
was difficult, and moreover, as described in Section 2.3.2 the
procedure suffers from poor precision with conventional injection
methods due to variations in force of injection, mixing etc.
Nevertheless, the results (Figure 5.6) suggest that the enhancement
effect may be due to an increase in the reaction rate and/or due
to an increase in the overall CL efficiency of the reaction.
This is because CL intensity (equation 1.6) is a product of
reaction rate and CL efficiency. The increase in the CL effi
ciency may be due to an increase in the fluorescence efficiency
of the antibody-bound labelled antigen. The possibility of uti
lising a change in the reaction or a change in the peroxyoxalate
CL efficiency has been described [30,39] for developing homogeneous
assays. Further work, however, is required to establish the exact
mechanism of the enhancement effect.
Experiments (Figure 5.7 and Table 5.3) performed with bovine
serum albumin, bovine serum, anti-a-2-macroglobulin, etc demon
strates that the enhancement assay is highly specific. Further
more, the effect is a consequence of specific antigen-antibody
complexation, because the effect is negligible (2-7%) in the pre
sence of anti-a-2-macroglobulin and reversible in the presence
of HSA. The negligible (ca.12%) reversal effect (Figure 5.7) with
BSA is possibly due to cross-reactivity of the antibodies and/or
due to contamination of the sample with HSA, on contact with the
sample loop and the fia tube. The CL enhancement effect (Table
5.3) observed with the anti-phenytoin antiserum may be due to
144
"
.........
........J LJ
60
50
aJ 40 > -re
QJ 30 cc:
20
10
-, D-HSA (F:P ratio, 1 :1)
o---(), D-HSA + anti-HSA antibodies
o~----~--~--~----~--~ 10 20 30 40 50 60
Time (Secs)
FIGURE 5.6: intensity-time curves showing the luminescence of D-HSA and D-HSA plus anti-HSA antibodies
145
--' u Qj
> .~ ...., '" ~
A
· .-
B
C
D
'--
FIGURE 5.7 Examples of recorder traces showing the luminescence
of (A) 28.1 x lD-9M D-HSA (F:P ratio!, 1:1);
(B) 28.1 x lD-9M D-HSA (F:P ratio, 1:1) + 85 x lD-9M anti-HSA antibodies; (C) 28.1 x lD-9M D-HSA (F:P ratio
1:1) + 39.8 x lD-9M BSA + 85 x lD-9M anti-HSA antibodies;
and (D) 28.1 x lD-9M D-HSA (F:P ratio, 1:1) + 40.2 x lD-9M HSA + 85 x lD-9M anti-HSA antibodies
146
Experimental CLl
28.1 -9 1 : 1 ) + 85 x 10-9M x 10 M D-HSA (F:P ratio, 71.0 anti-HSA antibodies
28.1 -9 -9 x 10 M D-HSA (F:P ratio, 1:1) + 85 x 10 M 6.0 anti-HSA antibodies + human serum (1:12000)
28.1 -9 1 : 1 ) + 85 x 10-9M x 10 M D-HSA (F:P ratio, 79.5 anti-HSA antibodies -9 -9 28.1 x 10 M D-HSA (F:P ratio, 1:1) + 85 x 10 M 78.5 anti-HSA antibodies + bovine serum (1:12000)
-9 33 x 10 t·1 FL-HSA (F:P ratio, 17.4:1 ) 14.5 -9 17.4:1 ) + anti-33 x 10 M FL-HSA (F:P ratio, 21.00 phenytoin antiserum (1:7500) ,
28.1 -9 x 10 M FITC-HSA (F:P ratio, 2.3:1) 19.0 -9 19.5 28.1 x 10 M FITC-HSA (F:P ratio, 2.3:1)
+ 4.3 x 10-9M anti-a-2-macroglobulin
-9 28.1 x 10 M MDPF-HSA (F:P ratio, 0.7:1) 15.0 -9 16.0 28.1 x 10 M ~1DPF-HSA (F:P ratio, 0.7:1)
+ 4.3 x 10-9M anti-a-2-macrog10bulin
TABLE 5.3: Effect on the luminescence of labelled HSA conjugates in the presence of each of the above samples
147
contamination of the antiserum with anti-HSA antibodies or
due to the use of HSA as an immunogenic carrier for raising of
the anti-phenytoin antibodies. The enhancement CLIA based on
the two-phase sequential technique and the simultaneous addition
technique are shown in Figure 5.8. Evidently, the two-phase
sequential assay has lower detection limits (HSA) than the simul
taneous assay. This is due to two factors: maximum binding of
HSA in phase-one of the assay and minimal dissociation of the
antibody-HSA complex in phase-two of the assay. The technique is
thus particularly useful for determining urinary albumin or when
the sample is available in limited amounts, for example sample from
a newly-born child.
The performance of the two-phase sequential enhancement CLIA
(Figure 5.9) was evaluated against a standard human serum (SHS)
sample and against the bromocresol green method. The precision
(CV of at least 5.6%) is acceptable for clinical measurements
and the results (Table 5.4) obtained agree favourably with the
value for the standard human serum sample and the bromocresol
green method.
148
....... --.J LJ
QJ
> 40-tU
--' QJ
0:::
40
30
20
10
• ., Two-phase sequential addition technique
- --, Simultaneous addition techn i que
\
o~--~----~--~--~----~ o 10 20 30 40 [HSA] X 10- 9 M
so
FIGURE 5.8: Comparison of the two-phase sequential addition enhancement CLIA and the simultaneous addition enhancement CLIA. The concentration of D-HSA (F:P ratio, 1:1) was 28.1 x 1O-9M
-1 Flow rate of H202: 4.8 m1 min
149
~
l1"I 0
50
40 ....... -.J LJ
<lJ 30 > .-
"'-It) ~
& 20
1
0' 6 io 4'0 6'0 . 8'0
[HSA] x 10-9 M
FIGURE 5.9: Standard dose-response curve based on the two-phase sequential addition technique. Error bars represent standard deviations for five determinations.
-1 Flow rate of H202: 5 ml min
Sample Two-phase sequential CV (%) BCG method CV (%) enhancement CLIA
SHS containing 47 g.l -1 albumin 47.7 g.l -1 [12,000] 4.2 (n=5) 46.7 g.l -1 [250] 4.1 (n=5) 49.3 g.l -1 [24,000] 5.6 (n=5)
Pooled human serum 45.3 g.l -1 [12,000] 4.5 (n=4 ) 45.6 g.l -1 [250] 5.0 (n=4)
TABLE 5.4: Comparison of the two-phase sequential enhancement CLIA, a standard human serum sample, and the bromocresol green method. Figures in square brackets represent dilution factors.
5.4 Conclusion
The study of analytical parameters involving comparison of
the luminescence of D-HSA (F:P ratio, 2.1 :1), FITC-HSA (F:P ratio,
2.3:1), and MDPF-HSA (F:P ratio, 2.4:1) conjugates demonstrated
that the D-HSA conjugate could be measured with the highest sensi
tivity and with the highest precision. The relatively high excita
tion efficiency of the D-HSA conjugate may partially explain the
results. The main criticism of this comparative study is that the
F:P ratio of the conjugate is not identical, and consequently,
the results are liable to misinterpretation. Further studies
involving comparison of the luminescence of a heavily (F:P ratio,
13.6:1) labelled FITC-HSA conjugate and a lightly (F:P ratio,
2.3:1) labelled FITC-HSA conjugate and also, D-HSA conjugates
confirmed that CL decreased with the heavily labelled conjugates.
The reduction in the CL signal is most likely a consequence of
decreased fluorescence efficiency, concentration quenching or
co11isiona1 interactions.
The CL enhancement effect as evident from titration curves
is due to antigen-antibody comp1exation. The magnitude of this
effect, however, depends on two major factors: concentration of
the anti-HSA antibodies, and the F:P ratio of the conjugates.
The effect of F:P ratio on the CL signal and the formation of
cross1inked aggregates (Ab-Ag-Ab, Ab-Ag-Ab-Ag, etc) may explain
the results. The CL enhancement is highly specific, because of
the negligible (Ca. 12%) reversal effect with bovine serum albumin.
This is possibly due to cross-reactivity and/or contamination of
152
the injected (into sample loop) with the human serum albumin.
Furthermore, the enhancement effect is negligible (2-7%) with
non-specific antibodies such as anti-a-~macroglobulin, and in
addition, the assay does not suffer from environmental inter
ferences (quenching). The mechanism of the enhancement effect is
uncertain. It is probably due to an increase in the reaction
rate and/or due to an increase in the overall CL efficiency.
This may be a consequence of an increase in the fluorescency
efficiency of the antibody-bound labelled antigen.
The two-phase sequential enhancement CLIA has lower limits
of detection than the simultaneous addition technique. This
is partially due to maximal binding of HSA in phase-one of the
assay and partially due to minimal dissociation of the antibody
bound HSA. The two-phase enhancement CLIA is thus particularly
useful for determination of albumin in urine, cerebrospinal
fluid, or when the sample is available in limited amounts, for
example samples from a newly-born child. Moreover, the technique
is valid (compares favourably with the BCG method), simple and
hence amenable to full automation, does not suffer from environ
mental interferences and flexible in approach, because labels such
as MDPF, FITC, etc may be employed.
153
CHAPTER SIX: GENERAL CONCLUSION
Luminescence procedures based on TCPO excitation of
fluorophors have thus far found limited utility in biochemical
analyses. This is primarily due to incompatibility of TCPO with
the aqueous, near-neutral environment required for biochemical
materials such as proteins. The procedures suffer from poor
reproducibility in static systems due to four major factors:
(a) limited water-solubility of TCPO in buffers such as phosphate,
barbital, etc, (b) hydrolysis of TCPO, (c) variations in force
of reagent injection, and (d) instantaneous production of CL.
These problems were partially circumvented in this work using
an optimised peroxyoxalate-fia luminometer and using 0.22M imi
dazole-HCl buffer, pH 7.0. The hydrolysis of TCPO, however,
presented a major practical problem, although it was minimised
by maintaining a constant time interval between TCPO addition
to the sample and injection of the sample plus TCPO into the
flowing hydrogen peroxide stream. McCapra [701 has recently
suggested that hydrolysis of oxalate derivatives may be minimised
using acridane phenyl ester derivatives with bulky protective
groups near the ester linkages. This may reduce the CL efficiency
of the oxalate-fluorescer system because of the substituent
(e.g. electronegative groups substituted with electron-donating
groups) effects. The precision of the described system may be
improved using sophisticated flow injection analysis with merging
zone principles. Alternatively. a centrifugal fast analyser may
154
be employed to minimise TCPO hydrolysis and to achieve better
mixing of the reagents [30]. The additional advantage of
utilising a centrifugal fast analyser is that a complete time
intensity curve can be generated and hence kinetics of a reaction
may be studied. The relatively high-cost of centrifugal fast
analysers may, however, at the present limit their wide use.
The use of the imidazole -HCl buffer (pH 7.0) eliminated
the non-specific quenching problem encountered in the preliminary
TCPO-excited LIA studies. The mechanism of the non-specific
quenching effect is obscure. It probably occurs via interaction
of protein groups with the high energy, l,2-dioxetanedione/charge
transfer complex (Scheme 1). Detailed investigations of the
dependence of this effect on the relative molecular weight of
added proteins is required to establish the exact mechanism and
its possible exploitation in analysis. The background serum
fluorescence problem, on the other hand, was reduced in this
study using adsorbents such as calcium hydroxylapatite, Blue
Sepharose CL-6B etc. Calcium hydroxylapatite is the most
efficient adsorbent because about 95% reduction in the serum
fluorescence was accomplished at the wavelengths studied. Tita
nium dioxide and titanium oxide are also promising materials,
but the laboratory grades are not suitable for column chromato
graphy. The spheroidal materials described by Thomson and
Miles [139] will be particularly useful in FIA techniques. This
is because molecular exclusion may be utilised to enhance the
fractionating properties of the adsorbents. These studies thus
155
provided a valuable insight into identification of the possible
endogeneous (serum and urine) fluorescent materials. Moreover,
the potential of this highly efficient, economical, and simple
approach in reducing serum fluorescence was demonstrated. The
use of this approach in homogeneous FIAs would significantly
improve the detection limits. This will, of course, be a
disadvantageous feature of the assay because the principal
advantage of homogeneous assays is their avoidance of any sepa
ration method.
The fluorescence enhancement utilising FL-T4 was unsuitable
for LIA studies due to poor reversibility of the assay. The
radioimmunoassay investigations suggested that incomplete
labelling of thyroxine may be responsible for the poor reversi
bility effect. The two-phase sequential addition FIA utilising
FITC-T4 has lower detection limits than the corresponding simul
taneous addition FIAs. This simple technique is thus particularly
useful for the determination of low analyte concentrations or
when the sample is available in limited amounts. The endogeneous
T4 binding serum proteins (TSG) presented a major problem in
developing a viable enhancement FIA, polarisation FIA, and the
TCPO-excited LIA utilising the fluorescence enhancement effect
of FITC-T4. These problems may be partially resolved using pre
extracted material from serum samples or using labels with minimal
affinity for serum proteins. Such a fluorescent label has appa
rently not been developed thus far, although a 125I-labelled
thyroxine derivative has recently been introduced in the Amerlex
156
T4 RIA kit, developed by Amersham International Limited, UK.
The ELISA technique as demonstrated in this study is highly
versatile, because the T4 assay was monitored with photo
and chemiluminescence (luminol, peroxyoxalate) detection.
The assay based on luminol CL compared favourably with the stan
dard colorimetric procedure. The TCPO-excited LIA utilising the
fluorescence reaction of tyramine-peroxidase-H202 may be improved
using fluorogenic substrates with high Ex/Em maxima and high
fluorescence efficiency. An alternative method of achieving
this would be the use of fluorogenic substrates with amino
groups. As an example of this simple approach the detection
limits [119] for aminofluoranthene is 83,500 lower than that of
fluoranthene.
The results obtained using the enhancement CLIA for human
serum albumin correlated well with a standard human serum sample
and the colorimetric method based on bromocresol green. The assay
exhibits excellent specificity and is not prone to environmental
interferences. The mechanism of the CL enhancement is valid for
both low- and high-molecular weight analytes, and furthermore,
the effect apparently occurred with a wide variety of fluorescent
labels such as fluorescein isothiocyanate, dansyl chloride, MDPF,
and fluorescamine. These studies thus suggest the possible exploi
tation of this simple technique in multicomponent analysis. One
possible approach would be to label an antigen, for example with
fluorescamine and a second antigen, for example. with rhodamine
B-isothiocyanate. The use of appropriate cut-off filters would
157
)
then enable measurement of the luminescence of these labels.
However, detailed investigation into the mechanism of the CL
enhancement effect is required for its exploitation in the
development of a generally-applicable CL enhancement immunoassay.
The combined merits of high intrinsic sensitivity and prac
ticality (ease of labelling, low-cost, etc) of LIAs ensures
increasing use and rapid expansion of these techniques in
analytical sciences. Further advances in LIAs are anticipated
in the following major areas.
Instrwnentation
a) Videofluorimeters [167,168] for improving the signal-to
noise ratio, and for the simultaneous representation of
excitation, and emission spectra in the form of three
dimensional spectra known as excitation-emission matrix (EEM)
or fluorogram. The EEM data would be particularly useful in
identifying fluorescent materials present in a complex
sample.
b) Photon-counting luminometers [30] for improving the signal
to-noise ratio.
c) Automation 069,170,171] and theutil isation of mi croprocessors
in data acquisition, storage etc. The use [l72]" of intelli
gence of computers in optimising an assay, for example,
selection of optimal operating parameters, corrections for
background interferences, etc. This would facilitate complete
automation of LIAs.
158
Monoelonal Antibodies
These antibodies, produced [173,174J by hybridoma fusion
of sensitised lymphocytes and myeloma cells are homogeneous.
Monoclonal antibodies are thus particularly valuable in enhan
cing specificity and sensitivity of an assay. Furthermore,
these antibody preparations will facilitate quality control.
It is quite evident that these techniques will revolu
tionise the areas of clinical diagonsis, forensic science, etc.
The full impact of these on LIAs, however, remains to be wit
nessed.
159
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