Development of a Membrane-Based Immuno-PCR Assay for Enhancing the Sensitivity of Lateral Flow

Detection

ii

Development of a Membrane-Based Immuno-PCR Assay for Enhancing the Sensitivity of Lateral Flow

Detection

By

Roni Nugraha

A thesis submitted in partial fulfillment of the Requirements for the Degree Masters of Science in Engineering

Department of Chemical Engineering Chung Yuan Christian University

2008

i

Abstract

Membrane-based immuno-PCR procedure was developed by applying

the immuno-PCR technique on membrane nitrocellulose to increase the sensitivity

of the lateral flow immunochromatography assay. The test procedure followed the

traditional lateral flow immunochromatography assay. But instead the gold

nanoparticles-conjugated antibody, this study employed DNA-conjugated

antibody as the signal reporter. The DNA reporter was amplified by PCR to

enhance the detection sensitivity.

The detector antibody was optimized its conjugation method for the

DNA-streptavidin complex. Two methodologies were tested the effect of the ratio

of DNA to streptavidin and the ratio antibody to DNA-streptavidin complex. The

1:1 ratio of antibody to DNA-streptavidin complex was found to give the best

result and therefore selected for further test.

Premixing of the detector antibody and DNA, 5 minutes waiting time,

and 0.5 mg/ml spraying concentration of capture antibody were concluded the

best condition to give the best signal. This condition was demonstrated on the

membrane-based immuno-PCR procedure to detected human IL-6 and reported

the detection sensitivity is

ii

reagents and assay formats is necessary to reduce the high background problem in

this study.

iii

PCR

DNA DNA

PCR

DNA streptavidin DNA-Streptavidin

DNA-Streptavidin

DNA-Streptavidin 11

DNA 5 0.5

mg/ml human IL-

6

iv

Acknowledgement

My foremost thank goes to my thesis adviser Dr. Jui Chuang Wu.

Without him, this thesis would not have been possible. I thank him for his

patience and encouragement that carried me on through difficult times, and for his

insights and suggestions that helped to shape my research skills. His valuable

feedback contributed greatly to this thesis.

I am grateful to the Head of Chemical Engineering Dept, Dr Tsair Wang

Chung, who introduced and helped me to start my graduate student life in

Chemical Engineering. I thank the rest of my thesis committee members: Dr.

Tzong-Yuan Wu and Dr. Hung-Ju Richard Su. Their valuable feedback helped me

to improve the thesis in many ways.

I thank all the students and staffs in Chemical Engineering department,

whose made my time was very enjoyable. I am grateful for time spent with

roommates and friends, Wetra, Irdham, Iqbal, Yusuf, Ratno, Osmand and all

international students in Chung Yuan Christian University. Also to my labmates,

Dan, Carol, Annie, Peter, Kevin, Syin, Anan and Vani, that had helped me a lot

and gave me support during my study in CYCU.

v

Last but not least, I thank to my parents, my sister, my brother and my

lovely wife for their understanding, endless patience and encouragement when it

was most required.

Chung Li, Taiwan, July 2008

Roni Nugraha

vi

Contents

Abstract ................................................................................................................ i

.............................................................................................................. iii

Acknowledgement.................................................................................................. iv Contents .............................................................................................................. vi

List of Figure........................................................................................................ viii

List of Table ........................................................................................................... ix Chapter 1. Introduction ........................................................................................... 1

1.1 Antibody-antigen interaction ...................................................................... 1 1.2 Immuno-PCR .............................................................................................. 9 1.3 Lateral flow immunochromatography assay............................................. 13

1.4 Motivation................................................................................................. 19 1.5 Objective ................................................................................................... 21

Chapter 2. Principle .............................................................................................. 22 2.1 Antibody-DNA conjugation...................................................................... 22 2.2 Lateral flow ............................................................................................... 24

2.3 Membrane-based immuno-PCR................................................................ 26 Chapter 3. Experimental ....................................................................................... 29

3.1 Materials.................................................................................................... 29 3.1.1 Polymerase Chain Reaction ................................................................ 29 3.1.2 Agarose gel electrophoresis ................................................................ 30 3.1.3 Lateral flow immuno-PCR.................................................................. 30

3.1.4 Buffer Solution.................................................................................... 31 3.2 Instruments................................................................................................ 33

3.3 Methods..................................................................................................... 35 3.3.1 Antibody-DNA conjugation................................................................ 35 3.3.2 Preparation of immunochromatographic test strips ............................ 38 3.3.3 Optimization of lateral flow-immuno-PCR assay............................... 40

3.3.4 Determination of the optimal concentration of capture antibody ....... 43 3.3.5 Sensitivity test of lateral flow immuno-PCR for human IL-6 ............ 44

vii

3.3.6 Analysis of gel image using ImageJ software..................................... 44 Chapter 4. Result and Discussion ......................................................................... 47

4.1 Preparation of antibody-DNA conjugate .................................................. 47 4.2 Optimization of lateral flow immuno-PCR............................................... 50 4.3 Sensitivity test of lateral-flow immuno-PCR for human IL-6 .................. 58

Chapter 5. Conclusion .......................................................................................... 60 Reference ............................................................................................................. 61 Appendix ............................................................................................................. 66

viii

List of Figure

Figure 1-3 Three proposed models described antibody-antigen interaction.. ......... 8 Figure 1-4 The format of immuno-PCR and ELISA.. .......................................... 10 Figure 1-5 The methods employed to conjugate DNA onto antibody.. ................ 12 Figure 1-6 Two formats of the lateral-flow immunochromatography assay. ....... 18 Figure 2-1 Formation of the DNA-conjugated antibody....................................... 22 Figure 2-2 Band distribution of DNA-streptavidin complex on agarose gel

electrophoresis. ..................................................................................... 23

Figure 2-3 Basic principle of the lateral flow immunochromatography assay ..... 26 Figure 2-4 The principle of membrane-based immuno-PCR................................ 28 Figure 3-2 DNA-conjugated antibody formation.................................................. 37 Figure 3-3 Preparation of immunochromatography strips. ................................... 39 Figure 3-4 Optimization of lateral flow immuno-PCR in order to get low

background with two different methods.. ............................................. 42 Figure 4-1 Analysis of DNA-streptavidin complex in 2% agarose gel ................ 48 Figure 4-2 Conjugation of biotinylated antibody with DNA-streptavidin

complex presented in 2% agarose gel electrophoresis. ........................ 49 Figure 4-3 Agarose gel electrophoresis of optimization of lateral flow

immuno-PCR........................................................................................ 52 Figure 4-4 Optimization of sample pre-treatment................................................. 52 Figure 4-5 Optimization of waiting time. ............................................................. 54 Figure 4-6 Optimization of test waiting time........................................................ 54 Figure 4-7 Optimization of spraying concentration of capture antibody.............. 56 Figure 4-8 Optimization of spraying concentration of the capture antibody........ 56 Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human

IL-6. ...................................................................................................... 59 Figure 4-10 The sensitivity test of the lateral-flow immuno-PCR on human

IL-6. ...................................................................................................... 59

ix

List of Table

Table 1-1 Overview of applications of lateral-flow immunochromatography assay...................................................................................................... 14

Table 3-1 Design of the concentration ratio of biotinylated DNA to streptavidin.. ......................................................................................... 35

Table Appendix 1 The Result of ImageJ measurement on the electrophoresis band of antibody-antigen interaction. ................................................... 66

Table Appendix 2 The Result of ImageJ measurement on the electrophoresis band of waiting time optimization........................................................ 66

Table Appendix 3The Result of ImageJ measurement on the electrophoresis band of the spraying concentration of capture antibody....................... 67

Table Appendix 4 The Result of ImageJ measurement on the electrophoresis band of the sensitivity test of the lateral-flow immuno-PCR ............... 67

1

Chapter 1. Introduction

1.1 Antibody-antigen interaction

Bodies normally develop immune reactions to protect organisms from

pathogens and tumors that can endanger their lives by identifying and killing the

pathogens and tumors cells. Antibody is one part of the immune system that

identifies and neutralizes foreign molecules such as bacteria and viruses.

Antibodies contribute to immunity in three main ways: they prevent pathogens

from entering or damaging cells by binding with them; they can stimulate removal

of a pathogen by macrophages and other cells by coating the pathogen; and they

can trigger direct pathogen destruction by stimulating other immune responses

such as the complement pathway [1].

Immune response can be stimulated when a body is invaded by foreign

molecules, called antigen. Substances that trigger an immune response generally

meet several criterias: the heterogeneity (different sequence); foreignness

(different from host); and the size (relatively large, e.g. 100 kDalton). Hapten

(small molecules) acts as an antigenic agent if conjugated with carrier proteins.

Most antigens are protein or polysaccharide, but only a small region of that

structure is recognized by an antibody. This small region called an antigenic

2

determinant or epitope. Nucleic acids and lipids are antigenic when these two

molecules conjugated with protein or polysaccharide.

Antibody has a four chain structure as the basic structure of antibody. It

contains two identical heavy chains and two identical light chains. The heavy

chain and the light chain are connected together by inter-disulfide bonds and non-

covalent interactions. The heavy and light chains can be separated in two domains,

variable and constant domains. Each antibody has a specific amino acid sequence

in variable domain. Most variability of sequences found in the variable region

called the hypervariable regions or the complementaritys determining regions.

This region determines the specificity of antibody to bind antigen (Figure 1-1).

Based on structure of heavy chain, antibody can be divided to five class

of immunoglobulin; immunoglobulin M (IgM), immunoglobulin D (IgD),

immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgG).

Their heavy chains are denoted by the corresponding lower-case Greek letter (, ,

, , and , respectively).

3

Figure 1-1 Basic structure of an antibody. 1. Fab region 2. Fc region 3.

Heavy chain with one variable (VH) domain followed by a constant domain

(CH1), a hinge region, and two constant (CH2 and CH3) domains. 4. Light

chain with one variable (VL) and one constant (CL) domain 5. Antigen

binding site (paratope) 6. Hinge regions.

S S S S

NH2

H2N

COOH

S S

H2N

NH2

HOOC

HOOC COOH

S S

1

2

6

3 5

4

4

Based on function relationship of immunoglobulin, an antibody can be

divided to two parts: Fab and Fc regions. The Fab fragment contains antigen

binding sites or called paratopes at the end of the region. Each Fab fragment

consists of one constant and one variable domain and each domain has heavy and

light chains. The Fc fragment is easily to be crystallized and has effectors function

of antibody. Both Fab and Fc fragments can be separated using papain enzyme.

This enzyme digests the immunoglobulin molecules from the hinge region. The

enzyme pepsin cleaves heavy chain of immunoglobulin at the inter-chain disulfide

bonds or below hinge region, resulting in a fragment that contains both antigen

binding sites. This fragment called F(ab')2 because it was divalent and the two

antigen binding sites remain linked together. The remaining of heavy chain was

digested by this enzyme into small part of peptide [2]. How an antibody can be

cleaved into parts is shown in Figure 1-2.

5

Figure 1-2 The Y-shaped immunoglobulin molecule dissected by partial

digestion with proteases. Papain cleaves the immunoglobulin molecule into

three pieces, two Fab fragments and one Fc fragment (upper panels). The Fab

fragment contains the V regions and binds antigen. The Fc fragment is

crystallizable and contains C regions. Pepsin cleaves immunoglobulin to yield

one F(ab)2 fragment and many small pieces of the Fc fragment, the largest of

which is called the pFc fragment (lower panels). F(ab)2 is written with a

prime because it contains a few more amino acids than Fab, including the

cysteines that form the disulfide bonds [2].

6

The most important characteristics of an antibody response are the

specificity, isotype or class, and affinity to its antigen. Antibody molecules are

very specific to their corresponding antigen. The binding strength of an antibody

to its antigen, in a single antigen-binding site binding to a monovalent antigen, is

termed its affinity; whereas the total binding strength of a molecule with more

than one binding site is called its avidity.

There are three models proposed to describe the interaction between

antibody and antigen; lock and key, induced fit models and preexisting

equilibrium/conformational selection model [3] as shown in Figure 1-3. Lock

and key model was first proposed 120 years ago by Fischer to describe the

interaction between enzymes and their respective substrates. This model then was

applied to explain the interaction between antibody and antigen. According to this

model, the antigen binding site of antibody was very rigid and the antigen had to

fit to the binding site. Unfortunately, this model couldnt explain the anomaly of

protein that can react with different shape of substrates [4]. The induced fit model,

proposed by Koshland in 1958 [4], try to solve this anomaly. Koshland introduced

postulate to describe his model that the binding site of protein must have precise

orientation and the substrate may generate the conformational change at the active

site so it would bring the proper orientation for interaction. This model is actually

7

similar in the principle that the protein must have precise orientation to fit with the

substrate except that the fitting only occurs after the changes induced by the

substrate itself. Study performed by Betts and Sternberg [5] on the conformational

changes for a set of 39 complexes and predominantly enzyme inhibitors, leading

to the conclusion that proteinprotein recognition occurs by the mechanism of

induced fit. However, Bosshard [6] noted that induced fit is possible only if the

match between the interacting sites is strong enough to provide the initial complex

strength and longevity so that induced fit takes place within a reasonable time. He

also pointed that there is an alternative mechanism to induced fit, called the

preexisting equilibrium/conformational selection model [6]. According to this

model, the antigen binding site on antibody doesnt need to make some change on

conformational to fit with the antigen. The antibody just simply selects the antigen

that has epitope with precise conformation with its binding site or in reciprocal

term, the antigen selects the antibody that has complementary conformation [7].

Studies conducted by Berger [8] and Foote & Milstein [9] validate this model.

However, Bosshard [6] stated that even the conformational selection model was

valuable and alternative for induced fit, it didnt mean that induced fit didnt occur

in protein-protein interaction. Actually, combination of these two models seems to

be the best to describe the interaction between molecules that apparently do not

actually fit to begin with.

8

Figure 1-3 Three proposed models described antibody-antigen interaction.

(A) Lock and key model proposed by Fischer in 1894, (B) induced fit model

proposed by Koshland to solve the anomaly that occurs in protein-protein

interaction, (C) proposed by Bosshard, is called the preexisting

equilibrium/conformational selection model. Based on this model, the antibody

has several conformational that can bind different antigen conformation.

antigen

Antigen binding site

A

B

C

9

1.2 Immuno-PCR

The development of immunoassay has grown rapidly since its first

establishment. After the introduction of radioimmunoassay (RIA), the first

quantitative immunoanalytical technique was proposed by Yalow and Barson [10]

in 1959, the methodologies of immunoassay using antibody have still continued to

expand to improve the sensitivity, shorten the assay time and make usage ease for

signal-generated molecules and alternative nonradioactive molecules.

Immuno-PCR, the immunoassay term introduced by Sano et al in 1992

[11], is one of ultimate quantitative immunoanalytical technique for protein

detection. This immunoassay technique utilizes the ability of DNA that can be

amplified exponentially from a single molecule. Immuno-PCR was similar with

ELISA in the basic working principle, but immuno-PCR uses DNA as target

marker instead of enzyme in ELISA (Figure 1-4). Because it possesses the

amplification ability of DNA, immuno-PCR allowed enhancement in sensitivity in

a 1000-10000 fold number of magnitude. But the sensitivity of immuno-PCR is

still limited by the sensitivity of the interaction of antibody and antigen and the

selectivity of the antibody to the target antigen [12].

10

Figure 1-4 The format of immuno-PCR and ELISA. In immuno-PCR, a DNA

is used as the target market instead of an enzyme in ELISA.

The immuno-PCR technique is constructed with three basic components;

the antigen recognition system, the DNA marker amplification system and the

signal manifestation [13]. The antigen recognition system involves the selection

of antigen-antibody pair with a high sensitivity and selectivity and an indirectly

DNA-marker with the antibody.

Linking the DNA marker to antibody is the most cumbersome step in

immuno-PCR. The early work of immuno-PCR, DNA was conjugated to antibody

via streptavidin-protein A chimera (Figure 1-5.A). The chimera has two

independent and specific binding sites: one is to biotin, derived from the

Immuno-PCR ELISA

PCR

Substrate

Product

Enzyme

1st antibody

2nd antibody

1st antibody

2nd antibody

DNA

11

streptavidin moiety, and the other is to the Fc portion of the immunoglobulin G

(IgG) molecule, derived from the protein A moiety. However, the use of this

chimera is limited to direct Immuno-PCR formats, which lack capture antibodies

with the same affinity as the detection antibody. Later, Ruzicka et al [14],

developed a new method to solve this problem. They conjugated biotinylated

dsDNA with biotinylated IgG by avidin. These three components were assembled

by the simple mixing of stoichiometric ratios (Figure 1-5.B). However, this

methodology has a potential drawback. That is the lack of homogeneity of the

avidin-DNA conjugates, which would reduce the accuracy and reproducibility of

the system. Besides, in situ assembly could create variable stoichiometry in the

reaction components. As well, extra steps are normally required for addition of

biotinylated reagents with binding proteins. Numerous wash steps are also needed

to remove excess reagents to free assay components from nonspecifically bound

reagents. Consequently, immuno-PCR assays become procedurally complex and

require considerable hands-on time [15]. Many researchers tried to reduce the

complexity of immuno-PCR by presynthesized antibody-DNA conjugates. Some

researchers [15], [16], [17] used direct covalent coupling to presynthesize

antibody-DNA conjugate (Figure 1-5.C), while others used biotin-streptavidin

coupling [18], [19].

12

Figure 1-5 The methods employed to conjugate DNA onto antibody. (A)

ProteinA-strevtavidin chimera is used to connect biotinylated DNA and the Fc

region of IgG. (B) Streptavidin conjugate biotinylated DNA and biotinylated

antibody. (C). DNA covalenty linked to antibody.

Since its first demonstration, immuno-PCR has been applied in

laboratory analysis and medical diagnosis. There are numerous examples of such

immuno-PCR applications, including detection of bacteria such as Clostridium

botulinum neurotoxin A [20] and Streptococcus pyogenes [21]. Immuno-PCR also

becomes major immunoanalytical method for analysis of viral infection. Barletta

reported on an improved sensitivity of real-time IPCR as compared with reverse

transcriptase-PCR (RT-PCR) for detection of HIV [22]. Low level of tumor and

disease-associated antigens [23], [24] also can be detected by immuno-PCR with

a good sensitivity.

DNADNA DNA

protein A chimera

chemical crosslinker

A B C

13

1.3 Lateral flow immunochromatography assay

Lateral flow immunochromatography is the most widely used membrane-

based immunoassay application. Compare to other methods, lateral flow gives an

easy of use and short assay time. The application of membrane on immunoassay

was started in 1979 by Towbin et al. in demonstrating that protein can be

transferred to microporous membrane nitrocellulose and detected using antibodies

[25]. Since that year, the application of membrane on immunoassay was

proliferated. The first test of lateral flow was made for detection of human

chorionic gonadotropin (GDP). Nowadays, the lateral flow has been used for

monitoring ovulation, detecting infectious disease organisms, analyzing drugs of

abuse, and measuring other analytes important to human physiology. Table 1-1

provides an overview on the variety of biomedical applications realized so far.

The application of membrane in immunoassay can be sorted in three

categories, antigen or antibody immobilized in specific location on a solid support

(ex. Western blot and dot blot immunoassay), a miniature of thin-layer

chromatography where the analyte flows transversely as the mobile phase and the

membrane as the stationary phase (ex. Lateral flow immunochromatography

assay), and the analyte flowing-through the porous membrane.

14

Table 1-1 Overview of applications of lateral-flow immunochromatography assay

Protein

class Antigen Year Remarks Ref.

Bacteria Vibrio harveyi 2007 Non-

competitive

Sithigorngul P

et al. [26]

Virus Canine distemper 2008 Non-

competitive An DJ, et al. [27]

Hormone 19-Nortestosterone 2007 Competitive Liu L, et al. [28]

Bacteria Cryptosporidium

parvum. 2000

Non-

competitive

Kozwich et al. [29]

Cardiac marker

Human heart-type fatty

acid-binding protein 2003

Non-

competitive Chan et al.[30]

Toxin Aflatoxin B1 2005 Competitive Delmulle et al.

[31] Hormone Clenbuterol 2006 Competitive Zhang et al. [32] Antibiotic Sulfonamides 2007 Competitive Wang et al. [33]

Bacteria Legionella

pneumophila 2006

Non-

competitive Horng et al. [34]

Toxin Microcystins 2003 Competitive Kim et al. [35]

Insecticide Carbaryl and endosulfan

2006 Competitive Zhang et al. [36]

Nucleic

acid Single stranded DNA 2003

Nucleic acid lateral flow

Corstjens et al. [37]

Enzyme Canine Trypsin-like immunoreactivity

2007 Non-

competitive

Waritani et al. [39]

Food additive

Glycyrrhizin 2005 Non-

competitive Putalun et al.[38]

Cells protein

Frataxin 2008 Non-

competitive Willis et al. [40]

15

Membrane-based immunoassay has been widely used, primarily because

it has high analyte-binding surface. The performance and capability of membrane-

based immunoassay are influenced by specific characteristics that build membrane

properties. Polymer type, porosity, surfactant content, and strength are several

key membrane characteristics [41]. The choice of membrane was considered

mostly based on the porosity of the membrane, capacity of protein-binding and

strength. Because the application of membrane for detection of protein uses

reactant that must flow through the membrane matrix, the porosity becomes an

important point in the selection of membrane type. Capacity of protein-binding

surface is corresponding with the ability of membrane immobilizing the protein.

A number of forces specifically hydrophobic interactions, hydrogen

bonding, and electrostatic force are known involved in the binding mechanism

of proteins, but the exact mechanism still not fully understood. There are two

proposed models to describe the mechanism of protein-binding on membrane. The

first model suggests that the electrostatic force involved in initial binding, while in

the long-term attachment, the binding is accomplished by combination between

hydrogen bonding and hydrophobic interaction. The second model suggests that

the initial binding is accomplished by hydrophobic interaction while the long-term

is caused by electrostatic force [42].

16

The lateral-flow immunochromatography assay is made up of a number

of components, including reagents, a sample pad, a conjugate pad, an aborbant

pad, and a porous membrane on which the reaction occurs. These components

interact together to affect many different aspects that influence the final

performance of lateral flow assay.

Generally there are two formats of lateral flow immunoassay,

competitive and non-competitive formats. The competitive format is mostly used

for testing small molecules with a single antigenic determinant that cannot be

bound with two antibodies simultaneously. While the non-competitive format is

used for testing the analyte with multiantigenic sites likes big molecules such as

hormone, bacteria or virus.

In the competitive format, the target analytes bind either to the reporter

particles or to the immobilized ligands. In the first case, the target analytes bind to

the ligands and block the ligands from binding to the reporters. This format was

used by Ho and Wauchope [43] to detect aflatoxin B1 (AFB1). The target analytes

was conjugated with liposome and the AFB1 antibody is immobilized at the

capture zone, where the competition occurs between AFB1-conjugated liposome

and AFB1. In the second format of competitive assay, the target analytes was

bound to the reporters and block these reporters from binding to the immobilized

17

ligand. Esch et al [44] used this format to detect water-borne Cryptosporidium

parvum oocysts. In both cases, the presences of the target analytes interfered the

binding of reporter to the test ligand and resulted in nonexisting signal which

indicated the presence of the target analytes.

The non-competitive assay or sandwich format uses two antibodies to

bind the analyte in between. The first antibody acts as a reporter, while the second

antibody captures the analytes at the test line. Different with the competitive assay,

which was interpreted analyte presence for a nonexistent signal; the single

intensity of the test line is equivalent with the concentration of the analyte for the

non-competitive assay.

18

Figure 1-6 Two formats of the lateral-flow immunochromatography assay. In

the competitive format, the protein-conjugated antigen is immobilized at test line,

while in the non-competitive assay, anti-antigen antibody is immobilized.

19

1.4 Motivation

Even though the lateral flow immunochromatography gives so many

advantages when it is applied on detection of proteomic substances in a user-

friendly format, very short time to get test result, a long-term stability over a wide

range of climates, and relatively inexpensive to make, the lateral flow

immunochromatography still has limitations in the sensitivity and quantification.

Currently, the lateral flow immunochromatography assay uses colloidal gold

nanoparticles or latex particles as the signal-generated molecule. These molecules

generate visual detection without additional readers but they also pay the cost that

a comparatively high sensitivity is not easy to achieve.

Polymerase chain reaction (PCR) has found a multitude of commercial

applications in the life sciences and in vitro diagnostics. In the PCR, the

researchers are enabled to produce millions of copies of a specific DNA sequence

in approximately two hours. The application of PCR technique in

immunoanalytical field has been proved increasing the detection limit of the assay.

On the other hand, immuno-PCR, immunoassay based PCR as the signal-

generated method, has been reported amplifying signal with 100-10000 folds

greater than that by ELISA.

20

By combining the lateral flow immunochromatography assay with a PCR

technique, this study hopes to increase the sensitivity of lateral flow detection.

The signal-generated molecules such as colloidal gold or latex particles are

changed with a nucleic acid in the immuno-PCR assay. By this assay the nucleic

acid on the membrane can be amplified to generate million copies of nucleic acid

and analyzed by gel electrophoresis. The intensity of the gel bands can be

quantified using available commercial software such as ImageJ or Photoshop.

This labeling method can therefore overcome the limitations mentioned above of

the lateral flow immunochromatography assay.

The membrane-based immuno-PCR also offers several advantages

compare with other immunoassay. Utilizing non-hazardous material on its

application makes membrane-based immunoassay can be applied at a laboratory

with population-dense environment. The first step of the assay, the presence of the

gene product is detected; whereas the second amplifies the single gene. Both steps

can be conducted on the case scene. A portable PCR machine makes its

application feasible. Employing immuno-PCR on the lateral flow assay besides

makes short assay time is achieved also bring the sensitivity higher than

conventional lateral flow.

21

1.5 Objective

The objective of this study was to develop a membrane-based immuno-

PCR to enhance the sensitivity of lateral flow immunochromatography assay. The

membrane-based immuno-PCR not only was expected to screen out the desired

antigen by the antibody-antigen interaction as the conventional lateral-flow

immunochromatography, but also provided a signal amplification methodology.

The specific detector of DNA-conjugated antibody could immunospecifically bind

the desired antigen from the sample, and the conjugated DNA could be amplified

using PCR technique. Its amplified intensity was considered to be relative with

the signal antigen.

22

Chapter 2. Principle

2.1 Antibody-DNA conjugation

In this study, DNA was conjugated with antibody using streptavidin as

the bridge. The conjugation was conducted by two steps. The first step,

biotinylated DNA was conjugated with streptavidin to form DNA-streptavidin

complex. The second step, biotinylated antibody was added to the previously

prepared DNA-streptavidin complex and formed DNA-conjugated antibody.

Figure 2-1 Formation of the DNA-conjugated antibody. Biotinylated DNA is

conjugated first with streptavidin to form DNA-streptavidin complex. The

complex is further conjugated with biotinylated antibody to form DNA-

conjugated antibody.

23

The DNA-streptavidin complex can be monovalent, divalent trivalent or

tetravalent and detectable from the band of gel electrophoresis. Each complex

shows a band at different position for different molecular weight level. The

monovalent complex shows a band on low molecular weight level but a little

higher than free DNA, whereas the divalent one shows the band about twice

higher than monovalent (Figure 2-2). Biotin-modified antibody can be added to

the DNA-streptavidin complex when the gel shows monovalent, divalent or

trivalent form, because there still have vacant sites on streptavidin available for

biotinylated antibody. As tetravalent form doesnt have any vacant site for biotin-

modified antibody, this form cannot be used to make DNA-conjugated antibody.

Figure 2-2 Band distribution of DNA-streptavidin complex on agarose gel

electrophoresis. A = marker, B = free DNA, C = monovalent DNA on

streptavidin, D = divalent, E= trivalent and F= tetravalent. Since the tetravalent

conformation has the highest molecular weight, it moves slower and its band

locates closer to the sample well than other conformations.

24

2.2 Lateral flow

The innovation of immunoanalytical method for diagnostic is more

evident than in therapeutics drug monitoring. Over the past decade, single-use

lateral flow immunoassays have been extremely successful in the laboratory and

in outpatient clinic and primary care environments. In the lateral flow

immunoassay, the reaction occurs in a porous solid phase materials such as

nitrocellulose membrane. All reaction components are impregnated and

immobilized on membrane and the sample is brought to contact with the

components by flowing its diluents.

In this study, the lateral flow immunochromatography assay is made

from four main components: sample pad, membrane, absorbent pad, and backing

pad. The sample pad is functionalized as a sample collector and made from glass

fiber. The membrane material is made from nitrocellulose. The membrane

provides the testing zone. The capture antibody is deposited as narrow band on

membrane and dried. The absorbent pad functions to absorb the overflow of the

sample to make the capillary flow continuous. The plastic backing pad with the

pressure-sensitive adhesive serves as a structural support for the layers above.

25

The basic working principle of lateral flow immunoassay was shown in

Figure 2-3. The antibody was immobilized on the membrane by the forces

hydrogen bond, hydrophobic interaction, and electrostatic force. With the

presences of high anti-chaotropic salt concentration, only antibody can bind to the

membrane, while other molecules are washed away. The analyte solution then

flows through the membrane and is captured by antibody.

The capillary migration is used to distribute antigen and detector

antibody on a rectangular membrane. The strip absorbs a finite volume of the

detector antibody/sample antigen mixture. As the detector antibody and sample

antigen migrate past the immobilized antibody at the membrane, they are

immunospecifically bound to the surface of the membrane. The migration process

is normally complete within 5 minutes and results in test strips in the form of bars

at the zone of immobilized antibody. The intensity of the zone is directly related

to the antigen concentration of the original sample.

26

Figure 2-3 Basic principle of the lateral flow immunochromatography assay.

The antigen is collected on sample pad and flows to the conjugate pad and catch

by labeled antibody. The antigen/antibody complecx flow through the membrane

and immunospecifically bind with the immobilized antibody.

2.3 Membrane-based immuno-PCR

The principle of membrane-based immuno-PCR is described in Figure 2-

4. The format of membrane-based immuno-PCR is similar with the indirect assay

of immuno-PCR. There are two antibodies used to capture the antigen. The first

antibody is immobilized on membrane nitrocellulose by spraying it onto the

surface of membrane. The membrane binds the antibody with several forces,

mainly hydrophobic interaction and electrostatic forces. The second antibody is

premixed first with the antigen. Premixing can reduce the stoichiometry

27

variability of the reaction between antigen and DNA-conjugated antibody when

the in situ reaction is applied.

The antigen/antibody mixture flows onto the membrane by the capillary

force and migrates through the immobilized capturing antibody on the membrane.

At this step, the antigen is immunospecifically bound with the capturing antibody

and the reaction is then complete.

For the amplification of DNA, the membrane piece, where the antibody

is immobilized, is cut off and put into a PCR tube to mix with PCR cocktail

containing primer, Taq DNA polymerase, dNTP, and water. The mixture is heated

to release the DNA from the membrane so that the amplification process will be

effective. The final product of PCR is then analyzed on gel electrophoresis.

.

28

Figure 2-4 The principle of membrane-based immuno-PCR. The principle of

membrane-based immuno-PCR is similar with the lateral flow assay. DNA-

conjugated antibody is used instead of gold nanoparticles-conjugated antibody.

The signal is analyzed from the product of PCR.

29

Chapter 3. Experimental

3.1 Materials

The following materials were used in the experiment:

3.1.1 Polymerase Chain Reaction

Double strands DNA 80 bp (5-biotin-TAG CAC GGA CAT ATA TGA

TGG TAC CGC AGT ATG AGT ATC TCC TAT GAC TAC TAA GTG

GAA GAA ATG TAA CTG TTT CCT TC-3) [45] as the target marker in

immuno-PCR was purchased from Purigo Biotech, Inc (Taiwan)

Forward primer 20 mer (TAG CAC GGT CAT ATA TGA TG) was

purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to

anneal to the template strand of DNA and allow the Taq Polymerase to

synthesize a strand complementary from the template strand.

Reverse primer 20 mer (GAA GGA AAC AGT TAC ATT TC) was

purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to

anneal to the template strand of DNA and allow the Taq Polymerase to

synthesize a strand complementary from the template strand.

dNTPs from Amersham (USA), for incorporating onto newly synthesized

DNA strands during the extension step of PCR

30

Taq Polymerase from New England Biolab (England) for synthesizing

new strand DNA during PCR.

PCR standard buffer from New England Biolab (England), to promote Taq

usage and maintain the pH of solution during PCR

3.1.2 Agarose gel electrophoresis

Molecular weight DNA ladder were purchased from New England Biolab

(England). It was used as a molecular weight marker in gel electrophoresis

5X TBE buffer was from Biobasic (Canada) as a buffer for electrophoresis

gel.

Agarose was from Biobasic (Canada) to make agarose gel to analyze DNA.

Ethidium bromide was from BioBasic (Canada) and was used to stain the

agarose gel, so that the gel bands can show on the Gel Imaging System.

3.1.3 Lateral flow immuno-PCR

Streptavidin was from Jackson ImmunoResearch Lab, Inc (USA). Used

for as a bridge to conjugate antibody and DNA

Human Interleukin-6 was from USBiological, as an antigen

Biotinylated mouse anti-human IL-6 antibody was from USBiological, for

capturing human IL-6 on lateral flow

31

Rabbit anti-mouse IgG was from USBiological, printed as control line on

lateral flow immuno-PCR to capture the mouse anti-human IL-6 antibody.

Nitrocellulose membrane 5 m AE98 was from Sartorius, on which the

reactions in lateral flow immuno-PCR took place.

Absorbent pad CS6 was from Whatman to keep the capillary force in the

lateral flow immuno-PCR assay.

Plastic backing pad was from Adhesive Research, Inc as a backing for

lateral flow materials.

Sample pad 33 Glass was from S&S, used to hold the sample of lateral

flow immuno-PCR.

3.1.4 Buffer Solution

Sodium chloride was from Bio Basic Inc, as material for making PBS

solution and protein lateral flow buffer

Potassium chloride was from Bio Basic Inc, as material for making PBS

solution

Sodium phosphate dibasic anhydrous was from Mallinckrodt, as material

for making PBS solution

Potassium phosphate was from Mallinckrodt, as material for making PBS

solution

32

Phosphate buffer saline (PBS)

Into a baker, 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4

were added with 800 ml of distilled H2O. The pH was adjusted to 7.4 with

HCl then added ddH2O to 1 liter. PBS buffer was used for routine

immunohistochemical staining and also used for diluting antibodies and

antigen.

Tween 20 was from Bio Basic Inc, as material for making protein lateral

flow buffer.

Bovine serum albumin was from Bio Basic Inc, as material for making

protein lateral flow buffer.

HEPES was from Calbiochem, as material for making protein lateral flow

buffer.

Tris (base) was from Bio Basic Inc, as material for making protein lateral

flow wash buffer.

Magnesium chloride was from Bio Basic Inc, as material for making

protein lateral flow wash buffer.

33

Protein lateral flow buffer

The lateral flow buffer was made by mixing 15 ml of Tween 20, 10 g BSA,

2.383 g HEPES, and 7.8894 g NaCl into 800 ml ddH20. The pH was

adjusted to 7.5 then added ddH20 until the final volume was 1 L.

Protein lateral flow wash buffer

The buffer was made to wash the nitrocellulose membrane. The

composition of this buffer was 6.057 g Tris, 2.922 g NaCl, and 5.0825 g

MgCl2. The materials were dissolved with ddH2O until the final volume

was 500 ml.

3.2 Instruments

The following instrument was used in the experiment:

TLC Linomat 5 (CAMAG Switzerland) for spraying antibody onto

nitrocellulose membrane.

Gel electrophoresis apparatus (Mupid Advance, Japan).

Microcentrifuge (Mikro 120 Hettich Zentrifugen, Germany) for

centrifuging protein and DNA.

PCR machine (Astec) for setting up temperature during PCR.

Biohazard safety hood was from High Ten Scientific Corp, for preparing

PCR and protein dilution.

34

Microwave for dissolving agarose powder into solution.

Balancer Precise 205A, for measuring weight of materials.

pH meter 330A from Orion, for measuring pH of solutions.

GeneFlash (Syngene, USA), as the gel imaging system.

Oven (Deng Yng, Taiwan) as an incubator.

Figure 3-1 Structure and the example of parameter input dialog of Linomat 5

TLC machine for a typical spraying procedure. Following the procedure shown

above to key in appropriate parameters, the TLC machine sprayed the desired

strips for the lateral flow test.

Syringe Plate

Display and function key

N2 Gas supply

The parameter input dialog

Globals Enter

Dosage Speed (70 nl/s) Predosage Volume (0.1 l)

Parameters Enter Plate Size (20 x 20 cm) Number of tracks (1) Number of Samples (1) Band Length (25 mm) First position X (21.5 mm) Track Distance (5 mm) App. Position Y

Track Assignment

Enter Track No 1 Track Volume ( 1l) Sample ID

Save Method Enter Default method Method No. 3

Syringe volume (100 l)

35

3.3 Methods

3.3.1 Antibody-DNA conjugation

The conjugation between antibody and DNA principally follow the

method of Niemeyer et al. [46]. The antibody was conjugated with DNA using

streptavidin (SA) as a bridge, utilizing the high affinity nature between biotin and

streptavidin (Figure 3-2). First, DNA was conjugated with streptavidin to formed

DNA-streptavidin complex. Conjugates of streptavidin and biotinylated dsDNA

fragment were typically prepared by adding DNA (3 M in ddH2O) to PBS and

subsequently added streptavidin (100 g/ml in PBS) until the total volume was 15

l (Table 3-1).

Table 3-1 Design of the concentration ratio of biotinylated DNA to

streptavidin. Several ratios was designed and tested to get the appropriate

concentration of DNA and streptavidin that give better result.

Molar ratio (DNA:SA) 5:1 4:1 3:1 2:1 1:1 1:2 1:3 1:4 1:5

DNA 3 M (l) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

Streptavidin 100 g/ml

(l) 0.5 0.65 0.9 1.35 2.7 5.4 8.1 10.8 13.5

PBS buffer (l) 13 12.85 12.6 12.15 10.8 8.1 5.4 2.7 0

36

The mixture of DNA and streptavidin was then incubated for 1 h at 37 C.

After DNA-streptavidin complex were prepared, the biotinylated mouse anti

human IL-6 antibody (7.5 l, 90 g/ml) was conjugated with the DNA-

streptavidin complex. The mixture was incubated for 30 min at 37 C. After

incubation, the product was stored at 4 C.

37

+

Biotinylated DNA Streptavidin

DNA-Streptavidin Complex

Biotinylated Antibody

Antibody-DNA conjugate

Figure 3-2 DNA-conjugated antibody formation. DNA-conjugated antibody

was formed using streptavidin as the bridge. DNA was first conjugated with

streptavidin to form the DNA-streptavidin complex. The antibody was then added

to the complex and formed the DNA-conjugated antibody.

38

3.3.2 Preparation of immunochromatographic test strips

The immunochromatographic strips was prepared with the following

procedures. First, membrane strips were sprayed with 0.5 mg/ml control capture

antibody, rabbit anti-mouse IgG, and the test capture antibody, mouse anti human

IL-6 antibody, using Linomat 5 machine with dosage speed 70 nl/s and N2

pressure 70 psi. The control line and test line were separately sprayed at two

different places near the top of the 50 x 25 mm membrane sheet, leaving a 5-mm

space in between. The membrane was stored in desiccators at room temperature

and 40% relative humidity for a day and then cut into 4 mm x 25 mm strips using

scissor. An absorbent pad was cut in sections of 4 x 15 mm and fixed on the

backing pad at the far end of the strip in the direction of the flow. The sample pad

with dimension 4 x 10 mm was fixed at the starting end of the strip. The

immunochromatographic strip was ready for use. The preparation procedure and

strip assemble were illustrated in Figure 3-3.

39

Figure 3-3 Preparation of immunochromatography strips. (A) The membrane

was sprayed with antibody at two separated line using Linomat 5. (B) the test line,

contained mouse anti-human IL-6 antibody and the control line contain rabbit anti

mouse IgG. (C) The sample pad and the absorbent pad were adhesed at the ends

of the device. The conjugate antibody was premixed with the sample; therefore

the conjugate pad was unnecessary.

40

3.3.3 Optimization of lateral flow-immuno-PCR assay

Lateral flow immuno-PCR was optimized the following two parameters

in order to get a low background: the conjugation condition between human IL-6

and DNA-conjugated anti human IL-6 antibody and the lateral flow test waiting

time.

Conjugation between antigen and its corresponding antibody was tested

in two different methods. The first method was premixing antigen with its

corresponding antibody. 10 l of 100 ng/ml antigen and 10 l DNA-conjugated

antibody (1:100 dilution) were mixed together and incubated for 5 min at 37 C.

After incubation, the mixture was added with 20 l lateral flow buffer then

applied onto the immunochromatography test strips. The second method was

adding the antigen onto the membrane first. After 5 min, the DNA-conjugated

antibody was added onto the membrane. After another 5 min the membrane was

washed using 1 ml lateral flow wash buffer. The result indicated that the first

method gave the better performance; therefore it was chosen to optimize the

second parameter.

The second parameter was the test waiting time for reducing background

after the immunochromatography strip was applied with a sample. The waiting

41

time was set at 2 min, 5 min, and 7 min. After applying the wash buffer, the

membrane around the test line was cut into about a 2mm piece. This membrane

piece was mixed with the PCR cocktail containing 10 l of 1 M of each primer,

10 l of 1 mM of dNTPs, 5 l of 10x standard Taq buffer and 0.4 l of Taq DNA

polymerase. The mixture was then subjected to PCR with the thermal cycling

conditions 95 C for 5 min as the initial denaturation, then 42 cycles of 95 C for

20 s, 53 C for 30 s and 72 C for 20 s. In the last cycle, 72 C was extended to 5

min and cooled to 4 C for 30 min. The PCR products were finally loaded in a 2%

agarose gel electrophoresis and the intensity of each band was measured using

ImageJ software.

42

Figure 3-4 Optimization of lateral flow immuno-PCR in order to get low

background with two different methods. Method A was premixing antigen and

corresponding antibody, whereas method B was with a step by step addition of

antigen and corresponding antibody. The result indicated method A was better.

43

3.3.4 Determination of the optimal concentration of capture antibody

The optimal concentration of capture antibody was determined using the

parameter from the previous optimization section. Four concentrations of capture

antibody: 1 mg/ml, 0.5 mg/ml, 100 g/ml, and 10 g/ml, were sprayed onto

nitrocellulose membrane. The membrane was stored in desiccators at room

temperature and 40% relative humidity for a day. The absorbent pad and sample

pad were assembled as described in section 3.3.2.

Ten microliters of antigen were mixed with 10 l DNA-conjugated anti

human IL-6 antibody and incubated for 5 min at 37 C. This premixed solution

then was mixed with 20 l lateral flow buffer and applied onto the membrane strip

and waited for 5 min. The strip was then washed by flowing 1 ml lateral flow

washing buffer and dried in room temperature for 5 min. The test line and the

control line were cut off using a knife and the membrane piece was moved into a

PCR tube and mixed with the PCR cocktail. The mixture was heated at 95 C for

5 min then subjected to PCR described in section 3.3.3. The PCR product was run

on 2% electrophoresis gel. The intensity of the band was measured using ImageJ

software.

44

3.3.5 Sensitivity test of lateral flow immuno-PCR for human IL-6

The sequential dilution of human IL-6, 10 pg/ml to 0.001 pg/ml, was

tested using lateral flow immuno-PCR to see its sensitivity. Ten microliters of

antigen were mixed with 10 l DNA-conjugated anti human IL-6 antibody and

incubated for 5 min at 37 C. This premixed solution then was mixed with 20 l

lateral flow buffer and applied onto the membrane strip and waited for 5 min. The

strip was then washed by flowing 1 ml lateral flow washing buffer and dried in

room temperature for 5 min. The test line and the control line were cut off using a

knife and the membrane piece was moved into a PCR tube and mixed with the

PCR cocktail. The mixture was heated at 95 C for 5 min then subjected to PCR.

The PCR product was run on an electrophoresis gel. The intensity of the band was

measured using ImageJ software.

3.3.6 Analysis of gel image using ImageJ software

ImageJ software was used to analyze the intensity of agarose gel image.

The software can be obtained freely from NIH website (http://rsb.info.nih.gov/ij/).

The tutorial for 1D gel image analysis using Image J can be loaded from internet.

In briefly, the band was selected using the rectangular selection tool by outlining

the first lane. In the ImageJ toolbar, selected Analyze>Gels>Select First Lane (or

45

press "1") and "Lane 1 selected" will be displayed in the status bar (Figure 3-5.A).

Analyze>Gels>Plot Lanes (or press "3") was selected to generate the lane profile

plots. The base lane was drawn in each peak using the straight line selection tool

(Figure 3-5.B) . The size for each peak was measured by clicking inside with the

wand tool (Figure 3-5.C). The data was saved and analyze further using Microsoft

Excel.

46

D) C)

A)

Figure 3-5 Analysis of gel image using ImageJ software. (A) Outlining the band using the rectangular selection tools. (B) Plotting

the line. (C) Measuring the area of each peak. (D) Saving the data.

B)

47

Chapter 4. Result and Discussion

4.1 Preparation of antibody-DNA conjugate

The conjugation between antibody and DNA was made using

streptavidin as a bridge. In a first set of experiment, DNA was preconjugated with

streptavidin. The preconjugation was prepared by mixing DNA and streptavidin in

several combination starts from 1:5 molar ratios to 5:1 molar ratio as described in

Table 3-1. The result of combination between DNA and streptavidin was

characterized using 2% agarose gel electrophoresis in the presence of standard

molecular weight as presented in Figure 4-1.

Characterization on gel electrophoresis of DNA-streptavidin complex

showed two bands at 80 bp and 300 bp for an equal or lower ratio of DNA to

streptavidin. The single band shown in the gel represented free DNA from the

excess of DNA used for the combination. Two bands at 80 bp and 300 bp shown

in the gel represented free DNA and tri-adduct of DNA-streptavidin complex,

respectively. The intensity of the band at 300 bp was overall higher than that at 80

bp. This proved that mostly DNA was conjugated with streptavidin in tri- adduct

of DNA-streptavidin complex and left small amount of free DNA in the solution.

One streptavidin molecule binds three molecules of DNA indicate that mostly

48

streptavidin behave trivalent linker molecule despite it has tetravalent binding

capacity. In addition, no appearance of other band at gel showed that streptavidin

only bind at trivalent format. The trivalent format left one free binding site that

can be occupied by biotinylated antibody so that antibody-DNA conjugate can be

made by the streptavidin bridge.

Lane 1 2 3 4 5 6 7 8 9 DNA ratio 5 4 3 2 1 1 1 1 1 SA ratio

M Free DNA 1 1 1 1 1 2 3 4 5

Figure 4-1 Analysis of DNA-streptavidin complex in 2% agarose gel. The

result indicated that the complex of DNA-streptavidin was formed when an

equal or lower ratio of DNA to streptavidin was used.

49

The DNA-streptavidin complex was further conjugated with the work

antibody to form the antibody-DNA conjugates. In the previous experiment the

DNA-streptavidin complex was successfully produced only when one or higher

molar excess of streptavidin was added. The resulting complex was in the agarose

gel with the band shown in 300 bp. The biotinylated antibody was then mixed

with each successful complex at equimolar ratio between antibody and DNA. The

result of conjugation was presented in the Figure 4-2.

Lane 1 2 3 4 5 6 7 8 9 10 DNA-SA

ratio 1-1 1-1 1-2 1-2 1-3 1-3 1-4 1-4 1-5 1-5

Antibody ratio

M Free DNA - 1 - 1 - 1 - 1 - 1

Figure 4-2 Conjugation of biotinylated antibody with DNA-streptavidin

complex presented in 2% agarose gel electrophoresis. The conjugation of the

antibody to DNA-streptavidin complex will result in the decreasing of the

intensity for the band at 300 bp.

50

The introduction of biotinylated antibody to the DNA-streptavidin

complex solution leaded the appearance of novel band with a high molecular

weight and reduction of the intensity of the band from DNA-streptavidin complex.

The reduction was clearly visible at equimolar ratio between DNA and

streptavidin. It was because one free binding site at DNA-streptavidin complex

was filled already by biotinylated antibody, formed antibody-DNA conjugate.

Antibody-DNA conjugate has high molecular weight so that it couldnt pass

through the pore of agarose gel. The intensity of the band was therefore reduced at

300 bp.

4.2 Optimization of lateral flow immuno-PCR

Antibody-DNA conjugate was applied to lateral flow immuno-PCR.

Lateral flow immuno-PCR term was referred to application of immuno-PCR on

lateral flow. The format of lateral flow immuno-PCR was similar with general

lateral flow, but the antibody-DNA conjugate was used instead of the antibody-

gold nanoparticles conjugate in general applications.

To get a high signal to noise ratio, the lateral flow immuno-PCR was

optimized. Two approaches were conducted for this optimization. In the first

approach, the reactions between antigen and antibody-DNA conjugate was

51

premixed at a tube; whereas in the second approach, they were step by step added

on membrane. Their immuno-PCR products were run on a 2% gel electrophoresis

as shown in Figure 4-3. In this Figure 4-3, the premixing approach gave a lower

background and higher test intensity than the step-by-step approach. In order to

more clearly indicate the optimization result, the intensity of each band was

further measured by ImageJ software and presented in signal to noise ratio as

shown in Figure 4-4. In the figure, premixing method gave about twice higher

ratio compared with the approach of step by step addition.

52

Figure 4-3 Agarose gel electrophoresis of optimization of lateral flow

immuno-PCR. The antigen and antibody-DNA conjugate reaction was 1 = step

by step addition or 2 = premixing. a = negative control , b = human IL6 sample

0

0.5

1

1.5

2

2.5

sequential Pre-mixed

Type of reaction between antibody and antigen

sign

al to

n

oise

ra

tio

Figure 4-4 Optimization of sample pre-treatment. The intensity was presented

in signal to noise ratio

o

M 1a 1b 2a 2b

53

The high background at the step-by-step method was believed caused

from a low capillary force. After addition of antigen onto the membrane, the

membrane became saturated with solution. Next addition of antibody-DNA

conjugate therefore flew slower by a lower capillary force. A low capillary force

made migration of antibody-DNA conjugate slow, such that the antibody-DNA

conjugate was trapped on the membrane to cause a high background.

Premixing method ensured the reaction between antigen and antibody

and also prevented from the reaction create stoichiometry variables. Since

molecules were not trapped on membrane, an additional wash to remove excess

reagent also can be avoided.

The second variable to be optimized to minimize background was the test

waiting time. The waiting time was set in 2 min, 5 min, and 7 min. After the

waiting time, the membrane was washed by the lateral flow wash buffer to

remove the excess reagent that could cause background. The membrane was then

cut around the test line and mixed with PCR cocktail and subjected to the PCR.

Sample and background signals were determined on a 2% agarose gel

electrophoresis as the previous optimization. The result was shown in

Figure 4-5 and Figure 4-6.

54

Figure 4-5 Optimization of waiting time. The immuno-PCR products were run

on a 2% gel electrophoresis. The waiting time set at (1) 7 min, (2) 5 min and (3) 2

min. Signals from (a) sample and (b) background were compared with each other.

0

0.5

1

1.5

2

2.5

2 min 5 min 7 minWaiting time

sign

al to

n

oise

ra

tio

Figure 4-6 Optimization of test waiting time. Analyis of 2% agarose gel

electrophoresis of immuno-PCR products. The intensity was reported as signal to

noise ratio. The highest value of signal to noise ratio was further chosen for the

next experiment.

M 1a 1b 2a 2b 3a 3b

o

55

The graphic of signal to noise ratio showed that 5 min waiting time was

the best choice for the next experiment. It should give enough time for antibody

and antigen to reach the stoichiometry for reaction. In other case, 2 minute waiting

time was not long enough for antigen and antibody to reach the stoichiometry for

reaction, such that when a wash was applied, the capture antibody was not strong

enough to bind with the antigen and therefore washed away. Two minutes were

also possibly not long enough to allow all samples to flow through the test line

and thus the residual samples were carried by the wash buffer to cause a

background. For the case of 7-minute waiting time, its sample signal was lower

than the 5-minute one, but its background signal was also higher. The higher

background was possibly caused by unwashed antibody-DNA conjugate on

nitrocellulose membrane. The antibody-DNA conjugate already bound strongly to

the membrane for a long waiting time, such that it couldnt be removed when the

wash was applied.

To obtain the best assay response and sensitivity, the spraying

concentration of capture antibody was optimized in this experiment. An optimal

spraying concentration supposedly offered a highest signal to noise ratio and also

saved raw material. The result of this test was presented in Figure 4-7 and Figure

4-8.

56

Figure 4-7 Optimization of spraying concentration of capture antibody. (a)

sample signal. (b) background

0

0.5

1

1.5

2

2.5

0.01 mg/ml 0.1 mg/ml 0.5 mg/ml 1 mg/ml

mass of capture antibody

sign

al to

n

oise

ra

tio

Figure 4-8 Optimization of spraying concentration of the capture antibody.

The result in figure 4-7 was shown in signal to noise. 0.5 mg/ml obviously gave a

better result.

a b b a b a b a 10 g/ml 100 g/ml 0.5 mg/ml 1 mg/ml

o

57

Figure 4-7 and Figure 4-8 optimized the spraying concentration of

capture antibody. The figure showed that the 0.5 mg/ml of capture antibody gave

the highest ratio. Concentrations higher or lower than 0.5 mg/ml didnt give a

better result, possibly due to the labeling efficiency of antigen to the immobilized

antibody and steric constraints of capture antibody. As the antibody concentration

was lower than the optimal one, the antibody/antigen interaction likely decreased

and resulted in the decreasing of the ratio. Furthermore, decreasing in the

concentration of the capture antibody also increased the empty spaces that can be

occupied by the reporter DNA-conjugated antibody. That was why the

background increased as the concentration of the capture antibody increased. For

the concentrations of the capture antibody above the optimal concentration, the

steric constraint of capture antibody was the main reason for ratio decreasing. Due

to random orientation of the capture antibody, overcrowding antibody in the

capture line will result in the decreasing of antibody/antigen contacts. The

background of 1 mg/ml obviously didnt contribute to the diminishment of the

ratio, because it was very low already.

58

4.3 Sensitivity test of lateral-flow immuno-PCR for human IL-6

The lateral-flow immuno-PCR was tested for human IL-6 detection

sensitivity. The conditions previously optimized were applied in this test. A serial

of ten fold dilution from 10 pg/ml to 0.001 pg/ml of human IL-6 was detected by

the membrane-based immuno-PCR methodologies. The result of the sensitivity

test was presented in Figure 4-9 and Figure 4-10. From the result of gel

electrophoresis analysis, membrane-based immuno-PCR was showed to be able to

detect

59

Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human

IL-6. The lateral-flow immuno-PCR was able to detect 0.001 pg/ml of human-

IL6. M = marker, 1-5) Human IL-6 with ten fold serial dilution from 10 pg/ml to

0.001 pg/ml, 6) = negative control,

0

500

1000

1500

2000

2500

3000

0 0.001 0.01 0.1 1 10

Original concentration of Human IL-6 (pg/ml)

Fin

al ge

l rea

d o

uts

Figure 4-10 The sensitivity test of the lateral-flow immuno-PCR on human

IL-6. membrane-based immuno-PCR was able to detect as low as 0.001 pg/ml

human IL-6

M 1 2 3 4 5 6

60

Chapter 5. Conclusion

A membrane-based immuno-PCR was developed in this study to increase

the sensitivity of the lateral flow immunochromatography assay. The method was

similar with the conventional lateral flow immunochromatography assay. But

instead the gold nanoparticles-conjugated antibody, this study employed a DNA-

conjugated antibody as the signal reporter. By the traditional PCR procedure, the

reporter DNA on membrane was amplified to improve the detection sensitivity.

The optimal condition for sample preparation was premixing antigen and

DNA-conjugated antibody and waiting for five minutes after sample application.

Premixing ensured the reaction between antigen and antibody and also prevented

the reaction from creating variable stoichiometry. Five minutes gave enough time

for antibody to react with antigen but did not trap the DNA-antibody in the pore

of the membrane. The concentration of receptor antibody was optimized as 0.5

mg/ml to achieve the highest signal to noise ratio. In addition, the membrane-

based immuno-PCR can detect

61

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Appendix

Table Appendix 1 The Result of ImageJ measurement on the electrophoresis

band of antibody-antigen interaction for Figure 4-4.

Sample intensity Background intensity Signal to Noise ratio

Premixed 12969.66 5597.024 2.317242 Step-by-step 13692.34 10979.65 1.247065

Table Appendix 2 The Result of ImageJ measurement on the electrophoresis

band of waiting time optimization for Figure 4-6.

Waiting time Sample intensity Background intensity Signal to Noise ratio

2 min 5521.225 4315.773 1.279313 5 min 7503.125 3222.468 2.328378

7 min 4453.225 2843.276 1.56623

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Table Appendix 3 The Result of ImageJ measurement on the electrophoresis

band of the spraying concentration of capture antibody for Figure 4-8.

Concentration of capture

antibody Sample intensity Background intensity Signal to Noise ratio

1 mg/ml 3975.912 2447.912 1.624205 0.5 mg/ml 6583.912 3154.397 2.087217 0.1 mg/ml 4789.962 3722.79 1.286659

0.01 mg/ml 5537.175 4015.347 1.379003

Table Appendix 4 The Result of ImageJ measurement on the electrophoresis

band of the sensitivity test of the lateral-flow immuno-PCR for Figure 4-10.

Concentration of human IL-6

Intensity

10 pg/ml 2441.397 1 pg/ml 1539.548

0.1 pg/ml 1534.598 0.01 pg/ml 2166.426

0.001 pg/ml 1749.669 0 pg/ml 962.012