Immunochemical Methods and Funtions
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Transcript of Immunochemical Methods and Funtions
![Page 1: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/1.jpg)
Immunochemical Methods in the Clinical Laboratory
Roger L. Bertholf, Ph.D., DABCCChief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville
Associate Professor of Pathology, University of Florida College of Medicine
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ASCP/Bertholf
Name The Antigen
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Early theories of antibody formation
• Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces.
• Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity.
• Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces.
• Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity.
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The “Template” theory of antibody formation
• Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor.
• Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation.
• Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor.
• Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation.
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Aminobenzene Sulphonate, a Hapten
NH2 NH2 NH2
SO3
SO3
SO3
Ortho Meta Para
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Classification of immunochemical methods
• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering• Nephelometry
• Turbidimetry
• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering• Nephelometry
• Turbidimetry
• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous
• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous
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Properties of the antibody-antigen bond
• Non-covalent
• Reversible
• Intermolecular forces
– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation
• Non-covalent
• Reversible
• Intermolecular forces
– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation
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Antibody affinity
AgAbAgAb
]][[
][
AgAb
AgAbKa
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Precipitation of antibody/antigen complexes
• Detection of the antibody/antigen complex depends on precipitation
• No label is involved
• Many precipitation methods are qualitative, but there are quantitative applications, too
• Detection of the antibody/antigen complex depends on precipitation
• No label is involved
• Many precipitation methods are qualitative, but there are quantitative applications, too
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Factors affecting solubility
• Size
• Charge
• Temperature
• Solvent ionic strength
• Size
• Charge
• Temperature
• Solvent ionic strength
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Zone of equivalence
The precipitin reaction
Pre
cipi
tate
Antibody/Antigen
etc.
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Single radial immunodiffusion
Ag
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Single radial immunodiffusion
][Agr r
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Double immunodiffusion
Örjan Ouchterlony
Developed double immunodiffusion technique in 1948
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Double immunodiffusion (Ouchterlony)
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Quantitative double immunodiffusion
S1
S2
S3 S4
S5
P
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Electroimmunodiffusion
• Why would we want to combine immunodiffusion with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”
• Why would we want to combine immunodiffusion with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”
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Electroimmunodiffusion
+
-
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Immunoelectrophoresis
• Combines serum protein electrophoresis with immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis
• Combines serum protein electrophoresis with immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis
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Immunoelectrophoresis
Specimen
-human serum
+
-
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Immunoelectrophoresis
P C P C P C
+
-
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Immunofixation electrophoresis
SPE IgG IgA IgM
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Particle methods involving soluble complexes
• The key physical property is still size
• Measurement is based on how the large antibody/antigen complexes interact with light
• The fundamental principle upon which the measurement is made is light scattering
• Two analytical methods are based on light scattering: Nephelometry and Turbidimetry
• The key physical property is still size
• Measurement is based on how the large antibody/antigen complexes interact with light
• The fundamental principle upon which the measurement is made is light scattering
• Two analytical methods are based on light scattering: Nephelometry and Turbidimetry
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Light reflection
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- -+
Molecular size and scattering
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Distribution of scattered radiation
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Nephelometry vs. Turbidimetry
0°-90°
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Inte
nsit
y of
sca
tter
ing
Time
Rate nephelometry
Rate
C2
C1
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Additional considerations for quantitative competitive binding immunoassays
• Response curve
• Hook effect
• Response curve
• Hook effect
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Competitive immunoassay response curve%
Bou
nd la
bel
Antigen concentration
%Bound vs. log concentration
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Logistic equation
%B
ound
labe
l
Log antigen concentration
a
d
c
Slope = b
d
cx
a
day b
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Logit transformation
%B
ound
labe
l
Log antigen concentration
a
d
y
yyY
1lnlogit
da
dyy
where
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Logit plotL
ogit
y
Log antigen concentration
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High dose “hook” effect%
Bou
nd a
ntig
en
Antigen concentration
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Analytical methods using labeled antigens/antibodies
• What is the function of the label?
– To provide a means by which the free antigens, or antigen/antibody complexes can be detected
– The label does not necessarily distinguish between free and bound antigens
• What is the function of the label?
– To provide a means by which the free antigens, or antigen/antibody complexes can be detected
– The label does not necessarily distinguish between free and bound antigens
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Analytical methods using labeled antigens/antibodies
• What are desirable properties of labels?
– Easily attached to antigen/antibody
– Easily measured, with high S/N
– Does not interfere with antibody/antigen reaction
– Inexpensive/economical/non-toxic
• What are desirable properties of labels?
– Easily attached to antigen/antibody
– Easily measured, with high S/N
– Does not interfere with antibody/antigen reaction
– Inexpensive/economical/non-toxic
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The birth of immunoassay
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
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Radioisotope labels
• Advantages
– Flexibility
– Sensitivity
– Size
• Advantages
– Flexibility
– Sensitivity
– Size
• Disadvantages
– Toxicity
– Shelf life
– Disposal costs
• Disadvantages
– Toxicity
– Shelf life
– Disposal costs
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Enzyme labels
• Advantages
– Diversity
– Amplification
– Versatility
• Advantages
– Diversity
– Amplification
– Versatility
• Disadvantages
– Lability
– Size
– Heterogeneity
• Disadvantages
– Lability
– Size
– Heterogeneity
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Fluorescent labels
• Advantages
– Size
– Specificity
– Sensitivity
• Advantages
– Size
– Specificity
– Sensitivity
• Disadvantages
– Hardware
– Limited selection
– Background
• Disadvantages
– Hardware
– Limited selection
– Background
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Chemiluminescent labels
• Advantages
– Size
– Sensitivity
– S/N
• Advantages
– Size
– Sensitivity
– S/N
• Disadvantages
– Hardware
– ?
• Disadvantages
– Hardware
– ?
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Chemiluminescent labels
+ 2H2O2 + OH -
COO -
COO -
O -
O -
+ h ( max = 4 3 0 nm )
+ N2 + 3H2O
NH2
L um i n o l
P e r o x i d a s e
O
O
N
NH
NH2
H
O
O*NH2
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Chemiluminescent labels
CH3
N+
CO2H
O O
B r -
Ac r i d i n i um e s t e r
O -
CO2H
+ H2O2 + OH -+ + CO2 + h
O
CH3
N
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Introduction to Heterogeneous Immunoassay
• What is the distinguishing feature of heterogeneous immunoassays?– They require separation of bound and free ligands
• Do heterogeneous methods have any advantage(s) over homogeneous methods?– Yes
• What are they?– Sensitivity– Specificity
• What is the distinguishing feature of heterogeneous immunoassays?– They require separation of bound and free ligands
• Do heterogeneous methods have any advantage(s) over homogeneous methods?– Yes
• What are they?– Sensitivity– Specificity
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Heterogeneous immunoassays
• Competitive
– Antigen excess
– Usually involves labeled competing antigen
– RIA is the prototype
• Competitive
– Antigen excess
– Usually involves labeled competing antigen
– RIA is the prototype
• Non-competitive
– Antibody excess
– Usually involves secondary labeled antibody
– ELISA is the prototype
• Non-competitive
– Antibody excess
– Usually involves secondary labeled antibody
– ELISA is the prototype
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Enzyme-linked immunosorbent assay
Microtiter well
E E E E E
Specimen 2nd antibodyE
Substrate
S P
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ELISA (variation 1)
Microtiter well
Specimen Labeled antigenE
EEE
S P
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ELISA (variation 2)
Microtiter well
Specimen Labeled antibodyE
E E E E
EEE
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Automated heterogeneous immunoassays
• The ELISA can be automated
• The separation step is key in the design of automated heterogeneous immunoassays
• Approaches to automated separation
– immobilized antibodies
– capture/filtration
– magnetic separation
• The ELISA can be automated
• The separation step is key in the design of automated heterogeneous immunoassays
• Approaches to automated separation
– immobilized antibodies
– capture/filtration
– magnetic separation
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Immobilized antibody methods
• Coated tube
• Coated bead
• Solid phase antibody methods
• Coated tube
• Coated bead
• Solid phase antibody methods
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Coated tube methods
Specimen Labeled antigen
Wash
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Coated bead methods
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Microparticle enzyme immunoassay (MEIA)
Labeled antibodyE
E E
S P
Glass fiber matrix
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Magnetic separation methods
Fe
Fe
FeFe
Fe
Fe
FeFe
Fe
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Magnetic separation methods
Fe Fe FeFe Fe
Aspirate/Wash
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Electrochemiluminescence immunoassay (Elecsys™ system)
Flow cell
Fe
Oxidized
Reduced
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ASCEND (Biosite Triage™)
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ASCEND
Wash
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ASCEND
Developer
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Solid phase light scattering immunoassay
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Introduction to Homogeneous Immunoassay
• What is the distinguishing feature of homogeneous immunoassays?
– They do not require separation of bound and free ligands
• Do homogeneous methods have any advantage(s) over heterogeneous methods?
– Yes
• What are they?
– Speed
– Adaptability
• What is the distinguishing feature of homogeneous immunoassays?
– They do not require separation of bound and free ligands
• Do homogeneous methods have any advantage(s) over heterogeneous methods?
– Yes
• What are they?
– Speed
– Adaptability
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Homogeneous immunoassays
• Virtually all homogeneous immunoassays are one-site
• Virtually all homogeneous immunoassays are competitive
• Virtually all homogeneous immunoassays are designed for small antigens
– Therapeutic/abused drugs
– Steroid/peptide hormones
• Virtually all homogeneous immunoassays are one-site
• Virtually all homogeneous immunoassays are competitive
• Virtually all homogeneous immunoassays are designed for small antigens
– Therapeutic/abused drugs
– Steroid/peptide hormones
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Typical design of a homogeneous immunoassay
No signal
Signal
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Enzyme-multiplied immunoassay technique (EMIT™)
• Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods
• Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods
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EMIT™ method
Enzyme
S
S P
No signal
SignalEnzyme
S
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EMIT™ signal/concentration curveS
igna
l (en
zym
e ac
tivi
ty)
Antigen concentration
Functional concentration range
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Fluorescence polarization immunoassay (FPIA)
• Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva
– Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market
• Like the EMIT, the first applications were for therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument
• Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva
– Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market
• Like the EMIT, the first applications were for therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument
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Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P
10-6-10-9 sec
10-4-10 sec
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Polarized radiation
z
y
x
Polarizingfilter
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Fluorescence polarization
OHO OH
C
O
O
Fluoresceinin
Orientation of polarized radiation is maintained!
out (10-6-10-9 sec)
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Fluorescence polarization
OHO
OH
C
O
O
Rotational frequency 1010 sec-1
in
Orientation of polarized radiation is NOT maintained!
out (10-6-10-9 sec)
But. . .
![Page 72: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/72.jpg)
Fluorescence polarization immunoassay
OHO OH
C
O
O
Polarization maintainedSlow rotation
OHO OH
C
O
O
Rapid rotation
Polarization lost
![Page 73: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/73.jpg)
FPIA signal/concentration curveS
igna
l (I
/I)
Antigen concentration
Functional concentration range
![Page 74: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/74.jpg)
Cloned enzyme donor immunoassay (CEDIA™)
• Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche)
• Both TDM and DAU applications are available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA applications in market penetration
• Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche)
• Both TDM and DAU applications are available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA applications in market penetration
![Page 75: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/75.jpg)
Cloned enzyme donor
Donor
Acceptor
Monomer(inactive)
Active tetramer
Spontaneous
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Cloned enzyme donor immunoassay
Donor
Acceptor
Donor
Acceptor
No activity
Active enzyme
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CEDIA™ signal/concentration curveS
igna
l (en
zym
e ac
tivi
ty)
Antigen concentration
Functional concentration range
![Page 78: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/78.jpg)
Other approaches to homogeneous immunoassay
• Fluorescence methods
• Electrochemical methods
• Enzyme methods
• Enzyme channeling immunoassay
• Fluorescence methods
• Electrochemical methods
• Enzyme methods
• Enzyme channeling immunoassay
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Substrate-labeled fluorescence immunoassay
Enzyme
S
S Fluorescence
No signal
SignalEnzyme
S
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Fluorescence excitation transfer immunoassay
Signal
No signal
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Electrochemical differential polarographic immunoassay
Oxidized
Reduced
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Prosthetic group immunoassay
Enzyme
Enzyme
P
P
S P
Signal
No signal
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Enzyme channeling immunoassay
Ag
E1
E2
Substrate
Product 1
Product 2
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Artificial antibodies
• Immunoglobulins have a limited shelf life
– Always require refrigeration
– Denaturation affects affinity, avidity
• Can we create more stable “artificial” antibodies?
– Molecular recognition molecules
– Molecular imprinting
• Immunoglobulins have a limited shelf life
– Always require refrigeration
– Denaturation affects affinity, avidity
• Can we create more stable “artificial” antibodies?
– Molecular recognition molecules
– Molecular imprinting
![Page 85: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/85.jpg)
History of molecular imprinting
• Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940
• Imparted antigen specificity on native globulin by denaturation and incubation with antigen.
• Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940
• Imparted antigen specificity on native globulin by denaturation and incubation with antigen.
![Page 86: Immunochemical Methods and Funtions](https://reader036.fdocuments.net/reader036/viewer/2022062305/563dba90550346aa9aa6b561/html5/thumbnails/86.jpg)
Fundamentals of antigen/antibody interaction
O
O-
O
O-
NH 3
+CH2-CH2-CH2-CH3
OH
N
NH2
Cl
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Molecular imprinting (Step 1)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Methacrylic acid+ Porogen
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Molecular imprinting (Step 2)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
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Molecular imprinting (Step 3)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Cross-linking monomerInitiating reagent
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Molecular imprinting (Step 4)
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Comparison of MIPs and antibodies
• In vivo preparation
• Limited stability
• Variable specificity
• General applicability
• In vivo preparation
• Limited stability
• Variable specificity
• General applicability
• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability
• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability
Antibodies MIPs
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Immunoassays using MIPs
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
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Aptamers
1014-1015 random sequences Target
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target