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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.
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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.
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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
• 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
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Antibody affinity
AgAbAgAb
]][[][
AgAbAgAbKa
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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
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Factors affecting solubility
• Size• Charge• Temperature• Solvent ionic strength
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Zone of equivalence
The precipitin reactionPr
ecip
itate
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”
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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
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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
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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
nsity
of s
catte
ring
Time
Rate nephelometry
Rate
C2
C1
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Additional considerations for quantitative competitive binding immunoassays
• 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%
Bou
nd la
bel
Log antigen concentration
a
d
c
Slope = b
d
cxa
day b
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Logit transformation%
Bou
nd la
bel
Log antigen concentration
a
d
yyyY
1lnlogit
da
dyy
where
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Logit plotLo
git 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
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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
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The birth of immunoassay
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
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Radioisotope labels
• Advantages– Flexibility– Sensitivity– Size
• Disadvantages– Toxicity– Shelf life– Disposal costs
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Enzyme labels
• Advantages– Diversity– Amplification– Versatility
• Disadvantages– Lability– Size– Heterogeneity
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Fluorescent labels
• Advantages– Size– Specificity– Sensitivity
• Disadvantages– Hardware– Limited selection– Background
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Chemiluminescent labels
• Advantages– Size– Sensitivity– S/N
• 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
NH2H
O
O*NH2
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Chemiluminescent labels
CH3N+
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
CH3N
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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
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Heterogeneous immunoassays
• 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
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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
EEES 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
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Immobilized 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 ES P
Glass fiber matrix
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Magnetic separation methods
Fe
Fe
Fe Fe
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
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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
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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
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EMIT™ method
Enzyme
S
S P
No signal
SignalEnzyme
S
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EMIT™ signal/concentration curveSi
gnal
(enz
yme
activ
ity)
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
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Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P10-6-10-9 sec
10-4-10 sec
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Polarized radiation
z
y
x
Polarizingfilter
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Fluorescence polarization
OHO OH
CO
O
Fluoresceinin
Orientation of polarized radiation is maintained!
out (10-6-10-9 sec)
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Fluorescence polarization
OHO
OH
CO
O
Rotational frequency 1010 sec-1
in
Orientation of polarized radiation is NOT maintained!
out (10-6-10-9 sec)
But. . .
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Fluorescence polarization immunoassay
OHO OH
CO
O
Polarization maintainedSlow rotation
OHO OH
CO
O
Rapid rotationPolarization lost
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FPIA signal/concentration curveSi
gnal
(I/I
)
Antigen concentration
Functional concentration range
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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
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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 curveSi
gnal
(enz
yme
activ
ity)
Antigen concentration
Functional concentration range
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Other approaches to homogeneous 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
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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.
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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
OH3C
CH3
N
NO N
NH
O
H3C
CH3
Methacrylic acid+ Porogen
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Molecular imprinting (Step 2)
N
NO N
NH
OH3C
CH3
N
NO N
NH
O
H3C
CH3
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Molecular imprinting (Step 3)
N
NO N
NH
OH3C
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 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
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Aptamers
1014-1015 random sequences Target
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target