Applications of Immunochemical Methods in the Clinical Laboratory
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Applications of Immunochemical Methods in
the Clinical Laboratory
Roger L. Bertholf, Ph.D.Associate Professor of Pathology
University of Florida College of Medicine
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The University of Florida
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University of Florida Health Science Center in Gainesville
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The University of Florida
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University of Florida Health Science Center/Jacksonville
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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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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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Types of labels
• Radioactive• Enzyme• Fluorescent• Chemiluminescent
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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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The birth of immunoassay• Rosalyn Yalow and
Solomon Berson developed the first radioimmunoassay in 1957
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Coated tube methodsSpecimen Labeled antigen
Wash
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Coated bead methods
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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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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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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 curve
Sign
al (e
nzym
e ac
tivity
)
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
• 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 radiationz
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 curve
Sign
al (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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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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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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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 sequencesTarget
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target
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Thank You!