Applications of Immunochemical Methods in
the Clinical Laboratory
Roger L. Bertholf, Ph.D.Associate Professor of Pathology
University of Florida College of Medicine
The University of Florida
University of Florida Health Science Center in Gainesville
The University of Florida
University of Florida Health Science Center/Jacksonville
Classification of immunochemical methods
• Particle methods– Precipitation
• Immunodiffusion• Immunoelectrophoresis
– Light scattering• Nephelometry• Turbidimetry
• Label methods– Non-competitive
• One-site• Two-site
– Competitive• Heterogeneous• Homogeneous
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
Types of labels
• Radioactive• Enzyme• Fluorescent• Chemiluminescent
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
The birth of immunoassay• Rosalyn Yalow and
Solomon Berson developed the first radioimmunoassay in 1957
Coated tube methodsSpecimen Labeled antigen
Wash
Coated bead methods
Enzyme-linked immunosorbent assay
Microtiter well
E E E E E
Specimen 2nd antibodyE
Substrate
S P
Microparticle enzyme immunoassay (MEIA)
Labeled antibodyE
E ES P
Glass fiber matrix
Magnetic separation methods
Fe
Fe
Fe Fe
Fe
Fe
FeFe
Fe
Magnetic separation methods
Fe Fe FeFe Fe
Aspirate/Wash
Electrochemiluminescence immunoassay
(Elecsys™ system)
Flow cell
Fe
Oxidized
Reduced
ASCEND (Biosite Triage™)
ASCEND
Wash
ASCEND
Developer
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
Typical design of a homogeneous immunoassay
No signal
Signal
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
EMIT™ method
Enzyme
S
S P
No signal
SignalEnzyme
S
EMIT™ signal/concentration curve
Sign
al (e
nzym
e ac
tivity
)
Antigen concentration
Functional concentration range
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
Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P10-6-10-9 sec
10-4-10 sec
Polarized radiationz
y
x
Polarizingfilter
Fluorescence polarization
OHO OH
CO
O
Fluoresceinin
Orientation of polarized radiation is maintained!
out (10-6-10-9 sec)
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. . .
Fluorescence polarization immunoassay
OHO OH
CO
O
Polarization maintainedSlow rotation
OHO OH
CO
O
Rapid rotationPolarization lost
FPIA signal/concentration curve
Sign
al (I
/I
)
Antigen concentration
Functional concentration range
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
Cloned enzyme donor
Donor
Acceptor
Monomer(inactive)
Active tetramer
Spontaneous
Cloned enzyme donor immunoassay
Donor
Acceptor
Donor
Acceptor
No activity
Active enzyme
Substrate-labeled fluorescence immunoassay
Enzyme
S
S Fluorescence
No signal
SignalEnzyme
S
Fluorescence excitation transfer immunoassay
Signal
No signal
Electrochemical differential polarographic immunoassay
Oxidized
Reduced
Prosthetic group immunoassay
Enzyme
Enzyme
P
P
S P
Signal
No signal
Enzyme channeling immunoassay
Ag
E1
E2
Substrate
Product 1
Product 2
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.
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.
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.
Fundamentals of antigen/antibody
interaction
O
O-
O
O-
NH 3+
CH2-CH2-CH2-CH3
OH
N
NH2
Cl
Molecular imprinting (Step 1)
N
NO N
NH
OH3C
CH3
N
NO N
NH
O
H3C
CH3
Methacrylic acid+ Porogen
Molecular imprinting (Step 2)
N
NO N
NH
OH3C
CH3
N
NO N
NH
O
H3C
CH3
Molecular imprinting (Step 3)
N
NO N
NH
OH3C
CH3
N
NO N
NH
O
H3C
CH3
Cross-linking monomerInitiating reagent
Molecular imprinting (Step 4)
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
Immunoassays using MIPs
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone• Neuropeptides: Leu5-enkephalin• Other: Atrazine, Methyl--glucoside
Aptamers
1014-1015 random sequencesTarget
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target
Thank You!
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