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 methods SpecimenLabeled antigen Wash

Coated bead methods

Enzyme-linked immunosorbent assay Microtiter well EEEEE Specimen 2nd antibody E Substrate SP

Microparticle enzyme immunoassay (MEIA) Labeled antibody E EE SP Glass fiber matrix

Magnetic separation methods Fe

Magnetic separation methods 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 SP No signal Signal Enzyme S

EMIT™ signal/concentration curve Signal (enzyme activity) 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 E0E0 E4E4 E3E3 E2E2 E1E1 Singlet Triplet A VR F IC P sec sec

Polarized radiation z y x Polarizing filter

Fluorescence polarization Fluorescein in Orientation of polarized radiation is maintained! out ( sec)

Fluorescence polarization O H O O H C O O Rotational frequency  sec -1 in Orientation of polarized radiation is NOT maintained! out ( sec) But...

Fluorescence polarization immunoassay Polarization maintained Slow rotation Rapid rotation Polarization lost

FPIA signal/concentration curve Signal (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 SFluorescence No signal Signal Enzyme S

Fluorescence excitation transfer immunoassay Signal No signal

Electrochemical differential polarographic immunoassay Oxidized Reduced

Prosthetic group immunoassay Enzyme P P SP Signal No signal

Enzyme channeling immunoassay E1E1 E2E2 Substrate Product 1 Product 2

Early theories of antibody formation Paul Ehrlich ( ) 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 ( ) 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 ( ) 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 O-O- NH 3 + CH 2 -CH 2 -CH 2 -CH 3 OH N NH 2 Cl

Molecular imprinting (Step 1) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C Methacrylic acid + Porogen

Molecular imprinting (Step 2) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C

Molecular imprinting (Step 3) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C Cross-linking monomer Initiating 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 AntibodiesMIPs

Immunoassays using MIPs Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (  2 -adrenoceptor antagonist) Hormones: Cortisol, Corticosterone Neuropeptides: Leu 5 -enkephalin Other: Atrazine, Methyl-  -glucoside

Aptamers random sequences Target Oligonucleotide-Target complex Unbound oligonucleotides Aptamer candidates PCR New oligonucleotide library + Target

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