Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D., DABCC Mark A. Bowman, Ph.D., MT(ASCP)

The University of Florida

University of Florida Health Science Centers in Gainesville and Jacksonville

The University of Iowa

University of Iowa College of Medicine                                                                                                   

Florida vs. Iowa                                                                

The American Society of Clinical Pathologists Marie Bass, MT(ASCP) Manager, ASCP Workshops for Laboratory Professionals Kathleen Dramisino, MT(ASCP) Workshop coordinator Tommie Ware A/V and materials support

Classification of immunochemical methods Particle methods Precipitation Immunodiffusion Immunoelectrophoresis Light scattering Nephelometry Turbidimetry Label methods Non-competitive One-site Two-site Competitive Heterogeneous Homogeneous

Properties of the antibody-antigen bond Non-covalent Reversible Intermolecular forces Coulombic interactions (hydrogen bonds) Hydrophobic interactions van der Waals (London) forces Clonal variation

Antibody affinity

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

Factors affecting solubility Size Charge Temperature Solvent ionic strength

The precipitin reaction etc. Precipitate Zone of equivalence Antibody/Antigen

Single radial immunodiffusion Ag

Single radial immunodiffusion

Electroimmunodiffusion Why would we want to combine immunodiffusion with electrophoresis? SPEED Specificity Carl-Bertil Laurell (Lund University, Sweden) Laurell Technique (coagulation factors) “Rocket electrophoresis”

Electroimmunodiffusion + -

Immunoelectrophoresis Combines serum protein electrophoresis with immunometric detection Electrophoresis provides separation Immunoprecipitation provides detection Two related applications: Immunoelectrophoresis Immunofixation electrophoresis

Immunoelectrophoresis + - -human serum Specimen

Immunoelectrophoresis + - P C P C P C     

Immunofixation electrophoresis Immunochemical Methods (handout addendum) Immunofixation electrophoresis SPE IgG IgA IgM   Bertholf and Bowman

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

Light reflection

Molecular size and scattering - +

Distribution of scattered radiation

Nephelometry vs. Turbidimetry 0°-90°

Rate nephelometry Intensity of scattering Time Rate C1 C2

Additional considerations for quantitative competitive binding immunoassays Response curve Hook effect

Competitive immunoassay response curve %Bound vs. log concentration %Bound label Antigen concentration

Log antigen concentration Logistic equation a %Bound label c Slope = b d Log antigen concentration

Log antigen concentration Logit transformation a %Bound label d Log antigen concentration

Log antigen concentration Logit plot Logit y Log antigen concentration

High dose “hook” effect %Bound antigen Antigen concentration

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

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

Radioisotope labels Advantages Disadvantages Flexibility Sensitivity Size Disadvantages Toxicity Shelf life Disposal costs

Enzyme labels Advantages Disadvantages Diversity Amplification Versatility Disadvantages Lability Size Heterogeneity

Fluorescent labels Advantages Disadvantages Size Specificity Sensitivity Disadvantages Hardware Limited selection Background

Chemiluminescent labels Advantages Size Sensitivity S/N Disadvantages Hardware ?

Chemiluminescent labels

Chemiluminescent labels

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

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

Enzyme-linked immunosorbent assay Substrate 2nd antibody E Specimen S P Microtiter well E

ELISA (variation 1) Specimen Labeled antigen E Microtiter well S P E

ELISA (variation 2) Labeled antibody E Specimen E Microtiter well

Human anti-animal antibodies Humans exposed to animals can produce antibodies to animal immunoglobulins Heterophilic antibodies Anti-isotypic Anti-idiotypic Human anti-mouse antibodies (HAMA) are most common Anti-animal antibodies can cross-link capture and detection reagent antibodies

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

Immobilized antibody methods Coated tube Coated bead Solid phase antibody methods

Coated tube methods Specimen Labeled antigen Wash

Coated bead methods

Microparticle enzyme immunoassay (MEIA) Labeled antibody E S P E Glass fiber matrix

Magnetic separation methods Fe

Magnetic separation methods Aspirate/Wash Fe

Electrochemiluminescence immunoassay (Elecsys™ system) Flow cell Oxidized Reduced Fe

ASCEND (Biosite Triage™)

ASCEND Wash

ASCEND Developer

Solid phase light scattering immunoassay

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

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 S Enzyme No signal S P Enzyme S Signal

EMIT™ signal/concentration curve Functional concentration range Signal (enzyme activity) Antigen concentration

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

Molecular electronic energy transitions Singlet A VR Triplet IC F P 10-6-10-9 sec 10-4-10 sec E0

Polarized radiation z y x Polarizing filter

Fluorescence polarization Fluorescein in out (10-6-10-9 sec) Orientation of polarized radiation is maintained!

Fluorescence polarization But. . . O H C Rotational frequency  1010 sec-1 in out (10-6-10-9 sec) Orientation of polarized radiation is NOT maintained!

Fluorescence polarization immunoassay Polarization maintained Slow rotation Rapid rotation Polarization lost

FPIA signal/concentration curve Functional concentration range Signal (I/I) Antigen concentration

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 Spontaneous Monomer (inactive) Active tetramer Acceptor Monomer (inactive)

Cloned enzyme donor immunoassay Acceptor No activity Acceptor Donor Active enzyme

CEDIA™ signal/concentration curve Immunochemical Methods (handout addendum) CEDIA™ signal/concentration curve Functional concentration range Signal (enzyme activity) Antigen concentration Bertholf and Bowman

Other approaches to homogeneous immunoassay Fluorescence methods Electrochemical methods Enzyme methods Enzyme channeling immunoassay

Substrate-labeled fluorescence immunoassay Enzyme No signal S Fluorescence Enzyme S Signal

Fluorescence excitation transfer immunoassay No signal Signal

Electrochemical differential polarographic immunoassay Oxidized Reduced

Prosthetic group immunoassay Enzyme No signal S P Enzyme P Signal

Enzyme channeling immunoassay Substrate Product 1 E1 Product 2 E2

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

Molecular imprinting

Immunochemical Methods (handout addendum) A final thought. . . “In science one tries to tell people, in such a way as to be understood by everyone, something that no one ever knew before. But in poetry, it's the exact opposite.” Paul Adrien Maurice Dirac (1902- 1984) Bertholf and Bowman