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 Ab Ag Ab Ag [ Ab Ag ] Ka [ Ab][ Ag ] 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 Precipitate The precipitin reaction etc. Zone of equivalence Antibody/Antigen Single radial immunodiffusion Ag Single radial immunodiffusion r r [Ag ] 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 SPE IgG IgA IgM 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 Rate C1 C2 Time Additional considerations for quantitative competitive binding immunoassays • Response curve • Hook effect %Bound label Competitive immunoassay response curve %Bound vs. log concentration Antigen concentration Logistic equation a %Bound label y c d ad x a c Slope = b Log antigen concentration b d Logit transformation a %Bound label y Y logit y ln 1 y y d where y a d d Log antigen concentration Logit y Logit plot Log antigen concentration %Bound antigen High dose “hook” effect 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 – Toxicity – Shelf life – Disposal costs Enzyme labels • Advantages – Diversity – Amplification – Versatility • Disadvantages – Lability – Size – Heterogeneity Fluorescent labels • Advantages – Size – Specificity – Sensitivity • Disadvantages – Hardware – Limited selection – Background Chemiluminescent labels • Advantages – Size – Sensitivity – S/N • Disadvantages – Hardware – ? Chemiluminescent labels NH 2 O NH 2 O* H N + N H 2 H 2 O 2 + OH - OO- Pe r ox i da se O + N2 + 3 H2O O L um i n o l NH 2 COO + COO - h ( ma x = 4 3 0 nm ) Chemiluminescent labels CH 3 Br - N+ O- CH 3 N O O + H 2 O 2 + OH - O CO 2 H A c r i d i n i um e s t e r + CO 2 + h + CO 2 H 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 E P E E Microtiter well E E ELISA (variation 1) Specimen Labeled antigen E S E P E Microtiter well E ELISA (variation 2) Labeled antibody E Specimen E E E E E E Microtiter well E 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 E Magnetic separation methods Fe Fe Fe Fe Fe Fe Fe Fe Fe Magnetic separation methods Aspirate/Wash Fe Fe Fe Fe 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 S No signal P S Signal Enzyme Signal (enzyme activity) EMIT™ signal/concentration curve Functional concentration range 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 E4 E3 E2 Triplet VR E1 IC A F 10-6-10-9 sec P E0 10-4-10 sec Polarized radiation z x Polarizing filter y Fluorescence polarization in Fluorescein out (10-6-10-9 sec) Orientation of polarized radiation is maintained! Fluorescence polarization in HO O O C O OH But. . . out (10-6-10-9 sec) Rotational frequency 1010 sec-1 Orientation of polarized radiation is NOT maintained! Fluorescence polarization immunoassay Slow rotation Polarization maintained Rapid rotation Polarization lost FPIA signal/concentration curve Signal (I/I) Functional concentration range 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 Donor Spontaneous Acceptor Monomer (inactive) Active tetramer Cloned enzyme donor immunoassay Donor Acceptor No activity Donor Active enzyme Acceptor Signal (enzyme activity) CEDIA™ signal/concentration curve Functional concentration range Antigen concentration Other approaches to homogeneous immunoassay • • • • Fluorescence methods Electrochemical methods Enzyme methods Enzyme channeling immunoassay Substrate-labeled fluorescence immunoassay S Enzyme S No signal Fluorescence S Signal Enzyme Fluorescence excitation transfer immunoassay No signal Signal Electrochemical differential polarographic immunoassay Oxidized Reduced Prosthetic group immunoassay P S Enzyme No signal P P Signal Enzyme Enzyme channeling immunoassay Substrate E1 Product 1 E2 Ag Product 2 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 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)