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Immunochemical Methods in the Clinical Laboratory Roger L Bertholf, Ph.D., DABCC Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville Associate Professor of Pathology, University of Florida College of Medicine Name The Antigen ASCP/Bertholf 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 Aminobenzene Sulphonate, a Hapten NH2 NH2 NH2 SO3 SO3 SO3 Ortho Meta Para 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 Substrate-labeled fluorescence immunoassay S Enzyme S No signal Fluorescence S Enzyme Signal Fluorescence excitation transfer immunoassay No signal Signal Electrochemical differential polarographic immunoassay Oxidized Reduced Prosthetic group immunoassay P S Enzyme No signal P P Enzyme Signal Enzyme channeling immunoassay Substrate E1 Product E2 Ag Product 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 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 O - Fundamentals of antigen/antibody interaction Cl O NH + N O- OH NH2 O CH2-CH2-CH2-CH3 Molecular imprinting (Step 1) Methacrylic acid + Porogen O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 2) O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 3) Cross-linking monomer Initiating reagent O H3C O NH N N CH3 N O H3C O NH N N CH3 N Molecular imprinting (Step 4) Comparison of MIPs and antibodies Antibodies MIPs • In vivo preparation • In vitro preparation • Limited stability • Unlimited stability • Variable specificity • Predictable specificity • General applicability • Limited applicability Immunoassays using MIPs • Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (α 2adrenoceptor antagonist) • Hormones: Cortisol, Corticosterone • Neuropeptides: Leu5-enkephalin • Other: Atrazine, Methyl-α-glucoside Aptamers 1014-1015 random sequences Target Oligonucleotide-Target complex Unbound oligonucleotides + Target Aptamer candidates PCR New oligonucleotide library ... structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation Aminobenzene Sulphonate, a Hapten NH2 NH2 NH2 SO3 SO3 SO3 Ortho Meta Para Classification of immunochemical