IMMUNOASSAYS Basic Concepts & Definitions Measurement of Antibody Affinity Quantitative Methods – competitive & noncompetitive assays Ref: Burtis & Ashwood; Tietz Fundamentals/Textbook Jan Klein & Vaclav Horejsi; Immunology (1997) Coleman Lombard Sicard; Fundamental Imm (1992) Gary D. Christian; Analytical Chem (1994)

1. BASIC CONCEPTS & DEFINITIONS Immunoassay: use of antibodies to detect analyte 1a. Antibodies Immunoglobulins that bind to Antigens 5 classes: IgG, IgA, IgM, IgD, IgE 1b. Immunogen Protein or a substance coupled to a carrier When introduced into foreign host -> induce Ab to form 1c. Antigen Any material which can react with Ab May not induce Ab formation

1d. Antigen-Antibody Binding Ab molecules have specific binding sites -> bind tightly to Ag -> cause pptn/neutralization/ death Binding of Ag to Ab due to van der Waals forces hydrophobic interactions charged group attractions Can measure Antibody affinity: strength of binding between Ab & Ag

2. MEASUREMENT OF ANTIBODY AFFINITY Binding of Ag to Ab is reversible -> association & dissociation Ag + Ab <-> AgAb Law of mass action: Rate of rxn  to concn of reactants ka[Ag][Ab] = kd[AgAb] K = ka/kd = [AgAb]/ [Ag][Ab] where K is equilibrium constant or affinity constant

r/c = nK – rK r = no. of molecules of bound Ag per Ab molecule c = concn of free Ag n = valency of Ab Plot r/c vs r => Scatchard Plot Straight line with slope k x intercept gives n y intercept gives nK K (liters/mole) measures affinity of complex

Why measure Affinity of an Antibody? To assess Ab specificity It influences the functional efficiencies of Abs eg. high-affinity Abs are very dependable for various applications: Diagnostic Therapeutic Analytical

Labeled Immunochemical Assays 3. QUANTITATIVE METHODS Read & Understand from Tietz Fundamentals: Radial Immunidiffusion Immunoassay Electroimmunoassay Turbidimetric & Nephelometric Assays Labeled Immunochemical Assays

COMPETITIVE vs NONCOMPETITIVE RXNS A. Competitive Immunoassays Used when have limited reagents (Ag) (i) Simultaneous Competitive Assay Labels Ag (Ag*) & unlabeled Ag compete for binding to Ab The probability of Ab binding to Ag* is inversely  to [Ag] Ab + Ag + Ag* <-> Ab:Ag + A-Ag*

(ii) Sequential Competitive Assay Step 1: unlabeled Ag mixed with excess Ab -> binding allowed to reach equilibrium Step 2: Ag* added sequentially -> equilibrate After separation -> det bound Ag* -> calculate [Ag] Larger fraction of Ag bound to Ab than in simultaneous assay If k1 >> k2 ->  in Ab:Ag ->  in Ag* binding Provide two- to four- fold improvement in detection limit

b. Noncompetitive Immunoassays Used when have excess reagent Immobilization of Ab to support Passively adsorption or bind covalently Direct or indirect attachment (Table 9-3) ii. Ag allowed to react with Ab -> wash other proteins iii. Add labeled Ab (conjugate) -> reacts with bound Ag Determine bound label -> [Ag*] or its activity is  [Ag]