Practice question

Radioimmunoassay RIA competitive binding assay pioneered Berson Yalow 1960 Nobel Prize Medicine 1977 enabled first quantification peptide hormones insulin glucagon pg concentrations revolutionizing endocrinology. Principle relies limited antibody binding sites fixed high-affinity polyclonal antibody specific analyte insulin 51 aa thyroxine T4 triiodothyronine growth hormone 191 aa incubated known quantity iodine-125 labeled antigen purified chloramine T oxidizing iodide iodine incorporating tyrosine achieving specific activity 50-100 uCi per ug variable unknown unlabeled sample antigen competing same paratope via mass action competitive inhibition law mass action. Equilibrium 24h 4 degrees balancing association dissociation. Separation free bound fractions essential via second antibody precipitation goat anti-rabbit Ig forming insoluble complexes centrifuged 3000 g dextran-coated charcoal adsorbing free small antigen <5 kDa leaving bound supernatant PEG precipitation magnetic beads coated Protein A facilitating magnetic separation automated platforms. Gamma scintillation counter NaI Tl crystal measures radioactivity cpm pellet inversely related sample antigen standard curve percent bound versus log concentration semilog spline interpolation calculation unknowns. Sensitivity detection limit 1 pg/mL due high specific activity tracer amplified high affinity Ka 10^10 per M specificity determined cross-reactivity related hormones. Radioisotope hazards gamma iodine-125 60 days half-life shielding short shelf life