Practice question

ELISA pioneered Engvall Perlmann 1971 exploits highly specific high-affinity antigen-antibody interaction KD nanomolar picomolar detecting analytes complex biological fluids serum urine CSF culture supernatant. Principle immobilization one component passive hydrophobic adsorption polystyrene 96-well high-binding blocking nonspecific sites BSA casein preventing background. Indirect sandwich formats most sensitive plate coated capture antibody monoclonal high-affinity against target antigen human chorionic gonadotropin hCG pregnancy marker HIV p24 capsid antigen cytokines IL-6 TNF-alpha allergen-specific IgE incubated specific immune complex formation governed mass action kinetics association rate 10^5 per M per s avidity bivalent enhancing stability. Washing PBS 0.05 percent Tween removing unbound proteins specific complex remains strong noncovalent hydrogen bonds van der Waals. Secondary antibody conjugated HRP enzyme 40 kDa via glutaraldehyde biotin-streptavidin amplification four biotin binding sites affinity 10^-15 M binding Fc primary antibody catalyzes oxidation chromogenic substrate TMB 3,3',5,5' presence H2O2 generating blue radical 650 nm sulfuric acid stop converts yellow diimine 450 nm measured spectrophotometrically microplate reader intensity directly proportional antigen concentration via 4PL standard curve interpolation recombinant standards. Sensitivity pg/mL dynamic range 3 logs high throughput screening hundreds samples daily low cost no radioisotope adaptability automation made ELISA cornerstone diagnostic infectious diseases autoimmunity allergy therapeutic monitoring.