Practice question

Indirect ELISA detects serum antibodies using antigen-coated wells. Initial coating immobilizes purified antigen to plastic overnight. Subsequent blocking with BSA or milk prevents nonspecific adsorption to empty spaces. Patient serum containing primary antibody is then added, allowing specific binding to coated epitope. After washing, enzyme-conjugated anti-human secondary antibody recognizes constant region of bound primary. Final addition of chromogenic substrate generates measurable product proportional to primary antibody titer. Reversing coating and blocking or adding detection before secondary would cause loss of signal or high background, breaking sequential molecular recognition cascade.