Enzyme-Linked Immunosorbent Assay of Peptides

Enzyme-linked immunosorbent assay (ELISA) is a member of the solid-phase immunoassay family that detects specific antigen-antibody binding reactions (1 ,2 ). A great many variables of antigen or antibody presentation, treatment, and detection are considered when performing ELISA (3 ,4 ). This chapter will describe the simplest form, a direct assay, in which the peptide antigen is immobilized on a polystyrene carrier, and a specific primary antibody in solution (detector) is added that recognizes the plate-bound antigen. A secondary, horseradish peroxidase (HRP)-coupled polyclonal antibody (reporter) binds to the primary antibody. The enzymatic activation of hydrogen peroxide releases a chromophore from o -phenylenediamine that is detected and quantified.