ELISA for Anti Peptide Antibodies

Because synthetic peptide immunogens used to generate antipeptide antibodies often are available in comparatively large amounts, it is straightforward to check antibody titres by an enzyme-linked colorimetric assay. This procedure also is useful for monitoring recovery of antibody after purification by affinity chromatography. The target antigen is passively adsorbed to the walls of microtitre wells, either as free peptide or as peptide conjugated to an irrelevant carrier protein (e.g., bovine serum albumin, if the immunogen was a KLH conjugate). Usually, the free peptide makes a perfectly effective antigenic target, but occasionally important determinants on some peptides can be masked by adsorption to the plate, in which case peptide-carrier conjugates should be used. Thus, if antibody titre on free peptide is low it is a good idea to try conjugated peptide as the target. Antibodies bound to the adsorbed peptide are detected with an appropriate enzyme-linked second-layer reagent, typically peroxidase-linked anti-immunoglo-bulin, and the assay developed with a colorimetric substrate.

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