Antisera are routinely screened for anti-peptide antibodies using an ELISA in which the peptide antigen is adsorbed onto the ELISA plate. The highest purity peptide available is used for screening. Antibody that binds to the peptide is normally quantified using an alkaline phosphatase labelled secondary antibody which is detected by the action of the enzyme on a chromogenic substrate (p-nitrophenyl phosphate). Antisera with the highest titres of anti-peptide antibody are selected for further testing and purification.
Occasional false negatives can occur in the ELISA because the adsorption of the peptide antigen to the plate may block the antigenic sites. This can be overcome by conjugating the peptide to a carrier protein (using a different protein from that used for immunisation) before adsorption onto the plate. Alternatively, the peptide antigen can be biotinylated and then attached to an avidin-coated plate. Where a cysteine residue has been added to the peptide for conjugation to the carrier protein, the same cysteine can conveniently be used to link the biotin.
An antigenic response to the peptide is not a guarantee that the antibodies will recognise the target protein. Ideally this should be determined using the assay method for which the antibody is intended (e.g. Western blotting or immunocytochemistry). Antibodies that detect the denatured target protein in Western blots will not necessarily bind to the native protein in tissue sections, cultured cells and protein microarrays.