Antigen attachment in ELISA.

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occurred during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems.