New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA.
A peroxidase saturation technique for the determination of coating antigen concentration necessary to saturate polystyrene plates with a wide range of different antigens is described. The same technique has also been used to compare the stability of antigen-polystyrene bonds for native and denaturated antigens. Furthermore, the inappropriate selection of solid-phase antigen concentration and its influence on ELISA results is analyzed and an experimental criterion to select the optimum antigen concentration is proposed. Two different antigens, BSA-Ar and hydatid antigen, were used for ELISA determination of specific antibodies as a model system. Optimum solid-phase antigen concentration was determined by two different methods: peroxidase saturation, in which binding of peroxidase to non-antigen-occupied polystyrene surface sites was used to evaluate the degree of coating by antigen; chequer-board titration, using several immune sera of different affinity. Optimum antigen concentration selected by chequer-board titration using low affinity sera was similar to that selected with peroxidase saturation. On the other hand, lower antigen concentration would be selected by chequer-board titration using high affinity sera. For this reason, the concentrations of low affinity antibodies would be underestimated using the chequer-board titration. These results indicate that peroxidase saturation should be used to avoid avidity-dependent artifacts in ELISA.
D000818:Animals; D000941:Antigens / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D010544:Peroxidases; D011137:Polystyrenes; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine
D000941:Antigens; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; D010544:Peroxidases