Caveats and suggestions for the ELISA.

This study identifies three categories of errors that may arise from faulty procedures utilized in the ELISA and offers suggestions for their recognition and avoidance. The first deals with the potential sources of 'background' values indicating that the practice of subtracting those values from the total OD measurements of the reactivity of a specific antigen-antibody system is not valid in all instances. A simple method of curve analysis has been devised for determining whether that correction is valid. The second consideration is that of stoichiometry of binding of second antibody to the test antibody-antigen complex which is shown to be restricted to a narrow range of concentrations and a short period of incubation of that reagent. The need and method to determine the conditions for stoichiometry for each ELISA protocol is demonstrated. Thirdly, the need for and properties of a suitable blocking agent to prevent binding of ambiguous reactants to the surfaces of the test wells are reiterated and the greater efficiency of BSA, compared with other frequently recommended blocking agents, is demonstrated.