Quantitative micro-determination of antibody by inhibition in single radial immunodiffusion

A modified method of single radial immunodiffusion was devised, providing a quantitating procedure of a small amount of antibody in antiserum. The method consisted of preincubation of the reference antigen with the antiserum, application of the mixture to the antigen well on the reference antibody agar plate, the measurement of the size of the precipitation ring, and comparison of the reduced ring size with that of the reference antigen alone. In this method, it has been found that a quantitative relationship exists between the concentration of antibody in the antiserum and the resulting ring size (-/d2) on the reference antibody agar plate. This method has the advantages that only a very small amount of antibody protein, such as a few micrograms, is required for the titration; and that appropriate antisera, even if they were prepared from different animal species, can most probably be used as the reference antiserum. The applicability of the modified method to the titration of antibody has been verified in this paper with several different antigen-antibody systems.