Antigen quantitation by a hemolytic radial immunodiffusion assay

The radial immunoprecipitin assay for antigen quantitation (Mancini's assay) was modified into a radial immunohemolytic assay, whereby the antigen lyses erythrocytes coated with the complementary antibody. Thus, when rabbit immunoglobulin G (IgG) diffused in Petri dishes from wells into agar gels containing erythrocytes coated with purified antibody specific for rabbit IgG, hemolytic areas developed around the wells. The lysis of the erythrocytes which had bound the IgG antigen required treatment with an antiserum to rabbit IgG and complement. When all the antigen molecules had been bound, the hemolytic areas appeared to be linearly related to the antigen concentrations and independent of temperature. Intradish coefficients of variation of replicate assays in 10 dishes ranged from 3.6 to 11.3. Interdish coefficient of variation from the intradish means was 3.8. Analysis of variance of the intradish means indicated no significant differences. This hemolytic radial immuno-diffusion assay compares well with the conventional radial immunodiffusion assay: it is very sensitive (the lowest concentration of antigen still mediating lysis is about 0.1 μg/ml), the edges of the hemolytic areas are sharper than the edges of the precipitin rings at any antigen concentration and non-precipitating antibody or horse antibody may he used.