1. The reaction horse serum and a pool of rabbit antihorse serum exhibiting four α procedure optima has been investigated with a view to analysing the antigens involved in the production of the multiple zones.
2. The following conclusions have been reached regarding the antigenic constitution of horse serum, a horse serum-albumin preparation and a horse serum-globulin preparation: Horse serum consists of at least five antigens. The horse serum-albumin preparation contains three antigens, two of which are probably impurities, one of these being a globulin. The horse serum-globulin preparation consists of three antigens and evidence was obtained of a fourth. Electrophoretic analysis of these protein preparations gave concordant result.
3. Multiple zones of rapid particulation in the precipitation reaction are due to the independent activity of multiple antigens and their homologous antibodies.
4. The absorption of precipitin from a mixture of antibodies by a mixture of antigens at a single α procedure optimum is both a useful and a practicable procedures.
5. Attention is drawn to the fact that the method of antigenic analysis used in the present series of experiments, involving the correlation of α; procedure optima occurring in the parallel titrations of a single antiserum against a mixed antigen and equivalent concentrations of physically or chemically prepared fractions of it supplemented by absorption of precipitins at individual and multiple α precedure optima and retitration of the absorbed sera, could be applied to the identification and nomenclature of proteins occurring in natural mixtures, and also to the quantitative and qualitative appraisal of the purity of proteins prepared by physical or chemical means. The method does not rely on the preparation of highly purified protein samples containing a single antigen.