A technique for the measurement of poliomyelitis virus D antigens by means of gel diffusion tests against antisera obtained from hyperimmunized calves is described in detail. 2. The precision of the method was estimated by performing several replicate tests with three or four dilutions of concentrated antigen of each of the three types. The D antigen content, if measured by means of twelve precipitation hexagons in agar can be calculated with a precision of about 10 per cent (95 per cent confidence limits). 3. The calf sera gave no precipitation with C antigens in the concentrations suitable for measuring D antigens. 4. The local reference antigens, the D antigen concentrations of which were adjusted to equal that of the reference antigens used in the Glaxo Laboratories, contained C as well as D antigens. This was observed in gel diffusion tests with specific anti-C sera from guinea-pigs. 5. The guinea-pig anti-C sera showed a precipitation with the immune calf sera as well as with normal calf serum. This indicated the presence of anti-calf antibodies in the guinea-pig sera, presumably elicited by traces of calf proteins in the vaccines. These antibodies might give rise to non-specific precipitation if antisera used in the test for D antigen were obtained from animal species other than those used to provide components in the tissue culture medium from which the antigens were derived.
