Preparation and standardization of an Australia antigen antibody of equine origin

Abstract

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.