The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.