Detection of culture-derived Babesia bovis exoantigen using a two-site enzyme immunoassay

Soluble exoantigens in the supernatants of Babesia bovis cultures have been shown to be efficient immunogens against bovine babesiosis. We used a two-site enzyme immunoassay to monitor the release of these antigens during in vitro cultivation. Bovine immunoglobulin G was isolated from serum of an adult cow previously immunized with culture-derived B. bovis exoantigens and challenged via needle with virulent parasites. The specific immunoglobulin G was used as a capture antibody and as an enzyme-conjugated recognizing antibody. The optimal protein concentration of capture antibody was 10 micrograms/ml. The 24-h cultures showed the greatest antigen concentration. The test was sensitive for detection of differences in species-specific antigenic activity among B. bovis isolates, for determining loss of antigenicity during storage and formalinization, and for monitoring the kinetics of exoantigen release during in vitro cultivation. Antigens cross-reactive with the other major Babesia species of cattle, Babesia bigemina, were also detected with this assay. The high specificity, sensitivity, and reproducibility of this technique should facilitate detection and quantitation of Babesia antigens during purification and in standardization of candidate immunogens.