Establishment and Application of an Indirect Enzyme-Linked Immunosorbent Assay for Measuring GPI-Anchored Protein 52 (P52) Antibodies in Babesia gibsoni-Infected Dogs

SIMPLE SUMMARY: In this study, a novel BgGPI52-WH antigen was identified and evaluated as an immunodiagnostic candidate. An indirect ELISA was established based on the BgGPI52-WH antigen. The results showed that the iELISA had good sensitivity, specificity and reproducibility, and the signal could be detected as early as the sixth day after infection. Clinical samples were tested using the established method, and 11.41% of the samples were positive. The results suggested that BgGPI52-WH is a good immunodiagnostic marker and the iELISA is a practical method for early diagnosis. ABSTRACT: Babesia gibsoni is a malaria-like protozoan that parasitizes the red blood cells of canids to cause babesiosis. Due to its high expression and essential function in the survival of parasites, the Glycosylphosphatidylinositol (GPI) anchor protein family is considered an excellent immunodiagnostic marker. Herein, we identified a novel GPI-anchored protein named as BgGPI52-WH with a size of 52 kDa; the recombinant BgGPI52-WH with high antigenicity and immunogenicity was used as a diagnostic antigen to establish a new iELISA method. The iELISA had a sensitivity of 1:400, and no cross-reaction with other apicomplexan parasites occurred. We further demonstrated that the degree of variation was less than 10% using the same samples from the same or different batches of an enzyme-labeled strip. It was found that the method was able to detect early infection (6 days after infection) in the sera of the B. gibsoni-infected experimental dogs in which antibody response to rBgGPI52-WH was evaluated. Clinical sera from pet hospitals were further tested, and the average positive rate was about 11.41% (17/149). The results indicate that BgGPI52-WH is a reliable diagnostic antigen, and the new iELISA could be used as a practical method for the early diagnosis of B. gibsoni.