Detection of equine antibodies to Babesia caballi by recombinant B-caballirhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was dev eloped for detection of equine antibodies specific for Babesia caballi, The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epi tope of a native 60-kDa B. caballi antigen. The gene encoding the recombina nt antigen was sequenced, and database analysis revealed that the gene prod uct is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-termina l repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardi zed complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results wer e CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonst rated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA- positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained anti bodies reactive with B. caballi when they were tested by IFA, These data in dicate that following infection with B. caballi, horses consistently produc e antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.