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

Abstract

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed 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 epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product 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-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated 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 antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce 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.

FIG. 1

Immunoprecipitated, metabolically labeled B. caballi proteins recognized by horse sera and MAbs. Lane 1, preinoculation serum from horse A2034; lane 2, immune serum from horse A2034; lane 3, anti-RAP-1 MAb 79/17.18.5; lane 4, IgG1 isotype control MAb 18.185. Molecular size markers (in kilodaltons) are given on the left (Kaleidoscope Standards; Bio-Rad Laboratories).

FIG. 2

Western-immunoblotted proteins treated with periodate followed by development with MAbs. (A) N. caninum 65-kDa protein; (B) B. caballi native RAP-1; (C) B. equi EMA-1/EMA-2 proteins. Lane 1, no treatment; lanes 2, 3, and 4, treatment with 0.1, 1.0, and 10 mM periodate, respectively. Numbers on the left are in kilodaltons.

FIG. 3

C-terminal repeats of B. caballi RAP-1. The residues in reverse type (black background) were conserved in three or more of the repeats. The truncated clone begins at the start of repeat 3 (residue 394). The asterisk indicates the stop codon for the protein. MAb 79/17.18.5 binds within this sequence of RAP-1. Residue numbering on the right is consistent with the numbering in the full-length clone (GenBank accession no. AF092736).

FIG. 4

Distribution of cELISA results by percent inhibition. Samples with discrepant results that were found to be positive by IFA have an additional mark: plus sign, the sample was IFA positive, in agreement with the cELISA; cross-hatching, the sample was IFA positive, in agreement with CFT; white blocks, the samples were cELISA positive irrespective of the CFT results; shaded blocks, the samples were cELISA negative, irrespective of the CFT results.