Submitted to: Clinical and Diagnostic Laboratory Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2002
Publication Date: 7/1/2002
Citation: Clinical & Diagnostic Lab. Immunol.9(6)2002 1301-1306 Interpretive Summary: These data show that reagents used in the developed cELISA for B. equi is sensitive in part because the antigen used in the assay is encoded by a gene highly conserved in B. equi isolates worldwide. Furthermore the epitope bound by the monoclonal antibody of this test is also highly conserved. These data provide the basis for its use as a national screening tool.
Technical Abstract: Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-Iinked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-Length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-Iength and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies, which inhibit binding ofMAb 36/133.97, also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region, which binds to both MAb 36/133.97 and inhibiting equine antibodies, has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.