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Clinical and Diagnostic Laboratory Immunology, November 2002, p. 1301-1306, Vol. 9, No. 6
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.6.1301-1306.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Conformational Dependence and Conservation of an Immunodominant Epitope within the Babesia equi Erythrocyte-Stage Surface Protein Equi Merozoite Antigen 1

Cristina W. Cunha,1,2 Lowell S. Kappmeyer,3 Travis C. McGuire,1 Odir A. Dellagostin,2 and Donald P. Knowles1,3,{dagger}*

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040,1 Center for Biotechnology, Federal University of Pelotas, RS, Brazil, 99010-900,2 Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164-66303

Received 14 January 2002/ Returned for modification 30 April 2002/ Accepted 1 July 2002

Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked 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-length 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 of MAb 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.

* Corresponding author. Mailing address: Animal Disease Research Unit, ARS-USDA, Washington State University, Pullman, WA 99164-6630. Phone: (509) 335-6022. Fax: (509) 335-8328. E-mail: dknowles{at}vetmed.wsu.edu.

{dagger} Present address: Center for Biotechnology, Universidade Federal de Pelotas, Campus Universitário, Pelotas, RS, Brazil, 99010-900.

Clinical and Diagnostic Laboratory Immunology, November 2002, p. 1301-1306, Vol. 9, No. 6
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.6.1301-1306.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ueti, M. W., Palmer, G. H., Kappmeyer, L. S., Scoles, G. A., Knowles, D. P. (2003). Expression of Equi Merozoite Antigen 2 during Development of Babesia equi in the Midgut and Salivary Gland of the Vector Tick Boophilus microplus. J. Clin. Microbiol. 41: 5803-5809 [Abstract] [Full Text]  


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