Conformational dependence and conservation of an immunodominant epitope within the babesia equi erythrocyte-stage surface protein equi merozoite antigen 1.
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.
Cunha
Cristina W
CW
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040, USA.
Kappmeyer
Lowell S
LS
McGuire
Travis C
TC
Dellagostin
Odir A
OA
Knowles
Donald P
DP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology*; D001403:Babesia / Q000276:immunology*; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000469:parasitology*; D006736:Horses; D016056:Immunodominant Epitopes; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; C085964:EMA-1 protein, Babesia equi; D016056:Immunodominant Epitopes; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins