Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis

Abstract

Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.

FIG. 1

MAb BEG3 binding to different forms of B. equi as observed with a confocal laser microscope. (a) Single ring form; (b) round form with cytoplasm; (c) double ring-like form; (d) Maltese cross form. Bar, 5 μm.

FIG. 2

Intensity of fluorescence on round form (trophozoite) of B. equi after reaction with the MAb. (a) Intensity of fluorescence was scanned along the yellow line. Bar, 5 μm. (b) Two peaks were observed in the scanning pattern of intensity.

FIG. 3

Electron micrograph of MAb BEG3 binding to the B. equi parasite. (a) The immunoreaction deposits (arrows) were observed on the surface of the B. equi parasite. (b) No immunoreaction deposits were observed in the cytoplasm of uninfected erythrocytes. Bar, 0.2 μm.

FIG. 4

Recognition of 19-kDa B. equi parasite antigen by MAb BEG3. Lanes: 1, B. caballi-infected erythrocyte lysate; 2, B. equi-infected erythrocyte lysate; 3, noninfected erythrocyte lysate. MW, molecular weight markers (in thousands).

FIG. 5

Morphological changes of B. equi parasites caused by MAb BEG3. (a) Parasites incubated with normal mouse IgG; (b) parasites incubated with MAb BEG3. Larger and vacuolated parasites in erythrocytes were observed. Bar, 5 μm.

FIG. 6

Immunoblot analysis of crude and partially purified antigen (fraction 9) obtained by HPLC. Crude (lanes 1 and 3) and partially purified (lanes 2 and 4) antigens were probed with anti-B. equi immune serum (lanes 1 and 2) and MAb BEG3 (lanes 3 and 4). MW, molecular weight markers (in thousands).

FIG. 7

Correlation of IFAT titers and ELISA values. Titer of <80 is negative in IFAT. Circles show values of individual horse sera; squares show mean values with standard deviations of horse sera at different IFA titers. Serum samples showing OD values more than those of the means plus 3 standard deviations of negative serum samples (dotted line) were considered positive by ELISA.