Membrane antigen of Toxoplasma gondii
Kawai, K.; Yaguchi, S.; Yata, K.; Kamei, K.
Nippon Ganka Gakkai Zasshi 84(9): 1107-1112
1980
ISSN/ISBN: 0029-0203 PMID: 7223548 Document Number: 164920
The antigenic activity of membrane antigen isolated from tachyzoites of T. gondii, strain RH, by sucrose density gradient ultracentrifugation and butanol extraction and the localization of the membrane antigen in the tachyzoite by immunoelectronmicroscopy, were studied. Clinical application of the serologic test with this antigen preparation by fluoropolarimetry in experimental toxoplasma infection was attempted. Analysis of total toxoplasmal antigen preparation by disc electrophoresis on polyacrylamide gel disclosed 10 protein bands, while soluble antigen preparation showed 6 separated protein bands and membrane antigen preparation, 2 distinct protein bands on staining. Gel filtration of the membrane antigen on a Sephadex G200 column yielded protein fractions with a peak at the position of ribonuclease as a marker for protein of MW 13,700. These fractions were pooled and lyophilized to be used as purified membrane antigen in the fluoropolarometric measurement. A 4-fold dilution of the membrane antigen blocked the latex agglutination with a porcine antitoxoplasma antiserum (reciprocal titer: 256 and a 2-fold dilution of the antigen inhibited the dye test with the same porcine antiserum. Immunoelectronmicroscopy with ferritin- or peroxidase-coupled antibody revealed localization of the membrane antigen in the outer membrane of the tachyzoite, but not at the intracellular organelle level. The purified membrane antigen was labeled with fluorescein isothiocyanate as described by Coons and the labeled membrane antigen, 0.5 .mu.g/ml, was combined with 1-20 .mu.g/ml of purified antibody (anti-membrane antigen IgG) and incubated at 37.degree. C for 30 min, followed by fluoropolarimetric measurement. The fluoropolarization increased progressively with the increase in amount of purified antibody added. The practicability of quantitative antibody detection by fluoropolarimetry with the use of membrane antigen is demonstrated, and the potential usefulness of the technique as a clinical laboratory test is indicated.