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Immunological properties of the 30 kDa antigen of toxoplasma gondii
D W Shin,*Y H Lee and T J Rho
Department of Parasitology, College of Medicine, Chungnam National University, Taejon, Korea.
Received January 16, 1997; Accepted February 28, 1997.
Abstract
The molecular weight 30 kDa membrane protein of Toxoplasma gondii (Toxoplasma 30 kDa) apparently conserved in most strains of T. gondii and sera of infected hosts. The present study aimed to elucidate Toxoplasma 30 kDa as a useful diagnostic antigen for serodiagnosis of toxoplasmosis by ELISA and for induction of protective immunity. Murine spleen cells immunized with the membrane antigen of T. gondii were fused with mouse Sp2/O-Ag14 myeloma cells. Out of 8 clones selected, five were IgG2b, the others belonged to IgG1 and IgG2a. The 30 kDa antigen was distributed mainly on the surface membrane of tachyzoites by indirect fluorescence method. Murine peritoneal macrophages which were activated by 30 kDa antigen produced more amounts of NO2 compared with crude antigen-treated group, however there were no significant differences in toxoplamacidal activity between the two groups. Higher specificity of Toxoplasma 30 kDa antigen was recognized for serodiagnosis of toxoplasmosis than the crude antigen. From these results, Toxoplasma 30 kDa antigen enhances the cytotoxic effect of macrophages as well as a more reliable means for the serodiagnosis of toxoplasmosis by ELISA.
Figures
Fig. 1A Western blot patterns of Toxoplasma crude antigen reacted with various kinds of antibodies. ① Toxoplasma antigens reacted with sera from mice infected with Beverley strain of T. gondii ② Toxoplasma antigens reacted with sera form mice immunized with Toxoplasma membrane antigen ③ Toxoplasma antigens reacted with culture supernatant secreting Toxoplasma 30 kDa monoclonal antibody ④ Toxoplasma antigens reacted with ascites secreting 30 kDa monoclonal antibody. Numbers represent molecular weight standards.
Fig. 1B Silver stain pattern of SDS-polyacrylamide gel applied to the antigens eluted by immunoaffinity chromatography. ① Molecular weight marker ②,③ and ④ represent antigenic fractions eluted by immunoaffinity chromatograph, respectively. Number represent molocular weight standard: α-lactoalbumin (14,200), trypsin inhibitor (20,100), carbonic anhydrase (29,000), ovalumin (45,000), albumin (66,000).
Fig. 2 Distribution of Toxoplasma 30 kDa antigen reacted with monoclonal antibody stained by indirect immunofluorescent antibody technique. A, RH strain of T. gondii reacted with normal mouse serum, there was no immunofluorescent; B, RH srain of T. gondii reacted with immune mouse serum as a positive control. Toxoplasma surface showed fluorescent entirely; C, RH strain of T. gondii reacted with Toxoplasma 30 kDa monoclonal antibody showed strong positive reaction on the surface membrane.
Fig. 3 Amounts of NO2 production by peritoneal macrophages from normal and activator-inoculated mice. Data shown are the mean ± standard deviation of 5 cases.
*p<0.05 compared to the normal mice
**p<0.05 compared to the Toxoplasma crude antigen-treated group
Fig. 4 Distribution of absorbance values of the sera from normal mice and Toxoplasma infected mice measured by ELISA. Sera from normal mice were react with Toxoplasma crude antigen (Normal-crude) or reacted with Toxoplasma 30 kDa antigen (Normal-30 kDa). Sera from Toxoplasma-infected mice were reacted with Toxoplasma crude antigen (Infected-crude) or reacted with 30 kDa antigen (Infected-30 kDa). Each dot means one case, and each bar means mean ± standard deviation of each group.
Tables
Table 1 Results of IgG antibody titers against 5 different parasitic antigens in supernatants of hybridomas by enzyme-linked immunosorbent assay
Table 2 Toxoplasmacidal activity of peritoneal macrophages from normal and activator-inoculated mice
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