Warning: mkdir(): Permission denied in /home/virtual/lib/view_data.php on line 81
Warning: fopen(upload/ip_log/ip_log_2024-09.txt): failed to open stream: No such file or directory in /home/virtual/lib/view_data.php on line 83
Warning: fwrite() expects parameter 1 to be resource, boolean given in /home/virtual/lib/view_data.php on line 84 Partially purified Toxoplasma gondii antigens by immunoaffinity chromatography
Partially purified Toxoplasma gondii antigens by immunoaffinity chromatography
M H Ahn,*1K H Hyun,1J O Kang,2 and D Y Min1
1Department of Parasitology, College of Medicine, Hanyang University, Seoul 133-791, Korea.
Received July 26, 1997; Accepted September 11, 1997.
Abstract
Tachyzoite antigens of Toxoplasma gondii (RH) were partially purified by immunoaffinity chromatography. The cultivated Toxoplasma in vivo (mouse) and in vitro (Hep-2 cell) and peritoneal fluid of T. gondii infected mice were collected for antigen analysis. Tachyzoite antigens collected from infected mouse showed positive bands of 76 kDa, 70 kDa, 64 kDa, 53 kDa, 46 kDa, 44 kDa, 41 kDa, 35 kDa, 25 kDa, 18 kDa, and 13 kDa on immunoblot with anti-Toxoplasma rabbit sera, and those from infected Hep-2 cells revealed reactive bands of 70 kDa, 64 kDa, 53 kDa, 35 kDa 28 kDa, and 13-10 kDa. After applying to an IgG-Sepharose column, two elution peaks, E-1 and E-2 fractions, were obtained from both soluble antigen of T. gondii and the peritoneal fluid of infected mice, respectively. Immunoblots of soluble antigen with immunized rabbit sera revealed positive bands of 97 kDa, 63 kDa, 53 kDa, and 35 kDa from E-1 fraction and 53 kDa and 35 kDa from E-2. In the case of the eluted peaks from mice peritoneal fluid, E-1 showed protein bands of 84 kDa, 76 kDa, 53 kDa, and 29 kDa bands and 53 kDa and 45 kDa from E-2 on immunoblots. Serum IgG antibody titer of mice immunized with T. gondii tachyzoites was increased on 1 week after booster immunization when analysed by ELISA using crude antigen, while it was elevated on 3 weeks after booster immunization by ELISA using purified antigen.
Figures
Fig. 1 SDS-PAGE (A) and immunoblot (B) analyses of T. gondii tachyzoite. M, marker; lane 1, T. gondii tachyzoite from infected mouse peritoneum; lane 2, T. gondii tachyzoite cultured in vitro with Hep-2 cell.
Fig. 2 SDS-PAGE (A) and immunoblot (B) analyses of crude or purified T. gondii antigen. M, marker; lane 1, T. gondii tachyzoite lysate; lane 2, the first elution peak of purified Toxoplasma antigen; lane 3, the second elution peak of purified Toxoplasma antigen.
Fig. 3 SDS-PAGE (A) and immunoblot (B) analyses of Toxoplasma infected mouse peritoneal fluid and purified antigen. M, marker; lane 1, ascitic fluid from infected mouse; lane 2, the first elution peak of purified antigen; lane 3, the second elution peak of purified antigen.
Tables
Table 1 Serum IgG antibody levels of T. gondii immunized mice by ELISA using crude antigen
Table 2 Serum IgG antibody levels of T. gondii immunized mice by ELISA using purified antigen
References
1.
Ahn MH, Son HJ, Leem MH, Min DY. [Antigen analysis of Toxoplasma gondii lysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB)]. Korean J Parasitol 1994;32(4):249–257.
2.
Asai T, Kim TJ, Kobayashi M, Kojima S. Detection of nucleoside triphosphate hydrolase as a circulating antigen in sera of mice infected with Toxoplasma gondii. Infect Immun 1987;55(5):1332–1335.
3.
Bessieres MH, Le Breton S, Seguela JP. Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens. Parasitol Res 1992;78(3):222–228.
4.
Charif H, Darcy F, Torpier G, Cesbron-Delauw MF, Capron A. Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites. Exp Parasitol 1990;71(1):114–124.
5.
Choi WY, Nam HW, Yoo JE. Membrane proteins and their antigenicity of Toxoplasma gondii. Korean J Parasitol 1988;26(3):155–162.
6.
Choi WY, Nam HW, Youn JH, Kim DJ, Kong Y, Kang SY, Cho SY. Korean J Parasitol 1992;30(2):83–90.
7.
Couvreur G, Sadak A, Fortier B, Dubremetz JF. Surface antigens of Toxoplasma gondii. Parasitology 1988;97(Pt 1):1–10.
9.
Im KI, et al. Yonsei Rep Trop Med 1991;22:15–20.
11.
Kasper LH, Bradley MS, Pfefferkorn ER. Identification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies. J Immunol 1984;132(1):443–449.
12.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.
13.
Makioka A, Suzuki Y, Kobayashi A. Recognition of tachyzoite and bradyzoite antigens of Toxoplasma gondii by infected hosts. Infect Immun 1991;59(8):2763–2766.
14.
Ryu JS, Min DY, Ahn MH, Choi HG, Rho SC, Shin YJ, Choi B, Joo HD. Toxoplasma antibody titers by ELISA and indirect latex agglutination test in pregnant women. Korean J Parasitol 1996;34(4):233–238.
15.
Shin DW, Lee YH, Rho TJ. [Immunological properties of the 30 kDa antigen of toxoplasma gondii]. Korean J Parasitol 1997;35(1):55–62.
16.
Sibley LD, Niesman IR, Asai T, Takeuchi T. Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole. Exp Parasitol 1994;79(3):301–311.
17.
Tenter AM, Johnson AM. Recognition of recombinant Toxoplasma gondii antigens by human sera in an ELISA. Parasitol Res 1991;77(3):197–203.
18.
Verhofstede C, Van Gelder P, Rabaey M. The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting. Parasitol Res 1988;74(6):516–520.
19.
Ware PL, Kasper LH. Strain-specific antigens of Toxoplasma gondii. Infect Immun 1987;55(3):778–783.
20.
Wee JL, Ho LC, Yap EH, Singh M. A monoclonal-based IgM capture ELISA for detection of antibodies to 22 and 41 kDa membrane antigens of Toxoplasma gondii. Parasitology 1992;104(Pt 1):25–31.