[Antigen analysis of Toxoplasma gondii lysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB)].
Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplasma lysate (RH strain), frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain). Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T. gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72 hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1:512 of 10 days after primary immunization, 1:2,048 of 10 days after secondary immunization, 1:1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplasma major antigens corresponding to MW of 24 kDa, 27 kDa, 30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T. gondii, the IgG antibody band of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory-secretory materials and lysate was important major antigen of T. gondii (RH).
Department of Parasitology, College of Medicine, Hanyang University Seoul, Korea.
M H
MH
Ahn
H J
HJ
Son
M H
MH
Leem
D Y
DY
Min
D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis*; D015151:Immunoblotting / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D014122:Toxoplasma / Q000276:immunology* / Q000378:metabolism
D000953:Antigens, Protozoan