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Korean J Parasitol > Volume 32(4):1994 > Article

Original Article
Korean J Parasitol. 1994 Dec;32(4):249-257. English.
Published online Dec 20, 1994.  http://dx.doi.org/10.3347/kjp.1994.32.4.249
Copyright © 1994 by The Korean Society for Parasitology
Antigen analysis of Toxoplasma gondii lysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot(EITB)
M H Ahn,*H J Son,M H Leem and D Y Min
Department of Parasitology, College of Medicine, Hanyang University, Seoul 133-791, Korea.
Received October 21, 1994; Accepted November 11, 1994.

Abstract

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).

Figures


Fig. 1
Coomasie blue stained SDS-PAGE pattern of T. gondii (RH) lysate. lane 1: RH tachyzoite lysate; lane 2: standard marker.


Fig. 2
Reaction of IgG antibody to T. gondii (RH) lysate antigen by EITB (lane 1-3).

lane 1: normal rabbit serum; lane 2, 3: T. gondii anti-serum of rabbit; lane 4: T. gondii lysate, amidoblack staining; lane 5: standard marker.



Fig. 3
Reaction of IgG and IgM antibodies to T. gondii lysate antigen by EITB.

(A and B) IgG and IgM antibodies to T. gondii (RH) lysate Ag. lane 1: normal rabbit serum; lane 2. 3, 4: T. gondii (RH) immunized rabbit serum; lane 5: T. vaginalis antigen. (C) IgG antibody to T. gondii (RH) lysate Ag. lane 1, 4: normal mouse serum; land 2, 3: serum infected with Fukaya and Beverly strains; lane 5: P. westermani antigen.



Fig. 4
Reaction of IgG antibody to excretory-secretory antigen of T. gondii by EITB (lane 1-4).

lane 1: normal mouse peritoneal fluid; lane 2: culture supernatant of T. gondii (RH) with CHL cell; lane 3: T. gondii (RH) infected mouse peritoneal fluid; lane 4: T. gondii (RH) lysate Ag; lane 5: T. gondii lysate Ag. amidoblack staining; lane 6: standard marker.


Tables


Table 1
Recognition of T. gondii (RH) lysate and excretory-secretory antigens from culture supernatant or infected mouse peritoneal fluid with rabbit or mouse antisera by EITB

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