Antigen analysis of <i>Toxoplasma gondii</i> lysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot(EITB)

, Ahn, M H; , Son, H J; , Leem, M H; , Min, D Y

doi:1994.32.4.249

Korean J Parasito > 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

References


1.	Achbarou A, Mercereau-Puijalon O, Sadak A, Fortier B, Leriche MA, Camus D, Dubremetz JF. Differential targeting of dense granule proteins in the parasitophorous vacuole of Toxoplasma gondii. Parasitology 1991;103(Pt 3):321–329.

2.	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.

3.	Bonhomme A, Thirion C, Boulanger F, Charton F, Burlet H, Pinon JM, Alix AJ. Toxoplasma gondii-structure variations of the antigen P30. Parasitology 1994;108(Pt 3):281–287.

4.	Brindley PJ, Gazzinelli RT, Denkers EY, Davis SW, Dubey JP, Belfort R Jr, Martins MC, Silveira C, Jamra L, Waters AP, et al. Differentiation of Toxoplasma gondii from closely related coccidia by riboprint analysis and a surface antigen gene polymerase chain reaction. Am J Trop Med Hyg 1993;48(3):447–456.

5.	Choi WY, Nam HW, Yoo JE. Membrane proteins and their antigenicity of Toxoplasma gondii. Korean J Parasitol 1988;26(3):155–162.

6.	Darcy F, Maes P, Gras-Masse H, Auriault C, Bossus M, Deslee D, Godard I, Cesbron MF, Tartar A, Capron A. Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen. J Immunol 1992;149(11):3636–3641.

7.	Duquesne V, Auriault C, Gras-Masse H, Boutillon C, Darcy F, Cesbron-Delauw MF, Tartar A, Capron A. Identification of T cell epitopes within a 23-kD antigen (P24) of Toxoplasma gondii. Clin Exp Immunol 1991;84(3):527–534.

8.	Fletcher S. Indirect Fluorescent Antibody Technique in the Serology of Toxoplasma Gondii. J Clin Pathol 1965;18:193–199.

9.	Hafid J, Raberin H, Pozzetto B, Akono ZY, Sung RT, Jana M, Gaudin O. Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting. Int J Parasitol 1992;22(8):1083–1088.

*10.*	Ise Y, Iida T, Sato K, Suzuki T, Shimada K, Nishioka K. Detection of circulating antigens in sera of rabbits infected with Toxoplasma gondii. Infect Immun 1985;48(1):269–272.

*11.*	Kasper LH, Currie KM, Bradley MS. An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii. J Immunol 1985;134(5):3426–3431.

*12.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*13.*	Lambin P. Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal Biochem 1978;85(1):114–125.

*14.*	Lappin MR, Bush DJ, Reduker DW. Feline serum antibody responses to Toxoplasma gondii and characterization of target antigens. J Parasitol 1994;80(1):73–80.

*15.*	Linder E, Thors C, Edberg F, Haglund S, von Bonsdorff CH. Generation of antibodies against Toxoplasma gondii antigen associated with dense granules and the parasitophorous vacuole of the host cell. Parasitol Res 1992;78(2):175–178.

*16.*	Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.

*17.*	Makioka A, Kobayashi A. Toxoplasmacidal activity of macrophages activated by recombinant major surface antigen (P30) of Toxoplasma gondii. Infect Immun 1991;59(8):2851–2852.

*18.*	Parmley SF, Gross U, Sucharczuk A, Windeck T, Sgarlato GD, Remington JS. Two alleles of the gene encoding surface antigen P22 in 25 strains of Toxoplasma gondii. J Parasitol 1994;80(2):293–301.

*19.*	Sharma SD, Mullenax J, Araujo FG, Erlich HA, Remington JS. Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J Immunol 1983;131(2):977–983.

*20.*	Sharma SD, Araujo FG, Remington JS. Toxoplasma antigen isolated by affinity chromatography with monoclonal antibody protects mice against lethal infection with Toxoplasma gondii. J Immunol 1984;133(6):2818–2820.

*21.*	Sibley LD, Sharma SD. Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice. Infect Immun 1987;55(9):2137–2141.

*22.*	Takahashi J, et al. Jpn J Parasitol 1985;34(2):87–92.

*23.*	Tenter AM, Johnson AM. Recognition of recombinant Toxoplasma gondii antigens by human sera in an ELISA. Parasitol Res 1991;77(3):197–203.

*24.*	Tomavo S, Martinage A, Dubremetz JF. Phosphorylation of Toxoplasma gondii major surface antigens. Parasitol Res 1992;78(7):541–544.

*25.*	Tomavo S, Couvreur G, Leriche MA, Sadak A, Achbarou A, Fortier B, Dubremetz JF. Immunolocalization and characterization of the low molecular weight antigen (4-5 kDa) of Toxoplasma gondii that elicits an early IgM response upon primary infection. Parasitology 1994;108(Pt 2):139–145.

*26.*	Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.

*27.*	Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.

*28.*	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.

*29.*	Ware PL, Kasper LH. Strain-specific antigens of Toxoplasma gondii. Infect Immun 1987;55(3):778–783.

*30.*	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.