Production and Evaluation of Toxoplasma gondii Recombinant Surface Antigen 1 (SAG1) for Serodiagnosis of Acute and ChronicToxoplasma Infection in Human Sera
AbstractBackground: The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis.Methods: This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain) was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyranoside (IPTG). A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA) as gold standard prior to testing them with the recombinant antigen.Results: Tested sera were divided into the following groups:(a) The 74 T. gondii IgG positive (b) 70 T.gondii IgM positive (c) 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5), malaria (N=14), leishmaniasis (N=7),fasciolasis (N=4 ), sterengyloidiasis (N=1 ). Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA) were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively.Conclusion: The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.
Tenter AM, Johnson AM. Recognition of recombinant Toxoplasma gondii antigens by human sera in an ELISA. Parasitol Res. 1991; 77: 197-203.
Robinson SA, Smith JE, Millner PA. Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding. Parasitology. 2004 ; 128: 391-396.
Manger Ian D, Hehl A, Boothroyd J. The Surface of Toxoplasma tachyzoites is dominated by a family of gly-cosylphosphatidylinositol- anchored antigens related to SAG1. Infect Immun. 1998; 66(5): 2237–2244.
Pietkiewicz H, Hiszczynska SE, Kur J, Petersen E, Nielsen H, Stankiewicz M, Andrzejewska I. Usefulness of Toxoplasma gondii recombinant antigens in serodiagnosism of human toxoplasmosis.J Clin Microbiol. 2004; 42: 2504-2508.
Kasper L, Crobb JH, Pfefferkorn ER. Isolation and characterization of a monoclonal anti-p30 antibody resistant mutant of T.gondii. Parasite Immunol. 1987; 9 (4):433-35.
Burg JL, Perelman D, Kasper LH, Ware PL. Molecular analysis of the gene encoding the major surface antigen of T. gondii. J Immunol. 1988; 144(10):3584-3591.
Windeck T, Gross U. Toxoplasma gondiistrain-specific transcript levels of SAG1and their association with virulence. Parasitol Res.1996; 82:715-719.
Harning D, Spenter J, Metsis A,Vuust J. Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in E. coli is recognized by human Toxoplasma –specific immunoglobulin M (IgM) and (IgG) antibodies. Clin Diagn Lab Immunol.1996; 3:355-7.
Kimbita En, Xuan X, Huang X. Serodiagnosis of Toxoplasma gondii infection in cats by ELISA using recombinant SAG1.Vet Parasitol. 2001; 102: 35-44.
Biemans RD, Gregoire MH. The confirmation of purified T. gondii SAG1 antigen, is adequate for serorecognition and cell proliferation. J Biotechnol. 1998; 66:137-146.
Kazemi B, Bandehpour M, Maghen L, Solgi GH. Gene Cloning of 30 kDa Toxoplasma gondii tachyzoites surface antigen (SAG1). Iranian J Parasitol.2007; 2:1–8.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning A laboratory manual. 2nd ed. Cold spring Harbor Laboratory Press. Plainview; 1989. P. 25-35.
Hiszczynska SE, Gabriela O, Lucyna HG. Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii. Vet Parasitol. 2011; 177: 281-289.
Manger A, Hehl A, Parmley S. Expressed Sequence Tag Analysis of the Bradyzoite Stage of Toxoplasma gondii: identification of developmentally regulated genes. Infect Immun. 1998 ; 66: 4 1632-1637.
Arabpour M, Bandehpour M, Niyyati M. Cloning and expression of Toxoplasma gondii tachyzoite P22 protein. African J of Biotechnol.2011; 10(40): 7746-7750.
Hettmann C, Soldati D. Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites. Oxford J Life Sciences Nucleic Acids Research.2002; 27 (22): 4344-4352.
Gamble H, Carolyn D, Dubey J. P. Use of recombinant antigens for detection of Toxoplasma gondii. J Parasitol.2000; 86(3): 459–462. Iranian J Parasitol: Vol. 7, No.3, 2012, pp.1-9 9 Available at: http://ijpa.tums.ac.ir
Johnson AM, Roberts H, Tenter AM. Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional antigen ELISAs. J Med Microbiol.1992; 37: 404-409.
Harning D, Spenter J, Metsis A. Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies. Clin Diagn Lab Immunol. 1996; 3(3): 355–357.
Jalallou N, Bandepour M, Khazan H. Recombinant SAG1 Antigen to detect Toxoplasma gondii Specific Immunoglobulin G in human Sera by ELISA Test. Iranian J Parasitol. 2010; 5(2):1-9.
Velmurugan G, Anup KT, Jammi R. High expression of SAG1 gene of Toxoplasma gondii (Izantnagar isolate) and their application in serodiagnosis of goat toxoplasmosis. Vet Parasitol. 2008; 154: 185-192.
Buffolano W, Beghetto D, Pezzo M, Spadoni A. Use of recombinant antigens for early postnatal diagnosis of congenital toxoplasmosis. J Clin Microbiol. 2005; 43:5916-24.
Aubert D, Maine GT, Villena I. Recombinant antigens to detect antigens to detect Toxoplasma gondii specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J Clin Microbiol. 2000; 38: 1144-50.
Pietkiewicz H, Hiszczynska SE, Kur J, Petersen E, Nielsen H. Usefulness of Toxoplasma gondii recombinant antigens in serodiagnosis of human toxoplasmosis. J Clin Microbial. 2004; 42:2504-2508.
Nigro M, Gutierrez A, Hoffer AM, Clemente M, Kaufer F, Carral L. Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis. Diagn Microbiol Infect Dis. 2003; 47:609-613.
Chen X, Li H, Rong L. High level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli. Pro Exp Purif. 2001; 23:33-37.
Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol.1998; 9:497-501.
Sawicka EH, Dazbrowska AB, Dazbrowski S, Pietkieicz H. High yield expression and single step purification of Toxoplasma gondiiSAG1,GRA1, and GRA7 antigens in Escherichia coli. Prot Exp Purif. 2003; 27: 150-157.