title: Characterization of Toxoplasma gondii SAG2 expressed in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.
abstract: A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Huang
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sugimoto
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Nagasawa
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mikami
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000276:immunology*; D002415:Cats; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000097:blood / Q000145:classification; D051379:Mice; D017924:Nucleopolyhedroviruses / Q000235:genetics; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012698:Serologic Tests; D018411:Spodoptera; D014122:Toxoplasma / Q000276:immunology*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C067547:surface antigen P22, Toxoplasma