title: Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus.
abstract: Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni2+ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.
authors.affiliation: Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China.
authors.forename: Min
authors.initials: M
authors.lastname: Cao
authors.forename: Hengbin
authors.initials: H
authors.lastname: Guo
authors.forename: Tang
authors.initials: T
authors.lastname: Tang
authors.forename: Changjun
authors.initials: C
authors.lastname: Wang
authors.forename: Xianfu
authors.initials: X
authors.lastname: Li
authors.forename: Xiuzhen
authors.initials: X
authors.lastname: Pan
authors.forename: Zhu
authors.initials: Z
authors.lastname: Jin
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Tang
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D017979:Gold Colloid; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D012285:Orientia tsutsugamushi / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012612:Scrub Typhus / Q000175:diagnosis*; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D017979:Gold Colloid; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins