Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus

Am J Trop Med Hyg. 2007 Mar;76(3):553-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Bacterial / blood*
  • Antigens, Bacterial / immunology*
  • Blotting, Western
  • Enzyme-Linked Immunosorbent Assay
  • Fluorescent Antibody Technique, Indirect
  • Gold Colloid
  • Humans
  • Immunoglobulin G / blood
  • Immunoglobulin M / blood
  • Orientia tsutsugamushi / immunology*
  • Recombinant Proteins / immunology
  • Scrub Typhus / diagnosis*
  • Sensitivity and Specificity

Substances

  • Antibodies, Bacterial
  • Antigens, Bacterial
  • Gold Colloid
  • Immunoglobulin G
  • Immunoglobulin M
  • Recombinant Proteins