Western Immunoblotting with FiveTreponema pallidumRecombinant Antigens for Serologic Diagnosis of Syphilis

Abstract

Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.

Laboratory diagnosis of syphilis with automated immunoassays

“…Several different immunoassays set up with whole-cell lysate (12)(13)(14)(15) or recombinant T. pallidum antigens (16)(17)(18)(19)(20)(21)(22) are described in the literature.…”

“…A WB test was considered positive when at least three bands out of TpN47, TmpA, TpN17, and TpN15 were clearly recognized. A test was considered negative when no bands or fewer than the three above-mentioned T. pallidum antigens were recognized (19,27). The reader was blinded of the identity and of the clinical status of the serum samples when the WB strips were examined.…”

“…Nontreponemal tests include the Venereal Disease Research Laboratory and the Rapid Plasma Reagin (RPR) card tests. Treponemal tests include the serum fluorescent treponemal antibody absorption test, the T. pallidum hemagglutination test (TPHA) (Radim, Pomezia, Italy), the enzyme-linked immunoassay (ELISA), and the Western Blot (WB) assay; both ELISA and WB tests can be based on either whole-cell lysate (12)(13)(14)(15) or recombinant (16)(17)(18)(19)(20) treponemal antigens. More recently, chemiluminescent immunoassays set up with recombinant antigens have been evaluated (21,22).…”

Evaluation of the BioPlex 2200 Syphilis System as a First-Line Method of Reverse-Sequence Screening for Syphilis Diagnosis

“…An IgG WB test was considered positive when at least three of the four bands corresponding to TpN47, TmpA, TpN17, and TpN15 were clearly recognized. IgM WB tests were considered positive when at least two of the four diagnostic bands were visible, including at least one with low molecular mass (15,31,32).…”

“…However, since positivity with respect to treponemal tests lasts a lifetime, they cannot be useful in followup. Treponemal tests include the serum fluorescent treponemal antibody absorption test (FTA-ABS), T. pallidum hemagglutination (TPHA) test, enzyme immunoassay (EIA), and Western blot (WB) assay; both EIA and WB tests can be based on either wholecell lysate (7)(8)(9)(10)(11) or recombinant (12)(13)(14)(15)(16) treponemal antigens. Recently, chemiluminescent immunoassays set up with recombinant antigens have been evaluated (17,18).…”