ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Sato, Noriharu; Hirata, Masato; Hirata, Ryogo; Zerbini, Lia C. M. S.; Silveira, Edilene P. R.; Melo, Carmen Silvia de; Ueda, Marli

doi:10.1590/s0036-46651999000200009

Cited by 13 publications

(7 citation statements)

References 16 publications

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“…Serum samples from blood donors and from patients with STD other than syphilis were all negative by IB-rTp, indicating a highly specific reactivity of these tested recombinant antigens. Similar results were obtained by Western blotting (13). Analysis by the IB technique showed that none of the samples studied reacted with GST, indicating that the reaction to the treponemal portion of the fusion protein is specific.…”

Section: A B Csupporting

confidence: 79%

“…pallidum have been cloned in fusion with GST and expressed in Escherichia coli (12). In the Western blot assay the purified recombinant antigens showed reactivity specifically against the treponemal fraction of the fusion protein, as no serum sample reacted with purified GST (13). In the present study we evaluated the GST-rTp47, GST-rTp17 and GST-rTp15 antigens in an immuno-slot blot (IB) assay for the diagnosis of syphilis.…”

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confidence: 99%

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Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Sato

Suzuki

Ueda

et al. 2004

Braz J Med Biol Res

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Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7%) and a specificity of 94.7% (95% CI, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

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Section: A B Csupporting

confidence: 79%

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confidence: 99%

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Sato

Suzuki

Ueda

et al. 2004

Braz J Med Biol Res

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“…Few data are available on the use of recombinant-antigenbased immunoblotting in the diagnosis of syphilis. Recently, a paper by Sato and coworkers (38) appeared describing the immune reactivity of three glutathione S-transferase fusion recombinant antigens (rTp47, rTp17, and rTp15), whereas Ebel et al (7) reported on the use of a single multiparametric line immunoassay, containing separated recombinant forms of four T. pallidum antigens, TpN47, TmpA, TpN17, and TpN15, for the confirmatory diagnosis of syphilis.…”

Section: Discussionmentioning

confidence: 99%

“…Among these polypeptides, at least five (TpN15, TpN17, TpN37, TmpA, and TpN47) proved to be of diagnostic relevance (4,23,28,31,32). While many different combinations of the above-mentioned immunogens in the recombinant form have been used in enzyme immunoassay methods (9,12,19,48,50), only a few preparations of recombinant T. pallidum polypeptides have been evaluated for the diagnosis of syphilis by WB (7,38). The use of recombinant antigens could avoid the difficulties in purifying specific T. pallidum proteins due to the complex antigenic structure of this spirochete, and it has the potential to increase the specificity of serologic investigations.…”

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confidence: 99%

Western Immunoblotting with FiveTreponema pallidumRecombinant Antigens for Serologic Diagnosis of Syphilis

Sambri¹,

Marangoni²,

Eyer³

et al. 2001

Clin Diagn Lab Immunol

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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.

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“…Recombinant rTp47, the recombinant antigen rTp47 produced in fusing with GST and purified by affinity chromatography was used [25]; native antigen of T. pallidum, soluble antigen of T. pallidum, Nichols strain, prepared by extraction with detergent Zwittergent® with protein concentration of 0.57 mg/mL, kindly provided by Wama Diagnostica.…”

Section: Antigensmentioning

confidence: 99%

Profile of Anti-Tp47 antibodies in patients with positive serology for syphilis analized by western blot

Miranda¹,

Sato²

2008

Braz J Infect Dis

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In Brazil, syphilis is still a great problem of public health. Serological test is essential for syphilis diagnosis and the current trend is the use of recombinant antigen in the treponemal tests, due to its confirmed higher sensibility and specificity. The purpose of the present study was to analyze the profile of anti-Tp47 antibodies in patients with positive serology for syphilis. One hundred positive sera samples were analyzed by Western Blot (WB) technique, using the recombinant antigen (rTp47). Ten of them did not present antibodies against the fraction rTp47, the results were confirmed by WB using native T. pallidum antigen. All ten samples had antibodies against the fractions Tp17 and Tp15 and presented low reactivity in VDRL, negative results or title below than 1:4. Considering that VDRL is used for therapeutic monitoring due to seroreversion of nontreponemal antibodies in response to the treatment, and that some studies reported loss of treponemal antibodies after treatment, we could speculate if these ten samples are cases of serological memory from patients previously treated for syphilis. In addition, although several features state the Tp47 fraction as one of the major antigenic components, based on our results we point out to the importance of including other antigenic proteins such as Tp17 and Tp15 in addition to Tp47 in tests for serological screening of syphilis.

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ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Cited by 13 publications

References 16 publications

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Western Immunoblotting with FiveTreponema pallidumRecombinant Antigens for Serologic Diagnosis of Syphilis

Profile of Anti-Tp47 antibodies in patients with positive serology for syphilis analized by western blot

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