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

Abstract

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.

Performance Evaluation of the Elecsys Syphilis Assay for the Detection of Total Antibodies to Treponema pallidum

“…Testing in the United States traditionally has been performed using a different algorithm, initially using nontreponemal tests for screening, but now many centers are adopting treponemal tests as the primary screening assay (23,24). In addition to their value in diagnosing syphilis at all stages, the use of treponemal tests has become more widespread due to the availability of T. pallidum recombinant polypeptides, which overcome the previous problem of antigenic variability of the proteins obtained from spirochetes grown in rabbit testes (25,47).…”

Sífilis: diagnóstico, tratamento e controle

“…[43][44][45] The Western-blot method identifies antibodies against IgM and IgG immunodeterminants with molecular masses (15kDa, 17kDa, 44kDa and 47kDa). 41,46 So far, these tests have shown a high level of sensitivity and specificity in all phases of syphilis, but are being used primarily in research projects. 40 In the early 1990´s, two PCR techniques were described and adopted, especially for detecting treponemic antigens in primary syphilis, with high levels of sensitivity and specificity.…”

Diagnóstico sorológico da sífilis

“…Currently, recombinant DNA techniques are used to obtain treponema antigen. 7 They are still under investigation, but will be the future commercial option, since its sensibility is 95.1% and specificity is 94.7%.…”