Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfatepolyacrylamide gel electrophoresis results from different laboratoriesAs the first step in a cooperative effort to standardize the identification of the polypeptides of Treponerna pallidum subsp. pallidurn, the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 ofthe major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T.pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T.pal1idum proteins. Abbreviations: CIE, crossed immunoelectrophoresis; 2DE, two-dimensional gel electrophoresis; IRS, infected rabbit serum; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; RIP, radioimmunoprecipitation: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; T, Tween 20 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0173-0835/87/0202-0077 %02.50/0 S. J. Norris et al. Electrophoresis 1987,8, 77-92 Figure 16. Reactivity of rabbit antiserum directed against cloned antigenTmpC (1301, seeFig. 6)with the 2DEpatternof T.paNidum.Theprocedure wasas described in Fig. 15. (A) Reaction with the TmpC-specific antiserum. (B) Antigenic profile following IRS counterstaining. The antiserum reacted specifically with a single band on the SDS-PAGE pattern (far left) and a single spot at the acid end of the 2DE pattern.Figure 17. Localization of cloned antigen TpD (1301, see Fig. 6) in the 2DE profile of T.pallidum, utilizing a specific rabbit antiserum prepared by affinity chromatography. (A) Reactivity of TpD-specific antiserum. Due to prolonged incubation with the color reagent, background reactivities of the antiserum to TmpC and the major 61 polypeptide were also detectable in this case. (B) Antigenic profile following IRS counterstaining. The positions of TpD and TmpC in the 2D and single dimension SDS-PAGE profiles are indicated by the arrows.

In syphilitic patients with or without CNS involvement the correlation of Treponema-specific IgG titre per milligram total IgG in CSF and serum (ITPA index) is a dependable source of information on the synthesis of treponemal IgG antibodies in the CNS. This index also provides a more reliable definition of asymptomatic neurosyphilis. Further, a discrimination between Treponema-specific and Treponema-non-specific IgG synthesis in the CNS is possible. Of 261 patients with clinical symptoms of neurosyphilis, 82% had a local production of treponemal IgG antibodies as shown by an elevated ITPA index. In patients with neurosyphilis and intrathecal synthesis of Treponema-specific IgG antibodies, 94% had oligoclonal IgG in the CSF. Comparison of different CSF protein alteration groups in untreated and treated neurosyphilitic patients showed that early diagnosis (and early treatment) led to improvement of the impairment of the blood-CSF barrier and reduction of the immune reaction in the CNS. However, synthesis of treponemal IgG antibodies in the CNS could persist as a 'scar syndrome' even after adequately cured infection.

Protein antigens of Treponema pallidum precipitated by immunoglobulin M (IgM) and IgG antibodies of sera from patients with untreated primary and secondary syphilis as well as treated secondary syphilis were characterized on a molecular basis. T. pallidum was labeled internally with [35S]methionine and solubilized in 0.1% sodium dodecyl sulfate-1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels followed by autoradiography revealed 32 distinct proteins with molecular weights between 13,500 and 200,000. Twenty-three proteins of T. pallidum with molecular weights between 15,500 and 115,000 were identified as antigens by double antibody radioimmunoprecipitation with IgM and IgG antibodies of sera from syphilitic patients. The molecular analysis of the IgM and IgG immune response to T. pallidum in human syphilis is in accord with earlier immunological observations. Finally, utilizing syphilitic human sera, we characterized 15 protein antigens of T. pallidum that are common to Treponema phagedenis by partial absorption of IgM and IgG antibodies with an ultrasonicate of T. phagedenis.

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