Purification of TR-b, a Reiter treponeme protein antigen precipitating with antibodies in human syphilitic sera

Abstract

Reiter treponeme antigen, cross-reacting with an antigen in Treponema pallidum (Nichols pathogenic strain). Sera from patients with secondary syphilis contain precipitating antibodies against TR-b. The isolation of TR-b from a bacterial sonic extract is described here. It involved five fractionation steps: anion-exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22 Ultrogel), and affinity chromatography on phenyl-Sepharose CL 4B, iminodiacetic acid-Sepharose CL 4B, and lysine-Sepharose 4B, respectively. The purified TR-b was enriched 199 times compared with the starting material, and the recovery was 12%. TR-b was shown to be a protein; it did not bind to a series of lectins, and by gel filtration and polyacrylamide gel electrophoresis, the molecular weight was determined to be 610,000 to 630,000. It was found by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be composed of identical 70,000-dalton subunits. The isolated TR-b was immunologically pure when tested in crossed immunoelectrophoresis against polyspecific anti-Reiter immunoglobulin. The purified TR-b antigen was used for the production of a monospecific rabbit antiserum, giving strong fluorescence with both the Reiter treponeme and T. pallidum in an indirect immunofluorescence test.

Molecular characterization of common treponemal antigens

“…Utilizing human syphilitic sera, we have characterized at least eight polypeptide antigens of 82,000, 73,000, 47,000, 45,000, 40,000, 35,500, 33,000, and 30,000 daltons that are common to both T. pallidum and Reiter. Similarly, Petersen et al (26) have identified six cross-reactive antigens utilizing a crossed immunoelectrophoresis technique, and Lukehart et al (15), using pooled rabbit anti-T. pallidum antiserum, have recently defined five common antigens of 69,000, 48,000, 40,000, 38,000, and 34,000 daltons.…”

“…Most of the studies have identified antigens common to both T. pallidum Nichols and the host-indigenous, nonpathogenic Treponema phagedenis biotype Reiter (referred to here as Reiter). Complement fixation (3-5, 7, 8, 12, 18, 20), gel diffusion (12,18,20), fluorescentantibody (6,13,17,27), crossed immunoelectrophoresis (20,(23)(24)(25)(26) and Western blot (15) techniques have revealed the presence of shared protein, polysaccharide, and/or protein-lipopolysaccharide complex antigens. Furthermore, absorption studies have shown that the presence of treponemicidal activity in normal human serum against T. pallidum and Reiter is stimulated by common T. pallidum and Reiter immunogens (P. A. Hanif and J. N. Miller, submitted for publication).…”

Molecular and immunochemical analysis ofTreponema pallidum

“…It has been hoped that such work would yield knowledge of the antigens of T. pallidum, primarily because the Reiter treponeme was known to cross-react antigenically with T. pallidum, and the organism has indeed been used as a source of both diagnostic antigen for the Reiter protein complement fixation test and of 'sorbent' for enhancing the specificity of immunofluorescence tests for T. pallidurn antibody. The Reiter treponeme has been very thoroughly investigated by Strandberg-Petersen and co-workers [32][33][34][35][36][37][38][39] who have extensively analysed the antigens of the Reiter treponeme by 2-dimensional immunoelectrophoresis using both homologous antiserum and rabbit and human syphilis antibody. While this technique does not necessarily separate antigens as individual polypeptides, and is thus not strictly analogous to the immunoblotting technique used in most of the work described so far, it has the advantage that native antigenic species can be analysed and compared.…”

“…While this technique does not necessarily separate antigens as individual polypeptides, and is thus not strictly analogous to the immunoblotting technique used in most of the work described so far, it has the advantage that native antigenic species can be analysed and compared. These authors have identified a number of immunoprecipitates formed by antibodies common to T. pallidum and the Reiter treponeme [32,33], and from these common immunoprecipitates they have characterized axial filaments [34], a heat-stable antigen, possibly a polysaccharide [35], protein antigens with subunits of 66 [36], 70 [37] and 48 kDa [38], and an RNA antigen [391. The protein of 48 kDa [38] relative to ovalbumin at 43 kDa may be analogous to P3.…”