Separation of Treponema pallidum from Tissue Debris Through Continuous-Particle Electrophoresis

Schmale, John D.; Kellogg, Douglas S.; Miller, Curtis E.; Schammel, Patricia; Thayer, James D.

doi:10.1128/aem.19.2.287-289.1970

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A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture.

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mentioning

confidence: 99%

“…Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture.…”

mentioning

confidence: 99%

Molecular characterization of common treponemal antigens

Hanff¹,

Miller²,

Lovett³

1983

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A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture. Thus, in an attempt to obtain information about treponemal antigenic interrelationships, investigators have concentrated their efforts toward the examination of cross-reactive antigens obtained from the nonpathogens. 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). However, with the exception of axial filament proteins (12, 21), the common treponemal antigens have not been well characterized in terms of their cellular location or molecular weight. As a prelude to isolation and purification of these particular cell components, we have characterized the common polypeptide antigens of T. pallidum and Reiter with normal and syphilitic human sera by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by electrophoretic transfer of the t Present address:

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“…Chandler and Clark (2) filtered testicular extracts through membranes of graded porosity, but this failed to separate treponemes from tissue components. Schmale et al (9) recovered much cleaner preparations by continuous particle curtain electrophoresis. However, this procedure damaged the organisms in that it converted one-third to one-half of the spirochetes in the cleaner fractions to spherical forms (i.e., ballooning of the outer envelope) and diminished the immunofluorescent staining of the rest, suggesting that they had lost some cellular constituent.…”

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confidence: 99%

Isolation and Purification of Treponema pallidum from Syphilitic Lesions in Rabbits

Hardy

¹

,

Nell

²

1975

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Treponema pallidum were extracted from testicular syphilomas of corticosteroid-treated rabbits and purified by differential centrifugation. The steroid therapy allowed a longer holding time for infected rabbits, which produced greater treponeme yields, averaging 1.58 × 10 10 treponemes per rabbit. The treatment, which also diminished cellular infiltration and increased the extracellular mucoid material in lesions, produced much cleaner suspensions than preparations from nontreated animals. Most of the treponemes in the purified suspensions were still motile, and none carried demonstrable host immunoglobulin. The preparations were free of recognizable host tissue debris and they contained, on the average, 1.9 × 10 −7 μg of protein per treponeme.

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“…Although Hunter et al (7) standardized methodology for preparing FTA-absorbed (ABS) antigen that was free of rabbit globulin, sensitive techniques revealed that these T. pallidum preparations were still contaminated with rabbit testicular material (5). The development of the continuous particle electrophoresis method for separation of T. pallidum from testicular material (16) resulted in the availability of a purified antigen for use in the FTA-ABS test. This continuous particle electrophoresis antigen is no longer available, and its preparation was technically difficult and expensive.…”

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confidence: 99%