Fresh and aged adherent T pallidum were used to study the accessibility of their outer membrane to antibodies by means of an indirect immunofluorescent technique. The integrity of the outer membrane was demonstrated by the non-reactivity with a monoclonal antibody directed against the axial filaments. Using the sera from patients with sero-positive primary and secondary syphilis no binding of IgG and IgM antibodies was observed. However, IgG and IgM antibody fractions isolated from the sera of patients with secondary syphilis, gave with the fresh fibroblast-adhering treponemes a mean of 14.5% IgG- and of 43.2% IgM positive treponemes. These means were 32.1% and 87.3% respectively for aged treponemes. Lower percentages were observed when fibronectin adhering treponemes were used. This demonstrates the inability of the outer membrane to bind antibodies in a majority of the fresh treponemes. This is partly lost on in vitro aging. Absence of IgG- and IgM fluorescence was also observed when sequential incubations with the antibody fractions and control sera were used. This was accompanied by the deposition of the third complement factor (C3) around the treponemes. Incubations of IgG- or IgM pre-coated adherent treponemes with heat-inactivated control sera or a C3 deficient serum did not result in the deposition of C3, and partially restored the detection of human antibodies. The most likely explanation for the absence of fluorescence is that antibodies become buried in an extra-cellular layer of serum proteins. The deposition of C3 from control sera alone most probably points to the classical pathway of complement activation and suggests that antibodies of rabbit origin constitute a part of the extracellular layer of treponemes.
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