Serological tests for syphilis.

Many of the nontreponemal or serological tests for syphilis involve the detection of serum antibodies (reagin) which are not directed against a component of the causative organism itself but may arise as a result of pathological changes brought about by proliferation of Treponema pallidum in host tissue. Test antigens for the detection of reagin are lipoidal, the main active component being the phospholipid, cardiolipin. It is known that reagin may be produced not only as a result of infection with T. pallidwn but also by a wide variety of other organisms and pathological conditions (3, 8). False-positive reactors have been defined as patients with no history or clinical evidence of syphilis or other treponematoses whose sera react with nontreponemal but not with treponemal antigens (5). In studies on the specificity of antibodies to bacterial cell membrane glycerol teichoic acids (11), we have used glycerol-phosphoryl-glycerolphosphoryl-glycerol (G3P2), derived from cardiolipin by the removal of the fatty acid residues, as a specific inhibitor of antibodies directed at the common polyglycerophosphate "backbone" of glycerol teichoic acids. It was therefore of interest to determine whether teichoic acid antibodies would react with cardiolipin itself in the serological tests used for the detection of reagin. Membrane glycerol teichoic acids appear to be ubiquitous in their occurrence in gram-positive bacteria (1), and this group of organisms is associated with many of the pathological conditions that may give rise to false-positive biological reactions for syphilis (8). W Antisera to teichoic acids may show serological specificities directed towards sugar substituents or to the polyolphosphate backbones of the polymer (11). The rabbit antisera used in this study were those previously prepared against membrane glycerol teichoic acids (lipoteichoic acid) or cell wall ribitol teichoic acids from various lactobacilli; the specificities of these sera are summarized and documented in Table 1. Human syphilitic sera were purchased (470-1) from the Commonwealth Serum Laboratories, Australia, or were a gift (C-007) from M. F. Garner (Institute of Clinical Pathology and Medical Research, Lidcombe, N.S.W. Australia). The Kolmer (KCF) and Reiter protein (RPCF) complement fixation tests used were standard procedures (9), all reagents being those commercially available (Commonwealth Serum Laboratories). Fluorescent treponemal antibody absorption (FTA-ABS) and treponema immobilization (TPI) tests were kindly performed by M. F. Garner. In the case of FTA-ABS tests on rabbit sera, a goat anti-rabbit gamma globulin fluorescein conjugate was used in place of the usual anti-human conjugate. For complement fixation tests with glycerol teichoic acid (GTA-CF), the lipoteichoic acid from Lactobacillus fermenti NCTC 6991 (10) at a concentration of 10 ,ug/ml in saline was used in place of diluted Kolmer cardiolipin antigen, all other reagents and their proportions being identical to the standard Kolmer procedure. For studies on the inhibition of complement fixation, sera were diluted to 4X their KCF titer and incubated for 1 hr at 37 C with either 1 ,ug of cardiolipin or 1 ,umole of G3P2 per ml of diluted serum. Absorbed sera were centrifuged, and the supernatant fluids were used immediately in KCF and GTA-CF tests. Diluted sera were also absorbed with glycerol teichoic acid by successive treatments with sheep red blood cells coated with L. fermenti membrane lipoteichoic acid (4) until no further agglutination occurred. Absorbed sera were clarified by centrifugation as before.