Sandwich immunoassay for the detection of lipoteichoic acid.

A sandwich immunoassay has been developed for the detection of lipoteichoic acid (LTA), a major cell wall constituent of gram-positive bacteria, from whole blood and ISOLATOR supernate. Monoclonal antibodies were produced to purified LTA from Streptococcus mutans, BHT and were further characterized for crossreactivity with gram-positive and negative bacteria and for reactivity to substituted and unsubstituted LTA. Eight monoclonal antibodies were identified that reacted exclusively with gram-positive bacteria. Those antibodies able to capture 3H-LTA were chosen to develop a sandwich immunoassay. The assay has a sensitivity of 0.2 ng LTA/mL in PBS, 0.5 ng/mL in whole blood and 2.0 ng/mL in whole blood that has been processed through the ISOLATOR. Further development of this assay may lead to the rapid detection of LTA from body fluids.

Immunoglobulin isotypes of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis.

Studies of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis were undertaken in attempts to identify the predominant antibody isotype produced and to delineate clinically significant antigens. The total antibody content of serum samples from 23 patients was determined by an enzyme-linked immunosorbent assay (ELISA) that employed anti-human immunoglobulin and isotype-specific antisera. The immunochemical reactivity of these antibodies was examined by Western blot analysis. The anti-T. vaginalis titer of all but two of these serum samples was greater than 200 (range, greater than 200 to 12,800). By using an ELISA titer of at least 200 as a criterion, 21 of the serum samples contained antibodies of the immunoglobulin G (IgG) isotype, 17 contained IgM antibodies, and 6 contained IgA antibodies directed to the protozoan. Western blot analyses of these serum samples revealed approximately 29 antigenic trichomonad polypeptides, with apparent molecular sizes ranging from 14 to greater than 100 kilodaltons and with individual serum samples possessing different patterns of reactivity. These results add to the current understanding of the serological and secretory immune responses to T. vaginalis, as well as define potential antigens for use in immunodiagnostics.

Rapid assay for immunological detection of Trichomonas vaginalis.

Trichomoniasis is a common sexually transmitted disease with an estimated incidence of 4 million to 8 million cases a year in the United States. The most commonly used method of diagnosis is a direct microscopic observation (wet mount) of vaginal secretions and, although both rapid and inexpensive, the sensitivity of this technique is generally 50 to 70%. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Trichomonas vaginalis which is both rapid and sensitive (detection limit of approximately 100 trichomonads per ml). This assay, which employs affinity-purified rabbit anti-T. vaginalis antibodies in a "sandwich" configuration, is simple to perform and is neither interfered with nor appears to cross-react with other microorganisms which are common inhabitants of the urogenital tract. One hundred and seventy-seven consecutive unselected patients attending a clinic for sexually transmitted diseases were evaluated for trichomoniasis by a broth culture technique monitored for up to 7 days (and considered here to be the standard for positivity), the conventional wet mount method, a solid culture procedure, and the ELISA. Of these, 84 were positive by culture; 33 were positive by the wet mount; and despite the fact that the vaginal specimens were diluted 20-fold during the culture procedures prior to testing in the ELISA, 65 were positive by ELISA. In addition to exhibiting a sensitivity of 77%, the specificity of the ELISA was 100%. These results demonstrate that the ELISA is a significant improvement over the wet mount method for the diagnosis of trichomoniasis.

Kinetics of antibody response to polypeptides of pathogenic and nonpathogenic treponemes in experimental syphilis.

Antigenic cross-reactivity between Treponema pallidum subspecies pallidum (TP) and nonpathogenic Treponema phagedenis biotype Reiter (TR), Treponema refringens strain Noguchi (TN), and Treponema vincentii (TV) was examined by the Western immunoblotting technique in pooled sera from five rabbits infected intratesticularly with T. pallidum. Sera were obtained before infection and on days 6, 12, 20, 30, 60, and 120 after infection. The pooled preinfection sera reacted with nine polypeptides of TV, nine of TS, and five of TR. The pooled sera did not show any clear-cut reactions with TP, but some individual rabbit sera did demonstrate visible reaction with three to five polypeptides of TP. Twelve days after infection, multiple serum antibodies reacting with polypeptides of all treponemes were detected. The number of antibodies reacting with polypeptides and the intensity of reaction increased with the duration of infection; for the nonpathogenic treponemes (TV, TS, TR) 21-26 polypeptides were identified on day 30, and by day 60 a total of 21 were detected for TP. By day 120 the reaction had become less pronounced, and fewer reactive polypeptides were seen. The extent of the cross-reactivity with the three nonpathogenic treponemes reflects the complex structure of T. pallidum, which should be viewed as a mosaic of more cross-reacting than strain- or species-specific antigens.

Antigenic contents of Treponema pallidum preparations.

In investigations of syphilis various Treponema pallidum antigens are used to study the immune responses of naturally or experimentally infected hosts. In the past these antigen preparations have rarely been examined for their antigenic contents and activity. In the present study, supernatant, sediment, and solubilised preparations of T pallidum Nichols strain (20 X 10(9) organisms/ml) and T phagedenis biotype Reiter were examined by modified counterimmunoelectrophoresis and immunoblotting for their antigenic contents. No T pallidum antigen was seen in the supernatant fraction, which contained cross reacting (Reiter) antigens and rabbit serum proteins. The remaining T pallidum preparations contained T pallidum antigens, cross reacting treponemal (Reiter) antigens, and rabbit serum proteins. These findings suggest that T pallidum preparations should be examined qualitatively and quantitatively before they are used for monitoring immune responses and interpreting data. Technology for these examinations is available.

Extensive cross reactivity between Treponema pallidum and cultivable treponemes demonstrated by sequential immunoadsorption.

Extensive cross reactivity between Treponema pallidum and three nonpathogenic species of treponemes was demonstrated by the Western blot technique. Rabbit antiserum produced by adjuvant immunization with solubilized T. pallidum antigens reacted with 34 T. pallidum antigens and with approximately 30 antigens each of T. phagedenis biotype Reiter, T. noguchii and T. vincentii. Adsorption of the antiserum with T. phagedenis Reiter removed only about half of the cross-reacting antibodies. Sequential adsorption with all three nonpathogenic treponemes removed antibodies to all but three polypeptides of 36,000, 34,000 and 27,000 daltons.

Antigenic evidence for host origin of exudative fluids in lesions of Treponema pallidum-infected rabbits.

Mucoid fluid accumulating within syphilitic lesions has been considered to be of Treponema pallidum origin. To test this assumption, we examined testicular exudative fluids from T. pallidum-infected rabbits for the presence of T. pallidum antigens by various sensitive immunochemical methods, including Western blot analysis. Antigenic analysis of these fluids revealed host components but not treponemal antigens. Prolonged immunization of rabbits, guinea pigs, and a goat with this material in complete Freund adjuvant elicited low titers (fluorescent-treponemal-antibody test titer, less than or equal to 10) of antitreponemal antibodies in the rabbits and guinea pigs but not in the goat. The data suggest that these mucoid fluids are of host origin. The presence of mucopolysaccharides in these fluids may be related to the infective process. The possible mechanism by which mucopolysaccharides protect T. pallidum from immune mechanisms and its potential relationship to the pathogenesis of the disease are discussed.