Trachomatis Antigen Research Articles

Enzyme immunoassay and enzyme-linked fluoroimmunoassay for detection ofChlamydia trachomatis antigen

An enzyme immunoassay (EIA) and an enzyme-linked fluoroimmunoassay (ELFIA) utilizing monoclonal antibody to major outer membrane protein of Chlamydia trachomatis L2 were developed for rapid detection of chlamydial antigen in clinical samples. The EIA and ELFIA could detect levels of purified chlamydial outer membrane protein as low as 1.0 and 0.2 ng/ml respectively. However, when EIA and ELFIA were compared to chlamydial isolation using 160 patient samples, the sensitivity rate was 68% and 85% respectively. The sensitivity of the antigen detection method might be increased by simply using less diluted samples than in the present study. Chlamydial antigen was also demonstrated by EIA and ELFIA in 15% and 23% of culture-negative samples. The reason for these false-positive findings remains undetermined.

Tear and serum antibody response to Chlamydia trachomatis antigens during acute chlamydial conjunctivitis in monkeys as determined by immunoblotting.

In this study, we examined the temporal antibody response by immunoblotting analysis in tears and sera of three cynomolgus monkeys (Macaca fasicularis) with primary acute Chlamydia trachomatis serovar B conjunctivitis. The objective was to identify chlamydial antigens stimulating antibody during the host responses in the course of this self-limiting infection with the rationale that they may be protective antigens. The major outer membrane protein (MOMP), lipopolysaccharide (LPS), and polypeptides of 60 and 68 kilodaltons (kDa) were the predominant antigens recognized by immunoglobulin A (IgA) in monkey tears. Tear IgA antibody specific for the MOMP was first detected 14 days postinfection, whereas tear IgA reactive with LPS or the 68- and 60-kDa polypeptides was first detectable on day 21. Tear IgA antibodies specific for each of these antigens persisted in tears through day 56, 4 weeks after both peak clinical disease and recovery of the organism from the conjunctivae. In contrast, tear IgG antibodies peaked at approximately 28 days postinfection, the time of maximal inflammatory response. The IgG response in monkey sera was similar to that observed for tear antibodies, in that the MOMP, 60-, and 68-kDa polypeptides were the primary immunogens. The exception was that IgG antibody against these antigens was detected 1 week later than that observed for tear IgA antibodies. Of three monkeys that responded with tear IgA antibody against LPS, one did not have detectable serum IgG LPS antibody. The specificity of the tear IgA antibody response of monkeys was determined by immunoblotting nine other C. trachomatis serovars in addition to the homologous B serovar. The tear IgA response to the MOMP was predominantly B complex subspecies-specific (serovars B, Ba, D, and E), whereas the response to chlamydial LPS was found to be species-specific. The significance of these observations in relation to previous vaccine studies in nonhuman primates is discussed.

Specific serum IgA antibodies in the diagnosis of Chlamydia trachomatis infection

Serologic diagnosis in pneumonia or pelvic inflammatory diseases by C. trachomatis was confirmed by demonstration of rising titer of specific serum IgM antibodies. The possiblity that the specific serum IgA antibodies might be a marker of active chlamydial infection had recently been suggested by Sarov. We discussed the usefulness of the serum specific IgA antibodies as a marker of active chlamydial infections.In monkeys inoculated with L2 strain of C. trachomatis, it was recognized that the specific serum IgA antibodies rised in primary infection and reinfection but the IgM antibodies did not appeared in secondary infection.Sera obtained from 17 men with C. trachomatis isolation-positive non-gonococcal urethritis (NGU) and 19 men with C. trachomatis isolation-negative NGU were studied for the presence of C. trachomatis specific IgG and IgA antibodies. The IgA positive-coincidence ratio was 94.1%(16/17) in isolation-positive NGU patients and the IgA negative-coincidence ratio was 78.9%(15/19) in the isolation-negative NGU patients.The specific srum IgA antibodies had been detected in three of four C. trachomatis antigen detected pneumonia infants and these four mothers had high level specific serum IgA and IgG antibodies.It is suggested from our studies that specific serum IgA antibodies may be used as a marker of active C. trachomatis infection.

Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women.

Chlamydia trachomatis antigens were detected in populations with the following infection prevalences: 26.5% (36 of 136) of men and 27.7% (48 of 173) of women attending a sexually transmitted disease clinic, 16.3% (53 of 324) of women attending a Planned Parenthood clinic, and 3.4% (4 of 117) of an obstetrics and gynecologic practice. Compared with cell culture of the combined female cervical specimens (15.8% prevalence), the respective sensitivities of Chlamydiazyme (Abbott Laboratories, North Chicago, Illinois) and Microtrak (Syva, Palo Alto, California) were 98.3% and 87.9%, specificities were 97.5% and 98.4%, positive predictive values were 87.7% and 92.7%, and negative predictive values were 99.7% and 97.5%. Both assays were 70.0% sensitive with male urethral specimens, and the other parameters of performance ranged between 84.0% and 97.2%. The antigen detection assays, compared with culture, performed equally well in subjects without or with clinical signs.

Solid-phase enzyme immunoassay for detection of Chlamydia trachomatis.

The prototype of a solid-phase enzyme immunoassay for the detection of Chlamydia trachomatis antigen was tested in 403 men and 135 women attending a venereal disease clinic. Culture on HeLa 229 cells was used as reference method. In men the overall sensitivity and specificity of the enzyme immunoassay was 70% and 98.5% respectively. In the 158 men with non-gonococcal urethritis the sensitivity and specificity was 73% and 96.5% respectively, in the 31 men with post-gonococcal urethritis 82% and 100%, in the 48 men with gonococcal urethritis 67% and 100%, in the 125 men with discharge but normal urinary sediment 54% and 99.1% and in the 41 men with no signs or symptoms 50% and 100%. In women the sensitivity and specificity of the assay was 70% and 92% respectively. On account of the low degree of sensitivity in both men and women and the low degree of specificity in women the prototype of the enzyme immunoassay is not yet considered suitable for clinical use. An improved model is being developed.

Enzyme immunoassay for the detection of Chlamydia trachomatis antigen in urethral and endocervical swabs.

An enzyme immunoassay technique based on the direct detection of Chlamydia trachomatis antigen in urethral or cervical swabs was used for the rapid diagnosis of chlamydial genital infection. Urethral and cervical samples from 140 patients were tested in parallel by enzyme immunoassay and cell culture using iodine staining. The direct test had a sensitivity of 92.5% and specificity of 97.2% when compared with the cell culture system. The enzyme immunoassay technique provides a rapid and simple method for diagnosing chlamydial genital infection and may be performed on a large number of samples in laboratories which do not have tissue culture facilities or a trained microscopist.

Characterization of Chlamydia trachomatis serotypes by human T-lymphocyte clones.

T cells primed to Chlamydia trachomatis serotypes A, F, and K were cloned by limiting dilution. All T-lymphocyte clones obtained reacted only with C. trachomatis antigens. The proliferative capacity of 89 clones was studied with autologous non-T cells as antigen-presenting cells and the chlamydia serotypes A, B, D, F, K, and LGV-2 as antigens. Most of the clones reacted to several of the chlamydia strains, indicating common antigenic determinants. Other T-cell clones reacted with only a few serotypes. On the basis of the proliferation of the T-cell clones to the chlamydia strains and to interleukin-2, different reactivity patterns were obtained, which possibly can be used to differentiate among the chlamydia strains.

Cellular immunity to the mouse pneumonitis agent.

During infection with the mouse pneumonitis biovar of Chlamydia trachomatis, heterozygous (nu/+) mice with relatively intact T cell function develop both delayed hypersensitivity to C trachomatis antigen and antigen-specific lymphocyte transformation, whereas athymic nude (nu/nu) mice do not. Nu/nu mice are protected against death from mouse pneumonitis by transfer of immune T cells from nu/+ mice, which are more resistant to C trachomatis. While this enables athymic mice to make antibody to C trachomatis (which does not occur without reconstitution), resistance correlates best with development of antigen-specific lymphocyte transformation in the recipient animals. During infection nu/+ mice develop activated alveolar macrophages (by both morphological and functional criteria) while nu/nu mice do not. Nu/+ mice that have been preinfected with Histoplasma capsulatum to activate cellular immunity become more resistant to C trachomatis than do nu/+ controls. Cell-mediated immunity to C trachomatis pneumonia is T cell dependent and is important in host defense.

Depression of the lymphocyte transformation response to microbial antigens and to phytohemagglutinin during pregnancy.

Lymphocyte transformation (LT) responses to Chlamydia trachomatis, to four other microbial antigens, and to phytohemagglutinin (PHA) were studied in 201 women during pregnancy and/or 3-18 wk postpartum. The LT responses to all stimulants tested were significantly depressed during pregnancy when compared with postpartum LT responses. This difference occurred whether LT assays were performed in autologous or pooled heterologous plasma collected from nonpregnant donors. Among women studied in the third trimester and again postpartum, the autologous LT stimulation index (LTSI) rose from 1.7 to 3.4 (P less than 0.001) with C. trachomatis elementary body antigen, from 3.7 to 7.9 (P less than 0.001) with Candida albicans cell wall extract, from 4.5 to 7.8 (P = 0.008) with streptokinase-streptodornase, from 1.7 to 3.0 (P = 0.007) with fluid tetanus toxoid, from 1.7 to 2.8 (P = 0.046) with mumps virus skin test antigen, from 35.5 to 87.0 (P less than 0.001) with PHA (2 micrograms/ml), and from 107.2 to 181.9 (P = 0.007) with PHA (10 micrograms/ml). LT responses to C. trachomatis were compared in 52 pregnant women and 58 nonpregnant women; all the women had C. trachomatis isolated at the time of LT assay. Using either plasma supplement, the mean LTSI with C. trachomatis antigen was significantly higher in nonpregnant women than in pregnant women, regardless of trimester (P less than 0.001). Among 12 women who were serially tested and remained culture positive for C. trachomatis throughout pregnancy and the postpartum period, the mean autologous LTSI rose from 1.9 in the third trimester to 7.8 postpartum (P = 0.0004). These data are the first to show that the immune response to an ongoing bacterial infection is depressed during pregnancy and to definitively document the depressed LT responses during human pregnancy.

Class-II HLA restriction of antigen-specific human T-lymphocyte clones. Evidence of restriction elements on both DR and MT molecules.

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. The class-II HLA restriction of T-lymphocyte clones (TLC) was studied by using allogeneic antigen-presenting cells (APC) carrying foreign class-II HLA antigens. Most of the TLC were restricted by one or the other of the D/DR determinants of the T-cell donor; that is, they did not respond when antigen was presented by APC expressing foreign D/DR determinants. Furthermore, heterogeneity of the DR4-expressing molecule could be demonstrated by T-cell clones from one person; APC from family members expressing DR4 gave high proliferative responses, whereas no proliferation was observed with most APC from unrelated persons expressing DR4. This heterogeneity of DR4 was confirmed by mixed lymphocyte culture (MLC) experiments, indicating a close relationship between restriction epitopes and those that activate allogeneic T cells. Other clones seemed to be restricted by other class-II HLA determinants, most probably MT determinants of the T-cell donor. The restriction specificities were confirmed by subcloning experiments.

Antigen-specific proliferative human T-lymphocyte clones with specificity for Chlamydia trachomatis.

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. A cloning efficiency of 20-25% was obtained. T-lymphocyte clones (TLC) with high proliferative responses were selected for further studies. Kinetic studies showed a peak response between 60 and 84 h after antigen stimulation. The TLC were OKT4+. They were specific for the chlamydial antigen and did not respond to other antigens when non-T cells were used as antigen-presenting cells (APC). Antigen-specific proliferation of the TLC required that the responding TLC and the APC shared class-II HLA determinants--that is, HLA-D/DR molecules. The true clonal nature of the TLC was confirmed by subcloning experiments.

Expression in E. coli of Chlamydia trachomatis antigen recognized during human infection.

Chlamydia trachomatis, a prokaryotic obligate intracellular parasite of eukaryotic cells, has long been recognized as the agent of trachoma, the major infectious cause of human blindness1. In the past decade, clinical investigations have shown that the frequency and severity of chlamydial genital infection parallels that of gonorrhoea, and that C. trachomatis is a major cause of non-gonococcal urethritis, epidymitis, proctitis, cervicitis and salpingitis, as well as conjunctivitis and pneumonia in neonates2. Despite the new awareness of its role in human disease, there is a need for improved methods of identification and control of C. trachomatis infections. Progress in these areas awaits a more detailed understanding of the pathogenesis of chlamydial infection, including definition of key antigenic structures recognized in human disease. We have studied the immune response to chlamydial polypeptides during human infection and have found that most infected patients have antibody to polypeptides of molecular weight (Mr) 67,000 (67K), 60K, 40K, 19K and 16K, regardless of infecting immunotype3. We report here the cloning and expression in Escherichia coli of the 19K C. trachomatis polypeptide, an immunogen in human chlamydial infection.

Simplified microimmunofluorescence test with trachoma-lymphogranuloma venereum (Chlamydia trachomatis) antigens for use as a screening test for antibody.

A simplified microimmunofluorescence test with trachoma-lymphogranuloma venereum (Chlamydia trachomatis) antigens has been devised as a screening test for antibody in human sera. The test differs from our standard procedure by amalgamating 15 different immunotypes into nine antigen pools, by using only three serum dilutions, and by dropping use of duplicate slides. The screening test could be performed on at least six times as many sera as the standard test for a given unit of effort. It was shown to have a sensitivity equal to the standard test and an ability to determine specific immunotype on the basis of antibody pattern (72%) only slightly reduced from the standard test (84%). The screening test was carried out on 876 patients from different population groups in the Seattle area. The tests showed that antibody was present frequently in persons attending venereal disease clinics (60%) and commonly (25%) in a group of adults without venereal disease. Nine percent of children under 15 years of age tested had antibody; the significance of this finding is unknown.