Serovar determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies.

A panel of 15 monoclonal antibodies was prepared that could distinguish among the 15 serovars of Chlamydia trachomatis. Twelve of these antibodies were specific for a single serovar (A, B, C, D, E, F, G, H, I, K, L1, and L2) and three were specific for two serovars (B/Ba, C/J, and C/L3). Ten of the serovar-specific and two of the bispecific antibodies were shown by immunoblotting to recognize epitopes on the major outer membrane protein. These data provide evidence that such epitopes are closely correlated with and may be partly responsible for the antigenic variations detected by microimmunofluorescence that distinguish the currently recognized serovars. When used in a radioimmunoassay, these antibodies correctly identified the serovar of 17 strains that had been serotyped by the microimmunofluorescence test. In addition, we found that the chlamydial antigen derived from 1.0 cm2 of an infected HeLa cell monolayer was sufficient to allow serotyping with these antibodies. Thus, these monoclonal antibodies may provide a rapid and reliable alternative to mouse immunization and microimmunofluorescence for serotyping of clinical isolates.     

Culture-independent diagnosis of Chlamydia trachomatis using monoclonal antibodies

To simplify the diagnosis of chlamydial genital infection, we used a fluorescein-conjugated monoclonal antibody in immunofluorescence tests on smears prepared from urethral or cervical secretions obtained directly from patients. This direct test, requiring less than 30 minutes to perform, was based on the detection of extracellular chlamydial elementary bodies. A comparison of the direct test with cultures stained with iodine on specimens from 926 patients demonstrated a sensitivity of 93 per cent and a specificity of 96 per cent. The direct test provides a rapid, simple, and sensitive method for the diagnosis of chlamydial infection, which can be performed in laboratories that do not have tissue-culture capability.     

Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies.

Seventeen monoclonal antibodies (MAs) were tested. Five of seven serovar-specific MAs from five different serovars and two of five subspecies-specific MAs showed neutralizing activity as well as serovar specificity. No species- or genus-specific MAs showed neutralizing activity. No neutralization occurred without complement. Results indicate neutralization was serovar specific and complement dependent.     

Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis.

We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle.     

Evaluation of serotyping using monoclonal antibodies and PCR-RFLP for Chlamydia trachomatis serotype identification

We compared genotyping by restriction fragment length polymorphism (RFLP) analysis of the amplified omp1 gene with serotyping by dot enzyme-linked immunosorbent assay (dot-ELISA) to determine the suitability of RFLP analysis for epidemiologic study. Fifteen prototypes of Chlamydia trachomatis and 30 clinical isolates were used in this study. To serotype with dot-ELISA, chlamydia antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with 11 mAbs that distinguish the 15 known serovars of C. trachomatis. For RFLP analysis, the amplified chlamydia omp1 gene was digested with AluI to differentiate serovars A to K and L1 to L3. Serovars of C, H, I, J, and L3 were further typed by RFLP analysis after digestion with HinfI, and a combination of EcoRI and DdeI. PCR-based RFLP could identify serotype of 28 among 30 clinical isolates tested. The remaining two untypical isolates were probably due to double infections or mechanical transferring error. Serotyping of C. trachomatis isolates shows that serovars E, D, F, and H are the most prevalent types found in urogenital samples in Korea. In this study, we show that RFLP analysis of amplified omp1 gene may be useful in genotyping C. trachomatis isolates.     

Comparison of two commercially available isolation systems for Chlamydia trachomatis

In a comparison of two commercially available chlamydial isolation systems in which cycloheximide-treated McCoy cell monolayers are used, the system from Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash., was found to be superior to that from M. A. Bioproducts, Walkersville, Md. for the detection of Chlamydia trachomatis by iodine staining. Of 288 clinical specimens run in parallel, 47 (16.3%) were positive, with 16 of 47 positive results detected in the Bartels system only and 1 of 47 positive results detected in the M. A. Bioproducts system only (P less than 0.001). A comparison of the number of inclusion-forming units per cover slip from clinical specimens and passaged isolates also showed that the Bartels cell system demonstrated higher inclusion counts than the M. A. Bioproducts system. In routine clinical use, overall isolation rates were higher (P less than 0.001) and contamination rates were lower (P less than 0.001) with the Bartels system as compared with results obtained in a previous time period in which the M. A. Bioproducts system was used.     

Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 23 isolates on passage 1 and 13 isolates on passage 2 were detected by Giemsa staining. Immunofluorescence staining was significantly more sensitive than Giemsa staining for detecting chlamydial inclusions, particularly from specimens containing low titers of Chlamydia.     

Unclassified serovars of Chlamydia trachomatis isolated from Japanese infants

Objective: To analyze antigenic and genetic variations of Chlamydia trachomatis among the serovars obtained from Japanese infants.
Methods: The polymerase chain reaction (PCR) was used to amplify a large part of the major outer-membrane protein gene, and restriction fragment length polymorphism (RFLP) was used to identify the serovars of C. trachomatis from nasopharyngeal and conjunctival swabs from Japanese infants and neonates.
Results: The typing of 10 nasopharyngeal isolates gave the following results: seven E, one H, and two unclassified serovars. The typing of seven conjunctival isolates gave the following results: five D, one F, and one unclassified serovar. Reactive patterns of these unclassified strains, determined by PCR-RFLP, to monoclonal antibodies were different from those of 15 reference serovars.
Conclusions: Characterization of unclassified variants will allow more detailed epidemiologic studies of perinatal C. trachomatis infections in Japan.     

Ultrastructural study of Chlamydia trachomatis surface antigens by immunogold staining with monoclonal antibodies.

Surface antigens of Chlamydia trachomatis were studied by immunogold staining with monoclonal antibodies and by electron microscopy. The serovar- and subspecies-specific epitopes were the most surface accessible. The species- and genus-specific epitopes were the least surface exposed. Similar serological specificity as that in the microimmunofluorescence test was demonstrated by immunogold staining.     

Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01). Improved sensitivity was most evident in specimens with low numbers of inclusions. Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture.     

Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay.

The technical complexity of determining the serovar of Chlamydia trachomatis strains has limited the use of serotyping in clinical and epidemiologic studies. We developed a simple method for rapidly serotyping isolates of C. trachomatis by using monoclonal antibodies in a dot-enzyme-linked immunosorbent assay (ELISA) system. Isolates were passaged three to six times in shell vial cultures to greater than 50% monolayer infection, and chlamydial elementary bodies were isolated by sonication and microcentrifugation. Chlamydial antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with C. trachomatis-specific monoclonal antibodies. Bound antibody was detected visually by a color reaction by using peroxidase-conjugated anti-mouse immunoglobulins. This method can be routinely applied to 60 or more specimens concurrently. We compared dot-ELISA serotyping with monoclonal antibody microimmunofluorescence serotyping of 124 clinical C. trachomatis isolates and found that dot-ELISA has sensitivity and serotyping accuracy comparable to that of monoclonal antibody microimmunofluorescence.     

Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.     

Simplified serological test for antibodies to Chlamydia trachomatis.

Three-hundred sixty sera from unselected patients attending two London venereal disease clinics were examined by a microimmunofluorescence test. Eleven egg-grown serotypes of Chlamydia trachomatis and the so-called "fast" strain SA2(f) were used as antigens. Of the 360 sera tested, 119 (33%) reacted to a titer of 1:16 or above with at least one antigen. Of these positive sera, over 50% cross-reacted with all 12 serotypes, and 95.5% reacted with SA2(f) in addition to other antigenic types. It is suggested that SA2(f) could be used as a single antigen for preliminary screening of a large number of sera for the presence or absence of chlamydial antibody.     

Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.

Monoclonal antibodies exhibiting Chlamydia trachomatis serovar specificity (serovar A, B-Ba, or C) and serogroup specificity (B, intermediate, or C serogroup) were produced and characterized. These antibodies reacted with the major outer membrane protein, recognized epitopes located at the chlamydial cell surface, and passively neutralized chlamydial toxicity for mice. The antibodies should be useful reagents for defining the molecular structure of these protective epitopes, a necessary step toward the development of a subunit or recombinant C. trachomatis vaccine.     

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.     

Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR.

Successful amplification of omp1 DNA by PCR is crucial in the genotyping of Chlamydia trachomatis when directly performed with clinical samples (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. McLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). Several primers flanking the four variable domains of the omp1 gene were selected and tested for sensitivity in several nested PCRs with serial dilutions of serovar G. The optimal sensitivity obtained was 0.1 to 0.01 inclusion-forming units, similar to that obtained in the C. trachomatis plasmid PCR. With this approach, any C. trachomatis PCR-positive sample can be typed.     

Optimization of a rapid test by using fluorescein-conjugated monoclonal antibodies for detection of Chlamydia trachomatis in clinical specimens.

A mixture of two fluorescein isothiocyanate-conjugated monoclonal antibodies (MAbs) was used to optimize a direct specimen test (Chlamydia Direct Specimen Test IF; Clonatec, Paris, France) for detection of chlamydial elementary bodies in clinical specimens. One MAb reacted with a subspecies-specific epitope of the major outer membrane protein (molecular weight 43,000) of Chlamydia trachomatis, whereas the other reacted with the periodate-sensitive genus-specific antigen (molecular weight 11,000) of Chlamydia spp. Nonfat dry milk was the most efficient additive at suppressing the fluorescent background and was included in the antibody preparation. Fc-dependent binding of fluorescein-conjugated MAbs to protein A-containing Staphylococcus aureus was inhibited by addition of purified rabbit immunoglobulin. The Chlamydia Direct Specimen Test IF was compared with tissue culture isolation by using 309 genital specimens. The sensitivity and specificity were 77.4 and 98%, respectively.     

Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies.

A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous.