INTRODUCTION

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Among the different antigens used to detect antibodies to Chlamydia trachomatis, large differences in sensitivity and specificity can be observed (3). The cysteine-rich protein outer membrane protein 2 (OMP2) has been found to be a major immunogen in chlamydial infections in an assay using denatured and truncated fusion proteins (9). As OMP2 had not been compared to other Chlamydia antigens, we investigated whether it was more sensitive and specific than four other antigens previously found to have the best diagnostic values in the serodiagnosis of C. trachomatis infection (3). Two of these antigens were used in commercially available enzyme-linked immunosorbent assays (ELISA). One detected antibodies to species-specific epitopes in variable domain IV of the major OMP (MOMP) of C. trachomatis (Labsystems Research Laboratory, Helsinki, Finland). The other was based on an exclusively Chlamydia-specific recombinant fragment of the total lipopolysaccharide (LPS) (3-deoxy-D-manno-2-octulopyranosonic acid) (Medac GmbH, Hamburg, Germany). The two remaining antigens were recombinant proteins: heat shock protein 60 (hsp60) and the polypeptide encoded by open reading frame 3 of the plasmid (pgp3), used also in ELISA (3). They were prepared and tested in exactly the same way as OMP2, except that OMP2 was prepared under denaturing conditions.

As OMP2 is a highly conserved structural protein among all Chlamydia species, anti-C. pneumoniae-specific antibodies were also determined with commercially available ELISA (Labsystems Research Laboratory) to investigate potential cross-reactivity.

The sensitivity was evaluated for samples from patients with well-defined disease (i.e., urethral or endocervical samples from which C. trachomatis DNA was amplified), and the specificity was evaluated for those from healthy blood donors.

	MATERIALS AND METHODS

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Patients. Serum samples were stored at 70°C until they were processed. The study subjects were categorized into one of the following two groups: group 1 subjects (n = 28; median age, 31 years; 25% were female) were patients with acute C. trachomatis urogenital infection as determined by C. trachomatis DNA amplification from urethral or endocervical samples by the Amplicor test (Roche Diagnostic Systems, Branchburg, N.J.); group 2 subjects (n = 56; median age, 45 years; 30% were female) were healthy blood donors.

Recombinant protein preparation. hsp60 and pgp3 antigen preparation was done as previously described (3). OMP2 was prepared in the same way. Template DNA for the PCR was obtained from C. trachomatis serovar D, strain UW-3/Cx, purchased from the American Type Culture Collection (catalogue no. VR-885). A 1,644-bp fragment (GenBank accession no. M23001) was amplified (1). Oligonucleotides used as primers were designed with 5' NcoI (5'-CATGccatggACAAACTCATCAGACGAGCAGTGACG-3') and 3' BamHI (5'-CGggatccATAGATGTGTGTATTCTCTGTATCA-3') restriction endonuclease sites (lowercase letters) and were synthesized by Microsynth (Balgach, Switzerland). The pQE-60 vector (Qiagen, Chatsworth, Calif.) was chosen to append a six-histidine tag to the C termini for large-scale purification via nickel chelate affinity chromatography. In order to change an incorrect base introduced at the cloning step, in vitro site-directed mutagenesis was performed with a QuickChange site-directed mutagenesis kit from Stratagene. For the amplification reaction, 5'- and 3'-end oligonucleotide primers containing the desired mutation were 5'-AGAAATTAACCATGAACAAACTCATCAGACGAGC-3', where the boldface A indicates the desired mutation and 5'-GCTCGTCTGATGAGTTTGTTCATGGTTAATTTCT-3', respectively.

Measurement of immunoglobulin G (IgG) and IgA antibodies against six-His-tagged recombinant proteins by ELISA and commercially available ELISA. Experimental conditions were as previously described (3).

Calculations. Sensitivity, specificity, positive predictive value, negative predictive value, false-positive and false-negative rates, and agreement were calculated as described previously (2).

	RESULTS

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Analysis of purified recombinant proteins. Protein solubility was determined according to the Qiagen protocol. OMP2 was found to be present with insoluble matter and was therefore purified under denaturing conditions.

View larger version (117K): [in this window] [in a new window]   FIG. 1.   The separation of expressed recombinant proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were visualized after Coomassie blue staining. Lanes from left to right: molecular mass standards, OMP2, hsp60, and pgp3.

Diagnostic values of different antibody determinations. When each assay was considered individually, the highest sensitivity was obtained with anti-OMP2 IgG (89%), but this antibody also showed the lowest specificity (57%); the highest specificity (89%) but the lowest sensitivity (57%) was obtained with anti-pgp3 IgG (Table 1).

Comparison of IgG responses to OMP2 with responses to other antigens. To determine what types of response accompanied IgG antibodies to OMP2, we examined the binding of IgG to the C. pneumoniae antigen and to genus-specific (LPS) and highly conserved (hsp60) antigens, as well as to C. trachomatis MOMP and pgp3, for each individual (Fig. 2). This analysis showed that among 49 samples positive for anti-OMP2 IgG, 18 (37%) had no anti-C. trachomatis MOMP or anti-pgp3 antibodies. Three samples (6%) had only anti-OMP2 IgG, three had only anti-C. pneumoniae antibodies (donors 8, 53, and 64), three had only IgG to a genus-specific (LPS) or highly conserved (hsp60) antigen (donors 33 and 39 and patient 34), and nine (18%) had IgG to both C. pneumoniae antigen and LPS or hsp60 antigen, in addition to anti-OMP2 IgG antibodies.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Sera from healthy blood donors or C. trachomatis-infected patients tested for anti-C. pneumoniae IgG (ELISA kit from Labsystems), anti-Chlamydia LPS antibodies (ELISA kit from Medac), and anti-C. trachomatis antibodies (ELISA kit for anti-MOMP antibodies from Labsystems; anti-OMP2, anti-hsp60, and anti-pgp3 antibody assays were developed in our laboratory). For anti-C. pneumoniae IgG, high reactivities (>100) are depicted as black boxes and lower reactivities (>45 to 100) are depicted as gray boxes. For anti-LPS, anti-MOMP, anti-OMP2, anti-hsp60, and anti-pgp3 IgG, high reactivities (result greater than or equal to the cutoff value with an optical density of >1.0) are depicted as black boxes and lower reactivities (result greater than or equal to the cutoff value with an optical density of <1.0) are depicted as gray boxes. For all tests, reactivities below the cutoff values are depicted as white boxes.

	DISCUSSION

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In order to improve the reliability of serological tests in the diagnosis of C. trachomatis infection, we compared the diagnostic values of different antigens used in immunoassays. The highest sensitivities (89 and 82%) were observed for IgG reactivity to OMP2 and LPS, respectively, but these antibodies also showed the lowest specificities (57 and 70%, respectively). These results confirm observations by Mygind et al. of OMP2 (9) and previous observations of LPS (3, 4). These low specificities were attributed to the high prevalence of anti-C. pneumoniae antibodies in the population (6, 8) and the fact that OMP2 (5, 10) and LPS are genus-specific antigens. The fact that 37% of anti-OMP2-positive samples had no anti-C. trachomatis MOMP or pgp3 antibodies supports the hypothesis that another species of Chlamydia (pneumoniae or maybe psittaci) might be the triggering bacterium.

Another antigen commonly used in immunoassays is hsp60. It is another conserved protein, and C. trachomatis hsp60 and C. pneumoniae hsp60 have been shown to be immunologically similar (7, 11). In the present study, anti-hsp60 IgG determination appears less specific (77%) than anti-MOMP IgG (84%) or anti-pgp3 IgG (89%) determination, and again, in the cases of four donors, anti-hsp60 IgG was associated with anti-C. pneumoniae IgG but not with anti-C. trachomatis MOMP or pgp3. These observations suggest that cross-reactivity with C. pneumoniae could be responsible for some anti-hsp60 responses.

A higher specificity was observed here than in a previous study, which also evaluated serum responses in patients with acute C. trachomatis infections and healthy blood donors (3), except with anti-hsp60 IgG. The difference might be explained by the fact that the healthy blood donor population of the present study was older (median age, 45 years, compared to 28 years for the blood donors of the previous study) and had a lower incidence of C. trachomatis infection.

In conclusion, the anti-OMP2 antibody test appears of marginal usefulness due to its nonspecificity. Two specific serological assays using MOMP and pgp3 as antigens are apparently able to discriminate among various Chlamydia species and may serve to avoid both overestimating and underestimating the prevalence of C. trachomatis infection.