JOURNAL OF CLINICAL MICROBIOLOGY, June 1994, P. 1419-1426 0095-1137/94/$04.00+0 Copyright C) 1994, American Society for Microbiology
Vol. 32, No. 6
Enzyme Immunoassay with Enhanced Specificity for Detection of Antibodies to Chlamydia trachomatis J. M. OSSEWAARDE,l* A.
DE
VRIES,1 J. A. R.
VAN DEN
HOEK,2
AND
A. M. vAN LOON'
Laboratory of Virology, National Institute of Public Health and Environmental Protection, Bilthoven,' and Municipal Health Service, Amsterdam,2 The Netherlands Received 16 November 1993/Returned for modification 24 January 1994/Accepted 25 February 1994
Two different methods for preventing the binding of cross-reacting antibodies to the genus-reactive chlamydial lipopolysaccharide (LPS) were used to improve the specificity of an enzyme immunoassay for the determination of antibodies to Chiamydia trachomatis. Coated elementary bodies were treated with either sodium periodate, to oxidize the antigenic sites of the LPS, or Triton X-100, to extract the LPS. By using these new enzyme immunoassays, the standard enzyme immunoassay, and the whole inclusion fluorescence (WIF) assay, antibodies to C. trachomatis were determined in sera from different groups of patients and controls. Paired serum samples from patients with culture-proven urogenital C. trachomatis infections showed similar responses in all three assays. Paired serum samples from patients with Chlamydia psittaci infections showed similar .responses in the WIF assay and the standard enzyme immunoassay, whereas significantly reduced titers were obtained in the enzyme immunoassays with treated antigen, especially in the convalescent-phase serum samples. Serum samples from patients with symptoms suggestive of infection with C. trachomatis, pregnant women, and blood donors were evaluated by all three types of assays. Eighty percent of the significant reductions in immunoglobulin G (IgG), IgA, and IgM titers were observed in sera with WIF assay titers in the lower classes (IgG, 1:s256; IgA, 1:s32; IgM, 1:.16). From these results we conclude that oxidation of the antigen by sodium periodate is a simple and effective method of producing an enzyme immunoassay with enhanced specificity that could be useful for diagnostic purposes and seroepidemiological studies.
Chiamydia trachomatis is considered one of the major causes of sexually transmitted diseases and, as such, is a major public health problem (48). Although knowledge of the impact of C. trachomatis infections in The Netherlands is considered patchy, the problem is generally believed to be of the same magnitude as in many other countries (45). Public health measures to control sexually transmitted disease are based on, among others, epidemiological and clinical studies. Since the symptoms of C. trachomatis infection are not specific, laboratory methods are required for a definitive diagnosis of C. trachomatis infections. These methods include several direct and indirect techniques (2). Direct techniques are the methods of choice for diagnosing most C. trachomatis infections. Isolation in cell culture is considered the "gold standard," although recently introduced assays based on PCR have been shown to be more sensitive (31). Serological methods are recommended only for use in assisting in the diagnosis of complications like salpingitis or perihepatitis or to be used as a tool in (sero)epidemiological studies. The complement fixation test (CFT) was the first available assay for the detection of antibodies to Chlamydia psittaci (3). Five years later, antigen preparation techniques also became available for C. trachomatis (36). CFT is still routinely performed for diagnostic purposes in many laboratories. However, the lack of sensitivity and specificity limits the use of CFT in seroprevalence studies. Two different immunofluorescence assays have been developed to improve on the CFT: (i) the microimmunofluorescence assay (MIF assay [47]) with purified elementary bodies and (ii) the whole inclusion fluorescence assay (WIF assay [39]). Since the fluorescence of the inclusions used in the WIF assay is easier to
read than that of elementary bodies, the WIF assay is preferred by many laboratories for use in routine diagnosis (38, 39, 46). It is, however, considered less specific than the MIF assay (32). To allow handling of larger numbers of samples and to provide more objective reading of the results, several enzyme immunoassays have been developed. Partially purified elementary bodies, purified major outer membrane protein, chlamydial lipopolysaccharide (LPS), or LPS from the Re mutant of Salmonella spp. have all been used as antigens (8, 9, 18, 20, 34, 44). In general, these enzyme immunoassays are considered very sensitive, but their specificities are variable and depend on the antigen source. Genus-specific epitopes are present on the major outer membrane protein, the 60-kDa proteins, and the LPS (24). The highest specificity is probably obtained with purified species-specific antigens (34), and the lowest specificity is probably obtained with the genus-specific LPS as antigen. Removal of the genus-specific epitopes on the LPS by oxidation or detergent extraction probably results in a more specific antigen (17, 29). Ehzyme immunoassays have many advantages over immunofluorescence assays, for example, the objective reading of the results and the possibility for automation. Recently, we developed an enzyme immunoassay in which the genus-specific epitopes on the LPS are removed by either oxidation with sodium periodate or extraction with Triton X-100 (29). Here we
report
our
evaluation of that enzyme immunoassay for
diagnostic and epidemiological purposes by using different groups of patients and controls.
sera from
MATERIALS AND METIIODS
Study groups. Sera were collected from five different groups of subjects: (i) 11 patients with culture-proven urogenital C. trachomatis infection (paired serum samples), (ii) 10 patients with C. psittaci infection (paired sertim samples), (iii) 50
Corresponding author. Mailing address: Laboratory of Virology, RIVM, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Phone: *
+31 30 743942. Fax: +31 30 281168. Electronic mail address:
[email protected].
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patients with symptoms suggestive of infection with C. trachomatis (single serum samples), (iv) 54 healthy pregnant women (single serum samples), and (v) 50 blood donors (single serum samples). The sera from subjects in group i were collected from patients attending an outpatient clinic for sexually transmitted diseases. The sera from subjects in groups ii, iii, and iv were sent to our laboratory for diagnostic or screening purposes. Group iii consisted of patients with symptoms of uncomplicated urogenital infection (n = 6), ascending infection (n = 17), infant pneumonia (n = 10), inguinal lymphadenopathy (n = 6), (reactive) arthritis (n = 8), and nonspecific systemic symptoms (n = 3). The sera from the blood donors (25 males and 25 females) were provided by the blood bank of the Red Cross, Utrecht, The Netherlands. WIF assay. C. trachomatis 434-B, serovar L2, and an avian strain of C. psittaci were inoculated onto 1-day-old DEAEdextran-pretreated monolayers of HeLa 229 cells in six-well microtiter plates. The plates were centrifuged at 4,800 x g for 1 h and 25°C and were incubated overnight at 37°C and 5% CO2. The next day, the cells were carefully trypsinized and 20 ,ul of a suspension containing 50,000 cells was spotted into each well of a microscope slide and incubated for 1 day at 37°C in 5% CO2. The slides were then fixed in methanol and were stored at -70°C until needed. To inactivate human immunodeficiency virus (37), all serum samples were pretreated with 0.5% Nonidet P-40 (NP-40) by mixing nine parts of serum with one part of 5% NP-40 in 0.01 M phosphate-buffered saline (PBS; pH 7.2). Before determining the presence of specific immunoglobulin G (IgG) antibodies, the sera were treated with a brain-liver-egg yolk suspension in order to remove nonspecific staining factors (12). Before testing for specific IgM or IgA antibodies, the sera were additionally treated with sheep anti-human IgG Fc fragment to prevent the interference of rheumatoid factor or a high titer of specific IgG antibodies (12). Fluorescein-labeled sheep anti-human IgG and IgM were obtained from the Department of Immunochemistry of the National Institute of Public Health and Environmental Protection. Fluorescein-labeled rabbit anti-human IgA was purchased from Dako (Glostrup, Denmark). The sera were diluted in Veronal buffer with 0.5% bovine serum albumin (BSA; Organon Teknika, Boxtel, The Netherlands). All sera were tested in twofold dilution series starting at 1:8. The last dilution yielding specific fluorescence was considered the titer. Enzyme immunoassays. Antigen of C. trachomatis serovar L2 and control antigen of noninfected HeLa 229 cells were prepared as described previously (29), with minor modifications. In short, C. trachomatis was inoculated onto 1-day-old DEAE-dextran-pretreated monolayers of HeLa 229 cells in six-well microtiter plates. The plates were centrifuged at 4,800 x g for 1 h and 25°C and were incubated for 3 days at 37°C in 5% CO2. The cells were then scraped from the surface of the wells, and the antigen was partially purified by sonication, differential centrifugation, and centrifugation through a layer of 35% sodium diatrizoate (Sigma, St. Louis, Mo.). The antigen was stored in aliquots of 50 [LI at -70°C (30). Polyvinylchloride microtiter plates (Falcon; Becton Dickinson, Oxnard, Calif.) were coated overnight with antigen or control antigen. The coated antigen was subsequently treated either with 1% sodium periodate in 0.01 M phosphate-buffered physiological saline (pH 6.0) or with 1% Triton X-100-5 mM EDTA in PBS as described previously (29). To inactivate human immunodeficiency virus (1), all serum samples were pretreated by mixing 95 ,ul of serum with 5 RI of 3-propiolactone prediluted 1:20 in PBS and incubating the mixture at 4°C overnight; this was followed by incubation for I h at 37°C to hydrolyze the remaining 3-propiolactone molecules. The sera
were then diluted in PBS with 0.5% Tween 20 (Sigma), 0.5% gelatin (Sigma), and 1% BSA and were tested in twofold dilution series starting at 1:100. Peroxidase-labeled rabbit anti-human IgG and IgA were purchased from Dako, and affinity-purified peroxidase-labeled goat anti-human IgM was purchased from Cappel (Organon Teknika). The last dilution with an absorption above the corresponding cutoff curve was considered the titer. The cutoff curves were constructed for each enzyme immunoassay separately by joining the calculated cutoff values at each dilution. The cutoff values were calculated by adding two times the standard deviation to the mean of the absorbance values of 75 serum samples that were all negative in the immunofluorescence test for specific IgG, IgA, and IgM antibodies to C. trachomatis. IgM-positive samples were tested by a latex agglutination assay (Behring, Marburg, Germany) for the presence of rheumatoid factor. Statistical analysis. The relationships between the titers obtained in the immunofluorescence tests and the enzyme immunoassays were analyzed by calculating the Spearman rank order correlation coefficient of the titers. Before analyzing the frequency distribution of IgG titers obtained in the different groups of sera, the results were reduced to four classes of IgG titers (immunofluorescence,
