Southeast Asian J Trop Med Public Health

A double antibody sandwich enzyme immunoassay (EIA) for chlamydial antigen detection was developed using a monoclonal antibody against lipopolysaccharide (LPS) of Chlamydia trachomatis as a coating antibody. Polyclonal rabbit antiserum against partially purified antigen from elementary body (EB) antibody and horse-radish peroxidase conjugated goat anti-rabbit antibody were used as the primary and secondary antibody respectively. The developed EIA could detect protein of partially purified EB at the lowest concentration of 250 ng/ml. The assay was evaluated against the cell culture (CC), DNA hybridization assay (PACE2 system: Gen-Probe,San Diego, CA, USA) and a commercial enzyme immunoassay (kEIA)(Bioquest, NSW,Australia). The sensitivity, specificity, positive and negative predictive values of the developed EIA (dEIA) were 87, 96.2, 80, 97.7 for the specimens from females and 90.9, 90.7, 71.4, 97.5 for the specimens from males repectively. Cross reaction was not found with Escherichia coli, Acinetobacter anitratus, β-Streptococcus group A, Enterobacter spp, Enterococcus, Lactobacillus spp, Neisseria spp, but it was found with Candida albicans and herpes simplex virus type 1. The developed EIA can be applied successfully for both genders, particularly males. The cost per test is less than those for CC, kEIA and PACE2. CHLAMYDIAL ANTIGEN DETECTION BY EIA Vol 31 No. 1 March 2000 97 the collection kits for PACE2 and kEIA. Three urethral swabs from male patients were taken and manipulated in the same way. Evaluation of specimens for chlamydial infection by cell culture, PACE2 assay and EIA Three methods were used for evaluation of chlamydial infection. For cell culture, cycloheximide-treated McCoy cell was used for Chlamydia trachomatis culture as described previously (Chomvarin et al, 1997). PACE2 assay using the chemiluminescent probe assay utilized an acridinium ester labeled single stranded DNA probe(s) complementary to rRNA of Chlamydia trachomatis. It was performed according to the manufacturer’s instruction. One positive and three negative references were included in each running (Chomvarin et al, 1997). The kEIA was performed using the HRP chlamydia EIA kit, which used a monoclonal antibody directed against a genus specific lipopolysaccharide (LPS). The method was performed as described previously (Chomvarin et al, 1997). A sample was considered true positive for chlamydial infection when it had at least one of the following criteria : 1) a positive standard culture with primary inoculation, 2) positive in both non culture tests (PACE2 and EIA). The sensitivity, specificity, positive and negative predictive values of each assay would be calculated based on the above criteria. Propagation of C. trachomatis C. trachomatis positive specimens were used for the propagation of C. trachomatis, using the same procedure for isolation (Chomvarin et al, 1997). After 48 hours culture, cells were removed, disrupted with glass beads and centrifuged at 500g for 5 minutes. The supernatant was collected and inoculated into two vials of confluent McCoy cells. One vial was used for staining with immunofluorescent reagent to evaluate the infection while another would be used for stock culture if at least 5.5x10 IFU/ml or 5-6 inclusions/ 400x power field was shown. Equal volume of stock media (4SP:0.4M sucrose and 0.02M phosphate) was added and the vial was stored at -70oC. Preparation and purification of chlamydial antigen The procedure was modified from Mahony et al (1983). Briefly, three ml of chlamydial stock solution was plated to cycloheximide-treated McCoy monolayers in 25 cm flasks and incubated at 37oC for 2 hours. The inoculate was then removed and replaced with maintenance medium [(85ml RPMI1640, 5 ml fetal calf serum, 2 ml vancomycin (5,000 μg/ml), 1 ml gentamicin (500 μg/ml), 40 μl fungizone (2,500 μg/ml), 5 ml glucose (0.11 μg/ ml), 0.2 ml cycloheximide (100 μg/ml)]. The culture was incubated further at 37oC in 5% CO 2 for 72 hours. The infected cells were harvested by scraping and disrupted with 5 mm glass beads. The cell debris was removed by centrifugation at 500g for 10 minutes. The supernatant was centrifuged at 30,000g for 20 minutes to collect chlamydial antigen, which was then pooled and resuspended in 3 ml PBS. The antigen was then partially purified by centrifugation at 20,000g through a column of 2 ml of 30% (v/v) urograffin-76 for 40 minutes. The pellet was collected, resuspended and assayed for protein concentration using Lowry’s method (Lowry et al, 1951). Preparation of immune serum to EB antigen Twenty, forty, sixty and eighty micrograms of partially purified EB antigen in incomplete Freund’s adjuvant was injected intravenously into a New Zealand white rabbit at three day interval respectively. A final dose of 200 μg of antigen in incomplete Freund’s adjuvant was injected intramuscularly on day 7 after the fourth injection. Two weeks later, the rabbit was bled and the serum was stored at -20oC until used. Detection of antibodies to C. trachomatis Antigens including partially purified EB, commercial EB (Syva company, San Jose, USA), chlamydial LPS (Bioquest, NSW, Australia), whole cell lysate of E. coli ATCC 25992, and A. anitratus at a concentration of 280 μg/ml were used to test the rabbit immune serum by dot immunoassay technique. Briefly, 20 μl of the antigen solution was blotted onto a 0.45 μm nylon blotting membrane (Costar, USA) under an adjustable vacuum. The membrane was air dried, washed for 3 times with 1x Tris-NaCl Tween (TNT) for ten minutes each, followed by blocking with 5% skim milk in 20mM Tris buffer saline (TBS), pH 7.5 at 4oC for overnight. The membrane was washed again for 3 times and incubated with 1:100 immune rabbit serum in 5% skim milk in TBS at 37oC for 2 hours. The membrane was then washed and incubated in HRP goat anti-rabbit IgG (Promega, USA) diluted to 1:1,000 for 2 hours. After 3 washes, the membrane was then incubated in 0.06 g% DAB substrate (3, 3 ́diaminobenzidine tetrahydrochloride, Sigma) at room temperature in the dark for 15 minutes. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Vol 31 No. 1 March 2000 98 The reaction was terminated by washing with tap water. The positive result was indicated by brown color. Dot immunoassay was also performed using mouse mAb against a commercial chlamydial LPS (Bioquest, NSW, Australia) diluted to 1:150, followed by HRP antimouse IgG (Dako, Denmark) as the comparison to rabbit immune serum. Development of an EIA assay Standardization of the assay: The optimal condition for the EIA was established by checkerboard titration. A range of 2-16 μg/ml of 100 μl mAb to chlamydial LPS antigen in coating buffer (carbonate-bicarbonate buffer) was used to coat EIA plates (Nunc, Denmark) at room temperature for overnight. The plate was further washed 3 times with PBS-Tween and blocked with EIA blocking solution (5% skim milk and 4% BSA in coating buffer) for 1 hour. The 100 μl of pooled positive and negative control sera were added to each well and incubated for 2 hours. After 3 washes, 100 μl of rabbit immune serum of various dilution (1:100 -1:800) in EIA diluting solution (1% skim milk and 4%BSA in PBS-Tween) was added and incubated at 37oC for 2 hours. The plate was washed and 100 μl of 1:1,000 to 1:6,000 HRP conjugated goat anti rabbit IgG was added, incubated for another 1 hour, followed by 6 washings. The 100 μl of OPD substrate solution was added to the wells and incubated for 30 minutes. The reaction was terminated by adding 50 μl of 2M H 2 SO 4 . The optical density was read by a MicroEIA Autoreader(Dynatech) at 490 nm. Analytical sensitivity and specificity of the developed EIA: Using the decided optimal conditions, the dEIA was used to detect chlamydial partially purified EB at the concentration of 0.0025 to 1 μg/ml. Various antigens were also tested, including E. coli, A. anitratus, Enterobacter spp, Enterococcus, β-Streptococcus group A, Lactobacillus spp, Candida albicans, Neisseria spp. These antigens were tested at the concentration equivalent to 1.5x10 organisms/ml. Herpes simplex virus (HSV) type 1 was cultured by using vero cells for 48 hours and the cell debris was removed by centrifugation. The supernatant was used as the antigen for testing as above. Evaluation of dEIA for clinical application Clinical application of the dEIA was carried out by testing against clinical specimens which were already evaluated by kEIA, cell culture and PACE2. Sensitivity, specificity and predictive values were calculated using a standard formula (Ilstrup, 1996). True positive result, which used in the formula, was obtained according to the criteria mentioned previously. Agreement between the HRP chlamydial EIA and the dEIA was assessed by using Kappa statistics (Landis and Koch, 1977). Within-run precision was determined by testing the pooled positive samples twenty times on the same plate. To determine the between-run precision, the same samples were tested two times on three plates running on the other day.