Human antisera

Following approval by the institutional review board at the University of Texas Health Science Center at San Antonio, 21 TFI, 15 IFC, and 29 FC patients were enrolled. All recruited women underwent diagnostic laparoscopy with chromotubation. Diagnosis of tubal infertility was based on 1 of the following findings: hydrosalpinx, fimbrial phimosis, or peritubal adhesions. Women with prior tubal ligation or a history of pelvic infection or inflammation other than pelvic inflammatory disease such as appendicitis were excluded. IFC patients were women with normal pelvic findings and tubal patency at laparoscopy. FC patients had no history of infertility with at least 1 live birth and normal pelvic findings at time of tubal ligation. All participants underwent a single blood draw. Serum samples were stored at –20°C until analyzed.

Cell culture and chlamydial infection

HeLa cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco PRL, Rockville, MD) with 10% fetal calf serum (FCS; Gibco BRL) at 37°C with 5% carbon dioxide (CO ₂ ) as previously described. C trachomatis serovar D or C pneumoniae AR39 organisms were grown, purified, and titrated as previously described.

After titration, organisms were stored at –80°C. For immunofluorescence assay, chlamydial organisms were used to infect HeLa cells grown on glass coverslips in 24-well plates. The subconfluent HeLa cells were treated with DMEM containing 30 μg/mL of diethylaminoethyl (DEAE)-dextran (Sigma, St Louis, MO) for 10 minutes. After removal of DEAE-dextran solution, chlamydial organisms were added to the wells for 2 hours at 37°C. The infected cells were continuously cultured in DMEM with 10% FCS and 2 μg/mL of cycloheximide (Sigma).

For preparing whole-cell lysates, infection was carried out in tissue culture flasks. Infected cultures were processed or harvested 48 hours after infection or as indicated in individual experiments.

Immunofluorescence assay

Antichlamydial organism antibodies in human sera were titrated using an immunofluorescence assay as previously described. Briefly, HeLa cells grown on coverslips were infected with C trachomatis or C pneumoniae organisms, fixed 48 hours after infection for C trachomatis and 72 hours for C pneumoniae with 2% paraformaldehyde, and permeabilized with 2% saponin. After blocking, human antisera were added to the chlamydia-infected cell samples. Goat antihuman immunoglobulin (Ig) G conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) was used to visualize human antibody binding and a Hoechst deoxyribonucleic acid (DNA) dye (blue; Sigma) to visualize HeLa and chlamydial DNA. The highest dilution of a serum that still gave a positive reactivity was defined as the titer of the given serum sample.

All human serum samples were serially diluted, and the appropriate dilutions were repeated multiple times based on the results obtained from prior dilutions to obtain a more accurate titer for each serum. For the time-course study, the C trachomatis –infected HeLa cells were processed as described above at various time points after infection as indicated in the data figure.

The processed samples were coimmunostained with a mouse anti-HSP60 (unpublished data) or anti-caseinolytic protease (Clp) P (unpublished data) plus rabbit anti– C trachomatis serovar D organisms. The primary antibody binding was visualized with a goat antimouse IgG conjugated with Cy3 (red) and a goat antirabbit IgG conjugated with Cy2 (green; both from Jackson ImmunoResearch Laboratories), respectively, and DNA by a Hoechst DNA dye.

Images were acquired with an Olympus AX70 fluorescence microscope equipped with multiple filter sets (Olympus, Melville, NY) as previously described. All microscopic images were processed using an Adobe Photoshop program (Adobe Systems, San Jose, CA).

Chlamydial fusion protein–arrayed microplate enzyme-linked immunosorbent assay (ELISA)

The glutathione S -transferase (GST) fusion protein ELISA for detecting human antibody recognition of chlamydial proteins was carried as previously described. The bacterial lysates containing individual chlamydial GST fusion proteins were added to 96-well microplates precoated with glutathione (Pierce, Rockford, IL).

The GST fusion protein lysates included all 14 chlamydial HSP family members: GST-CT110 (GroEL, HSP60); GST-CT111 (GroES, HSP10); GST-CT113 (ClpB, ClpB-related ATP-dependent protease); GST-CT286 (ClpC, Clp protease ATP-binding subunit); GST-CT341 (DnaJ protein); GST-CT395 (GrpE, HSP70 cofactor); GST-CT396 (DnaK, HSP70); GST-CT407 (DksA, probable DnaK suppressor); GST-CT431 (ClpP, ATP-dependent ClpP endopeptidase); GST-CT604 (GroEL, HSP60); GST-CT705 (ClpX, ATP-dependent ClpX-related protease; GST-CT706 (ClpP, ATP-dependent ClpP endopeptidase subunit); GST-CT709 (MreB, Rod shape determining protein MreB/HSP70 sugar kinase); and GST-CT755 (HSP60).

Lysates containing GST alone, as negative, and GST-chlamydial protease-like activity factor, as positive controls, were also included. After blocking, human antisera preabsorbed with a bacterial lysates containing GST alone were reacted with the plate-immobilized fusion proteins. The human antibody reactivity was detected with a goat antihuman-IgG conjugated with horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories) plus the substrate 2,2′-azino-bi(2-ethylbenzothiazoline-6-sulforic acid) diammonium salt (ABTS; Sigma). The optical density (OD) was measured at 405 nm using a microplate reader (Molecular Devices Corp, Sunnyvale, CA).

To confirm the antibody-binding specificity, all antisera were further absorbed with lysates made from either HeLa cells alone or C trachomatis serovar D–infected HeLa cells prior to reacting with the fusion protein-coated plates. The antibody binding that remained positive after HeLa-alone lysate absorption but significantly reduced by chlamydia-HeLa lysate absorption was considered true positive.

Western blot

Western blot with GST fusion proteins as antigens was carried out as previously described. GST fusion proteins (GST-HSP60, GST-HSP10, GST-ClpP) were purified from the corresponding bacterial lysates using glutathione agarose beads as previously described. The purified fusion proteins were resolved on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. Membrane-immobilized proteins were reacted with human sera pooled from each patient group and preabsorbed with bacterial lysates containing GST alone. Human antibody binding was detected with a goat antihuman IgG-HRP secondary antibody and visualized with an enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Data analyses

Data were analyzed using SPSS version 15.0 software (IBM, Chicago, IL). As a preliminary step, titer values were log transformed to produce a normal distribution and analyses were performed on transformed values. Analysis of variance was used to assess anti- C trachomatis and anti- C pneumoniae antibodies to evaluate overall mean differences among the 3 groups of patients.

The Student t test was utilized to compare differences between groups. Because the antibody titers had large variations within a given group, the serum titers were evaluated by ranges of less than 1:10 (negative), 1:10 to 1:10,000 (low), and greater than 1:10,000 (high). The χ ² and Fisher’s exact tests were used to compare TFI, IFC, and FC overall antibodies to C trachomatis and antibodies to C pneumoniae .

Finally, we evaluated pairwise differences between TFI vs IFC, TFI vs FC, and FC vs IFC in C trachomatis using logistic regression. ELISA results were analyzed also using χ ² and Fisher’s exact tests as appropriate.