To synchronize the menstrual cycle, one month before infection, all animals were treated intramuscularly with 1 mg of medroxyprogesterone acetate (MPA). Vaginal infection was performed by gently placing the inoculum on the os cervix through a speculum. One group of six rhesus monkeys received 107 inclusion forming units (IFU) and a second group of six animals received 105 IFU of C. trachomatis serovar D. Three additional monkeys were administered SPG and served as mock-infection controls. Three monkeys from the group infected with 105 IFU and three monkeys from the group infected with 107 IFU underwent scheduled necropsies at 10 weeks post-infection. The remaining nine animals received a second injection of MPA (1 mg) and at 14 weeks from the initial infection 7 of 9 monkeys were inoculated vaginally with 105 IFU of C. trachomatis serovar D. Blood was collected the day before each inoculation and at regular intervals following post-inoculation until the study end-point.
Growth protocol
The human C. trachomatis serovar D (UW-3/Cx) was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and was grown in HeLa-229 cells. Density gradient-purified elementary bodies (EB) were stored at -80 C in 0.2 M sucrose, 20 mM sodium phosphate (pH 7.4), and 5 mM glutamic acid (SPG).
Extracted molecule
protein
Extraction protocol
Serum samples were diluted 1:100 in a 3 mg mL-1 E. coli lysate solution in protein arraying buffer (Maine Manufacturing, Sanford, ME, USA) and incubated at room temperature for 30 min.
Label
biotin
Label protocol
Biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA)
Hybridization protocol
Serum samples were diluted 1:100 in a 3 mg mL-1 E. coli lysate solution in protein arraying buffer (Maine Manufacturing, Sanford, ME, USA) and incubated at room temperature for 30 min. Chips were rehydrated in blocking buffer for 30 min. Blocking buffer was removed, and chips were probed with pre-incubated serum samples using sealed, fitted slide chambers to ensure no cross-contamination of sample between pads. Chips were incubated overnight at 4°C with agitation. Chips were washed five times with TBS-0.05% Tween 20, followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:200 in blocking buffer at room temperature. Chips were washed three times with TBS-0.05% Tween 20, followed by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences, Frederick, MD, USA) at room temperature protected from light. Chips were washed three times with TBS-0.05% Tween 20, three times with TBS, and once with water. Chips were air dried by centrifugation at 1,000 x g for 4 min and dried overnight in dessicator before scanning.
Scan protocol
Probed microarrays (slides) were scanned using a GenePix 4300A High-Resolution Microarray Scanner (Molecular Devices, Sunnyvale, CA, USA) an image file (.tif) was saved for each array using GenePix Pro 7 software. The scanned image files were quantified using the Mapix software (Innopsys) auto gridding feature. For this process two input files are required: (1) a .gal file that defines the array and sub-array layout and (2) the tiff image file for an array. Once the auto-gridding is complete the overlay of the mapped array, subarray, and individual spot locations are shown in the graphical user interface (GUI). If the automatic gridding fails to map to the correct positions the mapping can be manually adjusted using the GUI. Once the gridding is confirmed to be correct the array spots are quantified and saved to an output .gpr file. For each spot on the slide the gpr file contains the foreground intensity (median of pixels inside of circle defining spot) and local background intensity (median of pixels just outside of circle defining spot).
Description
Serum samples were exposed to Chlamydia trachomatis protein microarrays and bound antibody to individual protein spots was measured by fluorescence. MK-0
Data processing
Foreground spot intensities were adjusted by local background by subtraction, and negative values were converted to 1. Data was normalized using the variance stabilization and calibration for microarray data (VSN) package implemented in R. The VSN model was built using only the 192 no-DNA control spot intensities for each sample.
