treatment group: 1/4 dose ETVAX® etec colony pcr result at v1 (d0): Negative etec colony pcr result at v7 (d97): Negative gender: Female
Extracted molecule
protein
Extraction protocol
not provided
Label
biotin
Label protocol
n/a
Hybridization protocol
Plasma 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
S114-2034.V1 anti-IgG antibody plasma samples were exposed to ETEC protein microarrays and bound antibody to individual protein spots was measured by fluorescence.
Data processing
Foreground spot intensities were adjusted by local background by subtraction, and negative values were converted to 1. Next, all foreground values were transformed using the base 2 logarithm. The dataset was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT controls for each sample. Since the IVTT control spots carry the chip, sample and batch-level systematic effects, but also antibody background reactivity to the IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding to the non-specific antibody binding to the IVTT controls (a.k.a. background). With the normalized data, a value of 0.0 means that the intensity is no different than the background, and a value of 1.0 indicates a doubling with respect to background. Normalization was applied separately to arrays 1 and 2.
