Sampling protocol: In PE, SW, USC, and USW, milk was collected using an electric breast pump. In all other cohorts, milk was manually expressed. Except for those collected in ETR, samples were immediately placed on ice, aliquoted within 30 min, and frozen at −20°C. Milk collected in ETR was preserved in a 1:1 ratio with Milk Preservation Solution (Norgen Biotek, Cat. 44800, Thorold, Ontario) and frozen within 6 d. We have shown previously that this method can maintain bacterial DNA integrity in human milk held at 37°C for at least 2 wk (34). All samples were shipped on dry ice to the University of Idaho, where they were immediately frozen at −20°C.
Label
Cy3-conjugated anti-human IgG
Label protocol
Cy3-conjugated anti-human IgG (Jackson ImmunoResearch, Cat. 709-165-098, West Grove, PA)
Hybridization protocol
Milk samples were diluted 1:5 in a 1.5 mg/mL E. coli lysate solution (Antigen Discovery, Inc., Irvine, CA) in protein arraying buffer (GVS, Cat. 10485356, Sanford, ME) and incubated at room temperature for 30 min. For milk samples previously diluted in preservative solution, samples were diluted 1:2.5 in the lysate solution. Microarray slides were hydrated in arraying buffer and then probed with 250 µL of the preincubated milk samples using sealed, fitted slide chambers to avoid cross-contamination between arrays. Arrays were incubated overnight at 4°C with agitation, washed three times with Tris-buffered saline (TBS)-0.05% Tween 20 (Thermo Scientific, J77500K8, diluted 20x in molecular grade water), and incubated with Cy3-conjugated anti-human IgG diluted 1:200 in arraying buffer and biotin-conjugated anti-Human IgA diluted 1:1000 (Jackson ImmunoResearch, Cat. 709-165-098 and Cat. 109-065-011, West Grove, PA). Arrays were washed three times with TBS–0.05% Tween 20 and incubated with streptavidin-conjugated SureLight P-3 (Columbia Biosciences, Cat. D7-2212, Frederick, MD) at room temperature, protected from light. Arrays were washed 3x with TBS–0.05% Tween 20, 3x with TBS, and once with water and then air dried by being centrifuged at 1,000 x g for 4 min and left overnight in a desiccator before scanning.
Scan protocol
Probed microarrays were scanned using a GenePix 4300A high-resolution microarray scanner (Molecular Devices, Sunnyvale, CA), and an image file (.tiff) was saved for each array using GenePix pro 7 software. The signals in the scanned images were quantified using Mapix software (Innopsys).
Data processing
All further data processing was performed in R (http://www.R-project.org). Data were normalized by first transforming raw values using the base 2 logarithm. Next, the data set was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT control spots from the IVTT antigen spots for each sample. Since the IVTT control spots carry not only the chip, sample, and batch-level systematic effects, but also antibody background reactivity to the E. coli cell-free IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding versus the nonspecific antibody binding to the IVTT controls. With the normalized data, a value of 0.0 means that the intensity is no different than that of the IVTT controls, a value of 1.0 indicates a doubling (2-fold) with respect to IVTT control spots, a value of 2.0 indicates 4-fold, 3.0 indicates 8-fold, etc. The normalized signal intensity of the purified protein spots is represented as the base 2 logarithm of the raw signal intensities.
Multipathogen analysis of IgA and IgG antigen specificity for selected pathogens in milk produced by women from diverse geographical regions: The INSPIRE Study [IgG]
Multipathogen analysis of IgA and IgG antigen specificity for selected pathogens in milk produced by women from diverse geographical regions: The INSPIRE Study
