Serum and NW samples were diluted 1:100 in a protein array blocking buffer (GVS, Sanford, ME, USA), supplemented with E. coli lysate (GenScript, Piscataway, NJ, USA) to a final concentration of 10 mg/mL, and preincubated at room temperature (RT) for 30 min. Concurrently, arrays were rehydrated in blocking buffer (without lysate) for 30 min. Blocking buffer was removed, and arrays were probed with preincubated serum samples using sealed chambers to prevent cross-contamination of samples between the pads. Arrays were incubated overnight at 4 C with gentle agitation. They were then washed at RT three times with Tris-buffered saline (TBS) containing 0.05% Tween 20 (T-TBS), biotin-conjugated goat anti-mouse IgA and Biotin-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) were diluted 1:400 in blocking buffer and applied to separate arrays for 1 h, RT with gentle agitation. Arrays were washed three times with T-TBS, followed by incubation with streptavidin-conjugated Qdot655 (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:200 in blocking buffer for 1 h, RT. Arrays were washed three times with T-TBS and once with water. Arrays were air dried by centrifugation at 500 g for 5 min. Images were acquired using the ArrayCAM imaging system from Grace Bio-Labs (Bend, OR, USA). Spot and background intensities were measured using an annotated grid (.gal) file. Mean fluorescence across antigens grouped by isotypes were used for subsequent analysis. The different antigens were acquired from Sino biological (Wayne, PA, USA).
Scan protocol
Images were acquired using the ArrayCAM imaging system from Grace Bio-Labs (Bend, OR) with gain and exposure times of 50 and 400-1000ms, respectively.
Description
Fig. 6 and 8
Data processing
Spot and background intensities were measured using an annotated grid (.gal) file. Signal intensities (SI) for each antigen on the array were first background corrected by subtracting sample-specific T-PBS buffer signals from purified protein spot signals. For statistical analysis, A two-way ANOVA was performed. A P value below 0.05 was considered significan. All data analyses and graphs were performed using GraphPad Prism software version 9 (GraphPad Software Inc., San Diego, CA, USA). We utilized the raw data for our analyses without any further corrections.
