Genepix pro image analysis software

Biotin labeling.All serological proteins were labeled with biotin using the modified procedure as previously described 28 (link), 29 (link). Briefly, 10 μL serum were diluted with 90 μL filtered 1×PBS (pH 7.4) followed by 1 μL of NHS-PEG4-Biotin (20 g/L in DMSO) (Thermo Fisher Scientific, MA, USA). After incubating for 1 h at room temperature, excess biotin molecules were removed using a Bio-Spin column via centrifugation at 1000 × g for 2 min. The collected biotinylated proteins were dissolved in 500 μL of PBS containing 5% milk (w/v) and stored at 4 ℃.
Sera screening. Antibody microarrays were assembled into an incubation tray (PEPperPRINT, Heidelberg, Germany) and blocked with 600 μL 5% milk (w/v) for 1 h at room temperature. After removing the milk, the arrays were incubated with pre-labeled serum proteins at 4 °C overnight. The slides were washed three times, 10 min per wash, with PBS containing 0.05% (w/v) Tween 20 (PBST). For detection, the arrays were incubated with 2 μg/mL streptavidin-PE for 1 h at room temperature in the dark and then washed three times with PBST. After centrifuging for 2 min at 1000×g, the slide was scanned using the GenePix 4000A microarray scanner (Molecular Devices, CA, USA). The fluorescent images were analyzed and the signal intensity was extracted using the GenePix Pro image analysis software (Molecular Devices, CA, USA).

Microarrays preparation: Properly diluted plasma samples (from 10 to 500-fold) and serially diluted standard immunoglobulins were printed onto a 3D modified slide surface (Capital Biochip Corp, Beijing, China) in two replicas using an Arrayjet microarrayer (Roslin, UK). Phosphate-buffered saline (PBS) and bovine serum albumin (BSA, 1 mg/mL) (Sigma-Aldrich, MO, USA) were used as negative controls. After printing, the plasma microarrays were stored at -20 ^∘C until use.
Microarrays detection: After equilibration to room temperature, the plasma microarrays were assembled into a microarray incubation tray (PEPperPRINT, Heidelberg, Germany) and blocked with 500 μL 1% BSA in each well for 1 h at room temperature. After removing the BSA, the arrays were incubated with the corresponding fluorescently-labeled antibody combinations for 1 h at room temperature. The resulting slides were washed three times with PBS containing 0.05% (w/v) Tween 20 (PBST), 5 min/each, and $H_{2}O$ tk min/each. The slide was scanned using a Genepix 4000A microarray scanner (Molecular Devices, CA, USA). The fluorescent images were analyzed and the signal intensity was extracted using a GenePix Pro image analysis software (Molecular Devices, CA, USA). The plasma Ig quantification includes IgA, IgA1-2, IgG, IgG1-4 and IgM.