Alkaline phosphatase conjugated goat anti mouse iga

Culture supernatants were tested for IgA antibodies by ELISA. 96 well EIA/RIA plates (Corning, Inc, Cat# 9018) were coated with 50 µl of a 1∶1000 dilution of goat anti-mouse IgA-UNLB (Southern Biotech, Cat# 1040-01) and incubated for 4 hours at 37°C. Plates were washed 4× with PBS and blocked overnight at 4°C with 200 µl of PBS containing 2% BSA (Sigma Aldrich, Cat#A8412). Blocking buffer was removed and 50 µl of a 1∶4 dilution of the samples were added to the wells. Mouse IgA (Southern Biotech, Cat# 0106-01) was used as a standard. Plates were incubated at 37°C for 2 hours. Plates were washed 6× with PBS-Tween 20 (0.05%) and incubated with a 1∶1000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgA (Southern Biotech, Cat# 1040-04) in PBS containing 1% BSA and 0.1% Tween 20 for 1 hour at 37°C. Plates were washed 6× with PBS-Tween 20 (0.05%) and developed by addition of p-nitrophenyl phosphate substrate (1 mg/ml) in diethanolamine buffer (0.1 M TRIS, 0.1 M NaCl, 5% Diethanolamine, 10 mM MgCl₂, pH9.8). The assays were read at OD 405 nm (E max precision microplate reader, Molecular Devices, Inc, Sunnyvale, CA).

ELISA plates were coated (16 h at 4°C) with 75 μl of unlabeled goat anti-murine IgA (Southern Biotech) at 5 μg/ml in PBS, washed four times with PBS 0.025% Tween 20, and saturated with 200 μl of PBS 10% fetal calf serum (FCS) for 2 h at room temperature. Fifty microliters of serial dilutions of stools was incubated for 4 h at room temperature. After four washes in PBS 0.025% Tween 20, 50 μl of alkaline-phosphatase-conjugated goat anti-mouse IgA (Southern Biotech) (1/500 in PBS 10% FCS) were added and plates incubated for 2 h at room temperature. The assay was developed with Sigma 104 phosphatase substrate.

The filtration plate (Millipore) was coated overnight at 4°C with 10 ug/ml VP1_237−249 peptide. After washing, the plate was blocked with complete RPMI-1640 for 2 h. Splenocytes or GALT lymphocytes were applied (5 × 10⁵/well) and incubated for 6 h at 37°C with LPS (100 ng/ml, Sigma-Aldrich) as positive control. After removing the supernatant, ice-cold deionized water was added and incubated on ice for 10 min to lyse the remaining cells. After washing, alkaline phosphatase-conjugated goat anti-mouse IgA (Southern Biotech) was added and incubated overnight. The enzyme activity was revealed by using Sigma Fast BCIP/NBT (Sigma-Aldrich). Spots were counted using ImmunoSpot analyzer 5.1.36 software (Cellular Technology).