Iga ap

Sera or fecal pellets were collected from CD19-cre Usp22 KO or WT littermate mice (around 7–8 weeks old), and fecal supernatant was prepared (10% wt/vol) with PBS. The Ig isotype-specific ELISA assays were performed as previously described^{55 (link)}. Briefly, 96-well ELISA plates (NUNC) were coated with goat antibodies against mouse IgM (catalog number: 1021–01; 1/1000 dilution), IgG1 (catalog number: 1071–01; 1/1000 dilution), IgG2b (catalog number: 1090-01; 1/1000 dilution), IgG3 (catalog number: 1101-01; 1/333 dilution), or IgA (catalog number: 1040-01; 1/500 dilution) (all from SouthernBiotech), respectively, overnight at 4 °C. After blocking with 3% bovine serum albumin (BSA), 1/50-diluted sera or 1/5-diluted fecal samples were added into the wells, followed by serial fourfold dilution. The bound antibodies from sera or fecal supernatant were detected by the appropriate isotype-specific goat anti-mouse Ig that was conjugated with alkaline phosphatase (AP), IgM-AP (catalog number: 1021-04; 1/2000 dilution); IgG1-AP (catalog number: 1070-04; 1/2000 dilution); IgG2b-AP (catalog number: 1090-04; 1/2000 dilution); IgG3-AP (catalog number: 1100-04; 1/4000 dilution); IgA-AP (catalog number: 1040-04; 1/2000 dilution) (all from SouthernBiotech), followed by the development with SIGMAFAST p-Nitrophenyl phosphate substrate.

The stool supernatant was collected by centrifugation of a 2 ml aliquot of stool sample. Then, Enzyme-linked Immunosorbent Assay (ELISA) with an alkaline-phosphatase system was performed. Corning 96-well half-area plates were coated with either Goat anti-human IgG-UNLB, IgA-UNLB or IgM-UNLB (Southern Biotech) in a final concentration of 5 µg/ml (total volume 30 µl). Plates were incubated overnight at 4 °C. All subsequent washing steps were performed with PBS 0.025% Tween 20 using a Microplate Washer. The plates were washed 4 times before being blocked with 75 µl PBS 10% FCS solution (blocking buffer) for 3 h. One to five dilutions of the supernatants were prepared. Standard dilution rows starting from 2 µg/ml were prepared using a human reference serum. Plates were incubated with 25 µl of the diluted supernatants and standards for 4 h at room temperature. After another washing 25 µl of the secondary antibodies diluted 1:500 in blocking buffer (Goat anti-human IgG-AP, IgA-AP and IgM-AP, Southern Biotech) were added for an incubation time of 2 h at room temperature. After washing 4 more times, 50 µl phosphatase substrate in a concentration of 1 mg/ml was added. After 30 min of incubation at 37 °C, the plates were read at 405 nm wavelength. The standard curve was calculated by the SkanIT software.