Igg1 ap

For detection of antibodies in serum, plates were coated with rat-anti-mouse IgE (R35-72; BD) or goat-anti-mouse IgG (Southern Biotech) overnight at 4°C. Purified mouse IgE (BD) or unlabelled mouse IgG1, IgG2a or IgG2b (all Southern Biotech) were used to generate a standard curve. Goat-anti-mouse IgE-AP, IgG1-AP, IgG2a-AP or IgG2b-AP (all Southern Biotech) were used for detection. For detection of parasite-specific antibodies, plates were coated with 20 μg/mL of HES suspension overnight at 4°C. After blocking for 2 hrs with 3% BSA, samples were incubated at room temperature for 2 hrs and the AP-coupled antibodies named above were used for detection. Absorption was measured on a Multiscan FC photometer (Thermo Fisher) at 405 nm and blank wells were used to subtract background.

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.