Anti mouse iga hrp

Mouse fecal IgA titers were measured using the Mouse IgA ELISA Quantitation Set (Bethyl Laboratories) following the manufacturer’s instructions. Mouse fecal albumin was measured using the Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories) following the manufacturer’s instructions. For both ELISAs, fecal samples were diluted 1/100 or 1/1,000, and concentration was determined based on a standard curve. For measurement of C. rodentium–specific antibodies in serum or fecal supernatants by ELISA, 10 µg/ml C. rodentium antigen was coated on 96-well plates, and sera were incubated in doubling dilutions. Antigen-specific IgG and antigen-specific IgA were detected using an anti-mouse IgG-HRP antibody (BD Biosciences) and an anti-mouse IgA-HRP (Bethyl Laboratories). For measurement of mouse IL-4 in cell culture, a capture anti-mouse IL-4 antibody (clone 11B11; donated by A. MacDonald, University of Manchester, Manchester, UK), recombinant mouse IL-4 (BioLegend) and a detection biotinylated anti-mouse IL-4 antibody (clone 24G2; BioLegend) were used. The mouse IL17A ELISA kit (Invitrogen) were used to detect cytokines in cells stimulated with C. rodentium antigen. Plates were developed with TMB peroxidase substrate (BD Biosciences), and optical densities were measured using a plate spectrophotometer.

Microplates (Nunc, Rochester, NY, USA) were first coated overnight with 5 μg/mL inactivated EV71 and blocked with Tris-buffered saline containing Tween 20/1% bovine serum albumin (BSA) before adding the serum, nasal-wash, BALF, or faecal extract samples. After a 2-h incubation at room temperature, the plates were washed, followed by the addition of goat anti-mouse IgG horseradish peroxidase (HRP; 1:10000; Bethyl) or anti-mouse IgA HRP (1:5000; Bethyl). For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA). Subsequently, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate was added. After colour development in the dark at room temperature for 20 min, the reaction was stopped by adding 100 μL of 1 M H₂SO₄. The absorbance at 450 and 550 nm was measured using a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA), and the results are expressed in ELISA units (E.U.): E.U. = (A_sample − A_blank) / (A_positive − A_blank).