Hrp conjugated goat anti mouse iga antibody

For the screen of monoclonal IgA antibodies against bacterial antigens, reciprocal dilutions of hybridoma-produced IgA antibodies were added to ELISA plates, which were pre-coated with whole bacteria of the 8M community, respectively, and blocked with 1% BSA. The captured IgA was detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA antibody (Sigma-Aldrich; A4789). ELISA plates were then developed by TMB microwell peroxidase substrate (KPL, Inc.; 50–76-03), quenched by 1 M H₂SO₄, and read using a BioTek Synergy HTX plate reader. The polyreactivity of hybridoma-produced IgA was tested by adding antibody to ELISA plates that were pre-coated with individual common antigen including lipopolysaccharide (Sigma-Aldrich; L4391), double stranded DNA (Sigma-Aldrich; D4522), insulin (Sigma-Aldrich; 91077C), flagellin (Sigma-Aldrich; SRP8029) and albumin (Sigma-Aldrich; A1653). ELISA plates were then washed, developed and read as described above.

Bacteria were cultured to OD₆₀₀ = 1~2 in appropriate condition and then fixed in 0.5% formaldehyde in PBS for 20 min at room temperature. After washing 3 times, bacteria density was adjusted to OD₆₀₀ = 1 in PBS. ELISA plates were coated with 30 μl of the adjusted bacterial suspension and incubated at 4°C overnight. After washing and blocking with 1% BSA, diluted fecal or seral samples containing polyclonal IgA were added and incubated overnight at 4°C. Captured IgA was detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA antibody (Sigma-Aldrich; A4789). ELISA plates were developed by 3,3’,5,5’-Tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL, Inc.; 50–76-03) and quenched by 1 M H₂SO₄. The colorimetric reaction was measured at OD = 450 nm by a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Inc.).