Polyclonal anti mouse ig hrp

For whole cell ELISA, bacteria were grown on BHI or GC plates for 16 h. Cells were harvested and resuspended in PBS at an OD₆₀₀ of 0.2. Microtiter plate wells were filled with 50 μl of the bacterial suspension and dried at room temperature overnight in the laminar flow cabinet. After the bacteria in the dried wells were heat-killed for 1 h in 56°C. For recombinant protein ELISA, wells of plates were coated with 100 ng of purified recombinant MsrA/B protein in 100 μl of coating buffer (0.5 M carbonate/bicarbonate buffer, pH 9.6) for 1 h at room temperature. All ELISAs were performed with mouse pre-immune or MsrA/B immunized sera, and secondary antibody as specified in the results [polyclonal anti-mouse Ig HRP (Dako) or IgG1, IgG2a, IgG2b, IgG3, or IgM HRP (Thermofisher Scientific)]. The substrate TMB (3,3′, 5,5;-tetramethylbenzidine) solution (Thermofisher Scientific) was used as per manufacture's instruction. Equal amount of 1 N hydrochloric acid was added to stop the reaction. Absorbance was read in a TECAN Model Infinite 200 Pro plate reader at 450 nm.

Bacteria were grown on BHI or GC plates for 16 h. Cells were harvested and resuspended in PBS at an optical density at 600 nm of 0.20. Microtiter plate wells were filled with 50 μl of the bacterial suspension and dried at room temperature overnight in the laminar flow cabinet. After the bacteria in the dried wells were heat killed for 1 h at 56°C, the wells were washed and ELISA was performed with MAb 6E4 at a dilution of 1:64. Secondary antibody (polyclonal anti-mouse Ig HRP; P044701; Dako) was used at a dilution of 1:2,000. The substrate TMB (3,3′,5,5-tetramethylbenzidine) solution (ThermoFisher Scientific) was used per the manufacturer’s instructions. Equal amounts of 1 N hydrochloric acid were added to stop the reaction. Absorbance was read in a Tecan model Infinite 200 Pro plate reader at 450 nm.