Alkaline phosphatase conjugated anti mouse ab specific for igg

Anti-Ft Ab production in response to immunization and/or Ft infection in immunized mice was measured through enzyme linked immunosorbent assay (ELISA). Briefly, ELISA plates were coated overnight at 4°C with 50 μl/well of inactivated Ft SchuS4 [5x10⁷ CFU/ml in carbonate buffer [4.3g/l sodium bicarbonate and 5.3 g/l sodium carbonate (Sigma-Aldrich, St. Louis, Missouri) at pH 9.4]. Plates were then blocked at 4°C for 2 hours with 200 μl/well of PBS containing 5% BSA and 0.02% sodium azide. Subsequently three-fold dilutions of sera (starting with 1:50) were added to the plates (50 μl/well) and incubated for 2 hours at 4°C. After washing, alkaline phosphatase conjugated anti-mouse Ab specific for IgG, IgA (Sigma-Aldrich, St. Louis, Missouri), or IgG2c (Abcam, Cambridge, MA), were added and incubated for 1 hours at 4°C. Following secondary Ab incubation and washing alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, Missouri) of 100 μl/well was added and plates were read at 405 nm using microplate reader following a 5-second (sec) shake.

Anti-Ft-Ab production in response to immunization with iFt-MHB versus iFt- BHI was measured by ELISA. Briefly, ELISA plates (Corning, Corning, NY, USA) were coated with 50 µl of Ft LVS (5 × 10⁷ CFU/ml) in carbonate buffer [4.3 g/l sodium bicarbonate (Sigma-Aldrich) and 5.3 g/l sodium carbonate (Sigma-Aldrich) at pH 9.4] for 16 h at 4°C. The plates were then washed with washing buffer [PBS (Sigma-Aldrich) containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich)] and blocked for 2 h with 200 µl of PBS containing 5% BSA. Serial threefold dilutions of sera (starting with 1/10) were added to the plates (100 µl/well) and incubated for 2 h at 4°C. After three washes with washing buffer, alkaline phosphatase-conjugated anti-mouse Ab specific for IgG (Sigma-Aldrich) was added and incubated for 1 h at 4°C. Plates were then washed three times with washing buffer, and then 100 µl of BCIP/NBT alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were subsequently incubated for 2 h, and absorbance at 405 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).