3 3 5 5 tetramethylbenzidine liquid substrate system for elisa

Maxisorp plates containing recombinant L. salivarius (pNZ, MMP, MMPsynth, MMP-GFP, and MMPsynth-GFP), as well as MAP K-10, whole cells and cell lysate were incubated at 37°C for 1 h and then blocked with a 5% (w/v) solution of dry skimmed milk powder in PBS. A 100-μl aliquot of purified monoclonal antibody 13E1 or 8G2, appropriately diluted in PBS plus 0.1% tween 20 (PBS/T) was added to each test well. Samples were incubated at 37°C for 1 h on a rocking platform. The wells were washed and 100 μl of secondary antibody (Peroxidase-labeled, Anti-Mouse IgG detection antibody) diluted in PBS/T containing 1% (w/v) milk powder was added. After a 1 h incubation at 37°C the wells were washed and 100 μl of 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate System for ELISA (Sigma) was added per well. The substrate was left to develop in the dark at room temperature for 30 min after which the reaction was stopped by addition of 50 μl of a 10% HCl solution. Absorbance readings were read at 450 nm using a microplate reader. The binding response values against each respective recombinant cell type were normalized by dividing the absorbance level obtained in test wells by that obtained in parallel control wells treated with diluent buffer without the addition of the primary monoclonal antibody.