Anti human igg antibody conjugated to hrp

Anti-MUC16 antibodies were expressed as described before. A portion of the supernatant was used to determine antibody concentration by ELISA. Serial dilution was performed for each antibody supernatant and samples were coated in Nunc MaxiSorp™ flat-bottom ELISA plates (Thermo Fisher Scientific) overnight at 4 ℃. After blocking for 1 hour at RT with 5% FBS, the plates were washed and incubated with anti-human IgG antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, Inc.) for 1 hour at RT. The plates were then washed and developed for 2.5 min. with 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma) before stopping the reaction with sulfuric acid. The plates were read at 450 nm using Epoch Microplate Spectrophotometer (Agilent BioTek). An antibody with a pre-determined concentration was used to construct a standard curve and calculate anti-MUC16 antibody expression levels.

Serial dilutions of recombinant MUC16 protein were performed and samples ranging from 100 nM to 0.195 nM were coated in Nunc MaxiSorp™ flat-bottom ELISA plates (Thermo Fisher Scientific) overnight at 4 ℃. After blocking for 1.5 hours at RT with 5% FBS, the plates were washed and incubated with anti-MUC16 antibodies in a concentration of 7 nM for 1h at RT. Afterwards, the plates were washed and incubated with anti-human IgG antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, Inc.) for 1 hour at RT. The plates were then washed and developed for 1 min. with 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma) before stopping the reaction with sulfuric acid. The plates were read at 450 nm using Epoch Microplate Spectrophotometer (Agilent BioTek). After subtracting the background of the primary antibodies to the blocking reagent or the background of the secondary antibody to the recombinant MUC16 (whichever was higher), a non-linear curve fitting to the one site, specific-binding equation was performed using GraphPad Prism 9 (Version 9.4.1 (458)) to estimate the Kd of each antibody.