Human NLRP3 Recombinant Rabbit Monoclonal Antibody [PSH11-09] - BSA and Azide free (Detector)

Sandwich ELISA analysis of human NLRP3 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA723275) diluted in carbonate/bicarbonate buffer, at a concentration of 2ug/ml overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1% BSA blocking buffer, and incubated with serial diluted Recombinant Human NLRP3 protein starting from 10000 pg/ml to 0 pg/ml and detect antibody (HA723278, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Interpolated concentrations of native NLRP3 in THP-1 and Jurkat extract samples based on a 1000 µg/ml extract load.

Interpolated concentration of native NLRP3 was measured in duplicate at different sample concentrations and interpolated from the NLRP3 standard curves. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean NLRP3 concentration was determined to be 888 pg/mL in THP-1 cell extract. There was no detectable signal in Jurkat cell extract.

Interpolated concentrations of spiked NLRP3 in cell culture media samples.

The concentrations of NLRP3 were measured in duplicates, interpolated from the NLRP3 standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).