Active NLR Family, Pyrin Domain Containing Protein 3 (NLRP3)

NLR Family, Pyrin Domain Containing Protein 3 (NLRP3), also known as NALP3, CIAS1, PYPAF or Cryopyrin, is a cytosolic multiprotein complex composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1 that responds to microbial infection, endogenous danger signals, and environmental stimuli. Aberrant activation of the NLRP3 inflammasome is associated with the pathogenesis of various inflammatory diseases, such as diabetes, cancer, and Alzheimer‘s disease. Interleukin 1 Beta (IL1b) can interact with NLRP3, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse NLRP3 and recombinant chicken IL1b. Briefly, NLRP3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IL1b-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NLRP3 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant mouse NLRP3 and recombinant chicken IL1b was shown in Figure 1, the EC50 for this effect is 0.16 ug/mL.