Active Neutrophil Cytosolic Factor 2 (NCF2)

Neutrophil Cytosolic Factor 2 (NCF2) ,as kown as as NCF-2, NOXA2, P67PHOX, and P67-PHOX， is a vital component of the NADPH oxidase complex in neutrophils, essential for immune defense against pathogens. Its dysfunction can lead to severe immune disorders like CGD.It has been reported that when the cell is properly stimulated, NCF2 binds to NOX1 and other necessary subunits such as p47phox and p22phox to form a complete NADPH oxidase complex.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse NCF2 and recombinant human NOX1. Briefly, NCF2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NOX1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NCF2 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 450/630nm immediately. The binding activity of recombinant mouse NCF2 and recombinant human NOX1 was shown in Figure 1, the EC50 for this effect is 0.05ug/mL.