Defensin Beta 2 (DEFb2) is a 7 kDa secreted protein. It exhibits antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria, with highest activity against Gram-negative bacteria. Antimicrobial activity against P.aruginosa seems to be salt-sensitive and is reduced with high salt concentrations greater than 25 mM. The human DEFb2 cDNA encodes a 64 aa protein with a 23 aa signal sequence and a 41 aa mature sequence. Besides, Toll Like Receptor 4 (TLR4) has been identified as an interactor of DEFb2, thus a binding ELISA assay was conducted to detect the interaction of recombinant human DEFb2 and recombinant human TLR4. Briefly, DEFb2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to TLR4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-DEFb2 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 50μL stop solution to the wells and read at 450/630nm immediately. The binding activity of DEFb2 and TLR4 was shown in Figure 1, the EC50 for this effect is 0.11 ug/mL.