TLR4 is a 100 kDa type I transmembrane glycoprotein that belongs to the mammalian Toll-Like Receptor family of pathogen pattern recognition molecules. TLRs plays a fundamental role in pathogen recognition and activation of innate immunity. Besides, TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. It is reported that RETN has been identified as an interactor of TLR4, thus a binding ELISA assay was conducted to detect the interaction of recombinant human RETN and recombinant human TLR4. Briefly, RETN was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl 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-RETN 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 TLR4 and RETN was shown in Figure 1. When recombinant human TLR4 is lmmobilized at 2 ug/mL (100 uL/well), the concentration of rhRETN that produces 50% optimal binding response is found to be approximately 6.25 ug/mL.