Active Cluster Of Differentiation 14 (CD14)

Figure. The binding activity of CD14 with LBP.
Cluster Of Differentiation 14 (CD14), also known as CD14, is a component of the innate immune system. CD14 acts as a co-receptor (along with the Toll-like receptor TLR 4 and MD-2) for the detection of bacterial lipopolysaccharide (LPS). CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its main ligand, CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid. Besides, Lipopolysaccharide Binding Protein (LBP) has been identified as an interactor of CD14, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CD14 and recombinant human LBP. Briefly, CD14 were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to LBP-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CD14 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 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 450nm immediately. The binding activity of CD14 and LBP was shown in Figure 1, and this effect was in a dose dependent manner.

CD14 also can enhance LPS-stimulated IL-8 secretion by THP-1 human acute monocytic leukemia cells. Therefore, THP1 cells were cultured in 24 well plates at a concentration of 106 cells/mL and stimulated by LPS (1μg/mL), then added of different concentrations of recombinant human CD14 (1μg/mL, 5μg/mL). After 24h later, the production of IL8 was determined in the supernatants by cytokine specific ELISA.
Result: When the concentration of CD14 was 5μg/mL, the secretion LPS-stimulated IL-8 can be significantly increased.

The standard curve was demonstrated as follows.

Figure. Effects of CD14 on IL8 production by THP1 cells.