The monoclonal antibody 4H1 reacts with soluble MD-2 (sMD-2) and the sMD-2/Toll-like receptor 4 (TLR4, CD284) complex. TLRs belong to a family of proteins that specifically recognizes and senses microbial products. They are highly conserved throughout evolution and act as innate immune recognition receptors against many pathogens. TLR4 is a functional receptor for gram-negative bacterial lipopolysaccharides (LPS). TLR4 associates with MD-2 which is absolutely required for LPS-induced activation of TLR4.
MD-2 exists as a cell surface protein in association with TLR4. It also exists as secreted forms consisting of MD-2 monomers and multimers. Circulating sMD-2 is mainly present as a doublet of ~20 and 25 kD, representing differentially glycosylated forms. Unlike TLR4, sMD-2 binds directly LPS without the need of soluble CD14 (sCD14). However, LPS-MD-2 interactions are increased when LPS is pretreated with CD14. Only monomeric sMD-2 is biologically active and able to associate with TLR4 and LPS. sMD-2 circulates in plasma of healthy individuals as a non-active, polymeric protein. In septic plasma, the total amount of sMD-2 was strongly elevated and contained both sMD-2 polymers and monomers. Soluble MD-2 is proposed to be an important mediator of organ inflammation during sepsis. During experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. This parallels enhanced TLR4 costimulatory activity. In vitro studies revealed that sMD-2 release appears to be restricted to endothelial and dendritic cells.
The monoclonal antibody 4H1 reacts with both the monomeric and the polymeric form of sMD-2. In addition, the monoclonal antibody 4H1 is able to inhibit LPS binding to MD-2.It does not cross-react with mouse MD-2.
Flow cytometry, Functional studies, Immuno assays, Immuno fluorescence, Western blot
For immuno assay, Western blotting and immunofluorescence dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.