Characterization of monoclonal antibodies to human soluble MD-2 protein.
Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4. MD-2 exists as a cell surface protein in association with TLR4 and as secreted forms consisting of MD-2 monomers and multimers. To facilitate the studies of MD-2 distribution, abundance, and function, we produced monoclonal antibodies (MAbs) to baculovirally expressed soluble MD-2 (sMD-2). Eleven MAbs were characterized by enzyme-linked immunosorbent assay (ELISA) with soluble TLR4/MD-2 complex (sTLR4/MD-2) and sMD-2, Western blotting against sMD-2 monomer and multimers, and inhibition of direct LPS binding to sMD-2. Four MAbs preferentially recognized mainly MD-2 oligomers, not monomers, as judged by Western blotting and ELISA. Anti-MD-2 MAbs useful for indirect immunofluorescent staining of cells expressing TLR4 and MD-2 were identified. One MAb that recognized all forms of MD-2 was used in an ELISA to measure sMD-2 in normal human sera as well as sera from intensive care patients with and without sepsis. Serum levels of sMD-2 were undetectable or very low in normal and in nonsepsis patients but significantly (p < 0.05) increased in sepsis patients. These MAbs should therefore be very useful new tools for studies of MD-2 expression and function in health and disease.
Viriyakosol
Suganya
S
Veterans Medical Research Foundation, San Diego, California, USA. sviriyak@ucsd.edu
McCray
Paul B
PB
Ashbaugh
Mark E
ME
Chu
Jayne
J
Jia
Hong Peng
HP
Weiss
Jerold
J
Kirkland
Theo N
TN
D000818:Animals; D000911:Antibodies, Monoclonal; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051218:Lymphocyte Antigen 96 / Q000097:blood / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000276:immunology; D018805:Sepsis / Q000097:blood / Q000276:immunology; D012995:Solubility; D051197:Toll-Like Receptor 4 / Q000378:metabolism
D000911:Antibodies, Monoclonal; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4