Characterization of monoclonal antibody HTA125 with specificity for human TLR4.
Binding of monoclonal antibody HTA125 to human toll-like receptor 4 (TLR4) was characterized by flow cytometry using MonoMac6 human monocytic cells. Data were obtained using direct binding to cell surface TLR4 by labeled HTA125, as well as inhibition of direct binding using purified reagents, and by two-step binding. HTA125 bound weakly to human TLR4, and could be inhibited by mouse Ig, mouse IgG Fc, and mouse IgG2a. In addition, purified human IgG Fc and purified human immunoglobulin of isotypes IgG1 and IgG4 could block binding of HTA125 to MonoMac6 cells. Furthermore, a mouse IgG1 monoclonal antibody possessing specificity for human CD64, which is a high affinity IgG Fc receptor, partially inhibited binding of HTA125 to MonoMac6 cells. Finally, co-stimulation via TLR4 and Fc receptor, resulted in cytokine production by MonoMac6 cells different than that induced via TLR4 alone. Therefore, the utility of HTA125 remains as a weak detector of human TLR4, and as an agent to block TLR4 ligands with an understanding that Fc receptor may be engaged also.
Wang
Rong
R
Corixa Corporation-Montana, 553 Old Corvallis Road, Hamilton, MT 59840, USA. jbaldridge@corixa.com
Stephens
Jeffrey
J
Lacy
Michael J
MJ
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D001681:Biological Assay; D002460:Cell Line; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007375:Interleukin-1 / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D009000:Monocytes / Q000166:cytology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D017452:Receptors, IgG / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D016207:Cytokines; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007375:Interleukin-1; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D017452:Receptors, IgG; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D013755:Tetradecanoylphorbol Acetate