Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen.
CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.
Moonsom
S
S
Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
Kasinrerk
W
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D019556:COS Cells; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D007114:Immunization; D016130:Immunophenotyping; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000276:immunology*; D008070:Lipopolysaccharides / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D009000:Monocytes / Q000276:immunology; D009504:Neutrophils / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011994:Recombinant Proteins