Generation and characterization of a human monoclonal IgM antibody that recognizes a conserved epitope shared by lipopolysaccharides of different gram-negative bacteria.
A hybridoma cell line secreting a human monoclonal antibody (humab) directed to an epitope in the lipid A region of lipopolysaccharides of Gram-negative bacteria was isolated. Peripheral blood lymphocytes (PBL) obtained from a healthy volunteer were immortalized by Epstein-Barr virus (EBV) transformation. Lymphoblastoid cell lines (LCL) secreting antibodies to the lipopolysaccharides of Gram-negative bacteria were determined by an enzyme-linked immunosorbent assay (ELISA) and subsequently fused with the human-mouse heteromyeloma cell line CB-F7 by polyethylenglycol (PEG)-mediated fusion. A hybridoma line producing a humab (LPD5H4), of the IgM/lambda isotype, which strongly reacted with the lipid A portion of Salmonella and E. coli spp. in ELISA, was established. The antibody was purified by hydrophobic interaction chromatography and gel filtration. Immunoblotting experiments showed a strong reactivity of the humab LPD5H4 with the lower molecular species of different rough and smooth lipopolysaccharide (LPS) types of the bacteria species Salmonella, E. coli, Klebsiella, and Neisseria meningitidis, whereas those of Pseudomonas spp. were negative. Binding of humab LPD5H4 to solid phase bound lipid A and different rough mutants of LPS could be inhibited by the corresponding antigens in solution. Competition assays with a murine monoclonal antibody to lipid A and with polymyxin B indicate that humab LPD5H4 recognizes its epitope in this extremely conserved part of the LPS molecule. In vitro tests demonstrated that the MAb is able to partially inhibit the LPS-induced release of TNF-alpha using isolated peripheral blood mononuclear cells (PBMC).
Seifert
M
M
Department of Medical Immunology, Medical School (Charité) of the Humboldt-University of Berlin, Germany.
Schoenherr
G
G
Roggenbuck
D
D
Marx
U
U
von Baehr
R
R
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D000918:Antibody Specificity; D002459:Cell Fusion; D000939:Epitopes / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000276:immunology*; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D011112:Polymyxin B / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D000939:Epitopes; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; C044203:anti-IgM; D011112:Polymyxin B