Generation and characterization of hamster-mouse hybridomas secreting monoclonal antibodies with specificity for lipopolysaccharide receptor.
Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS.
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66103.
S W
SW
Bright
T Y
TY
Chen
L M
LM
Flebbe
M G
MG
Lei
D C
DC
Morrison
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology*; D006224:Cricetinae; D000939:Epitopes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic