Characterization of a monoclonal antibody that specifically binds to choline phospholipids and its use in immunocytochemistry.
A monoclonal IgM (MC22-33F), raised in response to mouse embryonic dental papilla cells, was selected for further analysis on the basis of the unusual resistance of its epitope to detergent extractions and protease treatments of cell cultures. Binding of MC22-33F to cultured cells was abolished after either pre-treatment of the cells with phospholypase C or pre-incubation of the hybridoma culture supernatant with multilamellar phosphatidylcholine-containing vesicles. MC22-33F reacted with phosphatidylcholine, with the phosphatidylcholine analogue dimethylphosphatidylethanolamine, and with sphingomyelin immobilized on polystyrene surfaces or in thin-layer chromatograms. Crossreaction with other phospholipids was not observed. The surface of cultured epithelial cells was labeled by MC22-33F at sites of bleb formation. Combining immunostaining by MC22-33F and histochemical staining of cultured cells revealed codistribution of phospholipid-containing inclusions with either lysosomes or neutral fat droplets, and inhibition of lipid degradation by kanamycin resulted in a parallel accumulation of these inclusions and of neutral fats in the cytoplasm. Immunolabeling by MC22-33F of frozen mouse tissues was maximal in fat-storing and steroid-producing cells. Extracellular phospholipids present in calcifying cartilage septa strongly reacted with MC22-33F. This monoclonal antibody offers an interesting alternative to histochemical lipid stains for investigating fatty metamorphosis and extracellular lipid deposition under physiological and pathological conditions.
Mark
M P
MP
Institut de Biologie Médicale, INSERM-Université Louis Pasteur, Faculté de Médecine, Strasbourg, France.
Tsuji
T
T
Portoukalian
J
J
Rebbaa
A
A
Zidan
G
G
Ruch
J V
JV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody; D002855:Chromatography, Thin Layer; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016253:Microscopy, Immunoelectron; D012516:Osteosarcoma / Q000276:immunology / Q000648:ultrastructure; D010767:Phosphorylcholine / Q000276:immunology*; D051381:Rats; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010767:Phosphorylcholine