Monoclonal antibodies against the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli: preparation, characterization, and use in immunoaffinity chromatography.
Three mouse hybridoma cell lines are described that produce monoclonal antibodies directed against the membrane-bound, flavin adenine dinucleotide linked D-lactate dehydrogenase of Escherichia coli. In contrast to polyclonal antibodies produced in rabbits, none of the monoclonal antibodies inhibits enzyme activity. Immunoblots of D-lactate dehydrogenase proteolytic fragments indicate that each antibody is directed against a different region of the molecule. One monoclonal antibody, 1B2a, reacts with native, undigested D-lactate dehydrogenase only and is used to purify the enzyme in a single step. The protocol involves chromatography of a Triton X-100 extract of membrane vesicles containing D-lactate dehydrogenase on a column made with the monoclonal antibody coupled to a solid support. After the column is washed free of unadsorbed protein, elution at high pH in the presence of guanidine hydrochloride yields a fraction containing highly purified, catalytically active D-lactate dehydrogenase.
D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000201:enzymology; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010954:Plasmacytoma / Q000276:immunology
D000911:Antibodies, Monoclonal; D007770:L-Lactate Dehydrogenase