Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.
Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-alpha-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007700:Kinetics; D008214:Lymphocytes / Q000276:immunology; D026901:Membrane Transport Proteins / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D009004:Monosaccharide Transport Proteins; D010954:Plasmacytoma / Q000276:immunology; D011863:Radioimmunoassay; D027981:Symporters
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease