Production and characterization of monoclonal antibodies to rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

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Abstract
Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase], the key regulatory enzyme in cholesterol biosynthesis, was purified to apparent homogeneity. Purified HMG-CoA reductase yields a single diffuse band when sodium dodecylsulfate/polyacrylamide gels are stained with Coomassie blue and yields 2 adjacent bands when gels are stained with silver. Purified reductase was used to elicit the production of monoclonal antibodies. Spleen cells from BALB/c mice immunized with purified HMG-CoA reductase were fused with Sp-2/0 myeloma cells. Clones producing monoclonal antibodies to HMG-CoA reductase were identified by using a solid-phase radioimmunoassay and were subcloned in soft agar. The 3 relatively stable hybridoma lines isolated secrete different Ig as judged by their antibody subclasses and differing abilities to inhibit HMG-CoA reductase in solution. Efficient precipitation of solubilized HMG-CoA reductase was achieved with the 2 IgG antibodies but not with the IgM. A mixture of all 3 monoclonal antibodies immunoprecipitates more than 90% of the HMG-CoA reductase activity in solubilized rat liver extracts. These monoclonal antibodies should be useful probes for investigation of the regulation of HMG-CoA reductase and cholesterol synthesis.

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