Production and use of an inhibitory monoclonal antibody to human lipoprotein lipase

Abstract

Studies were performed to produce a monoclonal antibody to human lipoprotein lipase, verify the specificity of the antibody for lipoprotein lipase, and use this antibody for detection of lipoprotein lipase protein in human post-heparin plasma. Partially purified lipoprotein lipase from human milk was used as an antigen for the production of anti-lipoprotein lipase antibodies in mice. The spleen was removed from the animal having the highest titer of inhibitory antibodies to lipoprotein lipase and the cells were fused with mouse myeloma cells. Culture media from the resulting hybridomas were screened for their ability to inhibit lipoprotein lipase catalytic activity. This screening procedure thus identified only those hybridomas which produced antibodies directed against lipoprotein lipase. One monoclonal antibody, from one clone, was selected for detailed study. The specificity of this antibody for lipoprotein lipase protein was established by three methods. First, post-heparin plasma lipoprotein lipase activity and immunoreactivity detected by an enzyme-linked immunosorbent assay (ELISA) co-eluted during heparin-agarose and phenyl-Sepharose chromatography. Second, the antibody detected a protein which was released into the circulation after intravenous injection of heparin into humans. Third, both immunoreactive lipoprotein lipase protein and lipoprotein lipase enzymatic activity were lost by heat-inactivation of lipoprotein lipase. The use of active enzyme as an antigen and the procedure used to screen the monoclonal antibody-producing hybridomas allowed the production of an inhibitory anti-human lipoprotein lipase monoclonal antibody. This antibody is useful for detection of lipoprotein lipase protein in plasma and should allow for immunohistochemical staining of active lipoprotein lipase enzyme in tissues. Moreover, the methods described for screening hybridomas may be modified and used to produce specific antibodies against other partially purified enzymes.