A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes

Abstract

We have developed a sandwich-enzyme immunoassay (EIA) for the quantification of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in human postheparin plasma (PHP) using monoclonal antibodies (MAbs) directed against the corresponding enzymes purified from human PHP. The sandwich-EIA for LPL was performed by using the combination of two distinct types of anti-LPL MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of LPL was specifically measured using a beta-galactosidase-labeled anti-LPL MAb as an enzyme-linked MAb, an anti-LPL MAb linked with the bacterial cell wall as an insolubilized MAb, and purified human PHP-LPL as a standard. The sandwich-EIA for HTGL was carried out by using two distinct anti-HTGL MAbs that recognize different epitopes on HTGL. The limit of detection was 20 ng/ml for LPL and 60 ng/ml for HTGL. Each method yielded a coefficient of variation of less than 6% in intra- and inter-assays, and a high concentration of triglyceride did not interfere with the assays. The average recovery of purified human PHP-LPL and -HTGL added to human PHP samples was 98.8% and 97.5%, respectively. The immunoreactive masses of LPL and HTGL in PHP samples, obtained at a heparin dose of 30 IU/kg, from 34 normolipidemic and 20 hypertriglyceridemic subjects were quantified by the sandwich-EIA. To assess the reliability of the measured mass values, they were compared with the corresponding enzyme activities measured by selective immunoinactivation assay using rabbit anti-human PHP-LPL and -HTGL polyclonal antisera. Both assay methods yielded a highly significant correlation in either normolipidemic (r = 0.945 for LPL; r = 0.932 for HTGL) or hypertriglyceridemic subjects (r = 0.989 for LPL; r = 0.954 for HTGL). The normal mean (+/- SD) level of lipoprotein lipase mass and activity in postheparin plasma was 223 +/- 66 ng/ml and 10.1 +/- 2.9 mumol/h per ml, and that of hepatic triglyceride lipase mass and activity was 1456 +/- 469 ng/ml and 26.4 +/- 8.7 mumol/h per ml, respectively. The present sandwich-enzyme immunoassay methods make it possible to study the molecular nature of LPL and HTGL in PHP from patients with either primary or secondary hyperlipoproteinemia.