title: Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma.
abstract: We have developed a direct sandwich-enzyme-linked immunosorbent assay (ELISA) for quantification of the hepatic triglyceride lipase (HTGL) immunoreactive mass in human plasma. This direct sandwich-ELISA uses a combination of two distinct monoclonal antibodies (MAbs), which recognize different epitopes on the HTGL molecule: a horseradish peroxidase (HRP)-labeled anti-human HTGL MAb (2(4)F12C12) as an enzyme-linked MAb, and an anti-human HTGL MAb (1(11)A3H3) coated on a microtiter plate as a solid-phase MAb. Purified human post-heparin plasma (PHP)-HTGL was used as the standard material. The detection range of the sandwich-ELISA was 40-800 ng of HTGL protein per ml of plasma. The intra- and inter-assay coefficients of variation were less than 2.0% and 2.3%, respectively. The recovery tests resulted in variation only between 97.7% and 103.5%. No significant assay interference was caused by a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. The reliability of the HTGL mass values obtained with the direct sandwich-ELISA was assessed by comparison with the HTGL mass values determined by our earlier one-step sandwich-enzyme immunoassay (EIA). The two sets of values showed a highly significant correlation (r=+0.952, n=64). Strong correlation (r=+0. 959, n=50) was also found between the HTGL masses with the direct sandwich-ELISA and the HTGL activities determined with a selective immunoinactivation assay. The HTGL mass concentrations in PHP from 64 healthy subjects were 1916+/-841 ng/ml by the direct sandwich-ELISA and 1925+/-785 ng/ml (mean+/-standard deviation (SD)) by the one-step sandwich-EIA. The present direct sandwich-ELISA permits rapid identification of certain HTGL abnormalities in PHP samples from patients with hypertriglyceridemia or diseases such as hypothyroidism or renal failure, which affect HTGL.
authors.affiliation: The Division of Laboratory Products, Dainippon Pharmaceutical Co. Ltd., 33-94 Enoki, Suita, Osaka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nishimura
authors.forename: Y
authors.initials: Y
authors.lastname: Ohkaru
authors.forename: H
authors.initials: H
authors.lastname: Ishii
authors.forename: N
authors.initials: N
authors.lastname: Sunahara
authors.forename: A
authors.initials: A
authors.lastname: Takagi
authors.forename: Y
authors.initials: Y
authors.lastname: Ikeda
mesh_terms: D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D008049:Lipase / Q000097:blood*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: D000925:Anticoagulants; D006493:Heparin; D008049:Lipase