ELISA System for Human Endothelial Lipase

Abstract

BACKGROUND Endothelial lipase (EL) regulates the metabolism of HDL cholesterol (HDL-C). However, the role of EL in regulating plasma HDL-C concentrations and EL's potential involvement in atherosclerosis in humans has not been fully investigated due to the lack of reliable assays for EL mass. We developed an ELISA system for serum EL mass. METHODS Human recombinant EL proteins, purified from cultured media of human EL–transfected Chinese hamster ovary cells, were used as antigen and calibrator. Two specific monoclonal antibodies were generated in mice against recombinant EL protein for a sandwich ELISA. We measured EL mass in human serum using EL recombinant protein as a calibration standard. RESULTS The EL antibodies did not cross-react with lipoprotein lipase and hepatic triglyceride lipase. The detection limit of the ELISA was 20 pg/mL, which is approximately 10 times lower than that of previous ELISA systems. Recovery of spiked EL in serum was 90%–105%. Assay linearity was intact with a >4-fold dilution of serum. Intra- and interassay CVs were <5%. The serum EL mass in 645 human subjects was [mean (SE)] 344.4 (7.7) pg/mL (range 55.2–1387.7 pg/mL). Interestingly, serum EL mass was increased in patients with diagnosed cardiovascular disease and inversely correlated with serum HDL-C concentrations. There was no difference in EL mass between pre- and postheparin plasma samples. CONCLUSIONS This ELISA should be useful for clarifying the impact of EL on HDL metabolism and EL's potential role in atherosclerosis.

Metabolic Syndrome Modulates Association between Endothelial Lipase and Lipid/Lipoprotein Plasma Levels in Acute Heart Failure Patients

“…Although previous studies showed that pre- and post-heparin EL plasma levels are highly correlated 8 or similar 34 , the possibility cannot be excluded that post-heparin EL plasma levels would be differently affected by AHF-associated pathophysiological conditions and MetS compared to our findings with pre-heparin EL plasma levels. Furthermore, considering EL polymorphisms associated with decreased EL enzyme activity 15, 16 , together with possibly altered plasma levels of EL inhibitors, such as angiopoetin-like protein 3 35 or protein convertases 36 in AHF, the association of EL activity with plasma lipids/lipoproteins 9 might be different from what was found for the EL mass in this study.…”

Plasma Activity of Endothelial Lipase Impacts High-Density Lipoprotein Metabolism and Coronary Risk Factors in Humans

“…1D). Moreover, we recently reported that the plasma EL mass is similar between pre-and post-heparin plasma samples 12) . Therefore, the effects and dose-dependency of heparin treatment on the plasma EL mass and activity must be determined in detail in further studies.…”

“…EL-IP was performed using a monoclonal IgG antibody against human full-length EL proteins raised in mice (clone 26A1, Immuno-Biological Laboratories, Fujioka, Gunma, Japan) that reacts with the amino terminus of EL proteins 12) . 26A1 mouse IgG (10 μL, 1 μg) was crosslinked onto 40 μL of nProtein A and incubated overnight with the 300-μL conditioned plasma samples to immunoprecipitate EL.…”

“…Several studies have documented a significant inverse correlation between the plasma EL mass and the HDL-C level in humans 9,10,12) . However, the plasma EL mass in these studies showed a large amount (＞100 times) of variation, in contrast to the small amount of variation observed in the HDL-C level (25-100 mg/dL).…”

“…As a result, EL promotes the catabolism and remodeling of HDL particles and has a major influence on HDL metabolism, both in humans and mice [5][6][7][8][9][10] . Moreover, the EL concentrations are increased in patients with metabolic syndrome and inflammation and are associated with the development of coronary atherosclerosis [10][11][12] . On the other hand, high serum LDL-C concentration, according to the Japan Atherosclerosis Society Guidelines for the Prevention of Atherosclerotic Cardiovascular Diseases.…”