ELISA system for human endothelial lipase.
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.
Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan. ishida@med.kobe-u.ac.jp
Tatsuro
T
Ishida
Kazuya
K
Miyashita
Mamoru
M
Shimizu
Noriaki
N
Kinoshita
Kenta
K
Mori
Li
L
Sun
Tomoyuki
T
Yasuda
Shigeyuki
S
Imamura
Katsuyuki
K
Nakajima
Kimber L
KL
Stanhope
Peter J
PJ
Havel
Ken-ichi
K
Hirata
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D008076:Cholesterol, HDL / Q000097:blood; D004796:Clinical Enzyme Tests; D003327:Coronary Disease / Q000175:diagnosis / Q000201:enzymology; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008049:Lipase / Q000097:blood* / Q000276:immunology; D051379:Mice; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D008076:Cholesterol, HDL; D011994:Recombinant Proteins; C410382:LIPG protein, human; D008049:Lipase