ELISA system for human endothelial lipase

Tatsuro Ishida, Kazuya Miyashita, Mamoru Shimizu, Noriaki Kinoshita, Kenta Mori, Li Sun, Tomoyuki Yasuda, Shigeyuki Imamura, Katsuyuki Nakajima, Kimber Stanhope, Peter J Havel, Ken Ichi Hirata

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1656-1664
Number of pages9
JournalClinical Chemistry
Volume58
Issue number12
DOIs
StatePublished - Dec 2012

Fingerprint

Lipase
Enzyme-Linked Immunosorbent Assay
Serum
HDL Cholesterol
human LIPG protein
Metabolism
Atherosclerosis
Assays
Plasmas
Lipoprotein Lipase
Cricetulus
Recombinant Proteins
Calibration
Limit of Detection
Dilution
Ovary
Cardiovascular Diseases
Proteins
Monoclonal Antibodies

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Ishida, T., Miyashita, K., Shimizu, M., Kinoshita, N., Mori, K., Sun, L., ... Hirata, K. I. (2012). ELISA system for human endothelial lipase. Clinical Chemistry, 58(12), 1656-1664. https://doi.org/10.1373/clinchem.2012.187914

ELISA system for human endothelial lipase. / Ishida, Tatsuro; Miyashita, Kazuya; Shimizu, Mamoru; Kinoshita, Noriaki; Mori, Kenta; Sun, Li; Yasuda, Tomoyuki; Imamura, Shigeyuki; Nakajima, Katsuyuki; Stanhope, Kimber; Havel, Peter J; Hirata, Ken Ichi.

In: Clinical Chemistry, Vol. 58, No. 12, 12.2012, p. 1656-1664.

Research output: Contribution to journalArticle

Ishida, T, Miyashita, K, Shimizu, M, Kinoshita, N, Mori, K, Sun, L, Yasuda, T, Imamura, S, Nakajima, K, Stanhope, K, Havel, PJ & Hirata, KI 2012, 'ELISA system for human endothelial lipase', Clinical Chemistry, vol. 58, no. 12, pp. 1656-1664. https://doi.org/10.1373/clinchem.2012.187914
Ishida T, Miyashita K, Shimizu M, Kinoshita N, Mori K, Sun L et al. ELISA system for human endothelial lipase. Clinical Chemistry. 2012 Dec;58(12):1656-1664. https://doi.org/10.1373/clinchem.2012.187914
Ishida, Tatsuro ; Miyashita, Kazuya ; Shimizu, Mamoru ; Kinoshita, Noriaki ; Mori, Kenta ; Sun, Li ; Yasuda, Tomoyuki ; Imamura, Shigeyuki ; Nakajima, Katsuyuki ; Stanhope, Kimber ; Havel, Peter J ; Hirata, Ken Ichi. / ELISA system for human endothelial lipase. In: Clinical Chemistry. 2012 ; Vol. 58, No. 12. pp. 1656-1664.
@article{5a747681f33844ff82f3145732aae599,
title = "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.",
author = "Tatsuro Ishida and Kazuya Miyashita and Mamoru Shimizu and Noriaki Kinoshita and Kenta Mori and Li Sun and Tomoyuki Yasuda and Shigeyuki Imamura and Katsuyuki Nakajima and Kimber Stanhope and Havel, {Peter J} and Hirata, {Ken Ichi}",
year = "2012",
month = "12",
doi = "10.1373/clinchem.2012.187914",
language = "English (US)",
volume = "58",
pages = "1656--1664",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "12",

}

TY - JOUR

T1 - ELISA system for human endothelial lipase

AU - Ishida, Tatsuro

AU - Miyashita, Kazuya

AU - Shimizu, Mamoru

AU - Kinoshita, Noriaki

AU - Mori, Kenta

AU - Sun, Li

AU - Yasuda, Tomoyuki

AU - Imamura, Shigeyuki

AU - Nakajima, Katsuyuki

AU - Stanhope, Kimber

AU - Havel, Peter J

AU - Hirata, Ken Ichi

PY - 2012/12

Y1 - 2012/12

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84870324304&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84870324304&partnerID=8YFLogxK

U2 - 10.1373/clinchem.2012.187914

DO - 10.1373/clinchem.2012.187914

M3 - Article

VL - 58

SP - 1656

EP - 1664

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 12

ER -