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An ELISA method to measure inhibition of the COX enzymes

Nature Protocols volume 1, pages 1915–1921 (2006)Cite this article

Abstract

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).

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Figure 1: Overview of the metabolism of COX.
Figure 2: Determination of COX activity by measuring PGE2 production.
Figure 3
Figure 4: Concentration-dependent COX inhibition by indomethacin and celecoxib.

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Acknowledgements

The authors are grateful to the National Cancer Institute for support provided under the auspices of program project P01 CA48112 entitled “Natural Inhibitors of Carcinogenesis” and the Fogarty International Center for support provided under the auspices of an International Cooperative Biodiversity Group (ICBG) “Studies on Biodiversity of Vietnam and Laos.”

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Author notes

  1. John M Pezzuto

    Present address: University of Hawaii at Hilo, College of Pharmacy, Hilo, Hawaii, 96720, USA

Authors and Affiliations

  1. Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, College of Pharmacy, Nursing and Health Sciences, and The Purdue Cancer Center, Purdue University, West Lafayette, 47907, Indiana, USA

    Muriel Cuendet & John M Pezzuto

  2. Center for Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 60612, Illinois, USA

    Andrew D Mesecar

  3. Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, 48824, Michigan, USA

    David L DeWitt

Authors
  1. Muriel Cuendet
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  2. Andrew D Mesecar
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  3. David L DeWitt
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  4. John M Pezzuto
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Correspondence to John M Pezzuto.

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Cuendet, M., Mesecar, A., DeWitt, D. et al. An ELISA method to measure inhibition of the COX enzymes. Nat Protoc 1, 1915–1921 (2006). https://doi.org/10.1038/nprot.2006.308

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