title: A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology.
abstract: Cholesteryl ester transfer protein (CETP) is a serum component responsible for both cholesteryl ester and triglyceride trafficking between high-density lipoprotein (HDL) and the apolipoprotein B (apoB)-containing very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). Several fluorescence-based assays that monitor these transfers have been reported, but to date such assays have suffered from a low signal/background (S/B) ratio and have been described for use only in relatively purified in vitro systems. We have modified the more advanced of these assays to incorporate a noninterfering, nondiffusable fluorescence quencher into previously described cosonicate particles, often referred to as microemulsions. This simple improvement resulted in particles that had an average threefold enhanced S/B window over particles without quenchers but that continued to show the essential properties of a catalytic assay, including catalysis to a single endpoint, excellent linearity with protein and particle concentration, and an appropriate sensitivity to inhibition. This reduced assay noise allowed the subsequent development of protocols for the direct measure of cholesteryl ester (CE) transfer activity resident in human and animal serum as well as the development of 384- and 3456-well screening protocols with good precision and accuracy. Thus, by expanding the dynamic response window of the assay, we have created an assay generalizable to many settings.
authors.affiliation: Department of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Suzanne S
authors.initials: SS
authors.lastname: Eveland
authors.forename: Denise P
authors.initials: DP
authors.lastname: Milot
authors.forename: Qiu
authors.initials: Q
authors.lastname: Guo
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Sheryl A
authors.initials: SA
authors.lastname: Hyland
authors.forename: Laurence B
authors.initials: LB
authors.lastname: Peterson
authors.forename: Sylvie
authors.initials: S
authors.lastname: Jezequel-Sur
authors.forename: Gregory T
authors.initials: GT
authors.lastname: O'Donnell
authors.forename: Paul D
authors.initials: PD
authors.lastname: Zuck
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: John A
authors.initials: JA
authors.lastname: Wagner
authors.forename: Wesley K
authors.initials: WK
authors.lastname: Tanaka
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Hilliard
authors.forename: Omar
authors.initials: O
authors.lastname: Laterza
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Wright
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Matt S
authors.initials: MS
authors.lastname: Anderson
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D053480:Cholesterol Ester Transfer Proteins / Q000097:blood* / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008954:Models, Biological; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection
chemical_list: D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D005456:Fluorescent Dyes