A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology.
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.
Eveland
Suzanne S
SS
Department of Cardiovascular Diseases, Merck Research Laboratories, Rahway, NJ 07065, USA.
Milot
Denise P
DP
Guo
Qiu
Q
Chen
Ying
Y
Hyland
Sheryl A
SA
Peterson
Laurence B
LB
Jezequel-Sur
Sylvie
S
O'Donnell
Gregory T
GT
Zuck
Paul D
PD
Ferrer
Marc
M
Strulovici
Berta
B
Wagner
John A
JA
Tanaka
Wesley K
WK
Hilliard
Deborah A
DA
Laterza
Omar
O
Wright
Samuel D
SD
Sparrow
Carl P
CP
Anderson
Matt S
MS
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
D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D005456:Fluorescent Dyes