A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology.
Anal Biochem
; 368(2): 239-49, 2007 Sep 15.
Article
em En
| MEDLINE
| ID: mdl-17601482
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Bioensaio
/
Proteínas de Transferência de Ésteres de Colesterol
/
Corantes Fluorescentes
Tipo de estudo:
Evaluation_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Estados Unidos