Fluorescence assay for measuring the rate of cholesterol ester transfer

1. A method of measuring fluorescence, wherein said fluorescence is used to measure the CETP catalyzed transfer of a non-polar fluorescent ester between lipoprotein particles, comprising the steps of:

(A) Preparing a suspension comprising:

(1) A synthetic donor lipoprotein particle which comprises (a) a lipophilic non-polar cholesteryl ester of a fluorescent molecule which comprises the fluorescent heterocyclic BODIPY ring system, (b) an apolipoprotein, (c) a fluorescence quencher, said fluorescence quencher being effective in quenching fluorescence emitted by said lipophilic non-polar fluorescent ester, stable to diffusion, and not diffused by CETP; (d) a triglyceride, and (e) a phospholipid;

(2) An acceptor lipoprotein particle selected from the group consisting of a native lipoprotein particle and a synthetic high density lipoprotein particle; and

(3) A buffered solution which comprises CETP;

(B) Irradiating said suspension with light which stimulates fluorescence from said fluorescent ester; and

(C) Measuring the fluorescence from said fluorescent ester at one or more times after said suspension is prepared.

2. The method of measuring fluorescence as recited in claim 1 , wherein said lipophilic non-polar fluorescent ester is BODIPY-CE.

3. The method of measuring fluorescence as recited in claim 1 , wherein said synthetic donor particle is a synthetic HDL particle and said acceptor particle is selected from the group consisting of a native HDL particle, a synthetic HDL particle, a native LDL particle, a native VLDL particle, and a native IDL particle.

4. The method of measuring fluorescence as recited in claim 1 , wherein said synthetic donor particle is a synthetic HDL particle and said acceptor particle is a native LDL particle.

5. The method of measuring fluorescence as recited in claim 1 , wherein said fluorescence quencher is effective in quenching >85% of the fluorescence emitted by said lipophilic non-polar fluorescent ester, and is stable to diffusion so that >95% of said quencher remains in a donor particle for at least one month after said donor particle is prepared.

6. The method of measuring fluorescence as recited in claim 5 , wherein said fluorescence quencher is stable to diffusion so that >98% of said quencher remains in a donor particle for at least one month after said donor particle is prepared.

7. The method of measuring fluorescence as recited in claim 1 , wherein said buffered solution comprises native human CETP in plasma or serum.

8. The method of measuring fluorescence as recited in claim 1 , wherein said buffered solution comprises recombinant human CETP.

9. The method of measuring fluorescence as recited in claim 1 , wherein said donor particle is a synthetic HDL particle which comprises (a) BODIPY-CE, (b) apoHDL, (c) a fluorescence quencher, wherein said fluorescence quencher is effective in quenching >85% of the fluorescence emitted by said lipophilic non-polar fluorescent ester and is stable to diffusion so that >95% of said quencher remains in a donor particle at least one month after said donor particle is synthesized, (d) a triglyceride, and (e) a phospholipid;

said acceptor particle is a native LDL particle; and

said buffered solution comprises recombinant or native human CETP.

10. The method of measuring fluorescence as recited in claim 9 , wherein said fluorescence quencher is 4-(4-(dimethylaminophenyl)azo)benzoic acid dicetylamide.

11. A method of measuring the rate of CETP-catalyzed transfer of a lipophilic non-polar fluorescent cholesteryl ester between lipoprotein particles, comprising the steps of:

(A) Preparing a suspension which comprises:

(1) A synthetic donor lipoprotein particle which comprises (a) a lipophilic non-polar fluorescent cholesteryl ester containing a BODIPY group, (b) an apolipoprotein, (c) a fluorescence quencher, said fluorescence quencher being effective in quenching the fluorescence emitted by said lipophilic non-polar fluorescent ester, stable to diffusion, and not diffused by CETP, (d) a triglyceride, and (e) a phospholipid;

(2) An acceptor lipoprotein particle selected from a native lipoprotein particle and a synthetic high density lipoprotein particle; and

(3) A buffered solution which comprises CETP;

(B) Irradiating said suspension with light at a wavelength which stimulates fluorescence from said fluorescent ester;

(C) Measuring the fluorescence from said fluorescent ester at one or more times after said suspension is prepared; and

(D) Computing the rate of transfer of said cholesteryl ester containing a BODIPY group from said donor particle using the fluorescence measurements of step (C).

12. The method of measuring the rate of CETP-catalyzed transfer of a lipophilic non-polar fluorescent cholesteryl ester between lipoprotein particles as recited in claim 11 , wherein

said synthetic donor lipoprotein particle is a synthetic HDL particle which comprises apoHDL as the apolipoprotein;

said lipophilic non-polar fluorescent cholesteryl ester is BODIPY-CE; and

said acceptor lipoprotein particle is native LDL.

13. The method of measuring the rate of CETP-catalyzed transfer of a lipophilic non-polar fluorescent cholesteryl ester between lipoprotein particles as recited in claim 12 , wherein said fluorescence quencher is 4-(4-(dimethylaminophenyl)azo)benzoic acid dicetylamide.