Assays for measuring binding kinetics and binding capacity of acceptors for lipophilic or amphilphilic molecules

Patent metadata

Field	Value
Publication number	US-2016274134-A1
Application number	US-201615073941-A
Country	US
Kind code	A1
Filing date	Mar 18, 2016
Priority date	Mar 18, 2015
Publication date	Sep 22, 2016
Grant date	—

Abstract

Aspects of the invention relate to methods for measuring the binding constant of a lipophilic or amphiphilic molecule acceptor for a lipophilic or amphiphilic molecule. Methods involve rapid, cell-free competition assays including a labeled lipophilic or amphiphilic molecule and nanoparticle.

First claim

What is claimed is:

1 . A method for measuring the equilibrium constant of an acceptor for a molecule, comprising:
(a) providing a lipophilic or amphiphilic molecule, wherein the molecule can provide a detectable signal; (b) providing a structure, the structure comprising a nanostructure core and a lipid layer surrounding and attached to the nanostructure core, wherein the structure quenches the signal of the molecule when the structure and the molecule are proximate; (c) providing an acceptor; (d) allowing the acceptor to compete with the structure for binding with the molecule; and (e) measuring the signal, wherein the level of the signal correlates with the equilibrium constant of the acceptor for the molecule.

2 . The method of claim 1 , the method further comprising increasing the amount of acceptor provided.

3 . The method of claim 1 , the method further comprising increasing the amount of structure provided.

4 . The method of claim 2 , wherein increasing the amount of acceptor provided leads to an increase in signal.

5 . The method of claim 3 , wherein increasing the amount of structure provided leads to a decrease in signal.

6 . The method of claim 1 , wherein increasing or decreasing the amount of molecule improves the signal to noise ratio of the system.

7 . The method of claim 1 , wherein the method is a component of an assay.

8 . The method of claim 1 , wherein the method is a component of a diagnostic assay.

9 . The method of claim 1 , wherein the method is a method for assessing cardiovascular risk in a subject.

10 . A kit for measuring an equilibrium constant of an acceptor for a molecule, comprising:
(a) a lipophilic or amphiphilic molecule, wherein the molecule can provide a detectable signal; and (b) a structure, the structure comprising a nanostructure core and a lipid layer surrounding and attached to the nanostructure core, wherein the structure quenches the signal of the molecule when the structure and the molecule are proximate.

11 . The kit of claim 10 , further comprising instructions for use of the kit for measuring the equilibrium constant of the acceptor for the molecule.

12 . A system for measuring an equilibrium constant of an acceptor for a molecule, comprising:
(a) a sample, the sample comprising:
(i) a lipophilic or amphiphilic molecule, wherein the molecule can provide a detectable signal;
(ii) a structure, the structure comprising a nanostructure core and a lipid layer surrounding and attached to the nanostructure core, wherein the structure quenches the signal of the molecule when the structure and the molecule are proximate;
(iii) an acceptor; and
(b) a detector for measuring the signal.

13 . The system of claim 12 , further comprising a device configured to calculate the equilibrium constant of the acceptor for the molecule based on the detected signal.

14 . The system of claim 12 , further comprising a radiation source configured to induce the signal.

15 . The method of claim 1 , wherein the lipid layer is a bilayer.

16 . The method of claim 1 , wherein the molecule is a steroid or a derivative or analog thereof.

17 . The method of claim 1 , wherein the molecule is a lipopolysaccharide or a derivative or analog thereof.

18 . The method of claim 1 , wherein the molecule is a Cholestane, Cholane, Pregnane, Androstane or Estrane or a derivative or analog thereof.

19 . The method of claim 1 , wherein the molecule is BODIPY-cholesterol.

20 . The method of claim 1 , wherein the signal is fluorescence.

21 . The method of claim 1 , wherein the signal is fluorescence polarization.

22 . The method of claim 1 , wherein the nanostructure core is an inorganic material.

23 . The method of claim 1 , wherein the nanostructure core is a metal.

24 . The method of claim 1 , wherein the nanostructure core is gold.

25 . The method of claim 1 , wherein the structure further comprises apolipoprotein bound to at least the outer surface of the lipid layer.

26 . The method of claim 25 , wherein the apolipoprotein is apolipoprotein A-I, apolipoprotein A-II, or apolipoprotein E.

27 . The method of claim 1 , wherein the acceptor is a lipoprotein.

28 . The method of claim 1 , wherein the acceptor is a high-density lipoprotein (HDL).

29 . The method of claim 1 , wherein the acceptor is a component of serum.

30 . The method of claim 29 , wherein the serum is human serum.

31 . The method of claim 29 , wherein the serum is enriched for HDL.

32 . The method of claim 29 , wherein the serum is depleted for ApoB.

Assignees

• Univ Northwestern

Inventors

• Mutharasan Raja Kannan
• Thaxton C Shad

Classifications

• G01N2333/775

  Apolipopeptides · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• G01N2800/324

  Coronary artery diseases, e.g. angina pectoris, myocardial infarction · CPC title

• G01N33/553

  Metal or metal coated · CPC title