Methods, compositions and kits for assaying mitochondrial function

Patent metadata

Field	Value
Publication number	US-9513281-B2
Application number	US-201414519119-A
Country	US
Kind code	B2
Filing date	Oct 20, 2014
Priority date	Apr 8, 2011
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

The invention provides methods, compositions, devices, and kits relating to the use of cholesterol-dependent cytolysins (e.g., PFOs) for measuring intracellular mitochondrial activity.

First claim

What is claimed is:

1. A kit comprising:
a) container housing a purified cholesterol-dependent cytolysin; and
b) a container housing a reagent for evaluating an intracellular function of a cell or a cellular-respiration effector.

2. The kit of claim 1 , wherein the intracellular function is a mitochondrial function.

3. The kit of claim 1 , wherein the cellular-respiration effector is a nucleotide or a protonophore.

4. The kit of claim 3 , wherein the nucleotide is adenosine diphosphate (ADP).

5. The kit of claim 3 , wherein the protonophore is cyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

6. The kit of claim 3 , further comprising a container housing glutamate, pyruvate, malate, isocitrate, alpha-ketoglutarate or a combination thereof.

7. The kit of claim 3 , further comprising a container housing succinate, Glycerol 3-phosphate, N,N,N′,N′-Tetramethyl-p-Phenylenediamine (TMPD), or ascorbate.

8. The kit of claim 1 , further comprising a container housing a respiratory chain inhibitor, a oxidative phosphorylation inhibitor, an uncoupling agent, a transport inhibitor, an ionophore or a krebs cycle inhibitor.

9. The kit of claim 1 , further comprising a container housing rotenone, malonate, antimycin A, KCN, or oligomycin.

10. The kit of claim 1 , wherein the cholesterol-dependent cytolysin is Perfringolysin O (PFO).

11. The kit of claim 10 , wherein the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2.

12. The kit of claim 10 , wherein the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a cysteine to alanine substitution at amino acid position 459.

13. The kit of claim 10 , wherein the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a threonine to cysteine substitution at amino acid position 319 and a valine to cysteine substitution at amino acid position 334.

Assignees

• Univ Massachusetts
• Baystate Medical Center Inc

Inventors

• Yadava Nagendra
• Heuck Alejandro Pablo
• Kim Chul

Classifications

• G01N33/5079 Primary

  Mitochondria · CPC title

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• C07K14/33 Primary

  from Clostridium (G) · CPC title

• G01N2333/33

  from Clostridium (G) · CPC title