# US 2018/0023113 A1 · Acyl-CoA Dehydrogenases Micro/Nano Enzyme Assay

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- Number: US 2018/0023113 A1
- Document ID: US20180023113A1
- Status: Published application
- Kind: A1
- Application number: 15/656,414
- Filed: 2017-07-21
- Published: 2018-01-25
- Earliest claimed: 2016-07-22
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh - Of the Commonwealth System of Higher Education
- Inventors: Al-Walid A. Mohsen; Gerard Vockley
- Web page: https://patentgrep.com/patent/US20180023113A1

## CPC

- C12Q 1/32: involving dehydrogenase
- C12Y 103/99003: Acyl-CoA dehydrogenase (1.3.99.3) (C12Y103/08007, C12Y103/08008 and C12Y103/08009 take precedence)
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 2800/04: Endocrine or metabolic disorders
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein are methods of measuring acyl-CoA dehydrogenase activity in a biological sample in a multiwell microplate setting.

## Claims

### Claim 1

A method of quantifying acyl-coenzyme A dehydrogenase (ACAD) activity in a cell sample comprising:
a. determining fluorescence of an electron transfer flavoprotein (ETF) in a reaction mixture comprising lysed cells of the cell sample in a well of a multi-well vessel in an anaerobic environment;
b. adding a coenzyme A (CoA) substrate for a specific ACAD to the reaction mixture; and
c. monitoring a change in fluorescence of the ETF at one or more time points after addition of the CoA substrate to the reaction mixture,
wherein a loss of fluorescence of the ETF indicates the presence of an ACAD in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cell sample is a human cell sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the cell sample is free of red blood cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein oxygen is removed from the reaction mixture in step a. by vacuum.

### Claim 5 (depends on 1)

The method of claim 1, wherein steps a. through c. are conducted under an inert gas environment.

### Claim 6 (depends on 1)

The method of claim 1, wherein the ACAD is a medium-chain specific acyl-CoA dehydrogenase, a very long chain specific acyl-CoA dehydrogenase, an isovaleryl-CoA dehydrogenase, a short-chain specific acyl-CoA dehydrogenase, a long-chain specific acyl-CoA dehydrogenase, an isobutyryl-CoA dehydrogenase, a short/branched-chain acyl-CoA dehydrogenase, an Acyl-CoA Dehydrogenase Family, Member 9, or a glutaryl-CoA dehydrogenase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the ETF is a porcine ETF.

### Claim 8 (depends on 1)

The method of claim 1, wherein the reaction mixture in at least one well further comprises a drug that may affect ACAD activity, and wherein a difference in a change in fluorescence of the ETF in the at least one well in which the drug is present compared to one or more wells in which the drug is not present indicates differences in ACAD activity.

### Claim 9 (depends on 1)

The method of claim 1, wherein:
step b. further comprises adding a drug that may affect ACAD activity to at least one well, and wherein a difference in a change in fluorescence of the ETF in the at least one well in which the drug is present compared to one or more wells in which the drug is not present indicates differences in ACAD activity; and/or
the lysed cells in a well are contacted with a drug that may affect ACAD activity prior to lysis, and wherein a difference in a change in fluorescence of the ETF in the at least one well in which the lysed cells were contacted with the drug prior to lysis compared to one or more wells in which the lysed cells were not contacted with the drug prior to lysis indicates differences in ACAD activity.

### Claim 10

A method of quantifying acyl-coenzyme A dehydrogenase (ACAD) activity in a cell sample comprising:
a. adding the cell sample to a well of a multi-well vessel;
b. adding a lysis solution to the well to form a reaction mixture;
c. adding an electron transfer flavoprotein (ETF) to the reaction mixture;
d. removing oxygen from the reaction mixture;
e. determining fluorescence of an electron transfer flavoprotein (ETF) in the reaction mixture;
f. adding a coenzyme A (CoA) substrate for a specific ACAD to the reaction mixture; and
g. monitoring a change in fluorescence of the ETF at one or more time points after addition of the CoA substrate to the reaction mixture,
wherein a loss of fluorescence of the ETF indicates the presence of an ACAD in the sample.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cell sample is free of red blood cells.

### Claim 12 (depends on 10)

The method of claim 10, wherein steps e. through g. are conducted under an inert gas environment.

### Claim 13 (depends on 10)

The method of claim 10, wherein oxygen is removed from the reaction mixture by vacuum.

### Claim 14 (depends on 10)

The method of claim 10, wherein the lysis solution comprises a detergent, glucose, and a salt.

### Claim 15 (depends on 10)

The method of claim 10, wherein the ACAD is a medium-chain specific acyl-CoA dehydrogenase, a very long chain specific acyl-CoA dehydrogenase, an isovaleryl-CoA dehydrogenase, a short-chain specific acyl-CoA dehydrogenase, a long-chain specific acyl-CoA dehydrogenase, an isobutyryl-CoA dehydrogenase, a short/branched-chain acyl-CoA dehydrogenase, an Acyl-CoA Dehydrogenase Family, Member 9, or a glutaryl-CoA dehydrogenase.

### Claim 16 (depends on 10)

The method of claim 10, wherein the ETF is a porcine ETF.

### Claim 17 (depends on 10)

The method of claim 10, wherein a drug that affects ACAD activity is added to at least one well containing the reaction mixture in one of steps a., b., or c., and wherein a difference in a change in fluorescence of the ETF in the at least one well in which the drug is present compared to one or more wells in which the drug is not present indicates differences in ACAD activity.

### Claim 18 (depends on 10)

The method of claim 10, wherein a drug that affects ACAD activity is added to at least one well containing the reaction mixture in step f., and wherein a difference in a change in fluorescence of the ETF in the at least one well in which the drug is present compared to one or more wells in which the drug is not present indicates differences in ACAD activity.

### Claim 19

A method of quantifying acyl-coenzyme A dehydrogenase (ACAD) activity in a cell sample comprising:
a. adding the cell sample, comprising at least one ACAD, to a plurality of wells of a multi-well vessel;
b. adding a lysis solution to the plurality of wells to form a reaction mixture;
c. adding an electron transfer flavoprotein (ETF) to the reaction mixture;
d. adding a drug that may affect ACAD activity to at least one, but not all, of the plurality of wells containing the reaction mixture;
e. removing oxygen from the reaction mixture;
f. determining fluorescence of an electron transfer flavoprotein (ETF) in the reaction mixture;
g. adding a coenzyme A (CoA) substrate for the ACAD to the reaction mixture to initiate reaction; and
h. monitoring a change in fluorescence of the ETF at one or more time points after addition of the CoA substrate to the reaction mixture,
wherein a difference in a change in fluorescence of the ETF in the at least one well in which the drug is present compared to one or more wells in which the drug is not present indicates differences in ACAD activity.

### Claim 20 (depends on 19)

The method of claim 19, wherein the cell sample is derived from a lymphoblast or lymphocyte cell line.