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US 10,920,264 B2Granted
Acyl-CoA dehydrogenases micro/nano enzyme assay
AssigneeUniversity of Pittsburgh—Of the Commonwealth System of Higher Education
InventorsAl-Walid A. Mohsen, Gerard Vockley
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Abstract
Provided herein are methods of measuring acyl-CoA dehydrogenase activity in a biological sample in a multiwell microplate setting.
Claims18
A method of quantifying acyl-coenzyme A dehydrogenase (ACAD) activity in a cell sample comprising:
- a. measuring a baseline fluorescence of an electron transfer flavoprotein (ETF) in a reaction mixture comprising lysed cells of the cell sample in an open-to-air well of a multi-well vessel by exposing the ETF to light of an excitatory wavelength and measuring fluorescence at an emission wavelength;
- 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 by comparing the baseline fluorescence of the ETF to the fluorescence of the ETF at the 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, and
- wherein the reaction mixture of step a is subjected to deaeration prior to measuring the baseline fluorescence, and wherein measuring the baseline fluorescence, adding a CoA substrate, and monitoring the change in fluorescence are carried out in an air-containing environment.
3 more elements
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 and subjecting the reaction mixture to a deaeration process;
- c. adding an electron transfer flavoprotein (ETF) to the reaction mixture;
- d. exposing the reaction mixture to an air-containing environment;
- e. measuring baseline fluorescence of an electron transfer flavoprotein (ETF) in the reaction mixture by exposing the ETF to light of an excitatory wavelength and measuring fluorescence at an emission wavelength;
- 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 by comparing the baseline fluorescence of the ETF to the fluorescence of the ETF at the 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.
6 more elements
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 and subjecting the reaction mixture to a deaeration process;
- 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. exposing the reaction mixture to an air-containing environment;
- f. measuring baseline fluorescence of an electron transfer flavoprotein (ETF) in the reaction mixture by exposing the ETF to light of an excitatory wavelength and measuring fluorescence at an emission wavelength;
- 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 by comparing the baseline fluorescence of the ETF to the fluorescence of the ETF at the 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.
7 more elements
Dependencies are inferred from the claim text.