ELISA (Enzyme-Linked Immunosorbent Assay) to measure Acetyl Coenzyme A (Acetyl CoA) levels:

Materials Needed:

1. Coating antibody specific to Acetyl CoA
2. Blocking buffer (e.g., BSA or milk)
3. Acetyl CoA standards (prepared by serial dilution)
4. Samples containing Acetyl CoA (e.g., cell lysates, tissue extracts, or biological fluids)
5. Detection antibody specific to Acetyl CoA (conjugated to an enzyme)
6. Substrate solution for the enzyme conjugate (e.g., TMB substrate)
7. Stop solution (e.g., sulfuric acid or HCl)
8. Wash buffer (e.g., PBS-Tween)

Protocol:

1. Coating the Plate:

   • Dilute the coating antibody specific to Acetyl CoA in coating buffer and add it to the wells of a microtiter plate. Incubate overnight at 4°C or for 2 hours at room temperature.

2. Blocking:

   • Wash the plate with wash buffer to remove unbound antibody.
   • Block nonspecific binding sites on the plate by adding blocking buffer to each well. Incubate for 1 hour at room temperature or overnight at 4°C.

3. Sample and Standard Addition:

   • Prepare Acetyl CoA standards by serial dilution in sample diluent.
   • Add standards and samples (diluted in sample diluent) to the plate wells. Include blank wells with sample diluent only.
   • Incubate the plate for 2 hours at room temperature.

4. Washing:

   • Wash the plate 3-5 times with wash buffer to remove unbound analyte and antibodies.

5. Detection:

   • Add the detection antibody specific to Acetyl CoA (conjugated to an enzyme) to each well. Incubate for 1 hour at room temperature.

6. Washing:

   • Wash the plate again to remove unbound detection antibody.

7. Signal Development:

   • Add substrate solution to each well. Incubate for a specific time period (e.g., 15-30 minutes) until the color develops.
   • Monitor color development and stop the reaction by adding stop solution when the desired intensity is reached.

8. Measurement:

   • Measure the absorbance of each well at the appropriate wavelength (typically 450 nm) using a microplate reader.

9. Data Analysis:

   • Generate a standard curve using the absorbance values of the standards to determine the concentration of Acetyl CoA in the samples.
   • Calculate the concentration of Acetyl CoA in the samples based on the standard curve.

This protocol provides a basic guideline for performing an Acetyl CoA ELISA. Optimization of the assay conditions (e.g., antibody concentrations, incubation times)