• Pre-coated 96-Well Microplate
• Standard
• Standard Diluent Buffer
• Wash Buffer
• Detection Reagent A
• Detection Reagent B
• Diluent A
• Diluent B
• TMB Substrate
• Stop Solution
• Plate Sealer

• 37°C incubator
• Multi and single channel pipettes and sterile pipette tips
• Squirt bottle or automated microplate washer
• 1.5 ml tubes
• Distilled water
• Absorbent filter papers
• 100 ml and 1 liter graduated cylinders
• Microplate reader (wavelength: 450 nm)
• ELISA Shaker

• 1) Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the Standard Diluent buffer to carry out serial dilutions of the standard solution, as instructed in the Protocol.

• 2) Wash Buffer: Dilute the concentrated Wash Buffer with distilled water, as instructed in the Protocol.

• 3) Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, at 1:100.

• 1) Set standard, test samples and control wells.

• 2) Aliquot 100 µl of diluted standard into the standard wells.

• 3) Aliquot 100 µl of Standard Diluent buffer into control (zero) well.

• 4) Aliquot 100 µl of diluted samples into the sample wells. Incubate for 90 mins at 37 °C.

• 5) Aliquot 100 µl of Detection Reagent A to each well. Incubate for 1 hr at 37 °C.

• 6) Wash 3 times.

• 7) Aliquot 100 µl of Detection Reagent B to each well. Incubate for 30 mins at 37 °C.

• 8) Wash 5 times.

• 9) Aliquot 90 µl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C.

• 10) Aliquot 50 µl of Stop Solution.

• 11) Measure the OD at 450 nm.

• Equilibrate the kit components and samples to room temperature (18 – 25 °C) before use. It is recommended to plot a standard curve for each test.

• 1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample at least in duplicate.

• 2. Add 100 µL of each standard, control and sample into the appropriate wells. Seal the plate with a cover and incubate for 1 h at 37°C.

• 3. Remove the cover and discard the liquid.

• 4. Add 100 µl of the detection Reagent A working solution to each well. Seal the plate with a cover and incubate for 1 h at 37°C.

• 5. Remove the cover and discard the solution. Wash the plate 3 times with 1X Wash Buffer.

• 6. Add 100 µL of Detection Reagent B working solution into each well, seal and incubate at 37°C for 30 min.

• 7. Discard the solution and wash the plate 5 times with wash buffer as explained in previous step.

• 8. Aliquot 90 µl of TMB Substrate into each well. Seal the plate with a cover and incubate at 37°C for 10-20 min. Avoid exposure to light. The incubation time is for reference use only, the optimal time should be determined by end user. Do not exceed 30 min.

• 9. Add 50 µL of Stop Solution to each well. Read at 450 nm immediately.

Intra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of AICA Riboside / Acadesine (AICAR) were were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of AICA Riboside / Acadesine (AICAR) were tested on 3 different plates, 8 replicates in each plate.

CV (%) = (Standard Deviation / mean) × 100

Intra-Assay: CV<10%

Inter-Assay: CV<10%