—According to SIGMA ALDRICH® ALT Activity Assay Kit (MAK052) Product Information
a. Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents.
b. ALT Assay Buffer – Allow buffer to come to room temperature before use.
c. Fluorescent Peroxidase Substrate – Allow reagent to come to room temperature before use. Mix well by pipetting, then aliquot and store, protected from light and moisture, at –20 °C.
d. ALT Enzyme Mix – Reconstitute in 220 μL of water. Mix well by pipetting, then aliquot and store at –20 °C. Use within two months of reconstitution.
e. ALT Substrate – Reconstitute in 1.1 mL of ALT Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Keep cold while in use. Use within two months of reconstitution.
f. ALT Positive Control – Reconstitute in 100 μL of water. Mix well by pipetting, then aliquot and store at–20 °C. Keep cold while in use. Use within two months of reconstitution.
a. All samples and standards should be run in duplicate.
b. Pyruvate Standards for Colorimetric Detection
Dilute 10 μL of the 100 nmole/μL Pyruvate Standard with 990 μL of ALT Assay Buffer to prepare a 1 nmole/μL standard solution. Add 0, 2, 4, 6, 8, and 10 μL of the 1 nmole/μL standard solution into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add ALT Assay Buffer to each well to bring the volume to 20 μL (Add 20,18,16,14,12,10 μL of ALT Assay Buffer respectively).
c. Sample Preparation
Add 1–20 μL of samples (serium or plasma) into wells of a 96 well plate. Bring samples to a final volume of 20 μL with ALT Assay Buffer. For unknown samples, it is suggested to test several sample volumes to make sure the readings are within the standard curve range.
For the positive control (optional), add 5 μL of the ALT Positive Control to wells. Adjust well volume to 20 μL with ALT Assay Buffer.
d. Assay Reaction
①. Set up the Master Reaction Mix according to the scheme in Table 1.
100 μL of the Master Reaction Mix is required for each reaction (well). Prepare enough Master Reaction Mix for the number of samples, positive controls, and standards to be performed (prepare 2 × the number of samples, positive controls, and standards because of being run in duplicate).
②. Add 100 μL of the Master Reaction Mix to each of the standard, positive control, and test wells. Mix well using a horizontal shaker or by pipetting.
③. After 2–3 minutes, take the initial measurement (Tinitial). For colorimetric assays, measure the
absorbance at 570 nm (A570)1.
④. Incubate the plate at 37 °C taking measurements every 5 minutes. Protect the plate from light during
⑤. Continue taking measurements until the value of the most active sample is greater than the value of the highest standard. At this time the most active sample is near or exceeds the end of the linear range of the standard curve.
⑥. The final measurement for calculating the enzyme activity would be the penultimate reading or the value before the most active sample is near or exceeds the end of the linear range of the standard curve. The time of the penultimate reading is Tfinal.
⑦. Calculate the change in measurement from Tinitial to Tfinal.
ΔA570 = (A570)final – (A570)initial
Note: It is essential the initial and final measurements fall within the linear range of the reaction.
Correct for the background by subtracting the value obtained for the 0 (blank) standard from all readings. Plot the pyruvate standard curve.
Compare the Δmeasurement value (ΔA570) of each sample to the standard curve to determine the amount of pyruvate generated between Tinitial and Tfinal(B).
Note: A new standard curve must be set up each time the assay is run.
The ALT activity of a sample may be determined by the following equation: