Alt activity Assay protocol (Colorimetric Detection)

ALT Activity Assay protocol

(Colorimetric Detection)

—According to SIGMA ALDRICH^® ALT Activity Assay Kit (MAK052) Product Information

1. 
   Reagent preparation:
   

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.

2. Procedure

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).

Table 1.

Master Reaction Mix

Reagent	Volume
ALT Assay Buffer	86 μL
Fluorescent Peroxidase Substrate	2 μL
ALT Enzyme Mix	2 μL
ALT Substrate	10 μL

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 (T_initial). 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

the incubation.

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 T_final.

⑦. Calculate the change in measurement from T_initial to T_final.

ΔA570 = (A570)_final – (A570)_initial

Note: It is essential the initial and final measurements fall within the linear range of the reaction.

3. Results

Calculations

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 T_initial and T_final(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:

ALT Activity = [B × Sample Dilution Factor] / [ (T_final – T_initial) × V]

B = Amount (nmole) of pyruvate generated between T_initial and T_final

T_initial = Time of first reading in minutes.

T_final = Time of penultimate reading in minutes.

V = sample volume (mL) added to well.

ALT activity reported as nmole/min/mL = milliunit/mL,

where one milliunit (mU) of ALT is defined as the amount of enzyme that generates 1.0 μmole of pyruvate per minute at 37 °C.