Total Antioxidant Capacity Rapid Detection Kit (ABTS) FAQ

Total Antioxidant Capacity Rapid Detection Kit (ABTS, Micro Method) is a sensitive, miniaturized colorimetric assay kit designed to quantify the total antioxidant capacity of biological and food samples by measuring ABTS radical scavenging activity in a microplate format.

SKU

T1508390

Detection Method

ABTS radical scavenging colorimetric assay

Format

Microplate (micro method)

Application

Quantification of total antioxidant capacity in serum, plasma, tissue, cell lysates, and food/plant extracts

Measurement Wavelength

734 nm (typical for ABTS assay)

Supplier

Aladdin Scientific

Assay Principle and Overview

The Total Antioxidant Capacity Rapid Detection Kit (ABTS, Micro Method) is based on the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging method. In this assay, ABTS is oxidized to form the stable blue-green ABTS radical cation (ABTS•⁺). Antioxidants present in the sample reduce and decolorize the ABTS•⁺ radical in proportion to their concentration and antioxidant potency. The degree of decolorization is measured spectrophotometrically, typically at 734 nm, and total antioxidant capacity is expressed relative to a standard (commonly Trolox, a water-soluble vitamin E analogue), reported as TEAC (Trolox Equivalent Antioxidant Capacity).

The micro method format miniaturizes the assay for use with standard 96-well microplates, reducing reagent and sample consumption while enabling high-throughput processing. This makes the kit particularly suitable for laboratories working with limited or precious sample volumes.

Suitable Sample Types and Preparation

This kit is compatible with a wide range of biological and non-biological sample matrices, including:

• Serum and plasma
• Urine
• Tissue homogenates
• Cell lysates
• Plant and food extracts
• Beverages and other aqueous solutions

Proper sample preparation is critical for accurate results. Key recommendations include:

• Avoid hemolysis in blood-derived samples, as red blood cell components can interfere with absorbance readings.
• Deproteinization may be necessary for samples with high protein content to reduce background interference.
• Dilution: Samples with very high antioxidant capacity should be diluted within the linear range of the standard curve before measurement.
• Fresh samples are preferred; freeze-thaw cycles should be minimized. If storage is necessary, snap-freeze samples in liquid nitrogen and store at −80 °C.
• Lipid-rich samples may require additional processing steps to ensure complete solubilization and accurate measurement.

Step-by-Step Protocol Summary

The following is a general protocol outline for the ABTS Micro Method assay. Always refer to the official kit insert for exact volumes, incubation times, and reagent preparation instructions specific to lot T1508390.

1. Reagent preparation: Allow all kit components to equilibrate to room temperature. Prepare ABTS working solution according to kit instructions (typically by mixing ABTS stock with an oxidant such as potassium persulfate and allowing the reaction to proceed in the dark).
2. Standard curve preparation: Prepare a series of Trolox standard dilutions to generate a calibration curve covering the expected range of sample antioxidant capacity.
3. Sample and standard loading: Pipette standards, blank, and samples into designated microplate wells using the volumes specified in the kit insert.
4. Reagent addition: Add ABTS working solution to each well. Mix gently.
5. Incubation: Incubate the plate at the specified temperature and time (typically room temperature, protected from light).
6. Absorbance measurement: Read absorbance at 734 nm using a microplate reader.
7. Calculation: Determine sample antioxidant capacity from the Trolox standard curve and express results as mmol Trolox equivalents per liter (or per gram of tissue/protein, depending on sample type).

Storage, Stability, and Handling Precautions

Proper storage and handling of the kit components are essential to maintain reagent performance and assay reliability.

• Storage temperature: Refer to the kit insert for component-specific storage requirements. Typically, ABTS reagents should be stored at 2–8 °C or −20 °C (as specified per component), protected from light.
• Stability: Do not use reagents beyond the expiration date printed on the kit label. Degradation of ABTS radical solution can occur upon prolonged exposure to light, heat, or moisture.
• Avoid contamination: Use clean pipette tips for each reagent. Do not return unused working solution to the stock vial.
• Light sensitivity: ABTS radical cation is light-sensitive. Prepare working solutions immediately before use and keep plates covered during incubation.
• Safety: Handle all reagents according to standard laboratory safety practices. Consult the Safety Data Sheet (SDS) for each component before use.

Troubleshooting Common Issues

If unexpected results are obtained, consider the following troubleshooting guidance:

Problem	Possible Cause	Recommended Action
Low or no signal	ABTS working solution not properly prepared or degraded	Prepare fresh ABTS working solution; verify the blue-green color before use
High background / blank absorbance	Contamination of reagents or labware; light exposure of ABTS solution	Use fresh reagents; protect solutions from light throughout the assay
Non-linear standard curve	Incorrect Trolox dilutions; pipetting errors	Repeat standard preparation with calibrated pipettes; ensure accurate dilutions
High variability between replicates	Incomplete mixing; inconsistent incubation timing	Mix thoroughly after each addition; process all wells within the same time window
Sample readings exceed standard curve range	Sample antioxidant capacity too high	Dilute samples and re-assay; apply dilution factor to final calculation
Colored sample matrix interference	Intrinsic color of sample (e.g., plant extracts)	Run a sample blank (sample without ABTS reagent) and subtract background absorbance

Frequently asked questions

What is the detection principle of the ABTS Micro Method Total Antioxidant Capacity Kit?

The kit uses the ABTS radical scavenging principle: ABTS is oxidized to form the stable blue-green ABTS radical cation (ABTS•⁺), which is decolorized by antioxidants in the sample. The extent of decolorization, measured at 734 nm, is proportional to the total antioxidant capacity. Results are expressed as Trolox Equivalent Antioxidant Capacity (TEAC).

What sample types are compatible with this kit?

The kit is compatible with serum, plasma, urine, tissue homogenates, cell lysates, plant extracts, food extracts, and beverages. Samples should be fresh or properly frozen, and heavily pigmented or high-protein samples may require dilution or deproteinization prior to assay.

Why is my ABTS working solution not blue-green or losing color rapidly?

Loss of color in the ABTS working solution typically indicates degradation due to light exposure, contamination with antioxidants, or improper preparation. Ensure the ABTS radical solution is prepared strictly following the kit instructions, protected from light at all steps, and used within the recommended timeframe. If degradation is confirmed, prepare a fresh batch of working solution.

How should I express and calculate the final antioxidant capacity results?

Total antioxidant capacity is calculated by interpolating sample absorbance values against the Trolox standard curve. Results are typically expressed as mmol Trolox equivalents per liter (mmol TE/L) for liquid samples, or per gram of tissue or milligram of protein for solid samples. Apply any sample dilution factors to the final calculated value.

What are the recommended storage conditions for the kit components?

Storage conditions vary by component; please refer to the kit insert for component-specific instructions. In general, ABTS-based reagents should be stored protected from light at 2–8 °C or −20 °C as specified, and should not be used beyond the stated expiration date. Avoid repeated freeze-thaw cycles for frozen components.

References

1. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine. 1999;26(9-10):1231–1237. doi:10.1016/S0891-5849(98)00315-3
2. Aladdin Scientific. Product Insert: Total Antioxidant Capacity Rapid Detection Kit (ABTS, Micro Method), SKU T1508390.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.