Reactive oxygen species (ROS) mainly include hydroxyl radicals, superoxide radicals, and hydrogen peroxide. ROS are produced during normal physiological metabolism in cells or tissues, and can also be induced by certain environmental factors such as UV irradiation, γ‑ray irradiation, smoking, and environmental pollution. Once generated, ROS can cause oxidative damage to intracellular lipids, proteins, and DNA, inducing oxidative stress, which in turn leads to various tumors, atherosclerosis, rheumatoid arthritis, diabetes, liver injury, and central nervous system diseases. The body contains a variety of antioxidants, including antioxidant macromolecules, small‑molecule antioxidants, and enzymes, which can scavenge various ROS generated in vivo to prevent ROS‑induced oxidative stress. The total level of all these antioxidant macromolecules, small‑molecule antioxidants, and enzymes in a system reflects the total antioxidant capacity of that system. Therefore, measuring the total antioxidant capacity in various biological fluids such as plasma, serum, urine, and saliva, as well as in cell or tissue lysates, is of great biological significance.
Under the action of a suitable oxidant, ABTS is oxidized to the green‑colored ABTS•⁺. Its formation is inhibited in the presence of antioxidants. The total antioxidant capacity of the sample can be determined and calculated by measuring the absorbance at 414 nm. Trolox is an analog of vitamin E with antioxidant capacity similar to that of vitamin E, and is used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is defined as 1; at the same concentration, the total antioxidant capacity of other substances is expressed as the corresponding multiple of their antioxidant capacity relative to that of Trolox. This kit is suitable for detecting total antioxidant capacity in liquid samples such as serum (plasma), as well as in animal and plant tissues, cells, and other samples.
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 100T | Storage | Quantity Per Test | | T1508390WT | Total Antioxidant Capacity Rapid Detection Kit (ABTS, Micro Method)
(2-8℃ Components) | T1508390A | Buffer Solution | liquid | 24 mL | 2-8℃ | 152 μL |
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| T1508390B | ABTS Solution | liquid | 1 mL | 2-8℃. Protect from light | 10 μL |
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| T1508390C | H₂O₂ Solution | liquid | 0.5 mL | 2-8℃. | As needed |
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| T1508390D | Peroxidase | liquid | 0.2 mL | 2-8℃. | 2 μL | | T1508390DT | Total Antioxidant Capacity Rapid Detection Kit (ABTS, Micro Method)
(-20℃ Components) | T1508390E | Trolox Standard (5mM) | liquid | 0.6 mL | -20℃. Protect from light | As needed |
Notes: Each component is individually packaged according to its storage temperature (2-8°C /-20°C) upon shipment, with differentiated temperature-controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches: please refer to the actual received goods.Upon receipt, store components separately at the temperatures specified in the component list, The shelf-life is 12 months from the date of manufacture. |
Materials Required (Not Provided)
Instrument: Microplate reader (405-425 nm)
Reagents: Double-distilled water, PBS, or HBSS solution.
Protocol
1. Dilution of Hydrogen Peroxide Solution and Peroxidase, and Preparation of ABTS Working Solution
1.1 Dilute the hydrogen peroxide solution 800‑fold with double‑distilled water. For example, take 1 μL of hydrogen peroxide solution and add it to 799 μL of double‑distilled water; mix well to obtain a 1/800 hydrogen peroxide solution.
1.2 According to the number of samples to be tested, dilute an appropriate amount of peroxidase 10‑fold with Buffer Solution. For example, take 20 μL of peroxidase and add it to 180 μL of Buffer Solution; mix well to prepare 200 μL of Peroxidase Working Solution. The Peroxidase Working Solution should be prepared fresh before use.
1.3 Refer to the table below and prepare an appropriate amount of ABTS Working Solution according to the number of samples to be tested (including the standard curve).
| Component | 1 assay | 5 assays | 10 assays | 20 assays | Buffer Solution
| 152 μL | 760 μL | 1520 μL | 3040 μL | | ABTS Solution | 10 μL | 50 μL | 100 μL | 200 μL | | 1/800 H₂O₂ Solution | 8 μL | 40 μL | 80 μL | 160 μL | | ABTS Working Solution | 170 μL | 850 μL | 1700 μL | 3400 μL |
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Note: After preparation, the ABTS Working Solution should be stored at room temperature protected from light and used within 30 minutes. If the absorbance value of the zero standard is low during detection, it indicates that the hydrogen peroxide may have partially degraded. In this case, the dilution factor of hydrogen peroxide can be adjusted to 500‑fold or lower.
2. Preparation of Samples to Be Tested
2.1 Liquid samples such as serum (plasma): Each sample requires 10 μL and can be used directly for measurement.
2.2 Cell samples: Collect approximately 1 × 10⁶ cells (accurate counting is not necessary; cells can be scraped directly, and trypsin or EDTA digestion should be avoided). Place the cells in 200 μL of ice‑cold PBS or HBSS solution, and homogenize or sonicate to thoroughly disrupt the cells and release antioxidants. Centrifuge at 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for subsequent measurement.
2.3 Tissue samples: Add 100 μL of ice‑cold PBS or HBSS solution per 10 mg of tissue, and homogenize or sonicate to thoroughly disrupt the tissue and release antioxidants. Centrifuge at 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for subsequent measurement. All the above operations must be performed at 4 °C or on ice.
Note 1: For plasma preparation, heparin or sodium citrate can be used as anticoagulant; EDTA is not recommended.
Note 2: If the supernatant of liquid samples such as serum (plasma) or prepared cell or tissue samples is not used immediately, it can be stored at –80 °C. Samples stored at –80 °C show no significant change in measured data for at least one month.
Note 3: When measuring the total antioxidant capacity of cell or tissue samples, the protein concentration should also be determined. Aladdin’s BCA Protein Assay Kit (B665595) is recommended. The final total antioxidant capacity obtained is usually expressed as total antioxidant capacity per mg of protein, with the unit μmol/mg.
3. Sample Dilution
3.1 Before formal testing, to ensure that the sample values fall within the linear range (0.04–1 mM), it is recommended to perform a preliminary experiment with multiple dilution factors to determine the approximate total antioxidant capacity of the sample. If the values are not within the linear range, adjust the dilution factor accordingly.
3.2 If sample dilution is required, serum (plasma) and other liquid samples are recommended to be diluted with distilled water or PBS, while cell and tissue samples are recommended to be diluted with the solution used for cell or tissue homogenization.
4. Preparation for Standard Curve Determination
Dilute the standard with distilled water or the sample preparation solution. For serum, plasma, saliva, or urine samples, dilute the standard directly with distilled water or PBS. For cell or tissue samples, dilute the standard with the solution used for cell or tissue homogenization. Refer to the table below to dilute the standard: dilute the 5 mM Trolox standard solution to 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1 mM.
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | | Standard Concentration (mmol/L) | 0.1 | 0.2 | 0.3 | 0.4 | 0.6 | 0.8 | 1 | | 5 mmol/L Standard (μL) | 4 | 8 | 12 | 16 | 24 | 32 | 40 | | Diluent (μL) | 196 | 192 | 188 | 184 | 176 | 168 | 160 |
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5. Sample Measurement
5.1 Add 20 μL of Peroxidase Working Solution to each detection well of a 96‑well plate.
5.2 Add 10 μL of distilled water, PBS, or another appropriate solution to the blank control wells; add 10 μL of Trolox standard solutions at various concentrations to the standard curve wells; add 10 μL of each sample to the sample wells. Mix gently.
5.3 Add 170 μL of ABTS Working Solution to each well and mix gently.
5.4 Incubate at room temperature for 6 minutes and then measure A414. If measurement at A414 is difficult, it can also be measured within the range of 405–425 nm.
6. Calculation of Results
6.1 Preparation of the standard curve
Record the OD value of the blank well as Abₗₐₙₖ, the OD value of standard well as Aₛₜₐₙdₐᵣd, and the OD value of sample well as Aₛₐₘₚₗₑ. Calculate: ΔAₛₐₘₚₗₑ = Abₗₐₙₖ – Aₛₐₘₚₗₑ, ΔAₛₜₐₙdₐᵣd = Abₗₐₙₖ – Aₛₜₐₙdₐᵣd. Plot the standard curve using the standard concentrations as the x‑axis and the corresponding ΔAₛₜₐₙdₐᵣd values as the y‑axis. Obtain the linear regression equation, and then substitute ΔAₛₐₘₚₗₑ into the equation to obtain the total antioxidant capacity (T‑AOCₛₐₘₚₗₑ ) of the sample well (in mmol/L).
6.2 Calculation of total antioxidant capacity:
6.2.1 For liquid samples such as serum/plasma: Total antioxidant capacity (mmol/L) = T‑AOCₛₐₘₚₗₑ × f
6.2.2 For cell and tissue samples: Total antioxidant capacity (μmol/mg protein) = T‑AOCₛₐₘₚₗₑ × f ÷ Cₚᵣ
Notes:
f: dilution factor of the sample before addition to the assay system;
Cₚᵣ : protein concentration of the sample (mg/mL).
Precautions
1. Do not add substances that affect redox reactions, such as DTT or β‑mercaptoethanol, to the samples. Detergents such as Tween, Triton, and NP‑40 should also be avoided.
2. A microplate reader capable of measuring A414 (or 405–425 nm) or a spectrophotometer suitable for micro‑volume samples is required for the measurement.
3. If the absorbance value of the zero standard is low (e.g., below 1.5), it indicates that the hydrogen peroxide may have partially degraded. In this case, the dilution factor of hydrogen peroxide can be adjusted to 500‑fold or lower.
4. ABTS is irritating to the human body. Handle with care and take appropriate protective measures to avoid direct contact with the body or inhalation.
5. This product is for research use by professionals only. It must not be used for clinical diagnosis or treatment, nor for food or pharmaceutical products, and must not be stored in ordinary residential premises.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
Results Presentation
Typical standard curve:
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