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测定对象中各种抗氧化物质和抗氧化酶等构成总抗氧化水平。在生物学、医学和药学研究中常常检测血浆、血清、唾液、尿液等各种体液，细胞或组织等裂解液、植物或中草药抽提液及各种抗氧化物(antioxidant)溶液的总抗氧化能力。 测定原理：在酸性环境下，抗氧化物质还原Fe³⁺ -三吡啶三吖嗪(Fe³⁺-TPTZ)产生蓝色的Fe²⁺-TPTZ 的能力反映了总抗氧化能力。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 100T | 储存 |
| T1521765A | 提取液 | 液体 | 120mL | 2-8℃. |
| T1521765B | 试剂一 | 液体 | 20mL | 2-8℃.避光保存 |
| T1521765C | 试剂二 | 液体 | 2mL | 2-8℃.避光保存 |
| T1521765D | 试剂三 | 液体 | 2mL | 2-8℃.避光保存 |
| T1521765E | 标准品 | 粉末 | 10mg | 2-8℃.避光保存 |
试剂准备
混合液（现配现用）：临用前将试剂一、试剂二、试剂三按照10：1：1的比例混合，根据实验用量配制，用多少配多少，使用前可于37℃预热。
标准品（选做）：10 mg Trolox。临用前可取1 mg加入4 mL无水甲醇或无水乙醇溶解成0.25 mg/mL的母液（Trolox 浓度：1 µmol/mL），若重新做标曲则需用到该试剂，现配现用。
自备仪器和用品：
酶标仪、低温台式离心机、可调式移液器、96 孔板、研钵、冰和蒸馏水。
样品制备：
(1) 血清、血浆、唾液或尿液等液体样品
血浆 (制备时可以使用肝素或柠檬酸钠抗凝，不宜使用 EDTA 抗凝) 4 ℃，5000 rpm 离心 10 min，取上清待测。血清、唾液或尿液样品直接用于测定，也可以‑80 ℃冻存 (不宜超过 30 d) 后再测定。
(2) 组织样品
按照组织质量 (g) : 提取液体积 (mL) 为 1:5~10 的比例 (建议称取约 0.1 g 组织，加入 1 mL 提取液) 进行冰浴匀浆，然后 10000 g，4 ℃离心 10 min，取上清，置冰上待测。
(3) 细胞样品
按照细胞数量（ 10⁴ 个）：提取液体积（mL）为 500~1000：1 的比例（建议 500 万细胞加入 1 mL 提取液），冰浴超声波破碎（功率 200 W，超声 3 s，间隔 10 s，重复 30 次）；10000 g，4 ℃离心 10 min，取上清，置冰上待测。
测定步骤：
1、酶标仪预热 30 min 以上，调节波长至 590 nm。
2、样本测定
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总抗氧化能力计算公式：
单位定义：用从标准曲线上获得的抗氧化剂 Trolox 的量来表示样本的总抗氧化能力。
标准曲线：y = 0.9455x + 0.0004；
x:Trolox 浓度(μmol/mL)；
y:吸光值差值△A
（1）按样本质量计算
总抗氧化能力 (μmol Trolox/g 鲜重) =(△A测定 - 0.0004) ÷ 0.9455 × V反总 ÷ (V样 ÷ V样总 × W)= 21.15 × (ΔA - 0.0004) ÷ W
（2）按样本蛋白浓度计算 总抗氧化能力 (μmol Trolox/mg prot) = (△A测定 - 0.0004) ÷ 0.9455 × V反总 ÷ (V样 ÷ V样总 × Cpr)= 21.15 × (ΔA - 0.0004) ÷ Cpr
（3）按细胞计算
总抗氧化能力 (μmol Trolox/10⁴cell) = (△A测定 - 0.0004) ÷ 0.9455 × V反总 ÷ (V样 ÷ V样总 × 500)= 21.15 × (ΔA - 0.0004) ÷ 500
（4）按液体体积计算
总抗氧化能力 (μmol Trolox/mL) = (△A测定 - 0.0004) ÷ 0.9455 × V反总 ÷ V样= 21.15 × (ΔA - 0.0004)
V样总：加入提取液体积，1 mL；
V样：反应中样品体积，0.01 mL；
V反总 ：反应总体积，0.2 mL；
W：样品质量，g；
Cpr：样本蛋白浓度，mg/mL；
500：细菌或细胞总数，万。
注意事项：
1.正式测定前务必取2-3 个预期差异较大的样本做预测定。
2.试剂二对人体有刺激性，请采取适当的防护措施。为了您的安全和健康，请穿实验服并戴乳胶手套操作。
3.尽量避免使用在酸性条件下呈蓝色或接近蓝色的试剂，否则对本试剂盒的检测结果产生干扰。
4.样品中不宜添加Tween、Triton 和NP-40 等去垢剂和DTT、巯基乙醇等影响氧化还原反应的还原剂。
5.本法中，ΔA的线性范围为0.01~0.9，若大于0.9超出线性区间，需对样本进行稀释，最终计算结果需乘以稀释倍数；若小于0.01，可适当增加样本浓度。
6.若样本本身颜色较深，对显色会存在干扰，则需检测样本对照管的值，即在样本测定时同步做(样本+提取液)，计算A测定管-A对照管代入计算。
7.样本处理过程中施加了较高浓度的铁盐或亚铁盐会干扰测定，不宜用本测试方法。
8.提供的标准曲线及公式仅供参考，若有需要，可用提供的标准品自制标曲（计算公式及线性范围根据自制标准曲线需作对应修改），但需要注意，受仪器及个人操作影响，客户自制标准曲线可能与说明书中提供的标准曲线有差别。若无另外的实验要求，也可直接使用说明书所给标曲进行计算。
实例
| 样品 | 质量 g | 稀释倍数 | ΔA值 | μmol Trolox/g 鲜重 |
| 绿萝 | 0.1 | 1 | 0.488 | 103.13 |
| 桑叶 | 0.1 | 1 | 0.577 | 121.95 |
| 蒲公英 | 0.1 | 4 | 0.621 | 525.03 |
常见问题及解决方案
| 问题 | 可能原因 | 解决方法 |
| 样品读数重复性差 | 样品类型不合适 | 参考说明书了解不兼容的样品类型 |
| 组织样品匀浆不充分 | 延长超声时间/增加匀浆器的研磨次数 | |
| 样品反复冻融次数过多 | 将样品分装，不可反复冻融 | |
| 样品中含有干扰物质 | 排查干扰物质，必要时对样品进行脱蛋白处理 | |
| 样品读数超出线性范围 | 对样品进行浓缩或稀释，使读数处于线性范围内 | |
| 孔中有气泡 | 气泡会干扰读数，操作时尽量避免产生气泡，读数前务必去除气泡 | |
| 样品和标准品读数偏低/偏高 | 样品放置时间过长或储存不当 | 使用新鲜制备的样品，按推荐温度保存至使用前 |
| 试剂盒组分未完全解冻 | 待组分完全解冻后轻轻混匀再使用 | |
| 试剂在冰上放置时间过长 | 每次使用前新鲜配制反应混合液 | |
| 孵育时间/温度不当 | 参考说明书推荐的孵育时间和/或温度 | |
| 试剂加样量错误 | 检查移液枪是否校准（始终使用能吸取整体积的最小量程移液枪）；尽量避免吸取过小体积的试剂 | |
| 配制反应混合液时移液误差 | 始终配制混合母液 |
The total antioxidant level is constituted by various antioxidants and antioxidant enzymes in the sample. In biological, medical, and pharmaceutical research, the total antioxidant capacity is often measured in various body fluids such as plasma, serum, saliva, urine, as well as cell or tissue lysates, plant or herbal extracts, and various antioxidant solutions. Determination principle: Under acidic conditions, the ability of antioxidants to reduce Fe³⁺-tripyridyltriazine (Fe³⁺-TPTZ) to blue-colored Fe²⁺-TPTZ reflects the total antioxidant capacity.
Contents & Storage
| Component No. | Component Name | Appearance | 100T | Storage |
| T1521765A | Extraction Solution | Liquid | 120mL | 2-8℃. |
| T1521765B | Reagent Ⅰ | Liquid | 20mL | 2-8℃.Store in the dark. |
| T1521765C | Reagent Ⅱ | Liquid | 2mL | 2-8℃.Store in the dark. |
| T1521765D | Reagent Ⅲ | Liquid | 2mL | 2-8℃.Store in the dark. |
| T1521765E | Standard | Powder | 10mg | 2-8℃.Store in the dark. |
Reagent Preparation
Working Solution (prepare fresh before use): Mix Reagent Ⅰ, Reagent Ⅱ and Reagent Ⅲ at a ratio of 10:1:1 immediately before use. Prepare only the required volume according to experimental consumption. Pre-warm the mixture at 37 °C before use.
Standard (optional): 10 mg Trolox. Immediately before use, dissolve 1 mg standard substance in 4 mL anhydrous methanol or anhydrous ethanol to prepare a stock solution of 0.25 mg/mL (Trolox: 1 µmol/mL). This solution is needed for re-establishing the standard curve and must be freshly prepared.
Instruments and Consumables to Be Prepared
Microplate reader, low‑temperature desktop centrifuge, adjustable pipettors, 96‑well microplate, mortar, ice and distilled water.
Sample Preparation
(1) Liquid samples such as serum, plasma, saliva or urine
For plasma (prepare with heparin or sodium citrate as anticoagulant; EDTA anticoagulant is not recommended): centrifuge at 5000 rpm for 10 min at 4 ℃, collect the supernatant for test. Serum, saliva and urine samples can be assayed directly, or stored at −80 ℃ for no more than 30 d before measurement.
(2) Tissue samples
Homogenize tissue in ice bath at the ratio of tissue mass (g) : extraction solution volume (mL) = 1:5~10 (it is recommended to weigh about 0.1 g tissue and add 1 mL extraction solution). Centrifuge the homogenate at 10000 g for 10 min at 4 ℃, collect the supernatant and keep it on ice for test.
(3) Cell samples
Lyse cells by sonication in ice bath (power 200 W, sonicate 3 s, interval 10 s, repeat 30 times) at the ratio of cell number (104 cells) : extraction solution volume (mL) = 500~1000:1 (it is recommended to add 1 mL extraction solution for 5 million cells). Centrifuge at 10000 g for 10 min at 4 ℃, collect the supernatant and keep it on ice for test.
Assay Procedures
1. Pre‑warm the microplate reader for more than 30 min and set the wavelength to 593 nm.
2. Sample measurement
2. Sample measurement
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Calculation of Total Antioxidant Capacity
Unit Definition: The total antioxidant capacity of the sample is expressed as the amount of antioxidant Trolox obtained from the standard curve.
Standard Curve: y = 0.9455x + 0.0004
x: Trolox concentration (μmol/mL)
y: absorbance difference ΔA
(1) Calculation Based on Sample Mass
Total antioxidant capacity (μmol Trolox/g fresh weight) = (△Ameasured - 0.0004) ÷ 0.9455 × Vtotal reaction ÷ (Vsample ÷ Vtotal sample × W) = 21.15 × (ΔA - 0.0004) ÷ W
(2) Calculation Based on Sample Protein Concentration
Total antioxidant capacity (μmol Trolox/mg prot) = (△Ameasured - 0.0004) ÷ 0.9455 × Vtotal reaction ÷ (Vsample ÷ Vtotal sample × Cpr) = 21.15 × (ΔA - 0.0004) ÷ Cpr
(3) Calculation Based on Cell Number Total antioxidant capacity (μmol Trolox/10⁴ cell) = (△Ameasured - 0.0004) ÷ 0.9455 × Vtotal reaction ÷ (Vsample ÷ Vtotal sample × 500) = 21.15 × (ΔA - 0.0004) ÷ 500
(4) Calculation Based on Liquid Volume
Total antioxidant capacity (μmol Trolox/mL) = (△Ameasured - 0.0004) ÷ 0.9455 × Vtotal reaction ÷ Vsample = 21.15 × (ΔA - 0.0004)
Vtotal sample : volume of extraction solution added, 1 mL;
Vsample: sample volume in the reaction, 0.01 mL;
Vtotal reaction: total reaction volume, 0.2 mL;
W: sample mass, g;
Cpr: sample protein concentration, mg/mL;
500: total number of bacteria or cells, ten thousand.
Precautions
1.Prior to formal assay, it is necessary to perform pre‑test with 2‑3 samples with expected large differences.
2. Reagent Ⅱ is irritating to human body. Take appropriate protective measures. Wear lab coat and latex gloves for your safety and health during operation.
3. Avoid reagents that appear blue or near‑blue under acidic conditions, otherwise they will interfere with the test results of this kit.
4. Detergents including Tween, Triton and NP‑40, as well as redox‑interfering reductants such as DTT and β‑mercaptoethanol shall not be added to samples.
5. In this method, the linear range of ΔA is 0.01–0.9. If ΔA is greater than 0.9 and exceeds the linear range, the sample needs to be diluted, and the final calculation result must be multiplied by the dilution factor. If ΔA is less than 0.01, the sample concentration can be appropriately increased.
6. If the sample itself is deeply colored, it may interfere with color development. In this case, the value of the sample control tube needs to be measured, that is, a (sample + extraction solution) control should be run simultaneously with the sample measurement. Calculate A<sub>measuring tube</sub> - A<sub>control tube</sub> and substitute it into the calculation.
7. If a relatively high concentration of iron salts or ferrous salts is applied during sample processing, it will interfere with the measurement, and this assay method is not suitable.
8. The provided standard curve and formula are for reference only. If necessary, the provided standard can be used to prepare a self-made standard curve (the calculation formula and linear range should be modified accordingly based on the self-made standard curve). However, it should be noted that due to the influence of instruments and individual operations, the customer's self-made standard curve may differ from the standard curve provided in the instructions. If there are no other experimental requirements, the standard curve provided in the instructions can also be used directly for calculation.
Example
| Sample | Sample weight (g) | Dilution factor | ΔA value | μmol Trolox/g fresh weight |
| Pothos | 0.1 | 1 | 0.488 | 103.13 |
| Mulberry leaf | 0.1 | 1 | 0.577 | 121.95 |
| Dandelion | 0.1 | 4 | 0.621 | 525.03 |
Common Problems and Solutions
| Problems | Possible Causes | Solutions |
| Poor repeatability of sample readings | Inappropriate sample type | Refer to the manual for incompatible sample types |
| Insufficient homogenization of tissue samples | Prolong sonication time or increase grinding cycles with homogenizer | |
| Excessive repeated freeze-thaw cycles of samples | Aliquot samples; avoid repeated freeze-thaw operations | |
| Interfering substances present in samples | Check for interfering substances; perform deproteinization if necessary | |
| Sample readings exceed the linear range | Concentrate or dilute the sample to bring readings within the linear range | |
| Air bubbles in wells | Avoid bubble formation during operation; remove all air bubbles before measurement | |
| Low or high readings of samples and standards | Long sample standing time or improper storage | Use freshly prepared samples and store at recommended temperature before use |
| Kit components not fully thawed | Thaw components completely and mix gently before use | |
| Reagents kept on ice for too long | Prepare the reaction mixture freshly before each use | |
| Improper incubation time or temperature | Follow the incubation time and temperature recommended in the manual | |
| Incorrect reagent pipetting volume | Verify pipette calibration; always use the smallest-range pipette capable of aspirating the full volume and avoid pipetting very small volumes | |
| Pipetting errors during reaction mixture preparation | Prepare bulk master mix |
| 组分货号 | 组分名称 | 物理外观 | 100T | 储存 |
| T1521765A | 提取液 | 液体 | 120mL | 2-8℃. |
| T1521765B | 试剂一 | 液体 | 20mL | 2-8℃.避光保存 |
| T1521765C | 试剂二 | 液体 | 2mL | 2-8℃.避光保存 |
| T1521765D | 试剂三 | 液体 | 2mL | 2-8℃.避光保存 |
| T1521765E | 标准品 | 粉末 | 10mg | 2-8℃.避光保存 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939571 |
分析证书 | T1521765 |
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