BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
活性氧(Reactive oxygen species, ROS)主要包括羟基自由基、超氧自由基和过氧化氢。在细胞或组织的正常生理代谢过程中会产生活性氧，同时一些环境因子例如紫外照射、环境污染等也可以诱导活性氧的产生，活性氧产生后可以导致细胞内脂、蛋白和DNA等的氧化损伤,诱发氧化应激(Oxidative stress),继而导致各种肿瘤、动脉粥样硬化、风湿性关节炎、糖尿病、肝损伤、以及中枢神经系统疾病等。机体中存在多种抗氧化物,包括抗氧化大分子、抗氧化小分子和酶等，可以清除体内产生的各种活性氧，以阻止活性氧诱导的氧化应激(oxidative stress)的产生；一个体系内的各种抗氧化大分子、抗氧化小分子和酶的总的水平即体现了该体系内的总抗氧化能力，因此测定血浆、血清、尿液、唾液等各种体液，细胞或组织等裂解液中的总抗氧化能力具有非常重要的生物学意义。
本产品是一种采用ABTS作为显色剂,可以对血浆、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、或各种抗氧化物(antioxidant)溶液的总抗氧化能力进行检测的试剂盒；ABTS法测定T-AOC的检测原理是在氧化剂作用下ABTS被氧化成绿色的ABTS+，在抗氧化物存在时ABTS+的产生被抑制，可在734nm或405nm测定ABTS+的吸光度即可测定并计算出样品的总抗氧化能力。Trolox是一种维生素E的类似物，具有和维生素E相似的抗氧化能力，可作为其它抗氧化物总抗氧化能力的参考，例如Trolox的总抗氧化能力为1，相同浓度情况下，其它物质的抗氧化能力可用其抗氧化能力和Trolox相比的倍数来表示，该试剂盒快捷方便,加入待测样品3~6分钟即可进行吸光度测定，通常10~20个样品可以在20分钟内检测完毕。该试剂盒仅用于科研领域,不适用于临床诊断或其他用途。
组分表
| T1507359 | Component | 100 T | 300 T | 1000 T | Storage |
| T1507359A | ABTS 溶液 | 0.5ml | 1.5ml | 5ml | -20℃. Avoid freeze/thaw cycle. Store in the dark. |
| T1507359B | 氧化剂 | 0.5ml | 1.5ml | 5ml | 2-8℃. |
| T1507359C | Trolox | 5mg | 5mg | 5mg | 2-8℃. Store in the dark. |
自备材料:
1、实验材料:植物组织(苹果、香蕉、梨、玉米等)、血液、组织样本
2、蒸馏水、无水乙醇、二甲基亚砜、PBS等
3、研钵或匀浆器、离心机、离心管、酶标仪、96孔板
操作步骤(仅供参考):
1、准备样品:
① 植物样品:取正常或特殊条件下的新鲜植物组织,洗净擦干,切碎并迅速称取,按0.5g样品:4.5ml 蒸馏水的比例匀浆或研磨,4℃ 12000r/min离心5min,取上清备用。
② 血浆、血清、唾液和尿液样品:新鲜取样后可直接用该试剂盒进行测定,也可以-80℃冻存后再进行测定,一个月内没有显著变化【血浆制备时不宜使用EDTA抗凝】。
③ 组织样品:精确称取50mg组织,加入200μl预冷的PBS,超声或匀浆处理,充分破碎细胞使其抗氧化物释放出来,4℃ 12000r/min离心5min,取上清备用。
④ 细胞样品:收集约100万个细胞(无需准确计数,直接刮下,不用胰酶或EDTA消化处理),加入200μl预冷的PBS,超声或匀浆处理,充分破碎细胞使其抗氧化物释放出来,4℃ 12000r/min 离心5min,取上清备用。
⑤ 抗氧化物质:用水或其他溶剂配制成 0.1~1.5mM,即可用于测定。
⑥ 样品抗氧化能力较高时可用蒸馏水或相应溶剂稀释后再次测定。
2、配制ABTS工作液:应根据待测定样品的数量(含标准曲线)配制适量的 ABTS 工作液。
① 将 ABTS 溶液和氧化剂等体积混合即成ABTS工作母液,室温避光存放12h~16h后方可使用,配好的 ABTS工作母液室温避光存放,2~3天内稳定【20个待测样品取ABTS 溶液和氧化剂各0.1ml即可】。
② 临用前将 ABTS工作母液用无水乙醇、PBS或蒸馏水按要求稀释成ABTS工作液,当待测样品为非水溶性样品时,用无水乙醇稀释;当待测样品为水溶性样品时,用PBS或蒸馏水稀释;ABTS工作母液的稀释倍数约为30~55倍。注意:ABTS工作液的吸光度减去相应的空白对照(无水乙醇、PBS或蒸馏水)的吸光度后,酶标仪(300ul)A₇₃₄为0.5~1.5,对应的A₄₀₅为1.3~3.5;分光光度计(1cm光径)A₇₃₄为0.7~1.8,对应的A₄₀₅为 1.5~4.0。
3、配制Trolox溶液(10mM):向5mg Trolox 中加入2ml无水乙醇或二甲基亚砜,至全部溶解,即成。
4、配制Trolox标准梯度:用适当溶剂将Trolox溶液(10mM)稀释至0.05、0.15、0.3、06、0.9、1.2、1.5mM;对于血清、血浆、唾液或尿液样品可直接用蒸馏水或PBS稀释标准品;对于组织或细胞样品,使用PBS或相应的组织裂解液稀释标准品;其他样品用相应的样品配制液稀释标准品。
5、加样:按照下表设置空白孔、标准孔、测定孔,溶液应按照顺序依次加入96孔板中,并注意避免产生气泡,小心混匀【ABTS工作液与待测样品或标准品加入比例为40:1】。
室温反应3~6min即可测定。如果样品的吸光度值过高,可以减少样品用量或适当稀释后再进行测定,样品的检测最好能设置平行管。
加入物 (μl) | 空白孔 | 标准孔 | 测定孔 |
ABTS 工作液 | 280 | 280 | 280 |
蒸馏水 | 7 | - | - |
系列 Trolox标准(1~7号) | - | 7 | - |
待测样品液 | - | - | 7 |
6、测定:用酶标仪检测405nm或734nm处吸光值,依次记为A空白、A标准、A测定。
计算:
以系列 Trolox标准为横坐标,相应吸光度为纵坐标,绘制出总抗氧化能力的标准曲线。根据样品测定管的吸光度值计算出样品相当于多少浓度的Trolox标准的抗氧化能力。
细胞或组织样品在测定总抗氧化能力时需同时测定蛋白浓度,最后测定获得的总抗氧化能力通常表示为每毫克或每克蛋白重量中的总抗氧化能力,表示单位为 mmol/mg或mmol/g。例如某裂解液样品的蛋白浓度为0.15mg/ml,测得的抑制率和0.3mM Trolox相同,则该裂解液样品的总抗氧化能力为0.3mM/0.15mg/ml,即2mmol/g。
植物或中草药抽提液的抗氧化能力可以表示为每毫克或每克抽提物干重中的总抗氧化能力,表示单位为mmol/mg或mmol/g。例如某样品的浓度为0.1mg/ml,测得的抑制率和0.5mM Trolox相同,则该样品的总抗氧化能力为0.5mM/0.1mg/ml,即5mmol/g。
各种抗氧化物以摩尔浓度表示时,测得的总抗氧化能力用相对总抗氧化能力表示。例如0.2mM的某抗氧化物测得的OD值和1mM Trolox相同,则其相对总抗氧化能力为5。
血浆、血清、唾液或尿液等混合物的抗氧化能力,可直接用Trolox的摩尔浓度来表示。例如某样品测得的抑制率和0.6mM的Trolox相同,则该样品的总抗氧化能力为0.6mM;再如某样品稀释5倍后测得的抑制率和0.5mM的Trolox相同,则该样品的总抗氧化能力为2.5mM。
样品的自由基清除率(%)=(A空白-A测定)/A空白×100
注意事项:
1、实验材料应尽量新鲜,样品提取的整个过程最好在4℃或冰上操作,如取材后不能立即检测,也可以-80℃冻存后再进行测定(应在1个月内测定完毕)。
2、样品中不能添加DTT、巯基乙醇等影响氧化还原反应的物质,也不宜添加Tween、Triton和NP-40等去垢剂。
3、需使用可以测定734nm的酶标仪或可以测定微量样品的分光光度计进行检测。
4、Trolox 溶液低温下呈凝固状态,使用前应40℃左右水浴助溶。
5、如果样品在734nm检测有困难或受样品干扰,可尝试405nm检测;对于细胞或组织样品在405nm检测时,可能会因为样品本身的吸光度而对结果产生一定的干扰。
6、ABTS 对人体有刺激性,操作时请小心,并注意做好防护避免直接接触人体或吸入体内。
7、据文献报道,人血清或血浆的总抗氧化能力为0.5~2mM,唾液的总抗氧化能力为0.3~1mM,尿液的总抗氧化能力为0.2~3mM。维生素C的抗氧化能力为1.0,维生素E的抗氧化能力为1.0,GSH的抗氧化能力为1.3,鲜橙汁的总抗氧化能力为2.2。
8、该试剂盒仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
9、为了您的安全和健康,请穿实验服并戴一次性手套操作。
10、试剂开封后请尽快使用,以防影响后续实验效果。
附录：标准曲线制作：在室温条件下操作如下：
0.25ml ABTS工作母液用9.75ml无水乙醇50倍稀释成ABTS工作液，
A₇₃₄=0.692，A₄₀₅=1.445，
用无水乙醇将Trolox溶液(10mM)稀释至0.05、0.15、0.3、06、0.9、1、1.2、1.5mM，取280ul ABTS工作液再分别加入7ul蒸馏水或Trolox标准，混匀，室温放置6min，酶标仪测定405nm处各管的吸光度，其数值及标准曲线如下(仅供参考)：
| Trolox标准 | 吸光度 |
| 0 | 1.500 |
| 0.05 | 1.409 |
| 0.15 | 1.307 |
| 0.3 | 1.207 |
| 0.6 | 0.806 |
| 0.9 | 0.466 |
| 1.0 | 0.380 |
| 1.2 | 0.045 |
| 1.5 | 0.045 |
Reactive oxygen species (ROS) mainly include hydroxyl radicals, superoxide radicals, and hydrogen peroxide. ROS are generated during the normal physiological metabolism of cells or tissues; meanwhile, some environmental factors such as ultraviolet (UV) radiation and environmental pollution can also induce ROS production. Once produced, ROS can cause oxidative damage to lipids, proteins, DNA, and other substances in cells, trigger oxidative stress, and subsequently lead to various diseases, including tumors, atherosclerosis, rheumatoid arthritis, diabetes, liver injury, and central nervous system diseases. The body contains a variety of antioxidants, including antioxidant macromolecules, antioxidant small molecules, and enzymes. These antioxidants can scavenge various ROS produced in the body to prevent oxidative stress induced by ROS. The total level of antioxidant macromolecules, antioxidant small molecules, and enzymes in a system reflects the total antioxidant capacity (T-AOC) of that system. Therefore, determining the total antioxidant capacity in various body fluids (such as plasma, serum, urine, and saliva) and lysates (such as those from cells or tissues) holds significant biological importance.
This kit uses ABTS as a chromogenic reagent and is designed to determine the total antioxidant capacity (T-AOC) of various samples, including body fluids (such as plasma, serum, saliva, and urine), lysates (such as those from cells or tissues), extracts from plants or Chinese herbal medicines, and solutions of various antioxidants. The detection principle of T-AOC determination by the ABTS method is as follows: ABTS is oxidized to green ABTS⁺ under the action of an oxidizing agent; in the presence of antioxidants, the generation of ABTS⁺ is inhibited. The total antioxidant capacity of the sample can be determined and calculated by measuring the absorbance of ABTS⁺ at a wavelength of 734 nm or 405 nm. Trolox is an analog of vitamin E with antioxidant capacity similar to that of vitamin E, and it can be used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is defined as 1; under the condition of the same concentration, the antioxidant capacity of other substances can be expressed as a multiple relative to that of Trolox. This kit is fast and convenient: absorbance measurement can be performed 3–6 minutes after adding the sample to be tested, and usually 10–20 samples can be tested within 20 minutes. This kit is for research use only and not intended for clinical diagnosis or other purposes.
| T1507359 | Component | 100 T | 300 T | 1000 T | Storage |
| T1507359A | ABTS Solution | 0.5ml | 1.5ml | 5ml | -20℃. Avoid freeze/thaw cycle. Store in the dark. |
| T1507359B | Oxidizing agent | 0.5ml | 1.5ml | 5ml | 2-8℃. |
| T1507359C | Trolox | 5mg | 5mg | 5mg | 2-8℃. Store in the dark. |
Materials to Prepare by Yourself:
1. Experimental Materials: Plant tissues (e.g., apples, bananas, pears, corn), blood, tissue samples
2. Reagents/Solvents: Distilled water, absolute ethanol, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), etc.
3. Instruments/Equipment: Mortar or homogenizer, centrifuge, centrifuge tubes, microplate reader, 96-well plate
Operating Procedures (For Reference Only):
1. Sample Preparation:
① Plant Samples: Take fresh plant tissues (under normal or specific conditions), rinse and dry them thoroughly, then chop them into pieces and weigh quickly. Homogenize or grind the tissues at a ratio of 0.5 g sample : 4.5 mL distilled water. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.
② Plasma, Serum, Saliva, and Urine Samples:Freshly collected samples can be directly tested with this kit, or stored at -80°C for later testing (no significant changes will occur within one month).
Note: EDTA should not be used for anticoagulation during plasma preparation.
③ Tissue Samples:Accurately weigh 50 mg of tissue, add 200 μL pre-cooled PBS, and process via ultrasonication or homogenization to fully disrupt cells and release antioxidants. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.
④ Cell Samples:Collect approximately 1 million cells (no accurate counting required; scrape cells directly without trypsin or EDTA digestion). Add 200 μL pre-cooled PBS, and process via ultrasonication or homogenization to fully disrupt cells and release antioxidants. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.
⑤ Antioxidant Substances:Dissolve the antioxidant in water or other solvents to a concentration of 0.1–1.5 mM; the solution is then ready for testing.
⑥ If the antioxidant capacity of a sample is relatively high, dilute the sample with distilled water or the corresponding solvent before retesting.
2. Preparation of ABTS Working Solution:
Prepare an appropriate volume of ABTS working solution based on the number of samples to be tested (including the standard curve).
① Mix ABTS solution and oxidizing agent in equal volumes to prepare ABTS stock working solution. Store it at room temperature in the dark for 12–16 hours before use. The prepared ABTS stock working solution is stable for 2–3 days when stored at room temperature in the dark.【For 20 samples to be tested, take 0.1 mL each of ABTS solution and oxidizing agent.】
② Before use, dilute the ABTS stock working solution with absolute ethanol, PBS, or distilled water as required to prepare the ABTS working solution: For water-insoluble samples, use absolute ethanol for dilution; For water-soluble samples, use PBS or distilled water for dilution. The dilution factor of the ABTS stock working solution is approximately 30–55 times.
Note: After subtracting the absorbance of the corresponding blank control (absolute ethanol, PBS, or distilled water) from the absorbance of the ABTS working solution: For a microplate reader (300 μL reaction volume), the absorbance at 734 nm (A₇₃₄) should be 0.5–1.5, and the corresponding absorbance at 405 nm (A₄₀₅) should be 1.3–3.5; For a spectrophotometer (1 cm light path), the A₇₃₄ should be 0.7–1.8, and the corresponding A₄₀₅ should be 1.5–4.0.
3. Preparation of Trolox Solution (10 mM): Add 2 mL of absolute ethanol or dimethyl sulfoxide (DMSO) to 5 mg of Trolox and mix until fully dissolved.
4. Preparation of Trolox Standard Gradients: Dilute the 10 mM Trolox solution with an appropriate solvent to concentrations of 0.05, 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. Use the following solvents for dilution based on sample type: For serum, plasma, saliva, or urine samples: Dilute the standard with distilled water or PBS directly; For tissue or cell samples: Dilute the standard with PBS or the corresponding tissue lysis buffer; For other samples: Dilute the standard with the corresponding sample preparation solvent.
5. Sample Loading: Set up blank wells, standard wells, and test wells according to the table below. Add the solutions to the 96-well plate in sequence, avoid generating air bubbles, and mix gently.【The volume ratio of ABTS working solution to the sample/standard to be tested is 40:1.】
Incubate at room temperature for 3–6 minutes, then perform absorbance measurement. If the absorbance value of a sample is too high, reduce the sample volume or dilute the sample appropriately before retesting. It is recommended to set up duplicate tubes for sample testing.
Substances to Add (μl) | Blank Well | Standard Well | Test Well |
ABTS Working Solution | 280 | 280 | |
Distilled water | 7 | - | - |
Serial Trolox Standards (No. 1–No. 7) | - | 7 | - |
Test Sample Solution | - | - | 7 |
6. Measurement: Use a microplate reader to measure the absorbance at 405 nm or 734 nm, and record the values as ABlank、AStandard、ATest .
Calculations:
Plot a standard curve for total antioxidant capacity (T-AOC), with the serial Trolox standards as the x-axis (abscissa) and the corresponding absorbance values as the y-axis (ordinate). Based on the absorbance value of the sample test tube, calculate the antioxidant capacity of the sample in terms of the equivalent concentration of Trolox standard.
When determining the T-AOC of cell or tissue samples, the protein concentration must be measured simultaneously. The final T-AOC result is usually expressed as the T-AOC per milligram or gram of protein, with units of mmol/mg or mmol/g. Example: If the protein concentration of a lysate sample is 0.15 mg/mL, and its inhibition rate is equivalent to that of 0.3 mM Trolox, the T-AOC of the lysate sample is calculated as 0.3 mM ÷ 0.15 mg/mL = 2 mmol/g.
The antioxidant capacity of plant or Chinese herbal medicine extracts can be expressed as the T-AOC per milligram or gram of the extract’s dry weight, with units of mmol/mg or mmol/g. Example: If the concentration of a sample is 0.1 mg/mL, and its inhibition rate is equivalent to that of 0.5 mM Trolox, the T-AOC of the sample is calculated as 0.5 mM ÷ 0.1 mg/mL = 5 mmol/g.
When various antioxidants are expressed in molar concentration, their measured T-AOC is represented as relative total antioxidant capacity. Example: If the OD value of a 0.2 mM antioxidant is equivalent to that of 1 mM Trolox, its relative total antioxidant capacity is 5 (1 mM ÷ 0.2 mM = 5).
The antioxidant capacity of mixtures such as plasma, serum, saliva, or urine can be directly expressed in the molar concentration of Trolox.
Example 1: If the inhibition rate of a sample is equivalent to that of 0.6 mM Trolox, the T-AOC of the sample is 0.6 mM.
Example 2: If a sample is diluted 5-fold, and its inhibition rate is equivalent to that of 0.5 mM Trolox, the T-AOC of the original sample is 0.5 mM × 5 = 2.5 mM.
Free Radical Scavenging Rate (%) of the Sample = [(A₈ₗₐₙₖ − Aₜₑₛₜ) ÷ A₈ₗₐₙₖ] × 100
Precautions:
1. Experimental materials should be as fresh as possible. The entire sample extraction process is preferably performed at 4°C or on ice. If immediate detection is not possible after sample collection, the samples can be stored at -80°C for later testing (testing must be completed within 1 month).
2. Substances that affect redox reactions (such as DTT and mercaptoethanol) must not be added to the samples. Detergents (such as Tween, Triton, and NP-40) should also not be added.
3. A microplate reader capable of measuring at 734 nm or a spectrophotometer suitable for micro-sample measurement must be used for detection.
4. Trolox solution solidifies at low temperatures; before use, it should be dissolved with the assistance of a water bath at approximately 40°C.
5. If it is difficult to detect the sample at 734 nm or the sample causes interference, try detecting at 405 nm. For cell or tissue samples, detection at 405 nm may cause certain interference to the results due to the inherent absorbance of the samples themselves.
6. ABTS is irritating to the human body. Please operate with caution and take protective measures to avoid direct contact with the body or inhalation.
7. According to literature reports: The total antioxidant capacity (T-AOC) of human serum or plasma is 0.5–2 mM; The T-AOC of saliva is 0.3–1 mM; The T-AOC of urine is 0.2–3 mM; The antioxidant capacity of vitamin C is 1.0; The antioxidant capacity of vitamin E is 1.0; The antioxidant capacity of GSH is 1.3; The T-AOC of fresh orange juice is 2.2.
8. This kit is limited to scientific research use by professional personnel. It must not be used for clinical diagnosis or treatment, nor for food or pharmaceuticals. It must not be stored in ordinary residential areas.
9. For your safety and health, please wear a lab coat and disposable gloves during operation.
10. After opening, the reagents should be used as soon as possible to avoid affecting the results of subsequent experiments.
Appendix: Preparation of the Standard Curve
Operate at room temperature as follows:Dilute 0.25 mL of ABTS stock working solution with 9.75 mL of absolute ethanol at a 1:50 dilution ratio to prepare the ABTS working solution. The absorbance values are A₇₃₄ = 0.692 and A₄₀₅ = 1.445.
Dilute the 10 mM Trolox solution with absolute ethanol to obtain concentrations of 0.05 mM, 0.15 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1 mM, 1.2 mM, and 1.5 mM (note: corrected the possible typo "06" to "0.6" for concentration accuracy).
Take 280 μL of the ABTS working solution, then add 7 μL of distilled water (blank control) or each Trolox standard respectively. Mix well, incubate at room temperature for 6 minutes, and measure the absorbance of each tube at 405 nm using a microplate reader. The absorbance values and standard curve are provided below (for reference only):
| Trolox Standard | Absorbance |
| 0 | 1.500 |
| 0.05 | 1.409 |
| 0.15 | 1.307 |
| 0.3 | 1.207 |
| 0.6 | 0.806 |
| 0.9 | 0.466 |
| 1.0 | 0.380 |
| 1.2 | 0.045 |
| 1.5 | 0.045 |
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 |
|---|---|---|
 J2609471 |
分析证书 | T1507359 |
| F2622157 |
分析证书 | T1507359 |
| ZJ26F0131214 |
分析证书 | T1507359 |
危险品化学品经营许可证（带存储）