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POD（EC1.11.1.7）广泛存在于动物、植物、微生物和培养细胞中，可催化过氧化氢氧化酚类和胺类化合物，具有消除过氧化氢和酚类、胺类毒性的双重作用。
测定原理：
POD催化H₂O₂氧化特定底物，在470nm有特征光吸收。
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
正式测定前务必取2-3个预期差异较大的样本做预测定
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 50T | 储存 |
| P1521767A | 提取液 | 液体 | 60 mL | 2-8℃. |
| P1521767B | 试剂一 | 液体 | 50 mL | 2-8℃. |
| P1521767C | 试剂二 | 液体 | 100 μl | 2-8℃. Store in the dark. |
| P1521767D | 试剂三 | 液体 | 100 μl | 2-8℃. Store in the dark. |
自备仪器和试剂
粗酶液提取：
1、细菌、细胞或组织样品的制备：
细菌或培养细胞：先收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量（10⁴个）：提取液体积（ml）为500~1000：1的比例（建议500万细菌或细胞加入1ml提取液），超声波破碎细菌或细胞（冰浴，功率20％或200W，超声3s，间隔10s，重复30次）；8000g 4℃离心10min，取上清，置冰上待测。
组织：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0.1g组织，加入1ml提取液），进行冰浴匀浆。8000g 4℃离心10min，取上清，置冰上待测。
2、血清（浆）样品：直接检测。
测定步骤：
1、分光光度计预热30min以上，调节波长至470nm，蒸馏水调零。
2、工作液的配制：临用前将试剂一、试剂二、试剂三按照2.6（ml）：1.5（μl）：1（μl）的比例混匀；在37℃（哺乳动物）或25℃（其它物种）预热10min以上；现配现用。
3、在1ml玻璃比色皿中加入50μl样本和950μl工作液，混匀，记录470nm下1min时吸光值A1和2min后的吸光值A2。计算ΔA＝A2-A1。
注意：
如果ΔA小于0.005，可将反应时间延长到5min。如果ΔA大于0.5，可将样本用提取液稀释后测定，计算公式中乘以相应稀释倍数。
POD活性计算：
1、血清（浆）POD活性
单位定义：每ml血清（浆）在每ml反应体系中每分钟A470变化0.01为一个酶活力单位。
计算公式：
注意事项：
1. 如果样品浓度过高ΔA大于1.2，可将样本用提取液稀释后测定，计算公式中乘以相应稀释倍数。
2. 如果样本浓度过低ΔA小于0.01，可将反应时间延长或者增加样本浓度，最终的计算公式也要随之改变。
3. 工作液必须现用现配，可根据样本量等比例配制。
实验实例：
1. 样本为大豆粉，按照样本的处理流程，取上清按测定步骤操作，测得结果如下表，根据计算公式得：
| 样本-1 | 样本-2 | 样本-3 | 样本-4 | |
| 稀释倍数 | 2 | 2 | 4 | 4 |
| ΔA | 0.491 | 0.48 | 0.239 | 0.22 |
| POD (U/g 鲜重) | 32196.72 | 34909.09 | 31344.26 | 32000 |
2. 使用辣根过氧化物酶标准品，稀释至不同浓度，结果呈现一定的梯度，如下图：
辣根过氧化物酶浓度μg/mL
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 |
Peroxidase (POD, EC 1.11.1.7) widely exists in animals, plants, microorganisms and cultured cells. It catalyzes the oxidation of phenolic and amine compounds by hydrogen peroxide, and plays a dual role in eliminating the toxicity of hydrogen peroxide, phenols and amines.
Determination Principle:
POD catalyzes hydrogen peroxide to oxidate specific substrates, which shows characteristic light absorption at 470 nm.
Note:
This kit is only for scientific research, not for clinical diagnosis or other applications.
It is necessary to conduct preliminary tests with 2 to 3 samples with significant expected differences before formal detection.
Components and Storage:
Reagent 2: After opening, store at 4 °C in the dark and use within one month. Reagent 3: After opening, store at 4 °C in the dark and use within one month. |
Self-prepared Instruments and Reagents:
⁕Spectrophotometer (capable of absorbance measurement at 470 nm)
⁕Visible spectrophotometer, bench-top centrifuge, adjustable pipette, 1 mL glass cuvette, mortar
⁕Ice and distilled water
Crude Enzyme Extraction:
1. Preparation of bacterial, cellular and tissue samples
Bacteria or cultured cells: Collect bacteria or cells into centrifuge tubes and discard the supernatant after centrifugation. Mix extraction solution at the ratio of 500~1000 : 1 (10⁴ cells per mL extraction solution); it is recommended to add 1 mL extraction solution for 5 million bacteria or cells. Lyse samples by ultrasonication under ice bath with power of 20% or 200 W, 3-second ultrasonication with 10-second interval, repeat 30 times. Centrifuge at 8000 g and 4 ℃ for 10 minutes, take the supernatant and keep it on ice for test.
Tissues: Homogenize tissues under ice bath at the mass-to-volume ratio of 1 g tissue to 5~10 mL extraction solution; it is recommended to add 1 mL extraction solution to approximately 0.1 g tissue. Centrifuge at 8000 g and 4 ℃ for 10 minutes, take the supernatant and keep it on ice for test.
2. Serum or plasma samples: Direct detection
1. Preheat the spectrophotometer for no less than 30 minutes, set the wavelength to 470 nm and calibrate zero with distilled water.
2. Working solution preparation: Mix Reagent 1, Reagent 2 and Reagent 3 at the volume ratio of 2.6 mL : 1.5 μL : 1 μL before use. Preheat the mixture for over 10 minutes at 37 ℃ for mammalian samples or 25 ℃ for other species. Prepare freshly before use.
3. Add 50 μL sample and 950 μL working solution into a 1 mL glass cuvette and mix thoroughly. Record absorbance A1 at 1 minute and A2 at 2 minutes at 470 nm. Calculate ΔA＝A2-A1.
Precautions:
Extend reaction time to 5 minutes if ΔA is lower than 0.005. Dilute samples with extraction solution for retest if ΔA exceeds 0.5, and multiply the corresponding dilution factor in calculation.
Calculation of POD Activity:
1. POD activity in serum/plasma
Unit definition: One unit of enzyme activity is defined as the absorbance change of 0.01 at 470 nm per minute per milliliter serum/plasma in 1 mL reaction system.
Calculation formula:
Precautions:
1. If the sample concentration is too high and ΔA is greater than 1.2, dilute the sample with extraction solution before measurement, and multiply the corresponding dilution factor in the calculation formula.
2. If the sample concentration is too low and ΔA is less than 0.01, extend the reaction time or increase the sample concentration; the final calculation formula shall be modified accordingly.
3. The working solution must be freshly prepared before use. Prepare it proportionally according to the number of samples.
Experimental Example:
1. Soybean powder was used as the sample. Following the sample‑treatment procedure, the supernatant was taken and tested in accordance with the measurement steps. The measured results are shown in the table below. Calculated by the formula:
| Sample‑1 | Sample‑2 | Sample‑3 | Sample‑4 | |
| Dilution factor | 2 | 2 | 4 | 4 |
| ΔA | 0.491 | 0.48 | 0.239 | 0.22 |
| POD (U/g fresh weight ) | 32196.72 | 34909.09 | 31344.26 | 32000 |
2. Horseradish peroxidase standard was diluted to different concentrations, and the results showed a certain gradient, as shown in the figure below.
Horseradish peroxidase concentration (μg/mL)
Frequently Asked Questions and Solutions:
| Problem | Possible Cause | Solution |
| Poor repeatability of sample readings | Inappropriate sample type | Refer to the instructions for incompatible sample types. |
| Insufficient homogenization of tissue samples | Prolong sonication time or increase the grinding cycles of the homogenizer. | |
| Excessive repeated freeze‑thaw cycles of samples | Aliquot samples and avoid repeated freeze‑thawing. | |
| Interfering substances present in samples | Identify interfering substances; perform deproteinization treatment on samples if necessary. | |
| Sample readings outside the linear range | Concentrate or dilute samples to bring readings within the linear range. | |
| Air bubbles in wells | Air bubbles interfere with readings. Avoid bubble formation during operation and remove all air bubbles before measurement. | |
| Low‑ or high‑biased readings of samples and standards | Long sample standing time or improper storage | Use freshly‑prepared samples and store at the recommended temperature until use. |
| Incompletely‑thawed kit components | Thaw components completely and mix gently before use. | |
| Reagents kept on ice for too long | Prepare reaction mixtures freshly before each use. | |
| Incorrect incubation time or temperature | Follow the incubation time and/or temperature recommended in the manual. | |
| Incorrect reagent pipetting volume | Verify pipette calibration; always use the smallest‑range pipette capable of delivering the full volume; avoid pipetting very small volumes of reagents. | |
| Pipetting errors during reaction‑mixture preparation | Always prepare a master mix. |
| 组分货号 | 组分名称 | 物理外观 | 50T | 储存 |
| P1521767A | 提取液 | 液体 | 60 mL | 2-8℃. |
| P1521767B | 试剂一 | 液体 | 50 mL | 2-8℃. |
| P1521767C | 试剂二 | 液体 | 100 μl | 2-8℃. Store in the dark. |
| P1521767D | 试剂三 | 液体 | 100 μl | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636744 |
分析证书 | P1521767 |
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