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乳酸脱氢酶（lactate dehydrogenase，LDH或LD）属于氧化还原酶，能够催化氢氧原子或电子从一中底物转移到另一种底物上乳酸脱氢酶是糖酵解和糖异生的一个极其重要的酶，含有锌离子，广泛分布于人和动物组织、植物和微生物中，能可逆的催化乳酸（L）和丙酮酸（P）之间的氧化还原反应。其反应公式：乳酸+NAD⁺→丙酮酸+NADH+H⁺。其中：L→P为正向反应；P→L为逆向反应。
检测原理：利用乳酸脱氢酶催化上述正反应，即L-乳酸+NAD⁺→丙酮酸+NADH+H⁺，在上述反应过程中乳酸氧化成丙酮酸，同时NAD⁺氧化成NADH，引起340nm处吸光度的升高，其升高速率与样品中LDH活性呈正比关系，通过分光光度计或自动分析仪检测340nm处吸光度升高速率，通过计算获得乳酸脱氢酶的活性。该LD-L法的优点是：1、乳酸盐和NAD⁺底物溶液的稳定性比LD-P法中的丙酮酸和NADH底物溶液好；2、线性速率反应时间范围较宽；3、重复性比LD-P法和二硝基苯肼法好；4、准确性比二硝基苯肼法好；5、适用于自动分析仪。该试剂盒仅用于科研领域，不宜用于临床诊断或其他用途。
自备仪器和试剂
离心管或小试管、水浴锅、量筒、精密天平 、1mL石英比色杯
去离子水、生理盐水
操作步骤（仅供参考）
1、准备样品
1.1 血浆、血清样品
血浆、血清按照常规方法制备，可以直接用于本试剂盒的测定，室温保存3天，用于LDH的检测。
1.2 细胞或组织样品
取恰当细胞或组织进行匀浆，低速离心取上清，室温保存3天，用于LDH的检测。
1.3 长期保存样品
如果提取后的样品无法及时检测，需要放置时间较长，按下列方法操作：取LDH保护剂1支，加入1mL的去离子水，配制成LDH保护工作液，-20℃避光保存；按待测样品（如血清）：LDH保护工作液=9:1的比例混合，4℃避光保存。
注：样品准备完毕后可以用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内的LDH含量。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
2、配制LDH检测工作液
用精密天平称取42mg NAD，加入10mL LD-L Assay buffer混合溶解，即为LDH检测工作液，即配即用。
3、分光光度计测定
按照下表设置测定管，溶液应按照顺序依次加入，并注意避免产生气泡。如果样品中的LDH浓度过高，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（mL） | 测定管 |
待测样品（血清、血浆、体液等） | 0.05 |
LDH检测工作液（37℃提前预热） | 1 |
混匀，37℃孵育30s。
1mL石英比色杯光径1cm，立即以紫外分光光度计340nm处读取测定管吸光度，记录为A测定1。t min后再次读取吸光度，记录为A测定2。
注意：由于酶促反应时间较短，建议加样时间越短越好，其反应基本在1~3min内，其后反应趋于平缓，由于检测仪器、操作手法以及样品酶活性高低等条件的不同，参考值范围会有波动。
4、自动分析仪测定
如果样品中的浓度过高，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。根据实验室的自动分析仪性能，设置参数，下列参数仅供参考，记录待测样品管吸光度的升高速率（ΔA/min）。
温度 | 37℃ |
波长 | 340nm |
延迟时间 | 90s |
检测时间 | 180s |
待测样品 | 15μL |
LDH检测工作液 | 300μL |
5、结果计算
5.1 手工比色计算公式
LDH (U/L)=ΔA/min×(10⁶/6220)×(1.05/0.05)=ΔA/min×3376
参数说明：
ΔA/min=(A测定2−A测定1)/t
t=酶促反应的时间 (min)
6220=NADH的吸光度
2.1=反应液的总体积 (ml)
0.1=待测样品体积 (ml)
5.2 自动分析仪计算公式
LDH (U/L)=ΔA/min×(10⁶/6220)×(315/15)=ΔA/min×3376
参数说明：
ΔA/min=测定的340nm吸光度的升高速率
6220=NADH的吸光度
315=反应液的总体积 (μl)
15=待测样品体积 (μl)
注意：如果待测样品加入LDH保护工作液，其结果应除以0.9。
注意事项
1、处理后的样品应及时检测，否则LD4和LD5易失效。
2、血清或肝素抗凝血浆检测效果较好，草酸类、EDTA抗凝剂对LDH活性有抑制作用。
3、避免使用溶血样品。
4、酶促反应的时间一般为1~3min。时间延长，反应速率下降。
5、本反应需在340nm波长条件下测定，需要紫外分光光度计或全波长酶标仪、石英比色皿或UV酶标板进行测定。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
7、试剂开封后请尽快使用，以防影响后续实验效果。
Lactate dehydrogenase (LDH or LD) is an oxidoreductase that catalyzes the transfer of hydrogen atoms or electrons from one substrate to another. LDH is a crucial enzyme in glycolysis and gluconeogenesis, contains zinc ions, and is widely distributed in human and animal tissues, plants, and microorganisms. It catalyzes the reversible oxidation‑reduction reaction between lactate (L) and pyruvate (P). The reaction formula is: Lactate + NAD⁺ → Pyruvate + NADH + H⁺. The direction L → P is the forward reaction; P → L is the reverse reaction.
Detection Principle
This assay utilizes LDH to catalyze the forward reaction: L‑Lactate + NAD⁺ → Pyruvate + NADH + H⁺. During this reaction, lactate is oxidized to pyruvate while NAD⁺ is reduced to NADH, causing an increase in absorbance at 340 nm. The rate of increase is proportional to the LDH activity in the sample. The LDH activity is obtained by measuring the rate of absorbance increase at 340 nm using a spectrophotometer or automatic analyzer and performing calculations. Advantages of this LD‑L method include: 1. Lactate and NAD⁺ substrate solutions are more stable than the pyruvate and NADH substrate solutions used in the LD‑P method. 2. A wider linear rate‑reaction time range. 3. Better reproducibility compared to the LD‑P method and the dinitrophenylhydrazine method. 4. Better accuracy compared to the dinitrophenylhydrazine method. 5. Suitable for automatic analyzers. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
Operating Steps (For Reference Only)
1. Sample Preparation
1.1 Plasma and Serum Samples
Plasma and serum prepared by conventional methods can be used directly for assay with this kit. Store at room temperature for up to 3 days for LDH detection.
1.2 Cell or Tissue Samples
Homogenize appropriate amounts of cells or tissue, centrifuge at low speed, and collect the supernatant. Store at room temperature for up to 3 days for LDH detection.
1.3 Long‑Term Sample Storage
If extracted samples cannot be assayed promptly and require longer storage, proceed as follows: Reconstitute one vial of LDH Protectant with 1 mL of deionized water to prepare the LDH Protectant Working Solution. Store at –20°C protected from light. Mix the sample (e.g., serum) with the LDH Protectant Working Solution at a ratio of 9:1. Store at 4°C protected from light.
Note: After sample preparation, protein concentration can be measured using a BCA Protein Assay Kit to facilitate subsequent calculation of LDH content per unit protein weight in tissues or cells. Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready‑to‑Use BCA Protein Quantification Kit is recommended.
2. Preparation of LDH Assay Working Solution
Weigh 42 mg of NAD using a precision balance, add 10 mL of LD‑L Assay Buffer, mix to dissolve. This is the LDH Assay Working Solution. Prepare fresh for each use.
3. Spectrophotometric Measurement
Set up the test tube according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the LDH concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.
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Using a 1 cm pathlength 1 mL quartz cuvette, immediately read the absorbance of the test tube at 340 nm with a UV spectrophotometer. Record as ATest1. After t minutes, read the absorbance again and record as ATest2.
Note: Because the enzymatic reaction time is short, it is recommended to keep the sample addition time as brief as possible. The reaction essentially occurs within 1–3 minutes, after which it levels off. Reference ranges may vary due to differences in instruments, operational techniques, and sample enzyme activity levels.
4. Automatic Analyzer Measurement
If the sample concentration is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate samples. Set parameters according to the performance of the laboratory's automatic analyzer. The following parameters are for reference only. Record the rate of absorbance increase (ΔA/min) for the test sample tube.
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5. Result Calculation
5.1 Manual Colorimetric Calculation Formula
LDH (U/L) = ΔA/min × (10⁶ / 6220) × (1.05 / 0.05) = ΔA/min × 3376
Parameter Explanation:
ΔA/min = (ATest2 − ATest1) / t
t= Reaction time (min)
6220 = Molar absorptivity of NADH (L·mol⁻¹·cm⁻¹)
1.05 = Total reaction volume (mL)
0.05 = Sample volume (mL)
5.2 Automatic Analyzer Calculation Formula
LDH (U/L) = ΔA/min × (10⁶ / 6220) × (315 / 15) = ΔA/min × 3376
Parameter Explanation:
ΔA/min = Measured rate of absorbance increase at 340 nm
6220 = Molar absorptivity of NADH (L·mol⁻¹·cm⁻¹)
315 = Total reaction volume (μL)
15 = Sample volume (μL)
Note: If the sample was mixed with LDH Protectant Working Solution, divide the result by 0.9.
Precautions
1. Processed samples should be assayed promptly; otherwise, LD₄ and LD₅ may lose activity.
2. Serum or heparin‑anticoagulated plasma is preferred for detection. Oxalate and EDTA anticoagulants inhibit LDH activity.
3. Avoid using hemolyzed samples.
4. The enzymatic reaction time is generally 1–3 minutes. Prolonged time leads to a decrease in reaction rate.
5. This reaction must be measured at 340 nm, requiring a UV spectrophotometer or full‑wavelength microplate reader, quartz cuvettes, or a UV‑compatible microplate.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
7. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
| L1510342 | Component | 100T | Storage |
| L1510342A | NAD | 1EA | 2-8℃. Store in the dark. |
| L1510342B | LD-L Assay Buffer | 100 mL | 2-8℃. Store in the dark. |
| L1510342C | LDH保护剂 | 1EA | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0535503 |
分析证书 | L1510342 |
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