D-乳酸在D-乳酸脱氢酶的作用下氧化生成丙酮酸，在这一过程中 NAD⁺ 还原为NADH。生成的NADH在电子耦合试剂1-mPMS的作用下将WST-8还原成橙黄色的甲臜，在450nm左右有最大吸收峰。反应体系中生成的甲臜与样品中D-乳酸含量呈正比。在样品体积为50 μL时，本试剂盒可检测含量低至20 µM的D-乳酸，在0.02 mM-1 mM浓度范围内有良好的线性关系。
产品组分
D1501940 | 组分名称 | 物理外观 | 100 T | 储存 | 单次测试使用量 |
D1501940A | 裂解液 | 液体 | 20 mL | -20℃. | 按需 |
D1501940B | 检测缓冲液 | 液体 | 20 mL | -20℃. | 按需 |
D1501940C | 酶液 | 液体 | 200 μL | -20℃.避光 | 2 μL |
D1501940D | 辅因子 | 液体 | 1000 μL | -20℃.避光 | 10 μL |
D1501940E | 显色液 | 液体 | 200 μL | -20℃.避光 | 2 μL |
D1501940F | D-乳酸标准品 (100 mM) | 液体 | 200 μL | -20℃.避光 | 2 μL |
使用方法
1. 样本处理
(1) 血清 (浆)样品：直接测定。
(2) 细胞样品：每100万细胞加入200 μL裂解液，冰浴超声破碎细胞。4ºC，12,000×g离心5分钟，取上清用于检测。
(3) 组织样品：每10 mg组织加入100 μL 裂解液，冰浴匀浆或超声破碎细胞。4ºC，12,000×g离心5分钟，取上清用于检测。
(4) 细胞培养上清：离心后，直接测定。
2. 标准曲线设置
取2 μL D-乳酸标准品 (100 mM)，加入198 μL检测缓冲液，混匀，配制成200 μL浓度为1 mM 的D-乳酸溶液。分别取1 mM D-乳酸溶液 0、1、2.5、5、10、25、50 μL加入96孔板的标准品孔中，并用检测缓冲液补足至50 μL。此时，标准曲线的各孔D-乳酸浓度分别为0、0.02、0.05、0.1、0.2、0.5、1 mM。
3. 反应工作液的配制
根据标准品和样品的数量，按照每个反应50 µL的体积配制反应工作液。具体配制方法参考下表，建议现配现用。
样本数 | 1 | 10 | 20 | 50 |
检测缓冲液 | 36 μL | 360 μL | 720 μL | 1800 μL |
酶液 | 2 μL | 20 μL | 40 μL | 100 μL |
辅因子 | 10 μL | 100 μL | 200 μL | 500 μL |
显色液 | 2 μL | 20 μL | 40 μL | 100 μL |
总体积 | 50 μL | 500 μL | 1000 μL | 2500 μL |
注：NADH、NADPH等的存在会对D-乳酸的检测产生干扰。如果样品含有NADH、NADPH，需同时设置样品的背景对照孔，加入不含酶溶液的显色工作液，即配制反应工作液时2 µL 酶液用检测缓冲液替代。计算时样品孔的读数值需要减去背景对照孔的读数。
4. 标准品和样品测定
(1) 取50 μL梯度稀释的标准品和50 μL样本加入到96孔板中。
(2) 各孔加入反应工作液50 µL，混匀，37ºC避光反应30分钟。
(3) 反应结束后，450 nm测定吸光度。
5. 结果计算 注意有代码
测定孔、标准孔和空白孔的OD值分别记为A测定 、 A标准 和 A空白 ，其中 A空白 为标准曲线中不含D-乳酸的零浓度孔（标准品零浓度孔）的OD值。ΔA测定 = A测定 - A空白 ，ΔA标准 = A标准 - A空白 。
以D-乳酸标准品的浓度为横坐标， ΔA标准 为纵坐标，建立标准曲线，带入 ΔA测定 计算样品中D-乳酸的浓度(A)。如果样品的背景对照信号比较高，样品的信号值应减去样品背景对照的信号值。
D-乳酸浓度计算公式如下：C (mM) = A × n
注: A为根据标准曲线确定的D-乳酸浓度(mM)；n为稀释倍数。
6. 结果展示
注意事项
1. 酶液和辅因子需冰上解冻，使用前后避免室温长时间放置。
2. 正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
3. 生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护。
D-Lactate is oxidized by D-lactate dehydrogenase to generate pyruvate, during which NAD⁺ is reduced to NADH. The generated NADH reduces WST-8 to an orange-yellow formazan in the presence of the electron coupling reagent 1-mPMS, which has a maximum absorption peak at approximately 450 nm. The formazan generated in the reaction system is directly proportional to the D-lactate content in the sample. With a sample volume of 50 μL, this kit can detect D-lactate at levels as low as 20 µM, and exhibits a good linear relationship within the concentration range of 0.02-1 mM.
Components
D1501940 | Component Name | Appearance | 100 T | Storage | Quantity Per Test |
D1501940A | Lysis Buffer | Liquid | 20 mL | -20°C. | As needed |
D1501940B | Assay Buffer | Liquid | 20 mL | -20°C. | As needed |
D1501940C | Enzyme Solution | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
D1501940D | Cofactor | Liquid | 1000 μL | -20°C. Store in the dark | 10 μL |
D1501940E | Chromogenic Solution | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
D1501940F | D-Lactate Standard (100 mM) | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
Usage Protocol
1. Sample Preparation
1) Serum (plasma) samples: Measure directly.
2) Cell samples: Add 200 μL of lysis buffer per 1 × 10⁶ cells, and disrupt the cells by ultrasonication on ice. Centrifuge at 12,000 × g for 5 minutes at 4 ºC, and collect the supernatant for analysis.
3) Tissue samples: Add 100 μL of lysis buffer per 10 mg of tissue, and homogenize on ice or disrupt the cells by ultrasonication. Centrifuge at 12,000 × g for 5 minutes at 4 ºC, and collect the supernatant for analysis.
4) Cell culture supernatant: Centrifuge and measure directly.
2. Standard Curve Setup
Pipette 2 μL of D-Lactate Standard (100 mM), add 198 μL of Assay Buffer, and mix to prepare 200 μL of a 1 mM D-lactate solution. Then transfer 0, 1, 2.5, 5, 10, 25, and 50 μL of the 1 mM D-lactate solution into the standard wells of a 96-well plate, and bring the volume to 50 μL with Assay Buffer. The resulting D-lactate concentrations in the standard curve wells are 0, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM, respectively.
3. Preparation of Reaction Working Solution
Prepare the Reaction Working Solution according to the number of standards and samples, based on a volume of 50 µL per reaction. Refer to the table below for specific preparation methods. It is recommended to prepare it fresh before use.
Number of samples | 1 | 10 | 20 | 50 |
Assay Buffer | 36 μL | 360 μL | 720 μL | 1800 μL |
Enzyme Solution | 2 μL | 20 μL | 40 μL | 100 μL |
Cofactor | 10 μL | 100 μL | 200 μL | 500 μL |
Chromogenic Solution | 2 μL | 20 μL | 40 μL | 100 μL |
Total Volume | 50 μL | 500 μL | 1000 μL | 2500 μL |
Note: The presence of NADH, NADPH, etc. may interfere with the detection of D-lactate. If the sample contains NADH or NADPH, a sample background control well must be set up in parallel, and add the Chromogenic Working Solution without the enzyme solution, i.e., replace the 2 µL of enzyme solution with Assay Buffer when preparing the Reaction Working Solution. When calculating, subtract the reading of the background control well from that of the sample well.
4. Standard and Sample Assay
1) Add 50 μL of the serially diluted standards and 50 μL of samples to a 96‑well plate.
2) Add 50 µL of Reaction Working Solution to each well, mix, and incubate at 37 ºC in the dark for 30 minutes.
3) After the reaction, measure the absorbance at 450 nm.
5. Result Calculation
The OD values of the sample well, standard well, and blank well are recorded as Aₛₐₘₚₗₑ, Aₛₜₐₙdₐᵣd, and Abₗₐₙₖ, respectively, where Abₗₐₙₖ is the OD value of the zero-concentration well containing no D-lactate in the standard curve (the zero-concentration standard well). ΔAₛₐₘₚₗₑ= Aₛₐₘₚₗₑ - Abₗₐₙₖ ; Δ Aₛₜₐₙdₐᵣd= Aₛₜₐₙdₐᵣd - Abₗₐₙₖ.
Plot a standard curve with the concentration of the D-lactate standard as the x-axis and Δ Aₛₜₐₙdₐᵣd as the y-axis. Substitute ΔAₛₐₘₚₗₑ into the standard curve to calculate the D-lactate concentration in the sample (A). If the background control signal of the sample is relatively high, the signal value of the sample should be subtracted by the signal value of the sample background control.
The formula for calculating D-lactate concentration is as follows: C (mM) = A × n
Note: A is the D-lactate concentration (mM) determined from the standard curve; n is the dilution factor.
6. Results Presentation
Precautions
1. The Enzyme Solution and Cofactor should be thawed on ice. Avoid prolonged exposure to room temperature before and after use.
2. Before formal testing, it is recommended to perform a preliminary experiment using 2–3 samples with expected large differences.
3. Biochemical assay reagents generally have irritancy and biological toxicity. For your safety and health, please take proper biological safety precautions throughout the experiment.
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