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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。
酸性磷酸酶（acid phosphatase, ACP）是一类分布极为广泛的酶，存在于全身各组织中，主要定位于细胞的溶酶体内，因此常被用作溶酶体的标志酶。除此之外，在内质网和细胞质中也存在ACP。该酶的适宜pH范围为4.5至5.5，在不同动物体内其特性存在差异。
抗酒石酸酸性磷酸酶（Tartrate-resistant acid phosphatase, TRAP）是ACP的一种特殊形式。它存在于正常人肺泡巨噬细胞和白血病患者脾脏的细胞滤泡中，通常不释放入血液。血液中的TRAP主要来源于破骨细胞，因此通过检测血液中TRAP水平可以反映破骨细胞的活性，被广泛认为是评估机体破骨活性的唯一血液学指标。TRAP是一种糖基化的含金属蛋白酶，在破骨细胞（osteoclast）和破软骨细胞（chondroclast）中高表达，同时也在活化的巨噬细胞和神经元中表达。
检测原理：在酸性条件下，ACP可作用于pNPP，使其水解生成对硝基酚（p-nitrophenol, p-NP）。p-NP在碱性环境中呈黄色，黄色深浅与酶活性成正比，在405nm波长处有最大吸收峰。在检测体系中加入适量酒石酸后，可抑制其他ACP的活性，所测得的ACP活性即为TRAP的活性，通过测定p-NP的生成量可计算TRAP活性水平。
适用样本：细胞或组织裂解液、匀浆液、血浆、血清、尿液等。
| A1518291 | 组件 | 物理外观 | 100T | 储存温度 | 单次测试使用量 |
| A1518291A | 抗酒石酸酸性磷酸酶检测缓冲液 | 无色澄清透明溶液，无沉淀物 | 50 mL | 2-8°C. | 按需 |
| A1518291B | 对硝基苯磷酸二钠 | 淡黄色粉末 | 1EA×2 | -20°C, 避光. | 按需 |
| A1518291C | 酒石酸溶液 | 无色透明溶液，无沉淀 | 2.5 mL | 2-8°C. | 20 µL |
| A1518291D | 对硝基苯酚 (10 mM) | 无色/淡黄色透明溶液，无沉淀 | 0.5 mL | -20℃, 避光. | 100 µL |
| A1518291E | 终止液 | 无色透明溶液，无沉淀 | 85 mL | RT. | 800 µL |
自备仪器和试剂
操作步骤（仅供参考）
1. 样品准备
1.1 血浆、血清和尿液样品
血浆、血清按照常规方法制备后可直接用于测定，尿液通常也可以直接用于测定，来不及检测可-80℃冻存，但不宜反复冻融。
1.2 组织样品
按照组织样本质量（g）：生理盐水体积（mL）=1: 10匀浆，离心后取上清待测，留取部分上清进行BCA蛋白浓度测定。
1.3 细胞样品
按细胞数量（10⁶）：生理盐水体积（mL）=5: 1匀浆，离心后取上清待测，留取部分上清进行BCA蛋白浓度测定。
注：正式检测前，需选择2-3个预期差异大的样本稀释成不同浓度进行预实验，根据预实验的结果，结合本试剂盒的线性范围（1.0-200 U/L），使用PBS或生理盐水稀释后再行检测。
2. 配制显色工作液
取1支pNPP，使用前短暂离心，小心开启瓶盖，加入0.5 mL去离子水，盖紧盖子后涡旋震荡充分溶解，置于冰上待用，未用完部分-20℃避光保存2天；按1：20的比例加入TRAP Assay Buffer，混匀即成，置于冰上避光待用，新配制的显色工作液6h内有效。
3. 配制系列标准品
取出对硝基苯酚 (10 mM) 恢复至室温，按对硝基苯酚 (10 mM)： 抗酒石酸酸性磷酸酶检测缓冲液 =1：19 的比例混合，使浓度达到 0.5 mM，按下表继续稀释：
| 加入物（mL） | 1 | 2 | 3 | 4 | 5 | 6 |
| 对硝基苯酚 (0.5 mM) | 0.01 | 0.02 | 0.04 | 0.06 | 0.08 | 0.1 |
| 抗酒石酸酸性磷酸酶检测缓冲液 | 0.09 | 0.08 | 0.06 | 0.04 | 0.02 | 0 |
| 标准品浓度 (mM) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
4. TRAP加样
加入物（mL） | 空白管 | 标准管 | 测定管 | 对照管 |
抗酒石酸酸性磷酸酶检测缓冲液 | 0.1 | — | — | 0.2 |
系列标准品 (1-6号) | — | 0.1 | — | — |
待测样品 | — | — | 0.1 | 0.1 |
显色工作液 | 0.2 | 0.2 | 0.2 | — |
酒石酸溶液 | 0.02 | 0.02 | 0.02 | 0.02 |
注： 对照管用于校正样本本身及非特异性反应带来的背景吸光度。最终计算时，应采用测定管的吸光度减去对照管的吸光度（ΔA405）。
5. TRAP测定
轻轻混匀，37℃孵育10 min，每管加入0.8 mL 终止液终止反应；以空白管调零，比色杯光径 1 cm，分光光度计测定 405 nm处各管的吸光度。
6. 结果计算
以系列标准品 (1~6号) 浓度0.05、0.1、0.2、0.3、0.4、0.5 mM为横坐标，以对应的标准管吸光度为纵坐标，绘制标准曲线y=ax+b，根据测定管的吸光度在标准曲线上查得待测样品对硝基酚的生成量x=(ΔA405-b)÷a，单位为mM。
组织和细胞样本抗酒石酸酸性磷酸酶活性单位的定义
在pH 4.8、37℃条件下，每克组织或细胞蛋白每分钟催化底物生成1 μmol对硝基酚所需要的TRAP的酶量为一个活力单位。
公式如下：TRAP活力 (U/g prot)=x×1000×N÷t÷Cpr
血清(浆)和尿液样本抗酒石酸酸性磷酸酶活性单位的定义
在pH 4.8、37℃条件下，每升血清（浆）或尿液每分钟催化底物生成1 μmol对硝基酚所需要的TRAP的酶量为一个活力单位。
公式如下：TRAP活力 (U/L)=x×1000×N÷t
参数说明：
t：酶促反应时间，min
ΔA405：样本绝对OD值（测定OD值-对照OD值）
y：标准品OD值-空白管OD值
注意事项
1. 待测样品中不能含有磷酸酶抑制剂，同时需避免反复冻融。
2. 如果待测样品体积较少，可减少样品加样量，以抗酒石酸酸性磷酸酶检测缓冲液补至0.1 mL。
3. 如果无法检测405 nm，亦可检测400～415 nm范围内吸光度。
4. 建议每次测定时都做标准曲线，以使标准更准确，另外标准品避免反复冻融。
5. 待测样品中抗酒石酸酸性磷酸酶活性较低时，可适当延长孵育时间至30 min。
6. 检测组织和细胞样品时，需测定总蛋白浓度，推荐使用BCA蛋白定量试剂盒。
7. 配制1支显色工作液后应当日用完，因此请注意适当多准备一些样品一起检测。
8. 对硝基苯酚溶液对人体有害，终止液有腐蚀性，请小心操作。
9. 如果希望进行酶活性的绝对定量，进行酶反应时应精确计时，此时推荐采用孵育30 min或更长时间，以减小操作过程中的时间误差。
10. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Acid phosphatase (ACP) is a widely distributed enzyme found in various tissues throughout the body. It is primarily localized in cellular lysosomes and is therefore commonly used as a lysosomal marker enzyme. Additionally, ACP is present in the endoplasmic reticulum and cytoplasm. The optimal pH range for this enzyme is 4.5 to 5.5, and its characteristics vary among different animal species.
Tartrate-resistant acid phosphatase (TRAP) is a specific isoform of ACP. It is found within cytoplasmic vacuoles in normal human alveolar macrophages and in the spleen of patients with leukemia, and is not typically released into the bloodstream. TRAP in the blood originates mainly from osteoclasts. Therefore, measuring blood TRAP levels can reflect osteoclast activity and is widely regarded as the only hematological indicator for assessing bone resorption activity in the body. TRAP is a glycosylated, metal-containing enzyme highly expressed in osteoclasts and chondroclasts, and is also expressed in activated macrophages and neurons.
Assay Principle
Under acidic conditions, ACP acts on p-nitrophenyl phosphate (pNPP), hydrolyzing it to form p-nitrophenol (p-NP). In an alkaline environment, p-NP produces a yellow color, with the intensity being directly proportional to the enzyme activity, showing maximum absorbance at 405 nm. By adding an appropriate amount of tartrate to the reaction system, the activity of other ACP isoforms is inhibited, and the measured ACP activity specifically represents TRAP activity. TRAP activity levels are calculated by measuring the amount of p-NP generated.
Applicable Samples
Cell or tissue lysates, homogenates, plasma, serum, urine, etc.
Reagents, consumables and Equipments not provided
Assay Procedure (For Reference Only)
1. Sample Preparation
1.1 Plasma, Serum, and Urine Samples
Plasma and serum prepared by routine methods can be used directly for the assay. Urine can also typically be used directly. If not assayed immediately, samples can be stored at -80°C but avoid repeated freeze-thaw cycles.
1.2 Tissue Samples
Homogenize tissue samples using a ratio of tissue weight (g) : normal saline volume (mL) = 1:10. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.
1.3 Cell Samples
Homogenize cell samples using a ratio of cell number (10⁶) : normal saline volume (mL) = 5:1. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.
Note: Before formal testing, perform a preliminary experiment using 2-3 samples with expected activity differences, diluted to various concentrations. Based on the preliminary results and the kit's linear range (5.0-300 U/L), dilute samples appropriately with PBS or normal saline before the final assay.
2. Preparation of Working Color Solution
Briefly centrifuge one vial of pNPP before opening. Carefully open the vial, add 0.5 mL of deionized water, cap tightly, and vortex until completely dissolved. Keep on ice until use. Any unused portion can be stored at -20°C protected from light for up to 2 days.
Mix the dissolved pNPP with TRAP Assay Buffer at a ratio of 1:20 (e.g., for 1 part pNPP solution, add 19 parts TRAP Assay Buffer). Vortex to mix. This freshly prepared Working Color Solution is stable for 6 hours when kept on ice and protected from light.
3. Preparation of Standard Curve Dilutions
Allow the p-nitrophenol (10mM) to reach room temperature.
Prepare a 0.5 mM intermediate standard by mixing p-nitrophenol (10mM) with TRAP Assay Buffer at a ratio of 1:19 (e.g., 0.01 mL p-nitrophenol + 0.19 mL TRAP Assay Buffer).
Prepare serial dilution standards in tubes or a microplate according to the table below:
|
4. TRAP Assay Setup
Set up Blank, Standard, Test, and Control tubes as per the table below. Add solutions in the order listed, avoiding bubbles.
If sample TRAP activity is expected to be very high, reduce the sample volume or dilute the sample appropriately. It is recommended to run samples in duplicate or triplicate.
|
Note: The Control tube is used to correct for the background absorbance contributed by the sample itself and non-specific reactions. For the final calculation, use the absorbance difference: ΔA₄₀₅ = (Test Tube A₄₀₅) - (Control Tube A₄₀₅).
5. Measurement
Mix gently. Incubate at 37°C for exactly 10 minutes.
Add 0.8 mL of Stopping Solution to each tube to terminate the reaction.
Zero the spectrophotometer with the Blank tube at 405 nm using a 1 cm path length cuvette.
Measure the absorbance (A₄₀₅) of all tubes (Standard, Test, Control).
6. Calculation of Results
Standard Curve: Plot the absorbance (A<sub>405</sub>) of the Standard tubes (Y-axis) against their corresponding concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM) (X-axis). Perform linear regression to obtain the standard curve equation: y = ax + b.
Calculate p-NP Concentration (x) for Samples: Using the ΔA₄₀₅ value (Test A₄₀₅ - Control A₄₀₅) for the sample, calculate the concentration of p-nitrophenol generated: x = (ΔA₄₀₅- b) / a (unit: mM).
Definition of Enzyme Activity Unit
For Tissue and Cell Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per gram of protein at pH 4.8 and 37°C.
Formula: TRAP Activity (U/g prot) = x×1000×N÷t÷Cpr
For Serum (Plasma) and Urine Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per liter of serum (plasma) or urine at pH 4.8 and 37°C.
Formula: TRAP Activity (U/L) = x×1000×N÷t
Parameter Description:
a: Slope of the standard curve
b: Intercept of the standard curve
N: Dilution factor of the sample before addition to the reaction
1000: Unit conversion factor (1 mmol/L = 1000 μmol/L)
Cpr: Protein concentration of the sample (g prot/L)
t: Enzymatic reaction time (min)
ΔA₄₀₅: Absolute OD of the sample (Test OD - Control OD)
y: Standard OD - Blank OD
Notes
Sample Integrity: Test samples must not contain phosphatase inhibitors. Avoid repeated freeze-thaw cycles.
Low Sample Volume: If the sample volume is limited, reduce the sample volume and adjust the total volume to 0.1 mL with TRAP Assay Buffer in the Test and Control tubes.
Wavelength: If measuring at 405 nm is not possible, absorbance in the range of 400-415 nm is acceptable.
Standard Curve: It is recommended to generate a standard curve for each assay for the most accurate results. Avoid repeated freeze-thaw cycles of the p-nitrophenol standard.
Low Activity Samples: If sample TRAP activity is expected to be low, the incubation time can be extended up to 30 minutes.
Protein Assay: For tissue and cell samples, total protein concentration must be determined. A BCA Protein Assay Kit is recommended.
Working Solution Stability: Prepare only the amount of Working Color Solution needed for the immediate experiments, as it should be used on the day of preparation.
Safety: The p-nitrophenol solution is harmful to humans. The Stopping Solution is corrosive. Please handle with care.
Timing for Absolute Quantification: For precise absolute quantification, ensure accurate timing of the enzyme reaction. Using a longer incubation time (e.g., 30 min) can help minimize timing errors.
Personal Protection: For your safety and health, please wear laboratory appropriate clothing and disposable gloves.
| A1518291 | 组件 | 物理外观 | 100T | 储存温度 | 单次测试使用量 |
| A1518291A | 抗酒石酸酸性磷酸酶检测缓冲液 | 无色澄清透明溶液，无沉淀物 | 50 mL | 2-8°C. | 按需 |
| A1518291B | 对硝基苯磷酸二钠 | 淡黄色粉末 | 1EA×2 | -20°C, 避光 | 按需 |
| A1518291C | 酒石酸溶液 | 无色澄清透明溶液，无沉淀物 | 2.5 mL | 2-8°C. | 20 µL |
| A1518291D | 对硝基苯酚 (10 mM) | 无色/淡黄色透明溶液，无沉淀 | 0.5 mL | -20°C, 避光 | 100 µL |
| A1518291E | 终止液 | 无色澄清透明溶液，无沉淀物 | 85 mL | 室温 | 800 µL |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838352 |
分析证书 | A1518291 |
| ZJ26F0838351 |
分析证书 | A1518291 |
| ZJ26F0838350 |
分析证书 | A1518291 |
| ZJ26F0838349 |
分析证书 | A1518291 |
| ZJ26F0838348 |
分析证书 | A1518291 |
| ZJ26F0434310 |
分析证书 | A1518291 |
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