碱性磷酸酶 (S-AKP/ALP)广泛分布于动物组织、土壤及微生物中，可催化磷酸单酯水解脱磷酸反应。生物样本中，血清或组织 AKP/ALP 活性异常可反映骨骼代谢、肝胆损伤、细胞分化状态，常用于病理模型评估与细胞成骨分化功能检测。土壤中碱性磷酸酶是关键土壤酶指标，参与土壤有机磷矿化分解，将有机磷转化为植物可利用无机磷，其活性高低直接表征土壤磷素供应能力、微生物代谢水平及土壤肥力状况，是土壤质量评价、施肥效应与生态修复研究的重要参数。
本试剂盒检测原理为碱性磷酸酶（S-AKP/ALP）能够在碱性环境中催化磷酸苯二钠水解生成苯酚和磷酸氢二钠，在碱性条件下苯酚与氨基安替比林结合，并经氧化生成红色醌式结构物，呈深浅不一的红色，产物红色越深说明碱性磷酸酶活性越高，反之则酶活性越低，通过分光光度计测定510 nm 处吸光度，就可以计算出碱性磷酸酶活性水平。
A1528169 | 组件 | 物理外观 | 48T | 96 T | 储存条件 | 单次测试使用量 |
A1528169A | 检测缓冲液 | 无色液体 | 2.5 mL | 5.0 mL | 2-8℃. | 50 µL |
A1528169B | 提取液 | 无色液体 | 20 mL | 40 mL | 2-8℃. | 按需 |
A1528169C | 显色底物 | 无色液体 | 2.5 mL | 5.0 mL | 2-8℃.避光. | 50 µL |
A1528169D | 显色液 A | 浅黄液体 | 1.5 mL | 3.0 mL | 2-8℃.避光. | 30 µL |
A1528169E | 显色液 B | 浅黄液体 | 6.0 mL | 12 mL | 2-8℃.避光. | 120 µL |
A1528169F | 标准品(1mg/mL) | 无色液体 | 1.0 mL | 1.5 mL | 2-8℃.避光. | 按需 |
注意：显色液B和标准品有一定刺激性或毒性，请规范穿戴实验服和手套，避免接触到皮肤。
使用方法
1. 自备试剂耗材
(1) 96 孔板，PBS，去离子水。
(2) 37℃恒温培养箱或者水浴锅，酶标仪或可见分光光度计（能测 510 nm 处的吸光度）。
2. 样品准备
(1) 组织样本：请尽量使用新鲜的组织样本进行测定。每10 mg组织样本加入100-200 μL提取液，快速冰上匀浆（匀浆器提前放冰上预冷）。匀浆后的样本，4℃，8,000 g 离心10 min，取上清液，置冰上待测。
(2) 血液、血浆或者尿液样品：对于血清和血浆样品，按照常规方法制备后，可直接用于该试剂盒的检测；尿液样本通常也可以直接用于该试剂盒检测。
(3) 细胞 (菌) 样本：请尽量使用新鲜的细胞 (菌) 进行测定，而不要使用冻存的细胞 (菌) 进行测定。收集细胞 (菌)，首先用预冷的PBS清洗细胞 (菌) 2次 (PBS 重悬，4℃ 500 g 离心 5 min)，1×10⁶ 个细胞 (菌)建议加入100-200 μL提取液重悬，冰浴超声波破碎 (200 W，超声 2 s，间隔 3 s，5min)，4℃，8,000 g 离心 5 min，取上清液，置冰上待测。
3. 样品检测
建议正式实验前，选取2个样本做预测定，了解实验样品情况，熟悉流程，避免样本和试剂浪费。
(1) 标曲稀释：取50 μL标准品(1 mg/mL) + 950 μL去离子水，配制成0.05 mg/mL的标准品工作液，然后将标准品工作液按照下表稀释。
标准品 (0.05mg/mL) (µL) | 0 | 5 | 10 | 20 | 30 | 40 | 50 |
去离子水 (µL) | 50 | 45 | 40 | 30 | 20 | 10 | 0 |
相当于金氏单位 (U/L) | 0 | 5 | 10 | 20 | 30 | 40 | 50 |
(2) 显色工作液液配制：按照显色液A:显色液B=1:4比例混匀配制，以配制150 µL 显色工作液为例，取30 µL 显色液A和120 µL 显色液B混匀即可。显色工作液需现配现用。
(3) 上机检测：在96 孔板中按照下表，依次加入各试剂
试剂名称 | 测定管 | 对照管 | 空白管 | 标准管 |
待测样品 | 50 μL | 50 μL | - | - |
显色底物 | 50 μL | - | 50 μL | 50 μL |
去离子水 | - | 50 μL | 50 μL | - |
标准品 | - | - | - | 50 μL |
检测缓冲液 | 50 μL | 50 μL | 50 μL | 50 μL |
振荡混匀后于37 ℃避光孵育15 min。 |
显色工作液 | 150 μL | 150 μL | 150 μL | 150 μL |
混匀后，避光静置2 min后读取 A₅₁₀ 。ΔA=A测定 - A对照；Δ标准=A标准 - A空白 |
注意有上下标
4 结果计算
(1) 标准曲线：y=0.0204*X + 0.0293(仅供参考)；x是标准品浓度(μg/mL)，y 是△标准。算出样品ΔA的值，与标准曲线对比，即可算出样品中生成的苯酚浓度，然后换算成酶活单位。
(2) 碱性磷酸酶金氏活性单位定义：在 37℃条件下，100 mL待测样品与显色底物作用15 min，产生1 mg苯酚为一个金氏单位。
注意事项
1. 显色工作液需现用现配，需避光放置。
2. 显色液B和标准品有一定刺激性或者毒性，在操作中，请规范穿戴实验服和手套，避免接触到皮肤。
3. 本试剂盒仅用做科研研究，不得用于临床检测。
Alkaline phosphatase (S-AKP/ALP) is widely distributed in animal tissues, soil and microorganisms, and catalyzes the hydrolytic dephosphorylation of phosphate monoesters. In biological samples, abnormal AKP/ALP activity in serum or tissues reflects bone metabolism, hepatobiliary injury and cell differentiation status, and is frequently applied in pathological model evaluation and osteogenic differentiation assay. Soil-borne alkaline phosphatase serves as a critical soil enzyme indicator. It participates in the mineralization and decomposition of soil organic phosphorus, converting organic phosphorus into plant‑available inorganic phosphorus. Its activity directly characterizes soil phosphorus supply capacity, microbial metabolic level and soil fertility, acting as an essential parameter for soil quality assessment, fertilization‑effect investigation and ecological restoration research.
The assay principle of this kit is described as follows. Under alkaline conditions, SAKP/ALP catalyzes the hydrolysis of disodium phenyl phosphate to produce phenol and disodium hydrogen phosphate. Phenol reacts with 4aminoantipyrine under alkaline environment and is oxidized to form red quinonoid compounds. The red color intensity is positively correlated with alkaline phosphatase activity. Higher enzyme activity yields deeper red color, and vice versa. The absorbance is measured at 510 nm using a spectrophotometer to calculate alkaline phosphatase activity.
A1528169 | Components | Appearance | 48T | 96T | Storage | Usage volume per test |
A1528169A | Assay Buffer | Colorless liquid | 2.5 mL | 5.0 mL | 2-8℃. | 50 µL |
A1528169B | Extraction Buffer | Colorless liquid | 20 mL | 40 mL | 2-8℃. | As need |
A1528169C | Chromogenic substrate | Colorless liquid | 2.5 mL | 5.0 mL | 2-8℃.Store in the dark. | 50 µL |
A1528169D | Chromogenic Solution A | Light yellow liquid | 1.5 mL | 3.0 mL | 2-8℃.Store in the dark. | 30 µL |
A1528169E | Chromogenic Solution B | Light yellow liquid | 6.0 mL | 12 mL | 2-8℃.Store in the dark. | 120 µL |
A1528169F | Standard (1mg/mL) | Colorless liquid | 1.0 mL | 1.5 mL | 2-8℃.Store in the dark. | As need |
Note: Chromogenic Solution B and standard solution are irritant and toxic. Wear lab coat and gloves properly during operation to avoid skin contact.
Usage Method
1. Self-supplied Reagents and Consumables
(1) 96-well plates, PBS, deionized water.
(2) Incubator or water bath set at 37°C; microplate reader or visible spectrophotometer (capable of measuring absorbance at 510 nm).
2. Sample Preparation
(1) Tissue samples: Use fresh tissue samples for determination whenever possible. Add 100‑200 μL of Extraction Buffer per 10 mg tissue sample, and perform rapid homogenization on ice (pre‑cool the homogenizer on ice in advance). Centrifuge the homogenate at 8 000 g for 10 min at 4 °C. Collect the supernatant and keep it on ice for subsequent assay.
(2) Blood, plasma or urine samples: Serum and plasma samples can be directly assayed with this kit after routine preparation; urine samples are generally suitable for direct detection as well.
(3) Cell (bacterial) samples: Fresh cells/bacteria are recommended instead of frozen ones. Harvest cells/bacteria and wash twice with pre‑cooled PBS (resuspend in PBS, centrifuge at 500 g for 5 min at 4 °C). For 1×10⁶ cells or bacteria, resuspend in 100‑200 μL Extraction Buffer. Lyse by sonication on ice (200 W, 2 s pulse on, 3 s pulse off, total 5 min). Centrifuge the lysate at 8 000 g for 5 min at 4 °C. Collect the supernatant and keep on ice for testing.
3. Sample Detection
It is recommended to perform a pre-test with 2 samples prior to the formal experiment to characterize sample properties and get familiar with the operating procedure, so as to avoid waste of samples and reagents.
(1) Standard Curve Dilution: Pipette 50 μL of standard stock solution (1 mg/mL) and mix with 950 μL deionized water to prepare 0.05 mg/mL standard working solution. Then dilute the standard working solution according to the table below.
Standard (0.05mg/mL) (µL) | 0 | 5 | 10 | 20 | 30 | 40 | 50 |
Deionized water (µL) | 50 | 45 | 40 | 30 | 20 | 10 | 0 |
Corresponding King’s unit (U/L) | 0 | 5 | 10 | 20 | 30 | 40 | 50 |
(2) Preparation of Chromogenic Working Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a volume ratio of 1:4. For example, to prepare 150 μL chromogenic working solution, combine 30 μL Chromogenic Solution A with 120 μL Chromogenic Solution B and mix thoroughly. The Chromogenic Working Solution is recommended to be prepared fresh before use.
(3) Microplate Assay Setup: Add each reagent sequentially into the 96-well plate according to the table below.
Reagent Name | Sample Tube | Control Tube | Blank Tube | Standard Tube |
Sample to be tested | 50 μL | 50 μL | - | - |
Chromogenic substrate | 50 μL | - | 50 μL | 50 μL |
Deionized Water | - | 50 μL | 50 μL | - |
Standard | - | - | - | 50 μL |
Assay Buffer | 50 μL | 50 μL | 50 μL | 50 μL |
Mix thoroughly by shaking, then incubate at 37 °C in the dark for 15 min. |
Chromogenic Working Solution | 150 μL | 150 μL | 150 μL | 150 μL |
Mix well and stand for 2 min in the dark and then record absorbance at 510 nm ( A₅₁₀ ). ΔA = A<sub>sample</sub> − A<sub>control</sub>；Δstandard = A<sub>standard</sub> − A<sub>blank</sub>. |
4. Result Calculation
(1) Standard curve: (y = 0.0204*X + 0.0293) (for reference only); where X is standard concentration (μg/mL), and y is Δstandard. Calculate the ΔA value of the sample. Compare it against the standard curve to obtain the phenol concentration produced in the sample, and further convert it into enzyme activity units.
(2) Definition of King’s unit for alkaline phosphatase activity: Under 37 °C, one King’s unit is defined as the amount of enzyme that produces 1 mg of phenol after 15 min reaction between 100 mL test sample and chromogenic substrate.
Precautions
1. The chromogenic working solution shall be freshly prepared before use and stored away from light.
2. Chromogenic Solution B and standard solution are irritant and toxic. Wear lab coat and gloves properly during operation to avoid skin contact.
3. This kit is for research-only purposes and shall not be used for clinical diagnosis.
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