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腺苷脱氨酶(AdenosineDeaminase，ADA)是嘌呤核苷代谢中重要的酶类，属于一种巯基酶，每分子至少含2个活性巯基，ADA能催化腺嘌呤核苷转变为次黄嘌呤核苷，再经核苷磷酸化酶作用生成次黄嘌呤，其代谢缓和终产物为尿酸，ADA广泛分布于人体各组织中，以胸腺、脾和其他淋巴组织中含量最高，而肝、肺、肾和骨胳肌等含量低。
腺苷脱氨酶(ADA)检测试剂盒(波氏比色法)其检测原理是待测样品中的ADA催化腺嘌呤核苷水解脱氨，产生次黄嘌呤核苷和铵离子，利用波氏显色法测定氨离子生成量，其反应公式为：腺苷+ H₂O →次黄嘌呤+ NH₃ ，通过分光光度法(酶标仪)测定640nm处吸光度。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
注意事项：
1. 稀释样品和研磨样品所用水，均应为 ddH₂O，不可为普通的水。
2. 如果采用国际单位，需在测得活力单位基础上乘以1.19。
3. 如果没有分光光度计，也可用酶标仪测定，但应注意加入试剂量不同， 相应的检测次数会大大增加。
4. 该试剂盒测定下限在2-5 μg/mL之间；从肉眼观察，一般情况下浓度在 15-30 μg/mL即可显淡蓝色；浓度 ≥ 30 μg/mL可显蓝色。
5. 胸水标本经离心后取上清，置于4℃保存备用，ADA活性可稳定1周。
6. 血清样本应避免溶血，4℃保存3天。
附录：参考标准曲线范围：测定氨氮标准在2、4、6、8、10、15、20、25、 30、40、50、75、100 μg/mL时吸光度，据此作出其标准曲线如下:
使用说明：
1. 试剂准备
额外所需材料：
(1) 实验材料：血浆、血清、细胞或组织。
(2) 所需仪器：酶标仪、离心机、水浴锅。
(3) 其它：96孔板、离心管。
2. 准备样品
1) 血浆、血清样品：血浆、血清按照常规方法制备，可以直接用于该试剂盒的测定，-20℃冻存，用于ADA的测定。
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，-20℃ 冻存，用于ADA的测定。
3) 高活性样品：如果样品中含有较高活性的ADA，可以使用 ddH₂O 稀释。
4) (选做)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ADA含量。
3. 稀释标准品：用 ddH₂O 准确稀释氨氮标准(1mg/mL)至25 μg/mL，即为氨氮标准工作液(25 μg/mL)，4℃保存备用。
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4. ADA测定：以 ddH₂O 调零，酶标仪640 nm处测定吸光度(分别为A空白 、A标准 、A对照 、A测定 )。
5. 计算:
ADA活性单位的定义：在37℃ 1 mL血清中ADA 1小时催化底物产生1 μg 氨氮为一个ADA酶活力单位。
血清、血浆中ADA活力(A测定-A对照 )/(A标准-A空白 )×25
组织中ADA活力(U/mg)=[(A测定-A对照 )/(A标准-A空白 )×25]/待测样品的蛋白浓度(mg/mL)
式中：A测定=测定孔的吸光度
A对照 =对照孔的吸光度
A标准 =标准孔的吸光度
A空白=空白孔的吸光度
Adenosine Deaminase (ADA) is an essential sulfhydryl enzyme involved in purine nucleoside metabolism, with at least two active sulfhydryl groups per enzyme molecule. ADA catalyzes the conversion of adenosine to inosine, which is further decomposed into hypoxanthine by nucleoside phosphorylase; uric acid is the final metabolite. ADA is widely distributed in human tissues, predominantly enriched in thymus, spleen and other lymphoid tissues, with relatively low contents in liver, lung, kidney and skeletal muscle.
Based on the Boehringer colorimetric method, ADA in samples catalyzes hydrolytic deamination of adenosine to produce inosine and ammonium ions, and ammonium content is quantified via chromogenic reaction.
Reaction formula: Adenosine + H₂O → Inosine + NH₃
Absorbance is measured at 640 nm by spectrophotometry (microplate reader).
For research use only. Not for clinical diagnosis or other clinical applications.
Precautions
1. Double-distilled water ( ddH₂O ), instead of regular water, shall be adopted for sample dilution and tissue homogenization.
2. Multiply the determined activity value by 1.19 to convert results into international units.
3. A microplate reader can substitute for spectrophotometer; note that reagent addition volume differs, leading to a notable increase in detectable test numbers.
4. The lower limit of quantification ranges from 2 to 5 μg/mL. Visually, faint blue color appears at 15-30 μg/mL, and distinct blue develops at concentrations ≥30 μg/mL.
5. Supernatant of centrifuged pleural effusion can be stored at 4℃ for up to 1 week with stable ADA activity.
6. Hemolysis should be avoided for serum samples, which can be preserved at 4℃ for 3 days.
Appendix
Reference Calibration Curve Range:
The absorbance values of ammonia nitrogen standard solutions at concentrations of 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 75 and 100 μg/mL are measured for plotting the reference standard curve.
Operating Instructions
1. Preparation of Experimental Materials:
Additional required supplies:
(1) Test samples: plasma, serum, cells or tissues
(2) Instruments: microplate reader, centrifuge, thermostatic water bath
(3) Consumables: 96-well microplates, centrifuge tubes
2. Sample Pretreatment:
1) Plasma & serum: Prepare by standard protocols and test directly; store frozen at -20℃ for subsequent detection.
2) Cells & tissues: Homogenize specimens, collect supernatant after low-speed centrifugation and preserve at -20℃.
3) High-activity samples: Dilute samples with ddH₂O when ADA activity is excessively high.
4) Optional: Determine total protein concentration with BCA protein assay kit to calculate specific ADA activity per unit mass of total protein.
3. Preparation of Standard Working Solution:
Accurately dilute ammonia nitrogen stock standard (1 mg/mL) to 25 μg/mL with ddH₂O to prepare working standard solution; store at 4℃ before use.
4. Microplate Loading:
Set blank wells, standard wells, control wells and sample test wells in accordance with attached table. Add reagents sequentially and avoid air bubbles. Reduce sample volume or dilute samples appropriately if samples contain high ADA activity.
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5. ADA Measurement
Zero the microplate reader with ddH₂O , then measure absorbance at 640 nm to obtain Abₗₐₙₖ, Aₛₜₐₙdₐᵣd, A<sub>Control</sub> and Aₜₑₛₜrespectively.
6. Calculation
Definition of ADA activity unit
One unit (U) of ADA is defined as the amount of enzyme that catalyzes substrate to produce 1 μg ammonia nitrogen per hour in 1 mL serum at 37℃.
1) ADA activity in serum/plasma
2) ADA activity in tissue samples:
Nomenclature:
Aₜₑₛₜ: Absorbance of test well
AControl: Absorbance of control well
Aₛₜₐₙdₐᵣd: Absorbance of standard well
Abₗₐₙₖ: Absorbance of blank well
A1522300 | 组件 | 物理外观 | 100T | 储存 | 单次测试使用量 |
A1522300A | 氨氮标准 (1 mg/mL) | 无色液体 | 1 mL | 2-8°C | 按需 |
A1522300B | 底物缓冲液 | 无色液体 | 1.5 mL | 2-8°C | 12.5 μL |
A1522300C | 波氏ADA显色液 | 无色液体 | 15 mL | 2-8°C，避光 | 125 μL |
A1522300D | ADA检测缓冲液 | 无色液体 | 15 mL | 2-8°C，避光 | 125 μL |
A1522300E | ddH₂O | 无色液体 | 10 mL | 2-8°C | 按需 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737296 |
分析证书 | A1522300 |
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