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。
本试剂盒检测原理如下：底物腺苷在腺苷脱氨酶(ADA)作用下水解为次黄嘌呤核苷，次黄嘌呤核苷在嘌呤核苷磷酸酶催化下水解，生成次黄嘌呤和磷酸核糖。次黄嘌呤在黄嘌呤氧化酶作用下产生过氧化氢，过氧化氢在过氧化物酶、4-氨基安替比林及色源的作用下，产生红色物质，在波长550 nm 处有最大吸收，检测单位时间内红色物质产生的速率可以得到腺苷脱氨酶活力大小。
使用方法
1. 需自备试剂
0.9%氯化钠溶液。
2. 样本制备
血清血浆样本：可直接测定
组织样本：匀浆介质是生理盐水(0.9%氯化钠溶液)，匀浆离心后取上清进行测定，留取部分上清测蛋白。
3. 标准品的配制
按照下表用双蒸水将标准品稀释至0、0.2、0.4、0.5、0.6、0.7、0.8、1 mmol/L。
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
浓度（mmol/L） | 0 | 0.2 | 0.4 | 0.5 | 0.6 | 0.7 | 0.8 | 1 |
标准品（μL） | 0 | 20 | 40 | 50 | 60 | 70 | 80 | 100 |
双蒸水（μL） | 100 | 80 | 60 | 50 | 40 | 30 | 20 | 0 |
4. 样本测定
按照下表进行样本测定（在96孔板中反应）。
| 标准孔 | 测定孔 |
待测样本（μL） | - | 10 |
不同浓度标准品（μL） | 10 | - |
工作液（μL） | 180 | 180 |
显色剂（μL） | 90 | 90 |
37°C 下孵育7 min，酶标仪于550 nm 处，检测测定孔吸光度，记为A₁ 。 |
继续在37°C 下孵育10 min，酶标仪于550 nm 处检测标准孔与测定孔吸光度，记为A₂ 。注意：标准孔只需要检测A₂ ，测定孔需要检测A₁ 与A₂ 。 |
5. 结果计算
标准品拟合曲线：y = ax + b
（1）组织样本中腺苷脱氨酶活力计算公式：
定义：37°C 条件下，每克组织蛋白每分钟催化底物产生1 μmol 次黄嘌呤核苷的所需要的酶量为一个活力单位。
ADA 活力(U/gprot)=(A₂ - A₁ - b) ÷ a × 1000* ÷ T ÷ Cₚᵣ × f
（2）血清(浆)样本中腺苷脱氨酶活力计算公式：
定义：37°C 条件下，每升血清(浆)每分钟催化底物产生1 μmol 次黄嘌呤核苷的所需要的酶量为一个活力单位。
ADA 活力(U/L)= ( A₂ - A₁ - b) ÷ a × 1000* ÷ T × f
y：标准品OD值-空白OD 值(标准品浓度为0 时的OD 值)；
x：标准品的浓度；
a：标曲的斜率；
b：标曲的截距；
A₁ ：第一次孵育7 min 后检测的OD 值；
A₂ ：第二次孵育10 min 后检测的OD 值；
T：第二次孵育时间，10 min；
1000*：单位换算1 mmol=1000 μmoL；
Cₚᵣ ：待测样本的蛋白浓度(gprot/L)；
f：样本加入检测体系前的稀释倍数
注意事项
1. 加显色剂时，建议使用多道移液器快速加入，时间控制在2 min 之内。
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. 为了您的安全和健康，请穿实验服并戴乳胶手套操作。
Adenosine Deaminase (ADA) is an important enzyme in purine nucleotide metabolism and belongs to a type of sulfhydryl enzyme. Each molecule contains at least 2 active sulfhydryl groups. ADA can catalyze the conversion of adenosine nucleotide to hypoxanthine nucleotide, which is then phosphorylated by nucleoside phosphorylase to form hypoxanthine. The final metabolite of this process is uric acid. ADA is widely distributed in various tissues of the human body, with the highest content in thymus, spleen, and other lymphoid tissues, while the content is low in liver, lung, kidney, and skeletal muscle.
The detection principle of this kit is as follows: The substrate adenosine is hydrolyzed by adenosine deaminase (ADA) to form inosine, which is then hydrolyzed by purine nucleoside phosphorylase (PNP) to generate hypoxanthine and phosphoribose. Hypoxanthine is oxidized by xanthine oxidase to produce hydrogen peroxide, which, under the action of peroxidase, 4-Aminoantipyrine (4-AAP), and chromogenic reagents, generates a red substance with the maximum absorption at 550 nm. The rate of the production of the red substance within a certain period of time can be used to determine the activity of adenosine deaminase.
Instructions for Use
1. Reagents to be prepared by yourself
0.9% sodium chloride solution.
2. Sample preparation
Serum and plasma samples: can be directly measured
Tissue samples: homogenization medium is physiological saline (0.9% sodium chloride solution). After homogenization and centrifugation, the supernatant is taken for measurement, and a portion of the supernatant is retained for protein measurement.
3. Preparation of standards
Using deionized water, dilute the standards to 0, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, and 1 mmol/L according to the table below.
Serial Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Concentration (mmol/L) | 0 | 0.2 | 0.4 | 0.5 | 0.6 | 0.7 | 0.8 | 1 |
Standard solution (μL) | 0 | 20 | 40 | 50 | 60 | 70 | 80 | 100 |
Deionized water (μL) | 100 | 80 | 60 | 50 | 40 | 30 | 20 | 0 |
4. Sample Measurement
Carry out the sample measurement according to the table below (with the reaction taking place in a 96-well plate).
Reagent name | Standard well | Sample well |
Sample (μL) | - | 10 |
Standard solutions of different concentrations (μL) | 10 | - |
Working solution (μL) | 180 | 180 |
Chromogenic Agent (μL) | 90 | 90 |
Incubate for 7 minutes at 37°C, then measure the absorbance of the Sample wells at 550 nm using a microplate reader and record as A₁ . |
Continue incubating for an additional 10 minutes at 37°C, then measure the absorbance of both standard and sample wells at 550 nm and record as A₂ . Note: Only A₂ needs to be measured for the standard wells; both A₁ and A₂ must be measured for the sample wells. |
5. Result calculation
Standard curve equation: y = ax + b
(1) Adenosine deaminase activity calculation for tissue samples:
Definition: One unit of enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol inosine monophosphate per minute per gram of tissue protein under 37°C conditions.
ADA activity (U/gprot) = (A₂ - A₁ - b) ÷ a × 1000* ÷ T ÷ Cₚᵣ × f
(2) Adenosine deaminase activity calculation for serum (plasma) samples:
Definition: One unit of enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol inosine monophosphate per minute per liter of serum (plasma) under 37°C conditions.
ADA activity (U/L) = ( A₂ - A₁ - b) ÷ a × 1000* ÷ T × f
y: measured OD value minus blank OD value (OD at zero standard concentration);
x: standard concentration;
a: slope of the standard curve;
b: intercept of the standard curve;
A₁ : OD value measured after first incubation for 7 minutes;
A₂ : OD value measured after second incubation for 10 minutes;
T: duration of second incubation, 10 minutes;
1000*: unit conversion, 1 mmol = 1000 μmol;
Cₚᵣ : protein concentration of the sample (gprot/L);
f: dilution factor applied to the sample prior to addition into the assay system
Precautions
1. When adding the Chromogenic Agent, it is recommended to use a multi-channel pipette to add it quickly, with the time controlled within 2 minutes.
2. Before the formal test, it is recommended to select 2-3 samples with significant expected differences for a preliminary experiment.
3. For your safety and health, please wear a lab coat and latex gloves during the operation.
ZJ26F0737242
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