CN106967787B - 一种稳定的5’-核糖核苷酸水解酶检测试剂盒 - Google Patents
一种稳定的5’-核糖核苷酸水解酶检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种稳定的5'‑核糖核苷酸水解酶(过氧化物酶法)检测试剂盒，属于临床体外诊断检测试剂技术领域。本发明试剂盒包括试剂R1和试剂R2。通过选择100 mmol/L pH为7.6的HEPES缓冲液，同时在试剂R2中选择稳定性好的肌苷酸二钠作为底物，且添加适量的D‑海藻糖、甘露醇、Triton X100和叠氮钠，提高了试剂盒的热稳定性，试剂的准确度和抗干扰性也较好。因此，本发明提供的5'‑核糖核苷酸水解酶(过氧化物酶法)检测试剂盒有利于在市场中进一步的推广使用。
Description
技术领域
本发明涉及临床体外诊断检测试剂技术领域，特别涉及一种5'-核糖核苷酸水解酶检测试剂盒（过氧化物酶法）。
背景技术
5'-核糖核苷酸水解酶（5'-Nucleoticlase, 5'-NT）简称5'-核苷酸酶。5'-NT是一种对底物特异性不高的可作用于多种核苷酸的水解酶，能将次黄嘌呤核苷酸水解为次黄苷和磷酸。5'-NT广泛分布在人体和动物的组织中，例如肝、胆、胰、肠、心、脑、肺、肾、等脏器和组织中均有分布。5'-NT定位在细胞膜上，在肝内主要存在于胆小管和窦状间隙内。5'-NT在人体的运动、细胞生长发育、纤维蛋白合成、提高表皮或内皮屏障功能、神经传递及淋巴细胞的再循环及黏附、免疫应答等方面均发挥重要的作用。
本发明的试剂盒用于检测人体血清或血浆中5'-核糖核苷酸水解酶 （5'-NT）的活性，具有重要的临床意义。5'-NT活性增高常见于原发性和转移性肝癌、胆道癌、胰腺癌、胆道阻塞、胆管炎、急、慢性肝炎、肝硬化、药物性肝损伤，其活性增高可达2～6倍，且与病情严重程度呈正相关。5'-NT是诊断肝肿瘤非常灵敏的酶学指标。在病变早期，当肝功能、肝扫描和有关肝病检查阴性时本酶活性已明显增高。5'-NT经肝细胞膜进入胆汁，随之进入血清中，是检测肝胆疾病的指标之一。肝内外胆管堵塞和伴随循环转移的乳房切除术后的病人此酶升高明显。对于AFP阴性病例，其与AFP互补诊断肝癌的阳性率可达94％。ALP在肝胆系统疾病和骨骼系统疾病时均可增高。5'-NT除肝胆系统疾病增高外，骨骼系统疾病一般均不升高。因此5'-NT的检测能协助判断病人碱性磷酸酶（ALP）增高是由于肝胆系统疾病，还是骨髓系统疾病（如骨癌、骨肉瘤、佝偻病等）。5'-NT的检测也有助于鉴别诊断肝细胞性黄疸和阻塞性黄疸。
5'-核糖核苷酸水解酶（5'-NT）的检测方法主要有化学发光法、钼蓝比色法、过氧化物酶法等。本发明属于过氧化物酶法，优化反应体系，选用pH 7.6的HEPES缓冲液，选择稳定性更好的肌苷酸二钠为底物，并使用适量的D-海藻糖、甘露醇、Triton X100和叠氮钠为稳定剂，显著改善了检测试剂的热稳定性，发明了一种更稳定的5'-核糖核苷酸水解酶（过氧化物酶法）检测试剂盒。
发明内容
本发明的目的是提供一种稳定的用于检测血清或血浆中5'-核糖核苷酸水解酶（5'-NT）的试剂盒，该试剂盒采用过氧化物酶法。该试剂盒与常规的试剂盒相比，热稳定性和抗干扰性比常规的检测试剂盒要好，有利于保证试剂在夏季高温时节的长途运输中品质稳定，有利于在临床上的推广应用。
基本原理：
次黄嘌呤核苷酸(又名肌苷酸)在5'-核糖核苷酸水解酶（5'-NT）的催化下生成次黄苷和磷酸，次黄苷和磷酸在核苷酸磷酸化酶（PNP）作用下生成次黄嘌呤，次黄嘌呤在黄嘌呤氧化镁作用下氧化生成尿酸和过氧化氢，过氧化氢和TBHBA、4-AA在过氧化氢酶作用下生成红色醌类物质（Trinder's 反应）。
本发明是通过以下步骤得到的：
试剂R1组成为：
HEPES缓冲液，pH 7.6 100mmol/L
β-甘油磷酸钠 85mmol/L
氯化镁 53.4mmol/L
EDTA-Na2 12mmol/L
氯化钠 100mmol/L
亚铁氰化钾 0.01mmol/L
TBHBA 20mmol/L
叠氮钠 15mmol/L；
试剂R2组成为：
HEPES缓冲液，pH 7.6 100mmol/L
4-氨基安替比林 6mmol/L
肌苷酸二纳 6.8mmol/L
PNP 4.5kU/L
XOD 7.5kU/L
POD 30kU/L
D-海藻糖 5g/L
甘露醇 5%
Triton X100 1%
叠氮钠 15mmol/L。
所述的5'-核糖核苷酸水解酶检测试剂盒，试剂R1、R2均为100mmol/L pH 7.6的HEPES缓冲液。
所述的5'-核糖核苷酸水解酶检测试剂盒，其特征在于试剂R1含有镁离子激活剂，且有EDTA调节其浓度平衡。
所述的5'-核糖核苷酸水解酶检测试剂盒，其特征在于试剂R2选择较为稳定的肌苷酸二钠作为底物。
所述的5'-核糖核苷酸水解酶检测试剂盒，其特征在于试剂R2使用适量的D-海藻糖、甘露醇、Triton X100和叠氮钠来提高试剂的热稳定性。
所述的5'-核糖核苷酸水解酶检测试剂盒，其特征在于使用全自动生化分析仪采用速率法进行测定，检测主波长为546nm，副波长为800nm。
本发明的试剂盒在具有双试剂功能的全自动生化分析仪上进行，其具体使用方法如下：
加入生理盐水、样本或校准品5μl，再加入180μl 的R1试剂预孵育5min，加入90μl的R2试剂，延迟3min后读取吸光度A1，再反应2min后，读取吸光度A2，并计算ΔA/min。
本发明的有益效果：
1）试剂R1中含有镁离子作为5'-NT的激活剂，且有EDTA调节其浓度平衡，试剂的准确度良好；
2）试剂R1和试剂R2采用稳定性较好且具有适宜缓冲范围的HEPES缓冲体系，改善了试剂的缓冲性能，可提供更为稳定的pH环境；
3）试剂R2中选择更为稳定的肌苷酸二钠为底物，使用了适量的D-海藻糖、甘露醇、Triton X100和叠氮钠，因此试剂的热稳定性好，且不会对试剂的准确度产生影响，有利于保证试剂在夏季高温时节的长途运输中品质稳定；
4）试剂的抗干扰能力良好，成本也低，有利于该试剂在市场中进一步的推广。
附图说明
图1为两种试剂的相关性曲线图；
图2为本试剂盒具体使用方法；
图3为实施例1试剂与市场认可的5'-核糖核苷酸水解酶检测试剂盒对比检测结果；
图4为实施例试剂抗干扰性能比较；
图5为实施例1试剂热稳定性验证实验检测结果。
具体实施方式
下面结合具体实施例对本发明进行进一步说明。
实施例1
一种稳定的5'-核糖核苷酸水解酶（过氧化物酶法）检测试剂盒，它包括试剂R1和试剂R2；
其中试剂R1组成为：
pH 7.6 HEPES缓冲液 100mmol/L
β-甘油磷酸钠 85mmol/L
氯化镁 53.4mmol/L
EDTA-Na2 12mmol/L
氯化钠 100mmol/L
亚铁氰化钾 0.01mmol/L
TBHBA 20mmol/L
叠氮钠 15mmol/L；
试剂R2组成为：
pH 7.6 HEPES缓冲液 100mmol/L
4-氨基安替比林 6mmol/L
肌苷酸二纳 6.8mmol/L
D-海藻糖 5g/L
甘露醇 5%
Triton X100 0.3%
叠氮钠 15mmol/L
PNP 4.5kU/L
XOD 7.5kU/L
POD 30kU/L。
本实施例所述试剂R1和试剂R2，配制时需先加入缓冲物质HEPES，用氢氧化钠调到适当pH值后，再加入其它物质，各种酶类最后加入。本实施例所述试剂盒，在使用时，其测定方法是采用具有双试剂功能的日立7180全自动生化分析仪，利用速率法进行测定，检测主波长为546nm，副波长为800nm。
操作如下：
加入生理盐水、样本或校准品5μl，再加入180μl 的R1试剂预孵育5min，加入90μl的R2试剂，延迟3min后读取吸光度A1，再反应2min后，读取吸光度A2，并计算ΔA/min。
ΔA/min＝（A2- A1）/反应时间
5'-核糖核苷酸水解酶含量（U/L）＝（∆A/min测定÷∆A/min标准）×C标准
测定：检测样本 ∆A/min标准：标准品。
实施例2
准确度验证试验：将实施例1的5'-核糖核苷酸水解酶检测试剂作为实验组，市场上获得认可的一种准确度好、稳定性好的5'-核糖核苷酸水解酶检测试剂盒作为对照组进行检测，对22个临床血清样本进行检测，检测结果如图3所示。获得了两种试剂的相关性曲线如图1，结果表明，两组试剂盒的相关系数为0.999，说明两者相关性比较好。证明本发明试剂盒添加及更改的组分对其准确性不会造成影响，试剂盒依然保持较好的准确度。
实施例3
干扰性试验：取新鲜混合血清，分成2等份，然后将每等份再分成6等份，加入不同的干扰物质，使其在血清中的浓度达到表2的要求。然后分别用实施例1所得试剂，与市场常见并认可的5'-核糖核苷酸水解酶同时对比测定5'-核糖核苷酸水解酶的含量，对照组测定结果与加入不同干扰物质后各组的测定结果见图4。相对偏差(%)=(干扰样本的测定均值－对照样本的测定均值)/对照样本的测定均值×100%。
由图4可以看出，实施例1试剂在胆红素≤20mg/dL、血红蛋白≤500mg/dL、抗坏血酸≤20mg/dL、甘油三酯≤1250mg/dL、碱性磷酸酶≤1250U/L对测试结果没有明显干扰。与对照结果相比，本发明试剂的抗干扰能力更好。
实施例4
试剂热稳定性验证试验的具体操作方法：
将本发明实施例1中的试剂为试验组，一种市售5'-核糖核苷酸水解酶(过氧化物酶法)检测试剂盒的试剂为对照组，将试验组和对照组试剂分别封闭放置在37℃恒温水浴锅中（每天仅在检测的时候取出，检测完毕后，依然封口放回37℃水浴锅中，连续7天），作为37℃热稳定性验证。使用的全自动生化分析仪为日立7180。取冻干粉质控品，溶解均匀后，平均分成7份，-20℃储存，每天检测一个质控品，并且跟踪检测结果，其热稳定性结果见图5。
实验结果显示，实施例1试剂在37℃密封贮存7天，检测结果与初始检测结果的相对偏差未超过10%，较为稳定；而对照组试剂在37℃密封保存5天后与初始检测浓度的偏差开始超过10%，说明本发明试剂所选用的缓冲体系及添加适量D-海藻糖、甘露醇、TritonX100和叠氮钠后，试剂抗热稳定性增强。
综上所述，本发明提供的5'-核糖核苷酸水解酶（过氧化物酶法）检测试剂盒，选取GOODS缓冲体系中的100 mmol/L pH为7.6的HEPES缓冲液，同时在试剂R2中选择稳定性好的肌苷酸二钠作为底物，且添加适量的D-海藻糖、甘露醇、Triton X100和叠氮钠，提高了试剂盒的热稳定性，能够保证试剂在夏季高温时节的长途运输中品质稳定。此外，试剂的准确度和抗干扰性也较好。因此，本发明提供的5'-核糖核苷酸水解酶(过氧化物酶法)检测试剂盒有利于在市场中进一步的推广使用。
Claims (1)
1.一种稳定的5’-核糖核苷酸水解酶检测试剂盒，其特征在于，包括体积比为2：1的试剂R1和试剂R2，其组分和含量分别如下：
试剂R1：
HEPES缓冲液，pH 7.6 100mmol/L
β-甘油磷酸钠 85mmol/L
氯化镁 53.4mmol/L
EDTA-Na2 12mmol/L
氯化钠 100mmol/L
亚铁氰化钾 0.01mmol/L
TBHBA 20mmol/L
叠氮钠 15mmol/L；
试剂R2：
HEPES缓冲液，pH 7.6 100mmol/L
4-氨基安替比林 6mmol/L
肌苷酸二钠 6.8mmol/L
核苷酸磷酸化酶 4.5kU/L
黄嘌呤氧化酶 7.5kU/L
过氧化物酶 30kU/L
D-海藻糖 5g/L
甘露醇 5%
Triton X100 0.3%
叠氮钠 15mmol/L。
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