CN105420345B - 一种稳定、抗干扰能力强的血清5’-核糖核苷酸水解酶检测试剂及检测方法 - Google Patents
一种稳定、抗干扰能力强的血清5’-核糖核苷酸水解酶检测试剂及检测方法 Download PDFInfo
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Abstract
本发明涉及血清5’‑核糖核苷酸水解酶检测技术领域,特别涉及一种血清5’‑核糖核苷酸水解酶检测试剂。采用PIPES缓冲液,添加多种稳定剂,显著改善了试剂的稳定性;通过添加β‑甘油磷酸钠、胆红素氧化酶和抗坏血酸氧化酶,可以有效避免碱性磷酸酶、胆红素和抗坏血酸的干扰,大大增强试剂的抗干扰能力。此外,优选的新型两性表面活性剂十二烷基二甲基甜菜碱(BS‑12)的加入可防止反应体系浑浊,增强底物的稳定性,提高试剂的抗干扰能力。
Description
技术领域
本发明涉及血清5’-核糖核苷酸水解酶检测技术领域,特别涉及一种血清5’-核糖核苷酸水解酶检测试剂,还涉及使用此检测试剂的检测方法。
背景技术
5’-核糖核苷酸水解酶(5’-NT)是一种特殊的水解酶,能特异性地将次黄嘌呤核苷酸水解为次黄苷和磷酸。此酶广泛存在于人体组织,如肝、胆、肠、脑、心、胰等,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。肝内外胆管阻塞和伴随循环转移的乳房切除术后的病人此酶升高明显。诊断肝胆疾病5’-NT 较肝内其它酶类更敏感。
血清5'-NT 测定有化学比色法、速率法、酶比色法等,目前酶比色法较常用。 化学比色法虽然试剂易得,但操作麻烦,试剂种类多,不适宜自动分析难以自动化。酶比色法,采用多级偶联酶促反应,操作简单,适用自动化分析,但存在稳定性差,易受干扰等缺点。
鉴于此,本发明在酶比色法的基础上优化反应体系,添加稳定剂丙三醇、聚乙二醇6000、甘露醇、海藻糖、BSA、乙二醇等可有效提高试剂的稳定性;并且通过添加β-甘油磷酸钠、胆红素氧化酶和抗坏血酸氧化酶,可以有效避免碱性磷酸酶、胆红素和抗坏血酸的干扰,大大增强试剂的抗干扰能力。此外,优选的新型两性表面活性剂十二烷基二甲基甜菜碱(BS-12)的加入可防止反应体系浑浊,增强底物的稳定性,提高试剂的抗干扰能力。该试剂操作简便快速,适用于自动化分析,是一种更加稳定、抗干扰能力强的血清5’-核糖核苷酸水解酶(5’-NT)试剂。
发明内容
本发明的目的是提供一种用于检测血清5’-核糖核苷酸水解酶(5’-NT)的试剂及使用该试剂检测血清5’-核糖核苷酸水解酶活性的方法。该试剂盒采用酶比色法,可以有效检测血清5’-核糖核苷酸水解酶的活性,抗干扰能力强,稳定性好等优点。
基本原理:
5’-NT催化底物中次黄嘌呤核苷酸反应产生次黄苷和磷酸,再经过一系列催化反应最终生成红色醌类。在波长550nm处检测其吸光度,其变化程度与样本中的5’-NT活性成正比。
5’-NT
次黄嘌呤核苷酸+H2O次黄苷+磷酸
核苷酸磷酸化酶
次黄苷+磷酸次黄嘌呤+核酸-1-磷酸
黄嘌呤氧化酶
次黄嘌呤+2H2O+2O2 尿酸+2H2O2
POD
2H2O2+EHSPT+4-AA4H2O+红色醌类
注:EHSPT:N-乙基-N-(2-羟基-3硫代丙基)-3-甲基苯胺4-AA:4-氨基氨替吡啉POD:过氧化物酶
本发明是通过以下步骤得到的:
一种血清5’-核糖核苷酸水解酶检测试剂,包括试剂R1和试剂R2,所述试剂R1和试剂R2的组成如下:
试剂R1中含有
缓冲液········································100mmol/L,
4-氨基氨替吡啉································2.5mmol/L,
过氧化物酶····································2KU/L,
核苷酸磷酸化酶·······························2KU/L,
黄嘌呤氧化酶··································3KU/L,
β-甘油磷酸钠··································200mmol/L,
胆红素氧化酶··································2KU/L,
抗坏血酸氧化酶································3KU/L,
丙三醇········································5ml/L,
聚乙二醇6000··································10g/L,
乙二醇········································5ml/L,
甘露醇········································20g/L,
海藻糖········································10g/L,
BSA···········································2g/L,
十二烷基二甲基甜菜碱(BS-12)···················2g/L,
防腐剂········································0.5g/L;
2)试剂R2的组分为:
缓冲液········································100mmol/L,
5’-次黄嘌呤核苷酸·····························14mmol/L,
EHSPT·········································4mmol/L,
丙三醇········································5ml/L,
聚乙二醇6000··································10g/L,
乙二醇········································5ml/L,
甘露醇········································20g/L,
海藻糖········································10g/L,
BSA···········································2g/L,
十二烷基二甲基甜菜碱(BS-12)···················2g/L,
防腐剂········································0.5g/L;
所述的血清5’-核糖核苷酸水解酶检测试剂,试剂R1中缓冲液为25℃,pH为7.6的PIPES缓冲液。
所述的血清5’-核糖核苷酸水解酶检测试剂,试剂R2中缓冲液为25℃,pH为7.6的PIPES缓冲液。
所述的血清5’-核糖核苷酸水解酶检测试剂,所述防腐剂为NaN3。
所述的血清5’-核糖核苷酸水解酶检测试剂来检测血清5’-核糖核苷酸水解酶含量的检测方法,使用全自动生化分析仪利用速率法进行测定,检测主波长为550nm。
所述的检测方法,R1试剂和R2试剂的比例为2:1。
本发明的有益效果:
1)优化反应体系并添加丙三醇、聚乙二醇6000、甘露醇、海藻糖、BSA、乙二醇等多种稳定剂,可以显著改善试剂的稳定性;
2)新型两性表面活性剂十二烷基二甲基甜菜碱(BS-12)的加入可防止反应体系浑浊,增强底物的稳定性,提高试剂的抗干扰能力。
3)添加β-甘油磷酸钠、胆红素氧化酶和抗坏血酸氧化酶,可以有效避免碱性磷酸酶、胆红素和抗坏血酸的干扰,大大增强试剂的抗干扰能力。
4)试剂的准确度和稳定性良好,抗干扰性强,使用方便,完全可以满足临床需要。
附图说明
图1为两种试剂的相关性曲线图,
图2为两种试剂效期稳定性曲线图。
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
血清5’-核糖核苷酸水解酶的检测试剂,包试剂R1和试剂R2:
1)其R1的组成为:
PIPES(1,4-哌嗪二乙磺酸)缓冲液(pH=7.6,25℃) ·········100mmol/L,
4-氨基氨替吡啉······································2.5mmol/L,
过氧化物酶··········································2KU/L,
核苷酸磷酸化酶······································2KU/L,
黄嘌呤氧化酶········································3KU/L,
β-甘油磷酸钠········································200mmol/L,
胆红素氧化酶········································2KU/L,
抗坏血酸氧化酶······································3KU/L,
丙三醇··············································5ml/L,
聚乙二醇6000········································10g/L,
乙二醇··············································5ml/L,
甘露醇··············································20g/L,
海藻糖··············································10g/L,
BSA·················································2g/L,
十二烷基二甲基甜菜碱(BS-12)·························2g/L,
防腐剂NaN3··········································0.5g/L;
2)试剂R2的组分为:
PIPES(1,4-哌嗪二乙磺酸)缓冲液(pH=7.6,25℃) ·········100mmol/L,
5’-次黄嘌呤核苷酸···································14mmol/L,
EHSPT···············································4mmol/L,
丙三醇··············································5ml/L,
聚乙二醇6000········································10g/L,
乙二醇··············································5ml/L,
甘露醇··············································20g/L,
海藻糖··············································10g/L,
BSA·················································2g/L,
十二烷基二甲基甜菜碱(BS-12)·························2g/L,
防腐剂NaN3··········································0.5g/L;
3)本实施例试剂的使用方法:
本实施例描述的血清5’-核糖核苷酸水解酶检测试剂,在使用时采用具有双试剂功能的全自动生化分析仪,如日立7180全自动分析仪等,利用速率法进行测定。将R1和R2按照2:1的比例放置到对应的试剂位上,在样品盘的对应位置放置好蒸馏水、标准品和样本,操作如表1:
表1实施例1试剂检测方法
计算:血清5’-核糖核苷酸水解酶含量(U/L)=(∆A测定/min÷∆A标准/min)×C标准。
实施例2
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血清5’-核糖核苷酸水解酶(5’-NT)试剂同时对比测定血清中5’-NT的活性,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表2。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%。
由表2可以看出,实施例1试剂在胆红素≤20mg/dL、甘油三酯≤1250mg/dL、血红蛋白≤500mg/dL、抗坏血酸≤220mg/dL、碱性磷酸酶≤1250U/L对测试结果没有明显干扰。而对照组试剂在上述浓度干扰物质存在时,受到明显干扰,这说明通过优化反应缓冲体系、添加β-甘油磷酸钠、胆红素氧化酶和抗坏血酸氧化酶,及新型两性表面活性剂十二烷基二甲基甜菜碱(BS-12)后,实施例1试剂的抗干扰性能显著提高,远远优于对比试剂。
表2实施例试剂抗干扰性能比较
实施例3
相关性实验:利用实施例1配方配制试剂,与市场常见的国家食品药品监督管理局认可的某公司的5’-核糖核苷酸水解酶试剂盒进行对照检测,同时检测了20个临床血清样本,检测结果如表3所示。并获得了两种试剂的相关性曲线(如图1所示),通过检测结果显示,两个试剂盒的相关系数为0.9997,说明了两者有极大的相关性。
表3实施例1试剂与市场常见并得到认可的血清5’-核糖核苷酸水解酶测定试剂盒对比检测结果
实施例4
试剂的稳定性对比试验:对实施例1中的试剂,均匀分装13组,每组的试剂量为R1为20mL,R2为10mL;并且取13组市场常见的国家食品药品监督管理局认可的某公司的5’-核糖核苷酸水解酶(5’-NT)试剂盒作对照。放置到2-8℃冰箱中,每月的同一天取出一组试剂检测5’-NT质控品(靶值为12.5U/L),检测结果如图2所示,实施例1试剂在2-8℃储存条件下比市场常见的5’-核糖核苷酸水解酶(5’-NT)测定试剂盒更加稳定。
通过验证,本试剂与同类检测试剂对比相关性好,临床检测样本结果一致,能够达到市场对产品的应用要求,并且抗干扰性能好,是一种更加稳定、良好的5’-核糖核苷酸水解酶(5’-NT)检测试剂。
Claims (4)
1.一种血清5’-核糖核苷酸水解酶检测试剂,其特征在于包括试剂R1和试剂R2,所述试剂R1和试剂R2的组成如下:
试剂R1中含有
缓冲液·····················100mmol/L,
4-氨基氨替吡啉·················2.5mmol/L,
过氧化物酶···················2KU/L,
核苷酸磷酸化酶·················2KU/L,
黄嘌呤氧化酶··················3KU/L,
β-甘油磷酸钠··················200mmol/L,
胆红素氧化酶··················2KU/L,
抗坏血酸氧化酶·················3KU/L
丙三醇·····················5ml/L,
聚乙二醇6000··················10g/L,
乙二醇·····················5ml/L,
甘露醇·····················20g/L,
海藻糖·····················10g/L,
BSA······················2g/L,
十二烷基二甲基甜菜碱(BS-12)··········2g/L,
防腐剂·····················0.5g/L;
试剂R2的组分为:
缓冲液·····················100mmol/L,
5’-次黄嘌呤核苷酸···············14mmol/L,
EHSPT·····················4mmol/L,
丙三醇·····················5ml/L,
聚乙二醇6000··················10g/L,
乙二醇·····················5ml/L,
甘露醇·····················20g/L,
海藻糖·····················10g/L,
BSA······················2g/L,
十二烷基二甲基甜菜碱(BS-12)··········2g/L,
防腐剂·····················0.5g/L。
2.根据权利要求1所述的血清5’-核糖核苷酸水解酶检测试剂,其特征在于试剂R1中缓冲液为25℃,pH为7.6的PIPES缓冲液。
3.根据权利要求1所述的血清5’-核糖核苷酸水解酶检测试剂,其特征在于试剂R2中缓冲液为25℃,pH为7.6的PIPES缓冲液。
4.根据权利要求1所述的血清5’-核糖核苷酸水解酶检测试剂,其特征在于所述防腐剂为NaN3。
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