本发明专利技术涉及血清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。
【技术实现步骤摘要】
本专利技术涉及血清5' -核糖核苷酸水解酶检测
，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。
技术介绍
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，本专利技术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。
技术实现思路
本专利技术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核苷酸磷酸化酶黄嘌呤氧化酶POD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 本专利技术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_甘油憐酉爱纳..........................................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.................................................l〇g/L, BSA....................................................2g/L， 十二烷基二甲基甜菜碱（BS-12)...............................2g/L， 防腐剂.................................................〇· 5g/L; 2)试剂R2的组分为： 缓冲液..................................................100mmol/L, 5' -次黄噪呤核苷酉爱........................................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..................................................l〇g/L, BSA.....................................................2g/L， 十二烷基二甲基甜菜碱（BS-12)................................2g/L， 防腐剂..................................................〇· 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°C，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°C，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。 本专利技术的有益效果： 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， 增本文档来自技高网...
【技术保护点】
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； 2）试剂R2的组分为：缓冲液············································································································100mmol/L，5’‑次黄嘌呤核苷酸·············································...
【技术特征摘要】
【专利技术属性】
技术研发人员：赵新，李志明，谭柏清，罗维晓，王进，
申请(专利权)人：山东博科生物产业有限公司，
类型：发明
国别省市：山东;37
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