一种稳定、抗干扰能力强的5’-核糖核苷酸水解酶检测试剂及检测方法技术

本发明专利技术涉及5’-NT检测技术领域，特别涉及一种5’-NT检测试剂，试剂R1中含有缓冲液，β-甘油磷酸钠，MgCl2，Toos，防腐剂；试剂R2中含有缓冲液，5’-IMP，EHSPT，PNP，XOD，POD，抗坏血酸氧化酶，4-AA，防腐剂。采用磷酸盐缓冲液，添加稳定剂β-甘油磷酸钠、抗坏血酸氧化酶，大大增强了试剂的稳定性；不仅显著改善测定的性能，而且增强了试剂的稳定性和抗干扰能力。

Stable, strong anti-interference 5 '- ribose nucleotide hydrolase detection reagent and detection method

The present invention relates to the technical field of 5 -NT detection, in particular to a 5 '-NT kit, containing buffer reagent R1, beta glycerophosphate, MgCl2, Toos, preservative; buffer reagent containing R2, -IMP, EHSPT, 5' PNP, XOD, POD, 4-AA, ascorbic acid oxidase, preservatives. Using phosphate buffer, adding stabilizer sodium glycerophosphate and ascorbic acid oxidase, greatly enhanced the stability of the reagent; not only significantly improve the determination of performance, but also enhance the stability and anti-interference ability of reagent.

【技术实现步骤摘要】
一种稳定、抗干扰能力强的5’-核糖核苷酸水解酶检测试剂及检测方法
本专利技术涉及5’-核糖核苷酸水解酶检测
，特别涉及一种抗干扰能力强的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’-核苷酸酶全称5’-核糖核苷酸磷酸水解酶（5’-Nucleoticlase或5’-NT），广泛分布在肝、胆、胰、肠、心、脑、肺、肾、垂体、甲状腺、前列腺、睾丸等脏器和组织中，定位在细胞膜上，在肝内主要存在于胆小管和窦状间隙内，是一种催化核苷5’-单磷酸水解生成核苷和无机磷酸盐的酶，最适pH为6.6-7.0，受Mg2＋或Mn2＋激活，为Ni2＋所抑制。众多研究资料表明，血清5’-NT活性升高主要见于肝胆胰系统疾病及某些恶性肿瘤，故有较特异的诊断价值。胆管结石或肿瘤所致之肝外胆管阻塞，以及氯丙嗪、肝癌或肝硬变引起肝内胆汁郁积时，病人血清5’-NT活性可升高2-6倍。原发性或继发性肝硬变时，病人血5’-NT升高较在慢性活动型肝炎时敏感。肝细胞损伤发生肝昏迷时，病人血清5’-NT活性多呈正常。原发性或继发性肝癌患者此酶活性升高的阳性率为88.4％-97.2％。急性胰腺炎病人血清5’-NT略有升高，慢性胰腺炎时血清酶活性正常。发生肝继发癌灶的胰体或胰尾癌病人血清酶活性明显升高，以肝内有继发病灶的胰头癌病人，血清5’-NT活性升高最为显著。原发性乳腺癌患者约有65％血清5’-NT活性升高，在发生全血性广泛转移的病人血清5’-NT活性全部升高。测定血液、体液中的5’-NT及其同工酶水平对某些疾病的诊断、鉴别诊断、治疗及免疫功能的研究越来越受到临床的重视。目前，文献报道的5’-NT活性的测定方法有钼酸铵反应法、氨量测定法、化学发光法、NADPH测定法、尿素生成法等。这些方法准确性差、灵敏度低、仪器设备要求高、试剂成本高，难于全面推广应用。鉴于此，本专利技术在5’-NT催化次黄嘌呤核苷酸水解生成氨和次黄嘌呤核苷反应的基础上，再依次偶联三个酶促反应，建立了一种既能用于手工操作，又能用于自动生化分析仪的四酶偶联测定5’-NT活性的新方法。
技术实现思路
本专利技术的目的是提供一种用于检测5’-NT的试剂及使用该试剂检测5’-NT含量的方法。该试剂盒采用四酶偶联法，可以有效检测5’-NT的含量，抗干扰能力强，稳定性好等优点。基本原理：次黄嘌呤核苷酸在5’-NT作用下，生成次黄嘌呤核苷和磷酸，5’-NT次黄嘌呤核苷酸+H2O次黄嘌呤核苷+磷酸次黄嘌呤核苷在嘌呤核苷磷酸化酶（PNP）催化下，与磷反应生成次黄嘌呤和核糖1-磷酸PNP次黄嘌呤核苷+Pi次黄嘌呤+核糖1-磷酸次黄嘌呤在黄嘌呤氧化酶（XOD）催化氧化生成尿酸和H2O2：XOD次黄嘌呤+2H2O+O2尿酸+2H2O2再偶联由过氧化物酶催化的反应POD2H2O2+4-AA+EHSPT4H2O+醌染料通过测定红色醌化合物在550nm处吸光度的变化速率（ΔA/min）可测得5’-NT活性。本专利技术是通过以下步骤得到的：一种5’-NT检酸测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：1）试剂R1的组分为：缓冲液··········································································100mmol/Lβ-甘油磷酸钠····························································6g/LMgCl2··········································································10g/LToos·············································································0.5g/L防腐剂·········································································0.5g/L；2）试剂R2的组分为：缓冲液········································································100mmol/L5’-IMP·········································································11mmol/LPNP·············································································0.3U/mLXOD···········································································0.4U/mLPOD············································································0.1U/mL抗坏血酸氧化酶························································2.7U/mLEHSPT····································································4mmol/L4-AA·············································································4mmol/L；以上试剂均用pH7.4的磷酸盐缓冲溶液。以上5’-NT检测试剂，所述防腐剂为NaN3。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。所述的5’-NT检测试剂来检测5’-NT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）采用新的缓冲体系和稳定剂，显著改善了试剂的稳定性；2）采用四酶偶联法，不仅显著改善测定的性能，而且增强了抗干扰能力，适宜批量试样全自动分析；3）试剂的准确度和稳定性良好，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专利技术进行进一步说明：实施例15’-NT的检测试剂，包试剂R1和试剂R2：1）试剂R1的组分为：缓冲液··········································································100mmol/Lβ-甘油磷酸钠····························································12g/LMgCl2·························································本文档来自技高网...

【技术保护点】
一种5’‑NT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：1）试剂R1的组分为：缓冲液

【技术特征摘要】
1.一种5’-NT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：1）试剂R1的组分为：缓冲液··········································································100mmol/Lβ-甘油磷酸钠····························································12g/LMgCl2··········································································20g/LToos·············································································0.5g/L防腐剂·········································································0.5g/L；2）试剂R2的组分为：缓冲液········································································100mmol/L5’-IMP·········································································11mmol/LPNP··················································...

【专利技术属性】
技术研发人员：甘宜梧，史秀明，谢清华，谭柏清，
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