技术领域
本发明涉及5’-核糖核苷酸水解酶检测技术领域，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。
背景技术
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，本发明在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。
发明内容
本发明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
POD
2H2O2 + 4-AA + EHSPT 4 H2O+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。
本发明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/L
β-甘油磷酸钠····························································6g/L
MgCl2··········································································10g/L
Toos·············································································0.5g/L
防腐剂·········································································0.5g/L；
2）试剂R2的组分为：
缓冲液········································································100mmol/L
5’-IMP·········································································11mmol/L
PNP·············································································0.3U/mL
XOD···········································································0.4U/mL
POD············································································0.1U/mL
抗坏血酸氧化酶························································2.7U/mL
EHSPT ····································································4mmol/L
4-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。
本发明的有益效果：
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发明进行进一步说明：
实施例 1
5’-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/L
β-甘油磷酸钠····························································12g/L
MgCl2··········································································20g/L
Toos·············································································0.5g/L
防腐剂·········································································0.5g/L；
2）试剂R2的组分为：
缓冲液········································································100mmol/L
5’-IMP·········································································11mmol/L
PNP·············································································0.3U/mL
XOD···········································································0.4U/mL
POD············································································0.1U/mL
抗坏血酸氧化酶························································2.7U/mL
EHSPT ·······································································4mmol/L
4-AA·············································································4mmol/L；
3）本实施例试剂的使用方法：
本实施例描述的5’-NT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： 5 ’ -NT 含量（ U/L ）＝（ ∆A 测定÷ ∆A 标准）× 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5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’-NT试剂同时对比测定血清中5’-NT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试剂在抗坏血酸≤1704μmol/L、胆红素≤684μmol/L、血红蛋白≤10 g/L、甘油三酯≤22.6 mmol/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，这说明实施例1试剂的抗干扰性能远远优于对比试剂。
表 2 实施例 1 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’-NT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0.9929，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 5 ’ -NT 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’-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，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’-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’-NT检测试剂。