一种稳定、抗干扰能力强的尿酸试剂及检测方法与流程

本发明涉及尿酸检测技术领域，特别涉及一种尿酸检测试剂，还涉及使用此检测试剂的检测方法。

背景技术：

尿酸是嘌呤分解代谢的最终产物。由肾脏随尿液排出体外。健康成人体内尿酸含量约为1.1g，其中约15%存在于血液中，血液中尿酸经肾小球过滤后，大部分由肾小管重吸收。尿酸是血浆中非蛋白氮重要成分之一，在严重肾脏损害时，血中尿酸可显著升高，而轻度受损时变化不大。故血尿酸测定是诊断肾重度受损的敏感指标。

目前，尿酸(UA) 的检测方法有磷钨酸还原法、尿酸酶法和HPLC法。钨酸还原法是目前临床比较常用的方法，利用无蛋白滤液中的尿酸在碱性环境中可被磷钨酸氧化成尿囊素及二氧化碳，磷钨酸被还原成钨蓝(tungsten blue)，根据钨蓝颜色深浅，用710nm波长滤光板或红色滤片，与同样处理的标准比较，进行比色，以空白调零点，可求出其浓度值。该方法操作简单，但有溶血样本时不能用该法测试，且易受其他还原性物质干扰等缺点。HPLC检测法虽然简单，但样品预处理比较复杂，设备昂贵难以普及。而现在最流行的方法是尿酸酶-过氧化物酶偶联法，该法灵敏且不需要去蛋白，但容易受维生素C 和胆红素等干扰物质的影响。

鉴于此，本发明在尿酸酶-过氧化物酶偶联法的基础上，优化反应体系，采用PIPES缓冲液，添加多种抗干扰物质和稳定剂，显著改善了试剂的抗干扰能力和稳定性，是一种更加稳定、抗干扰能力强的尿酸（UA）试剂。

技术实现要素：

本发明的目的是提供一种用于检测 尿酸（UA）的试剂及使用该试剂检测尿酸含量的方法。该试剂盒采用尿酸酶-过氧化物酶偶联法，可以有效检测尿酸的含量，具有抗干扰能力强、稳定性好等优点。

基本原理：

尿酸在尿酸酶作用下，生成尿囊素和过氧化氢（H₂O₂），再利用Trinder反应系统，过氧化氢在过氧化物酶（POD）催化下，与4-氨基安替比林（4-AAP）和N-乙基-间甲苯胺丙磺酸钠（TOOS），生成红紫色色素。反应液的颜色与尿酸浓度成正比。

。

本发明是通过以下步骤得到的：

一种尿酸检测试剂，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-氨基安替比林_{································································}1.3mmol/L

BSA_{···················································································}1g/L

蔗糖_{···················································································}5g/L

海藻糖_{···············································································}2g/L

过氧化物酶_{·······································································}12KU/L

抗坏血酸氧化酶_{·······························································}5KU/L

胆红素氧化酶_{···································································}8KU/L

烷基酚聚氧乙烯醚( APEO)_{·············································}2g/L

防腐剂_{················································································}0.5g/L。

试剂R2的组分为：

缓冲液_{················································································}100mmol/L

TOOS_{··················································································}13mmol/L

尿酸酶_{·················································································}2KU/L

BSA_{·····················································································}1g/L

蔗糖_{·····················································································}5g/L

海藻糖_{·················································································}2g/L

烷基酚聚氧乙烯醚( APEO)_{···············································}2g/L

防腐剂_{··················································································}0.5g/L。

所述的尿酸检测试剂，试剂R1中缓冲液为25℃，pH为7.6的哌嗪-1,4-二乙磺酸缓冲液。

所述的尿酸检测试剂，试剂R2中缓冲液为25℃，pH为7.6的哌嗪-1,4-二乙磺酸缓冲液。

所述的尿酸检测试剂，所述防腐剂为NaN₃。

所述的尿酸检测试剂检测尿酸的检测方法，使用全自动生化分析仪利用终点法进行测定，检测主波长为546nm。

所述的检测方法，R1试剂和R2试剂的比例为4:1。

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）采用新型表面活性剂烷基酚聚氧乙烯醚( APEO)，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；

3）试剂的准确度和稳定性良好，价格便宜，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：

尿酸的检测试剂，包括试剂R1和试剂R2。

其R1的组成为：

PIPES（哌嗪-1,4-二乙磺酸）缓冲液(pH=7.6，25℃) _{····························}100mmol/L

4-氨基安替比林_{····························································································}1.3mmol/L

BSA_{···············································································································}1g/L

蔗糖_{···············································································································}5g/L

海藻糖_{···········································································································}2g/L

过氧化物酶_{···································································································}12KU/L

抗坏血酸氧化酶_{···························································································}5KU/L

胆红素氧化酶_{·······························································································}8KU/L

烷基酚聚氧乙烯醚( APEO)_{·········································································}2g/L

防腐剂NaN_{3·································································································}0.5g/L。

试剂R2的组分为：

PIPES（哌嗪-1,4-二乙磺酸）缓冲液(pH=7.6，25℃) _{··························}100mmol/L

TOOS_{···········································································································}13mmol/L

尿酸酶_{··········································································································}2KU/L

BSA_{··············································································································}1g/L

蔗糖_{··············································································································}5g/L

海藻糖_{··········································································································}2g/L

烷基酚聚氧乙烯醚( APEO)_{·········································································}2g/L

防腐剂NaN_{3·································································································}0.5g/L。

本实施例试剂的使用方法：

本实施例描述的尿酸检测试剂，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，利用终点法进行测定。将R1和R2按照4:1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：尿酸含量（μmol/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尿酸（UA）试剂同时对比测定血清中UA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尿酸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0.9992，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尿酸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5mL；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尿酸（UA）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UA质控品（靶值为340μmol/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 尿酸（UA）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，是一种更加稳定的尿酸（UA）检测试剂。