CN104792718A - 一种血清γ-谷氨酰氨基转移酶试剂及检测方法 - Google Patents

Abstract

本发明涉及一种生物检测试剂，具体涉及一种稳定的、抗干扰能力强的血清γ-谷氨酰氨基转移酶检测试剂及检测方法，属于临床体外检测技术领域。试剂R1中含有缓冲液，双甘氨肽，BSA，海藻糖，APG(烷基糖苷)，防腐剂；试剂R2中含有缓冲液，L-γ-谷氨酰-3-羧基-4-苯基重氮酸，BSA，海藻糖，APG(烷基糖苷)，防腐剂。采用PIPES（哌嗪-1,4-二乙磺酸）缓冲液，添加稳定剂BSA和海藻糖，大大增强了试剂的稳定性；采用新型表面活性剂烷基糖苷，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。

Description

一种血清γ - 谷氨酰氨基转移酶试剂及检测方法

技术领域

本发明涉及一种生物检测试剂，具体涉及一种稳定的、抗干扰能力强的血清γ-谷氨酰氨基转移酶检测试剂及检测方法，属于临床体外检测技术领域。

背景技术

GGT，即谷氨酰转肽酶，是存在于肾、胰、肝、脾、肠、脑、肺、骨骼肌和心肌等组织中，肾内最多，其次为胰和肝，胚胎期则以肝内最多，在肝内主要存在于肝细胞浆和肝内胆管上皮中。正常人血清中GGT主要来自肝脏。正常值为3～50U/L。此酶在急性肝炎、慢性活动性肝炎及肝硬化失代偿时仅轻中度升高。但当阻塞性黄疸时，此酶因排泄障碍而逆流入血，原发性肝癌时，此酶在肝内合成亢进，均可引起血中转肽酶显著升高，甚至达正常的10倍以上。酒精中毒者GGT亦明显升高，有助于诊断酒精性肝病。

目前国内测定GGT主要采用IFCC和欧洲常规SZASZ法。二者均是以γ谷氨酰-3-羧基-4-对硝基苯胺和双甘肽为底物的酶动力。GOT作用于γ-谷氨酰-3-羧基-4-对硝基苯胺和双甘肽产生γ一谷氨酰双甘肽和5-氨基2-硝酸苯甲酸盐，在405nm处检测吸收峰，计算出血清中GGT的浓度。

但以上方法均存在以下缺点：

1）底物L-γ-谷氨酰-3-羧基-对硝基苯胺随放置时间的延长出现自然水解，导致水空白上升，影响试剂的应用；

2）影响GGT活性的成分较多，试剂的抗干扰能力较差。

针对上述缺点，本发明在IFCC和欧洲常规SZASZ法的基础上，优化缓冲体系，采用哌嗪-1,4-二乙磺酸缓冲液（PIPES），添加稳定剂和抗干扰物质，显著改善了试剂的稳定性和抗干扰能力，是一种更加稳定、抗干扰能力强的γ-谷氨酰氨基转移酶（GGT）的试剂。

发明内容

本发明的目的是提供一种用于检测血清γ-谷氨酰氨基转移酶（GGT）的试剂及使用该试剂检测血清γ-谷氨酰氨基转移酶活性的方法，采用SZASZ法，可以有效检测血清 γ-谷氨酰氨基转移酶的活性，抗干扰能力强，稳定性好等优点。

本发明所采用的技术方案是：

以L-γ-谷氨酰-3-羧基-对硝基苯胺为底物，双甘肽为γ-谷氨酰基的受体。在GGT的催化下，谷氨酰基转移到双甘肽分子上，同时释放出黄色的2-硝基-5-氨基苯甲酸，引起405 nm波长处吸光度的增高，吸光度增高速率与GGT活性成正比例关系。

。

一种稳定的、抗干扰能力强的血清γ-谷氨酰氨基转移酶检测试剂，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的组分为：

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

双甘氨肽_{·······································································}100mmol/L

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

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

APG(烷基糖苷)_{··································································}2g/L

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

试剂R2的组分为：

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

L-γ-谷氨酰-3-羧基-4-苯基重氮酸_{········································}4mmol/L

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

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

APG(烷基糖苷)_{··································································}2g/L

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

所述的γ-谷氨酰氨基转移酶检测试剂，试剂R1中缓冲液为25℃，pH为7.7的哌嗪-1,4-二乙磺酸缓冲液。

所述的γ-谷氨酰氨基转移酶检测试剂，试剂R2中缓冲液为25℃，pH为7.7的哌嗪-1,4-二乙磺酸缓冲液。

所述的γ-谷氨酰氨基转移酶检测试剂，所述防腐剂为NaN₃。

所述的γ-谷氨酰氨基转移酶检测试剂来检测γ-谷氨酰氨基转移酶的检测方法，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405nm。

所述的检测方法，R1试剂和R2试剂的比例为3:1。

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糖苷，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为实施例1与市场上常见的试剂检测结果相关性分析。

图2为实施例1与市场上常见的试剂检测结果有效稳定性对比。

具体实施方式

下面结合具体实施例来进一步详细描述本发明。

实施例 1

一种稳定的、抗干扰能力强的血清γ-谷氨酰氨基转移酶检测试剂，包含试剂R1和试剂R2：

试剂R1的组分为：

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

双甘氨肽_{········································································································}100mmol/L

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

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

APG(烷基糖苷)_{··································································································}2g/L

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

L-γ-谷氨酰-3-羧基-4-苯基重氮酸_{········································································}4mmol/L

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

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

APG(烷基糖苷)_{··································································································}2g/L

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

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

本实施例试剂的使用方法为：

本实施例描述的γ-谷氨酰氨基转移酶检测试剂，在使用时采用具有双试剂功能的全自动生化分析仪，如东芝40全自动分析仪等，利用速率法进行测定。将R1和R2按照3:1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：γ - 谷氨酰氨基转移酶活性（ 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GGT试剂同时对比测定血清中GGT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≤50mg/dL、胆红素≤40mg/dL、血红蛋白≤200 mg/dL、甘油三酯≤500 mg/dL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 γ-谷氨酰氨基转移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0.9985，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γ-谷氨酰氨基转移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18mL，R2为6mL，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 γ-谷氨酰氨基转移酶（GGT）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GGT质控品（靶值为51U/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 γ-谷氨酰氨基转移酶（GGT）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γ-谷氨酰氨基转移酶（GGT）检测试剂。

Claims (6)

1.一种稳定的、抗干扰能力强的γ-谷氨酰氨基转移酶检测试剂，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的组分为：

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

双甘氨肽_{·······································································}100mmol/L

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

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

APG(烷基糖苷)_{··································································}2g/L

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

试剂R2的组分为：

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

L-γ-谷氨酰-3-羧基-4-苯基重氮酸_{········································}4mmol/L

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

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

APG(烷基糖苷)_{··································································}2g/L

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

2.根据权利要求1所述的γ-谷氨酰氨基转移酶检测试剂，其特征在于试剂R1中缓冲液为25℃，pH为7.7的哌嗪-1,4-二乙磺酸缓冲液。

3.根据权利要求1所述的γ-谷氨酰氨基转移酶检测试剂，其特征在于试剂R2中缓冲液为25℃，pH为7.7的哌嗪-1,4-二乙磺酸缓冲液。

4.根据权利要求1所述的γ-谷氨酰氨基转移酶检测试剂，其特征在于所述防腐剂为NaN₃。

5.一种使用权利要求1-4中任一项所述的γ-谷氨酰氨基转移酶检测试剂来检测γ-谷氨酰氨基转移酶的检测方法，其特征在于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405nm。

6.根据权利要求5所述的检测方法，其特征在于R1试剂和R2试剂的比例为3:1。