CN104988207A - 一种稳定、抗干扰能力强的α-羟丁酸脱氢酶试剂及检测方法 - Google Patents
一种稳定、抗干扰能力强的α-羟丁酸脱氢酶试剂及检测方法 Download PDFInfo
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Abstract
本发明涉及α-羟丁酸脱氢酶检测技术领域,特别涉及一种α-羟丁酸脱氢酶检测试剂,试剂R1中含有缓冲液,乙二胺四乙酸二钠(EDTA-2Na),α-酮丁酸,十二烷基二甲基甜菜碱,防腐剂;试剂R2中含有缓冲液,NADH,BSA,蔗糖,海藻糖,甘露醇,十二烷基二甲基甜菜碱,防腐剂。采用PIPES(哌嗪-1,4-二乙磺酸)缓冲液,添加稳定剂BSA,蔗糖,海藻糖,甘露醇,大大增强了试剂的稳定性;采用两性表面活性剂十二烷基二甲基甜菜碱,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
技术领域
本发明涉及α-羟丁酸脱氢酶检测技术领域,特别涉及一种α-羟丁酸脱氢酶检测试剂,还涉及使用此检测试剂的检测方法。
背景技术
α-羟丁酸脱氢酶(α-HBDH),心肌酶谱中酶的一种,实质上是LDH1型同功酶,主要来源于心肌、肾脏和红细胞,在心肌中含量最丰富。α-羟丁酸脱氢酶活性的测定常用于急性心肌梗死(AMI)辅助诊断,为AMI的临床诊断、病情观察、疗效分析和愈后估计提供了有力的定量指标。其测定对AMI的诊断比LDH更为专一,更有价值。此外,α- 羟丁酸脱氢酶增高主要见于心肌梗塞、活动性风湿性心肌炎、急性病毒性心肌炎、溶血性贫血等。
α-羟丁酸脱氢酶(α-HBDH)常用的测定方法主要有比色法和连续监测法。比色法是以前较为常用的检测方法,该法利用α-HBDH作用于过量的α-酮丁酸生成α-羟丁酸,则剩余者与2,4-二硝基苯肼形成α-酮丁酸苯腙,在碱性溶液中呈棕色,其颜色深浅与酶活性成反比。该法操作简单,但试剂存在污染性较大,易受其他物质干扰等缺点。连续监测法是目前较为常用的方法,利用α-HBDH催化α-酮丁酸还原成α-羟丁酸,同时使NADH氧化成NAD,在340nm监测NADH的氧化速率与该酶活性成正比。该法简便快速、适用于全自动生化分析,但该法试剂底物NADH稳定性不佳,需进一步改进稳定性。
鉴于此,本发明在连续监测法的基础上,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α-羟丁酸脱氢酶(α-HBDH)试剂。
发明内容
本发明的目的是提供一种用于检测 α-羟丁酸脱氢酶(α-HBDH)的试剂及使用该试剂检测α-羟丁酸脱氢酶活性的方法。该试剂盒采用连续监测法,可以有效检测α-羟丁酸脱氢酶的活性,抗干扰能力强,稳定性好等优点。
本发明的基本原理:
α-HBDH催化α-酮丁酸还原成α-羟丁酸,同时使NADH氧化成NAD,在340nm监测NADH的氧化速率与该酶活性成正比。
本发明是通过以下步骤得到的:
一种α-羟丁酸脱氢酶检测试剂,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···········································································50mmol/L,
乙二胺四乙酸二钠(EDTA-2Na)·········································3mmol/L,
α-酮丁酸······································································5mmol/L,
十二烷基二甲基甜菜碱···················································2g/L,
防腐剂············································································0.5g/L;
2)试剂R2的组分为:
缓冲液···········································································50mmol/L,
NADH················································································0.3mmol/L,
BSA···················································································1g/L,
蔗糖·················································································10g/L,
海藻糖···············································································3g/L,
甘露醇··············································································10g/L,
十二烷基二甲基甜菜碱····················································2g/L,
防腐剂··············································································0.5g/L。
所述的α-羟丁酸脱氢酶检测试剂,试剂R1中缓冲液为25℃,pH为7.4的哌嗪-1,4-二乙磺酸缓冲液。
所述的α-羟丁酸脱氢酶检测试剂,试剂R2中缓冲液为25℃,pH为7.4的哌嗪-1,4-二乙磺酸缓冲液。
所述的α-羟丁酸脱氢酶检测试剂,所述防腐剂为NaN3。
所述的α-羟丁酸脱氢酶检测试剂来检测α-羟丁酸脱氢酶活性的检测方法,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340nm。
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,可以防止底物NADH氧化,显著改善了试剂的稳定性;
2)采用两性表面活性剂十二烷基二甲基甜菜碱,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为两种试剂有效期稳定性曲线图。
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
α-羟丁酸脱氢酶的检测试剂,包试剂R1和试剂R2:
1)其R1的组成为:
PIPES(哌嗪-1,4-二乙磺酸)缓冲液(pH=7.4,25℃) ··························50mmol/L
乙二胺四乙酸二钠(EDTA-2Na)·····································································3mmol/L,
α-酮丁酸···································································································5mmol/L,
十二烷基二甲基甜菜碱··············································································2g/L,
防腐剂NaN3 ·······························································································0.5g/L
2)试剂R2的组分为:
PIPES(哌嗪-1,4-二乙磺酸)缓冲液(pH=7.4,25℃) ··························100mmol/L
NADH············································································································0.3mmol/L,
BSA·············································································································1g/L,
蔗糖···········································································································10g/L,
海藻糖·······································································································3g/L,
甘露醇·······································································································10g/L,
十二烷基二甲基甜菜碱··············································································2g/L,
防腐剂NaN3 ································································································0.5g/L。
3)本实施例试剂的使用方法:
本实施例描述的α-羟丁酸脱氢酶检测试剂,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:α
-
羟丁酸脱氢酶活性(
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4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α-羟丁酸脱氢酶(α-HBDH)试剂同时对比测定血清中α-HBDH的活性,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≤1026μmol/L、甘油三酯≤2000mg/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,这说明通过优化反应缓冲体系、添加两性表面活性剂后,实施例1试剂的抗干扰性能显著提高,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α-羟丁酸脱氢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0.9994,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α
-
羟丁酸脱氢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 α-羟丁酸脱氢酶(α-HBDH)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α-HBDH质控品(靶值为260U/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 α-羟丁酸脱氢酶(α-HBDH)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α-羟丁酸脱氢酶(α-HBDH)检测试剂。
Claims (7)
1.一种α-羟丁酸脱氢酶检测试剂,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···········································································50mmol/L,
乙二胺四乙酸二钠(EDTA-2Na)··································3mmol/L,
α-酮丁酸······································································5mmol/L,
十二烷基二甲基甜菜碱···················································2g/L,
防腐剂············································································0.5g/L;
2)试剂R2的组分为:
缓冲液···········································································50mmol/L,
NADH················································································0.3mmol/L,
BSA···················································································1g/L,
蔗糖·················································································10g/L,
海藻糖···············································································3g/L,
甘露醇··············································································10g/L,
十二烷基二甲基甜菜碱····················································2g/L,
防腐剂··············································································0.5g/L。
2.根据权利要求1所述的α-羟丁酸脱氢酶检测试剂,其特征在于试剂R1中缓冲液为25℃,pH为7.4的哌嗪-1,4-二乙磺酸缓冲液。
3.根据权利要求1所述的α-羟丁酸脱氢酶检测试剂,其特征在于试剂R2中缓冲液为25℃,pH为7.4的哌嗪-1,4-二乙磺酸缓冲液。
4.根据权利要求1所述的α-羟丁酸脱氢酶检测试剂,其特征在于试剂R1和R2中表面活性剂为十二烷基二甲基甜菜碱。
5.根据权利要求1所述的α-羟丁酸脱氢酶检测试剂,其特征在于所述防腐剂为NaN3。
6.根据权利要求1所述的α-羟丁酸脱氢酶检测试剂来检测α-羟丁酸脱氢酶的检测方法,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340nm。
7.根据权利要求6所述的检测方法,其特征在于R1试剂和R2试剂的比例为4:1。
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