CN103558397A - 一种缺血修饰白蛋白检测试剂及检测方法 - Google Patents
一种缺血修饰白蛋白检测试剂及检测方法 Download PDFInfo
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Abstract
本发明涉及缺血修饰白蛋白检测技术领域，特别涉及一种缺血修饰白蛋白检测试剂，试剂R1中含有缓冲液，氯化钴，十二烷基二甲基甜菜碱，防腐剂；试剂R2中含有缓冲液，噻二唑偶氮二甲氨基苯酚，十二烷基二甲基甜菜碱，防腐剂。采用新显色剂噻二唑偶氮二甲氨基苯酚，有效避免了Pd、Ni、Zn和Cu等金属离子的干扰，更加稳定、低毒、易于保存，大大增强了试剂的稳定性；采用新型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，而且对维护体系的稳定，防止出现脂浊，效果明显，并且显著改善了试剂的稳定性和抗干扰能力。
Description
技术领域
本发明涉及缺血修饰白蛋白检测技术领域，特别涉及一种缺血修饰白蛋白检测试剂，还涉及使用此检测试剂的检测方法。
背景技术
缺血修饰白蛋白（IMA）是由于血清白蛋白的氨基酸序列在缺血时受到自由基等破坏，从而致使与过渡金属的结合能力改变的白蛋白。缺血修饰白蛋白主要用于急性心肌缺血的辅助诊断、对于排除急性心肌梗塞、不稳定心绞痛等急性冠状动脉综合症（ACS）具有重要意义，是一种理想的对心肌缺血敏感的新型标记物。与传统的心肌损伤标志物相比，缺血修饰白蛋白（IMA）在缺血发作的短时间内血液中浓度就会升高，但此时心肌细胞的不可逆性损伤还未发生，因此IMA更具早期诊断价值，能够辅助临床医生尽早明确心肌缺血的诊断，从而进行早期干预和治疗。
目前，IMA的测定方法多数采用ACB比色分析法。采用白蛋白钴结合试验的原理，依据IMA较正常白蛋白结合金属钴离子能力降低，在反应系统中加入一定量的钴离子，并通过测定游离钴离子的浓度，推算出IMA的含量。最早美国Bar-Or等创立了终点法白蛋白钴结合试验，因受实验反应系统背景干扰的原因，动力学曲线不佳，线性范围狭窄，IMA阴阳性数值重叠，影响了实验的重现性和特异性。
发明内容
为了解决以上现有技术中缺血修饰白蛋白检测方面存在的重现性和特异性不好、受干扰严重的问题，本发明提供了一种灵敏度高，抗干扰性能好，稳定的缺血修饰白蛋白检测试剂。
本发明还提供了所述使用上述缺血修饰白蛋白检测试剂对缺血修饰白蛋白进行检测的检测方法。
本发明是通过以下步骤得到的：
一种缺血修饰白蛋白检测试剂，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··········································································0.1mol/L，
氯化钴···········································································24mmol/L，
十二烷基二甲基甜菜碱················································3g/L，
防腐剂············································································0.5g/L；
2）试剂R2的组分为：
缓冲液·············································································0.1mol/L
噻二唑偶氮二甲氨基苯酚····························24mmol/L
十二烷基二甲基甜菜碱···················································3g/L
防腐剂················································································0.5g/L。
所述的缺血修饰白蛋白检测试剂，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。
所述的缺血修饰白蛋白检测试剂，试剂R2中缓冲液为25℃，pH为5.0的MES缓冲液。
所述的缺血修饰白蛋白检测试剂，所述防腐剂为NaN3。
所述的缺血修饰白蛋白检测试剂来检测缺血修饰白蛋白的检测方法，使用全自动生化分析仪利用双试剂终点法进行测定，检测主波长为548nm。
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）双试剂比色法测定IMA，采用新显色剂噻二唑偶氮二甲氨基苯酚（TDAMAP），有效避免了Pd、Ni、Zn和Cu等金属离子的干扰，而且TDAMAP比二硫苏糖醇（DTT）更加稳定、低毒、易于保存，大大增强了试剂的稳定性；
2）采用新型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，而且对维护体系的稳定，防止出现脂浊，效果明显，相比之前所用的TRITON X-100（曲拉通X-100）、APEO（烷基酚聚氧乙烯醚）等有更好的效果，并且显著改善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为不同表面活性剂对试剂稳定性影响曲线图，
图3为不同显色剂对试剂稳定性影响曲线图。
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
缺血修饰白蛋白的检测试剂，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=7.6，25℃) ··························0.1mol/L
CoCl2 (氯化钴) ·······················································································24mmol/L
十二烷基二甲基甜菜碱···········································································3g/L
防腐剂NaN3····························································································0.5g/L
2）试剂R2的组分为：
MES缓冲液(pH=5.0，25℃) ·································································0.1mol/L
噻二唑偶氮二甲氨基苯酚（TDAMAP）·················································24mmol/L
十二烷基二甲基甜菜碱···········································································3g/L
防腐剂NaN3·····························································································0.5g/L。
3）本实施例试剂的使用方法：
本实施例描述的缺血修饰白蛋白检测试剂，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，利用双试剂终点法进行测定。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：缺血修饰白蛋白含量（U/mL）＝（??A测定/min÷??A标准/min）×C标准。
对比实施例1
缺血修饰白蛋白的检测试剂盒，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=7.6，25℃) ··························0.1mol/L
CoCl2 (氯化钴) ·······················································································24mmol/L
TRITON X-100（曲拉通X-100）·······························································3g/L
防腐剂NaN3····························································································0.5g/L
2）试剂R2的组分为：
MES缓冲液(pH=5.0，25℃) ·································································0.1mol/L
噻二唑偶氮二甲氨基苯酚（TDAMAP）·················································24mmol/L
TRITON X-100（曲拉通X-100）·······························································3g/L
防腐剂NaN3·····························································································0.5g/L。
对比实施例2
缺血修饰白蛋白的检测试剂盒，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=7.6，25℃) ··························0.1mol/L
CoCl2 (氯化钴) ·······················································································24mmol/L
APEO（烷基酚聚氧乙烯醚）···································································3g/L
防腐剂NaN3····························································································0.5g/L
2）试剂R2的组分为：
MES缓冲液(pH=5.0，25℃) ·································································0.1mol/L
噻二唑偶氮二甲氨基苯酚（TDAMAP）·················································24mmol/L
APEO（烷基酚聚氧乙烯醚）·····································································3g/L
防腐剂NaN3·····························································································0.5g/L。
对比实施例3
缺血修饰白蛋白的检测试剂盒，包试剂R1和试剂R2（检测方法与使用二硫苏糖醇作为显色剂的缺血修饰白蛋白检测试剂盒相同）：
1）其R1的组成为：
PIPES（哌嗪-1,4-二乙磺酸）缓冲液(pH=7.6，25℃) ··························0.1mol/L
CoCl2 (氯化钴) ·······················································································24mmol/L
十二烷基二甲基甜菜碱············································································3g/L
防腐剂NaN3····························································································0.5g/L
2）试剂R2的组分为：
MES缓冲液(pH=5.0，25℃) ·································································0.1mol/L
DTT（二硫苏糖醇）················································································24mmol/L
十二烷基二甲基甜菜碱···········································································3g/L
防腐剂NaN3·····························································································0.5g/L。
检测性能测试
一、干扰性试验：
取新鲜混合血清，分成6等份，然后将每等份分成10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。然后分别测定血清中IMA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试剂在总胆红素小于560μmol/L，Vc含量小于0.45g/L，乳酸含量小于4.5mmol/L，血红蛋白含量小于4.5g/L，甘油三酯含量小于8mmol/L时对测试结果没有明显干扰。而其他实施例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其他实施例试剂。
表2不同实施例试剂抗干扰性能测试结果
二、精密度试验：
取高、低值分别为81.0和62.0U/mL的缺血修饰白蛋白标准品，用实施例1和对比实施例1、2、3中得到的试剂，连续检测20次，求出批内精密度。实施例1和对比实施例1、2、3各配制10份试剂，每天分别对高、低值缺血修饰白蛋白标准品进行检测，连续检测10天，获得批间精密度.结果如表3和表4所示，实施例1试剂的批内CV为2.19-2.69%，批间精密度为2.29-3.60%，明显优于其他实施例试剂，并且远远满足试剂相关行业标准对精密度的要求（重复性CV≤10%；批间相对极差R≤10％）。
表3不同实施例试剂批内精密度检测结果对比
表4不同实施例试剂批间精密度检测结果对比
三、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缺血修饰白蛋白测定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5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5实施例1试剂与市场常见并得到认可的缺血修饰白蛋白测定试剂盒对比检测结果
四、试剂稳定性试验：
（1）2~8℃稳定性试验：
将如下4种试剂：
A试剂：按实施例1配方配制，共13组，每组的试剂量为R1为20mL，R2为10 mL。
B试剂：按对比实施例1配方配制，共13组，每组的试剂量为R1为20mL，R2为10 mL。
C试剂：按对比实施例2配方配制，共13组，每组的试剂量为R1为20mL，R2为10 mL。
D试剂：除R1和R2中不加表面活性剂十二烷基二甲基甜菜碱外，其余均与实施例1相同，共13组，每组的试剂量为R1为20mL，R2为10 mL。
放置到2-8℃冰箱中，每月的同一天分别取出A、B、C、D中的一组来检测定值血清（理论浓度为75.4U/mL），检测结果如图2所示：可以看出，加入表面活性剂可显著改善试剂的稳定性，但十二烷基二甲基甜菜碱比TRITON X-100、APEO（烷基酚聚氧乙烯醚）效果更好，试剂更稳定。
（2）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；并且取对比实施例3的试剂，均匀分装13组，每组的试剂量为R1为20mL，R2为10mL。放置到2-8℃冰箱中，每月的同一天取出一组试剂检测定值血清（理论浓度为75.4U/mL），检测结果如图3所示，实施例1试剂在2-8℃储存条件下比市场常见的缺血修饰白蛋白（IMA）测定试剂盒更加稳定，说明新型两性表面活性剂十二烷基二甲基甜菜碱可以显著改善试剂的稳定性，并且说明新的显色剂TDAMAP的稳定性较传统显色剂DTT（二硫苏糖醇）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，并且灵敏度高，抗干扰性能好，是一种更加稳定、良好的缺血修饰白蛋白检测试剂。
Claims (6)
1.一种缺血修饰白蛋白检测试剂，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··········································································0.1mol/L，
氯化钴···········································································24mmol/L，
十二烷基二甲基甜菜碱················································3g/L，
防腐剂············································································0.5g/L；
2）试剂R2的组分为：
缓冲液·············································································0.1mol/L，
噻二唑偶氮二甲氨基苯酚····························24mmol/L，
十二烷基二甲基甜菜碱···················································3g/L，
防腐剂················································································0.5g/L。
2.根据权利要求1所述的缺血修饰白蛋白检测试剂，其特征在于试剂R1中缓冲液为25℃，pH为7.6的哌嗪-1,4-二乙磺酸缓冲液。
3.根据权利要求1所述的缺血修饰白蛋白检测试剂，其特征在于试剂R2中缓冲液为25℃，pH为5.0的MES缓冲液。
4.根据权利要求1所述的缺血修饰白蛋白检测试剂，其特征在于所述防腐剂为NaN3。
5.一种使用权利要求1-4中任一项所述的缺血修饰白蛋白检测试剂来检测缺血修饰白蛋白的检测方法，其特征在于使用全自动生化分析仪利用双试剂终点法进行测定，检测主波长为548nm。
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2:1。
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