CN105543336A - 一种稳定、抗干扰能力强的血清磷脂检测试剂及检测方法 - Google Patents

一种稳定、抗干扰能力强的血清磷脂检测试剂及检测方法 Download PDF

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CN105543336A
CN105543336A CN201510971392.0A CN201510971392A CN105543336A CN 105543336 A CN105543336 A CN 105543336A CN 201510971392 A CN201510971392 A CN 201510971392A CN 105543336 A CN105543336 A CN 105543336A
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李志明
甘宜梧
赵新
李静
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Abstract

本发明涉及血清磷脂检测技术领域，特别涉及一种血清磷脂检测试剂，试剂R1中含有缓冲液，磷脂酶D，DAOS，抗坏血酸氧化酶，胆红素氧化酶，聚乙二醇6000，蔗糖，黄原胶，甘露醇，海藻糖，BSA，聚氧丙烯氧化乙烯甘油醚（Pluranic？L64），防腐剂；试剂R2中含有缓冲液，4-氨基氨替吡啉，过氧化物酶，胆碱氧化酶，聚乙二醇6000，蔗糖，黄原胶，甘露醇，海藻糖，BSA，聚氧丙烯氧化乙烯甘油醚（Pluranic？L64），防腐剂。采用HEPES缓冲液及Trinder反应新色原DAOS，添加多种稳定剂，显著改善了试剂的稳定性；通过添加胆红素氧化酶和抗坏血酸氧化酶，可以有效避免胆红素和抗坏血酸的干扰，大大增强试剂的抗干扰能力。此外，优选的新型非离子表面活性剂聚氧丙烯氧化乙烯甘油醚（Pluranic？L64）的加入可防止反应体系浑浊，增强底物的稳定性，提高试剂的抗干扰能力。

Description

一种稳定、抗干扰能力强的血清磷脂检测试剂及检测方法
技术领域
本发明涉及血清磷脂检测技术领域，特别涉及一种血清磷脂检测试剂，还涉及使用此检测试剂的检测方法。
背景技术
血清磷脂主要包括卵磷脂、溶血卵磷脂、神经磷脂和脑磷脂等四部分。临床工作中一般只测定血清总磷脂，血清磷脂的增高见于糖尿病、肾病综合症、慢性出血性贫血、肝硬化、肝坏死、胆道阻塞、甲状腺功能减退、原发性高血压、梅毒等；减退见于甲状腺功能亢进、急性感染性发热、营养不良性肝硬化等。
目前，血清总磷脂的检测有化学法和酶法等，现以酶法最为常用。化学法，方法稳定，但操作繁琐，试剂对操作人员危害较大，易造成环境污染，且难以实现自动化操作。而酶法试剂，操作简便，灵敏度高，适用自动化分析，但试剂稳定性稍差，容易受胆红素和抗坏血酸等干扰物质干扰。
鉴于此，本发明在酶法的基础上采用Trinder反应新色原DAOS，添加稳定剂聚乙二醇6000、蔗糖、甘露醇、海藻糖、BSA、黄原胶等，有效提高了试剂的稳定性；并且通过添加胆红素氧化酶和抗坏血酸氧化酶，可以有效胆红素和抗坏血酸的干扰，大大增强试剂的抗干扰能力。此外，优选的新型非离子表面活性剂聚氧丙烯氧化乙烯甘油醚（PluranicL64）的加入可防止反应体系浑浊，增强底物的稳定性，提高试剂的抗干扰能力。该试剂操作简便快速，适用于自动化分析，是一种更加稳定、抗干扰能力强的血清血清磷脂（PLIP）试剂。
发明内容
本发明的目的是提供一种用于检测血清磷脂（PLIP）的试剂及使用该试剂检测血清磷脂含量的方法。该试剂盒采用酶法，可以有效检测血清磷脂的含量，抗干扰能力强，稳定性好等优点。
基本原理：
磷脂在磷脂酶D的作用水解生成胆碱和磷脂酸，胆碱被胆碱氧化酶氧化生成过氧化氢，过氧化氢与4-氨基安替比林、DAOS反应生成蓝色染料。此染料在600nm有最大吸收峰，吸收强度与血清中磷脂含量成正比。
磷脂酶D
磷脂＋H2O胆碱＋磷脂酸
胆碱氧化酶
胆碱＋2O2+H2O甜菜碱＋2H2O2
POD
2H2O2＋DAOS＋4-AA4H2O＋蓝色染料
注：DAOS：N-乙基-N-(2-羟基-3-磺丙基)-3'5-二甲氧基苯胺钠盐
4-AA：4-氨基氨替吡啉
POD：过氧化物酶
本发明是通过以下步骤得到的：
一种血清磷脂检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
1)试剂R1中含有
缓冲液······················································100mmol/L，
磷脂酶D·····················································1KU/L，
DAOS························································1.4mmol/L，
抗坏血酸氧化酶··············································2KU/L，
胆红素氧化酶················································2KU/L，
聚乙二醇6000················································10g/L，
蔗糖························································20g/L，
黄原胶······················································0.5g/L，
甘露醇······················································20g/L，
海藻糖······················································10g/L，
BSA·························································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）························1g/L，
防腐剂······················································0.5g/L；
2）试剂R2的组分为：
缓冲液······················································100mmol/L，
4-氨基氨替吡啉··············································2.5mmol/L，
过氧化物酶··················································20KU/L，
胆碱氧化酶··················································8KU/L，
聚乙二醇6000················································10g/L，
蔗糖························································20g/L，
黄原胶······················································0.5g/L，
甘露醇······················································20g/L，
海藻糖······················································10g/L，
BSA·························································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）························1g/L，
防腐剂······················································0.5g/L；
所述的血清磷脂检测试剂，试剂R1中缓冲液为25℃，pH为7.2的HEPES缓冲液。
所述的血清磷脂检测试剂，试剂R2中缓冲液为25℃，pH为7.2的HEPES缓冲液。
所述的血清磷脂检测试剂，所述防腐剂为NaN3。
所述的血清磷脂检测试剂来检测血清磷脂含量的检测方法，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600nm。
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）优化反应体系，采用Trinder反应新色原DAOS，并添加聚乙二醇6000、蔗糖、甘露醇、海藻糖、BSA、黄原胶等多种稳定剂，可以显著改善试剂的稳定性；
2）添加胆红素氧化酶和抗坏血酸氧化酶，可以有效避免胆红素和抗坏血酸的干扰，大大增强试剂的抗干扰能力；
3）新型非离子表面活性剂聚氧丙烯氧化乙烯甘油醚（PluranicL64）的加入可防止反应体系浑浊，增强底物的稳定性，提高试剂的抗干扰能力；
4）试剂的准确度和稳定性良好，抗干扰性强，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
血清磷脂的检测试剂，包试剂R1和试剂R2：
1）其R1的组成为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.2，25℃)·100mmol/L，
磷脂酶D·······································1KU/L，
DAOS··········································1.4mmol/L，
抗坏血酸氧化酶································2KU/L，
胆红素氧化酶··································2KU/L，
聚乙二醇6000··································10g/L，
蔗糖··········································20g/L，
黄原胶········································0.5g/L，
甘露醇········································20g/L，
海藻糖········································10g/L，
BSA···········································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）··········1g/L，
防腐剂NaN3····································0.5g/L；
2）试剂R2的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.2，25℃)·100mmol/L，
4-氨基氨替吡啉································2.5mmol/L，
过氧化物酶····································20KU/L，
胆碱氧化酶····································8KU/L，
聚乙二醇6000··································10g/L，
蔗糖··········································20g/L，
黄原胶········································0.5g/L，
甘露醇········································20g/L，
海藻糖········································10g/L，
BSA···········································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）··········1g/L，
防腐剂NaN3····································0.5g/L；
3）本实施例试剂的使用方法：
本实施例描述的血清磷脂检测试剂，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：血清磷脂含量（mg/dL）＝（?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血清磷脂（PLIP）试剂同时对比测定血清中PLIP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、甘油三酯≤1250mg/dL、血红蛋白≤400mg/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，采用Trinder反应新色原DAOS，并添加胆红素氧化酶和抗坏血酸氧化酶及新型非离子表面活性剂聚氧丙烯氧化乙烯甘油醚（PluranicL64）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磷脂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0.9997，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血清磷脂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磷脂（PLIP）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PLIP质控品（靶值为215mg/dL），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磷脂（PLIP）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、良好的磷脂（PLIP）检测试剂。

Claims (7)

1.一种血清磷脂检测试剂，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·················································100mmol/L，
磷脂酶D················································1KU/L，
DAOS···················································1.4mmol/L，
抗坏血酸氧化酶·········································2KU/L，
胆红素氧化酶···········································2KU/L，
聚乙二醇6000···········································10g/L，
蔗糖···················································20g/L，
黄原胶·················································0.5g/L，
甘露醇·················································20g/L，
海藻糖·················································10g/L，
BSA····················································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）···················1g/L，
防腐剂·················································0.5g/L；
2）试剂R2的组分为：
缓冲液·················································100mmol/L，
4-氨基氨替吡啉·········································2.5mmol/L，
过氧化物酶·············································20KU/L，
胆碱氧化酶·············································8KU/L，
聚乙二醇6000···········································10g/L，
蔗糖···················································20g/L，
黄原胶·················································0.5g/L，
甘露醇·················································20g/L，
海藻糖·················································10g/L，
BSA····················································2g/L，
聚氧丙烯氧化乙烯甘油醚（PluranicL64）···················1g/L，
防腐剂·················································0.5g/L。
2.根据权利要求1所述的血清磷脂检测试剂，其特征在于试剂R1中缓冲液为25℃，pH为7.2的HEPES缓冲液。
3.根据权利要求1所述的血清磷脂检测试剂，其特征在于试剂R2中缓冲液为25℃，pH为7.2的HEPES缓冲液。
4.根据权利要求1所述的血清磷脂检测试剂，其特征在于试剂R1和R2中表面活性剂为聚氧丙烯氧化乙烯甘油醚（PluranicL64）。
5.根据权利要求1所述的血清磷脂检测试剂，其特征在于所述防腐剂为NaN3。
6.一种使用权利要求1-5中任一项所述的血清磷脂检测试剂来检测血清磷脂的检测方法，其特征在于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600nm。
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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