CN111808918A - 一种测定5`-核甘酸酶的试剂盒 - Google Patents
一种测定5`-核甘酸酶的试剂盒 Download PDFInfo
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- CN111808918A CN111808918A CN202010727098.6A CN202010727098A CN111808918A CN 111808918 A CN111808918 A CN 111808918A CN 202010727098 A CN202010727098 A CN 202010727098A CN 111808918 A CN111808918 A CN 111808918A
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- phosphate
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Abstract
一种测定5’‑核苷酸酶的试剂盒,由彼此独立的试剂R1和试剂R2组成;所述试剂R1的组分包括:缓冲液、防腐剂、保护剂、抗干扰剂、表面活性剂、反应增强剂、过氧化物酶、嘌呤核甘磷酸化酶、黄嘌呤氧化酶、色原物质和水;所述试剂R2的组分包括:缓冲液、保护剂、反应增强剂、防腐剂、肌苷酸二钠、4‑氨基安替比林和水。本发明分析灵敏度高,线性可高达400U/L,重复性好,抗干扰能力强,并且对异常标本结果的检测与实际浓度相符,且不影响正常标本测试结果准确性。
Description
技术领域
本发明涉及体外诊断试剂领域,具体涉及一种5’-核苷酸酶检测试剂盒。
背景技术
5’-核苷酸酶是一组与碱性磷酸酶不同生化性质的磷酸水解酶,主要分布于心、肝、脑、肌肉、肾和肺等,在肝内分布于胆小管、肝窦和枯否氏细胞,在肝细胞浆中含量最高,由肝脏代谢。5’-核苷酸酶在血液中活性的升高主要见于肝胆系统疾病,肝癌、肝炎以及肝硬化,特别是肝癌,常伴随甲胎蛋白、癌胚抗原和α-岩藻糖苷酶以及其它肝功能项目同时检测,对肝胆疾病进行综合分析。
由于血清5’-核苷酸酶测定对肝脏疾病的诊断有较好的特异性和灵敏度,在近20年中,测定方法得到不断发展和改进,先后发展了五代方法和的检测试剂,其主要特点可简述如下:
第一代:5’-核苷酸酶将5-肌苷一磷酸脱氨产生次黄苷和磷酸。通过动态测量5-肌苷一磷酸265nm处吸光度,可以测算5’-核苷酸酶活力大小。由于高底物浓度造成吸光度浓度过高,该法仅适用于5-肌苷一磷酸浓度低于15umol。如此低的底物浓度达不到底物饱和要求,造成5’-核苷酸酶活性检测失真。因此,该法不适用于临床应用。
第二代:5’-核苷酸酶将5-肌苷一磷酸脱氨产生次黄苷和磷酸,后者与钼酸铵作用生成磷钼酸,再被还原剂还原成钼蓝,颜色深浅与释放的磷酸根成正比。测磷比色法试剂种类多,操作繁琐、费时,不适宜自动化分析,内源性磷酸对该测定也有干扰,而且血清中碱性磷酸酶也能水解5-肌苷一磷酸生成相同的产物,反应过程中需加入Ni2+离子抑制碱性磷酸酶的非特异性反应,否则会影响测定结果的准确性。
第三代:Kalcker氏应用5’-核苷酸酶偶联嘌呤核苷磷酸化酶和黄嘌呤氧化酶反应连续检测法。通过测量293nm处尿酸盐吸光度上升的速度来计算5’-核苷酸酶活性。但是,293nm时血清吸光度太高,造成临床应用不便。
第四代:通过5’-核苷酸酶偶联嘌呤核苷磷酸化酶和黄嘌呤氧化酶,过氧化氢酶和醛脱氢酶,借助过氧化氢反应测量334nm处还原型辅酶Ⅱ吸光度上升的速率来计算5’-核苷酸酶活性。该法克服了以往5’-核苷酸酶测试的困难,然而,鉴于反应步骤多,试剂成本高,阻碍实际应用。
第五代:5’-核苷酸酶经Mg2+或Mn2+激活后酶解次5-肌苷一磷酸产生次黄苷;次黄苷再通过嘌呤核苷磷酸化酶的作用,与PO4 3—生成次黄嘌呤;后者在黄嘌呤氧化酶氧化下生成尿酸和过氧化氢;最后过氧化物酶分解水产生原子氧,氧化苯胺类显色剂和4-氨基安替比林反应,生成紫红色的有色醌。550nm处吸光度上升的速度来测算5’-核苷酸酶的活性。反应具有抗干扰能力强。适合自动化快速测定的特点,为临床常规开展5’-核苷酸酶检测试剂创造有利条件。目前市场上基本上是用的该方法。但是,市售试剂盒中存在着较多问题,比如,对异常值测定结果偏低、线性范围偏窄、重复性和抗干扰能力较差。
发明内容
鉴于以上所述现有技术的缺点,本发明提供了一种线性范围高、重复性好、抗干扰能力强的、能真实反映5’-核苷酸酶的活性的检测试剂盒,并且该试剂盒对异常值测定更符合实际浓度,不影响正常标本测试结果准确性。
本发明公开了一种测定5’-核苷酸酶的试剂盒,由彼此独立的试剂R1和试剂R2组成;
试剂R1的组分:
试剂R2的组分:
进一步地,所述反应磷酸盐为磷酸钾、β-甘油磷酸钠和磷酸氢二钠中的一种或几种。
还进一步地,所述试剂R2中还包括甘油,甘油浓度为0.5~30wt%。
再进一步地,所述防腐剂为叠氮钠、ProClin300和庆大霉素硫酸盐中的一种或几种。
更进一步地,所述缓冲液为Tris、PB、MOPSO/Na和Hepes缓冲液中的一种或几种。
更进一步地,所述色原物质为TOPS、TOOS、4-氯酚、TBHBA、MADB和EHSPT中的一种或几种。
更进一步地,所述表面活性剂为TritonX-100、PEG6000、Tween20和Tween80中的一种或几种。
更进一步地,所述试剂R1所含组分及浓度为:100mmol/L Tris缓冲液,1.2mol/L天门冬氨酸,0.15mol/L谷氨酸,0.8KU/L嘌呤核苷酸磷酸化酶,1.6KU/L黄嘌呤氧化酶,3KU/L过氧化物酶,5mmol/L TOPS,0.05wt%Proclin300,1.2KU/L抗坏血酸氧化酶,5μmol/L亚铁氰化钾,0.5wt%Triton X-100,1wt%Tween80,100mmol/L氯化镁,50mmol/L氯化钾,20mmol/Lβ-甘油磷酸钠,所述试剂R1的pH为7.0;
所述试剂R2所含组分及浓度为:100mmol/L磷酸盐缓冲液,80mmol/L肌苷酸二钠,2mmol/L 4-AAP,0.05wt%proclin300,20wt%甘油,150mmol/L磷酸氢二钠,所述试剂R2的pH为7.0。
与现有试剂配方相比,本发明具有如下有益效果:分析灵敏度高,线性可高达400U/L,重复性好,抗干扰能力强;大部分市售试剂对异常值测定结果偏低,若提高异常标本的测定结果,也会影响正常标本的测定值,使之按比升高,本试剂添加表面活性剂和反应增强剂后,可使测定异常标本结果与实际浓度相符,且不影响正常标本测试结果准确性。
附图说明
图1为本发明提出的一种测定5’-核苷酸酶的试剂盒的线形图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制各方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中己有完善说明,具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,Second edi tion,Cold Spring HarborLaboratory Press,1989and Third edi tion,2001:Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates:theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego:Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edi tion,Academic Press,San Diego,1998:METHODSIN ENZYMOLOGY,Vo l.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999:和]METHODS IN MOLECULAR BIOLOGY,Vo l.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
实施例1:
试剂R1的制备:
试剂R2的制备:
对比例1:(空白组:去除天门冬氨酸、谷氨酸、氯化镁、磷酸盐)
试剂R1的制备:
试剂R2的制备:
对比例2:(空白组+天门冬氨酸)
试剂R1的制备:
试剂R2的制备:
对比例3:(空白组+谷氨酸)
试剂R1的制备:
试剂R2的制备:
对比例4:(空白组+天门冬氨酸、谷氨酸)
试剂R1的制备:
试剂R2的制备:
对比例5:(空白组+天门冬氨酸+谷氨酸+磷酸盐)
试剂R1的制备:
试剂R2的制备:
对比例6:(空白组+天门冬氨酸+谷氨酸+氯化镁+氯化钾)
试剂R1的制备:
试剂R2的制备:
使用实施例1中的试剂盒,检测市售的5’-核苷酸酶。用接近线性区间上限的高浓度5’-核苷酸酶样品和接近线性区间下限的低浓度5’-核苷酸酶样品或纯化水混合成至少5个稀释浓度的5’-核苷酸酶样品,试剂校准后重复测定3次稀释后的5’-核苷酸酶样品,根据测定均值和理论值进行线性拟合。
实施例1的线性结果:
实施例1的线性范围为400U/L以上,线性相关系数为R2=0.999。
针对实施例1、对比例1、对比例2以及对比例3进行稳定性测试。将待检试剂分成两份,一份放2-8℃保存,一份放37℃加速,7天后取出同时用因子法(理论因数F=829)进行梯度稀释样本测试,进行稳定性比对分析。结果如下:
根据国家规定,企业制定的试剂有效期应在超期一个月内仍稳定,实施例1结果符合要求。将每组的未加速数据与37℃加速7天数据进行对比后,相对于对比例1、2和3,实施例1的加速后数据与未加速数据差距最小,因此实施例1是本发明稳定最好的;对比例1由于缺乏保护剂,加速后数据与未加速数据差距最大,稳定性最差;对比例2中含有天门冬氨酸,对比例3中含有谷氨酸,加速后数据与未加速数据差距相较对比例1较小,但大于实施例1,这是由于实施例1中的天门冬氨酸和谷氨酸的协同作用使其稳定性有显著的提升。
用实施例1、对比例1~6以及市售试剂盒为对照试剂做对比实验,其中标本号1~16为临床血清,标本号17~19为加入了市售5’-核苷酸酶的临床样品。其中对照试剂为北京利德曼生化股份有限公司生产的5’核苷酸酶(5’NT)测定试剂盒(过氧化物酶法),该试剂盒的成分未知,为同领域高质量产品,检测结果精度高,因此作为对照标准。
结果表明,本发明制备的试剂盒与对照试剂相关性较好,测值一致。对比例1~4中均不含反应增强剂,其中对比例1不含保护剂,对比例2和3分别含有天门冬氨酸和谷氨酸,对比例4中含有天门冬氨酸和谷氨酸,将对比例1~4的数据与对照试剂数据进行对比,差距较大,尤其高浓度标本(17~19号)的数据明显偏低;对比例5中含有磷酸盐,对比例6中含有氯化镁,相对于对比例1~3,对比例5和6的数据在高浓度范围与对照试剂数据差距缩小,但是相较实施例1与对照试剂的差距还是偏大,这是由于氯化镁和磷酸盐的联用,显著地缩小了实施例1与对照试剂的结果差距,尤其是对于高浓度标本的检测。
以上所述仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
2.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述磷酸盐为磷酸钾、β-甘油磷酸钠和磷酸氢二钠中的一种或几种。
3.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述试剂R2中还包括甘油,甘油浓度为0.5~30wt%。
4.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述防腐剂为叠氮钠、ProClin300和庆大霉素硫酸盐中的一种或几种。
5.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述缓冲液为Tris、PB、MOPSO/Na和Hepes缓冲液中的一种或几种。
6.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述色原物质为TOPS、TOOS、4-氯酚、TBHBA、MADB和EHSPT中的一种或几种。
7.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:所述表面活性剂为TritonX-100、PEG6000、Tween20和Tween80中的一种或几种。
8.根据权利要求1所述测定5’-核苷酸酶的试剂盒,其特征在于:
所述试剂R1所含组分及浓度为:100mmol/L Tris缓冲液,1.2mol/L天门冬氨酸,0.15mol/L谷氨酸,0.8KU/L嘌呤核苷酸磷酸化酶,1.6KU/L黄嘌呤氧化酶,3KU/L过氧化物酶,5mmol/L TOPS,0.05wt%Proclin300,1.2KU/L抗坏血酸氧化酶,5μmol/L亚铁氰化钾,0.5wt%Triton X-100,1wt%Tween80,100mmol/L氯化镁,50mmol/L氯化钾,20mmol/Lβ-甘油磷酸钠,所述试剂R1的pH为7.0;
所述试剂R2所含组分及浓度为:100mmol/L磷酸盐缓冲液,80mmol/L肌苷酸二钠,2mmol/L 4-AAP,0.05wt%proclin300,20wt%甘油,150mmol/L磷酸氢二钠,所述试剂R2的pH为7.0。
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CN105420345A (zh) * | 2015-12-16 | 2016-03-23 | 山东博科生物产业有限公司 | 一种稳定、抗干扰能力强的血清5’-核糖核苷酸水解酶检测试剂及检测方法 |
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CN106967787A (zh) * | 2017-02-13 | 2017-07-21 | 山东博科生物产业有限公司 | 一种稳定的5’‑核糖核苷酸水解酶检测试剂盒 |
CN108690868A (zh) * | 2017-04-10 | 2018-10-23 | 广州市伊川生物科技有限公司 | 一种腺苷脱氨酶测定试剂盒及其测定方法 |
CN109112181A (zh) * | 2017-06-24 | 2019-01-01 | 浙江亚培生物技术有限公司 | 一种5`-核苷酸酶活性测定方法及5`-核苷酸酶检测试剂盒 |
CN111139284A (zh) * | 2020-01-16 | 2020-05-12 | 浙江夸克生物科技有限公司 | 一种高准确度的5’-核苷酸酶测定试剂盒 |
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CN111662955A (zh) * | 2020-07-26 | 2020-09-15 | 武汉中太生物技术有限公司 | 一种腺苷脱氨酶测定试剂盒 |
CN111662955B (zh) * | 2020-07-26 | 2023-05-02 | 武汉中太生物技术有限公司 | 一种腺苷脱氨酶测定试剂盒 |
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