CN117604068A - 一种甘油激酶活性测定方法及测定试剂盒 - Google Patents
一种甘油激酶活性测定方法及测定试剂盒 Download PDFInfo
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Abstract
本发明涉及检测技术领域,具体涉及一种甘油激酶活性测定方法及测定试剂盒,将样品与主要由缓冲液、ATP、还原型辅酶Ⅰ、磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶、七水合硫酸镁、还原型谷胱甘肽及稳定剂组成的试剂依次混合,检测最终显色产物在波长为340nm处吸光度下降的速度,从而测算甘油激酶活性大小,由此利用酶学方法,不易受到内、外源其他物质的干扰,酶方法简便、易于操作,酶反应的特异性促使测试结果准确,具有灵敏度高、特异性强的特点,酶反应都是在缓冲液条件下进行,没有污染环境问题、酶法测定方法不需要特殊、昂贵的仪器,测试成本低廉,因此可以保证具有较高的测试准确性,更便于推广应用。
Description
技术领域
本发明涉及检测技术领域,尤其涉及一种甘油激酶活性测定方法及测定试剂盒。
背景技术
甘油到磷酸二羟丙酮的分解代谢包括两步酶促反应。在甘油激酶(CK)的催化下,甘油激酶酸化形成3-磷酸甘油,3-磷酸甘油在3-磷酸甘油脱氢酶(mtGPD)的催化下氧化成磷酸二羟丙酮,进一步返回至糖酵解途径。此外。甘油还可以在甘油氢化酶的作用下生成D-甘油醛,进一步生成D-甘油酸(乙醛(AND+)脱氢酶)和3-磷酸甘油醛(丙糖激酶);也可以在甘油脱水酶的作用下,单向地生成2-羟基丙醛。
甘油激酶(GK)是利用甘油的限速酶,催化甘油磷酸化反应生成3-磷酸甘油,反应中要有Mg2+和ATP的参与,1,6-二磷酸果糖(FBP)抑制该酶的活性。
测定甘油激酶目前尚无成熟的生化比色方法酶联免疫法利用抗原、抗体特异性结合来测定,但其测定出的只是甘油激酶的含量,不能测定出酶活力,且价格昂贵。
发明内容
本发明的目的在于提供一种甘油激酶活性测定方法及测定试剂盒,解决现有技术中测定甘油激酶目前尚无成熟的生化比色方法酶联免疫法利用抗原、抗体特异性结合来测定,但其测定出的只是甘油激酶的含量,不能测定出酶活力,且价格昂贵的问题。
为实现上述目的,本发明提供了一种甘油激酶活性测定试剂盒,40-200mmol/L缓冲液、2-20mmol/L甘油、5-20mmol/LATP、1-5mmol/L还原型辅酶Ⅰ、2-5mmol/L磷酸烯醇式丙酮酸、10-20U/ml乳酸脱氢酶、5-10U/ml丙酮酸激酶、10-40mmol/L七水合硫酸镁、10-40mmol/L还原型谷胱甘肽和10%-50%稳定剂。
其中,所述稳定剂为乙二醇、丙三醇、甘油、聚糖、聚醇、硫酸铵、盐或腺苷二磷酸中的至少一种。
其中,所述缓冲液为三羟甲基氨基甲烷-盐酸缓冲液、Tris-盐酸/EDTA缓冲液、磷酸盐缓冲液、三乙醇胺缓冲液中的一种。
其中,所述缓冲液的pH范围为6.0~11.0。
其中,试剂配成单试剂、双试剂或三试剂。
本发明还提供一种甘油激酶活性测定方法,采用上述所述的甘油激酶活性测定试剂盒,包括如下步骤:
将样品与主要由缓冲液、ATP、还原型辅酶Ⅰ、磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶、七水合硫酸镁、还原型谷胱甘肽及稳定剂组成的试剂依次混合;
检测最终显色产物在波长为340nm处吸光度下降的速度,从而测算甘油激酶活性大小。
其中,温度控制在20℃至50℃,反应时间控制在分钟6-8分钟。
其中,被测样品与试剂的比例控制在1/10至1/250。
本发明的一种甘油激酶活性测定方法及测定试剂盒,将最终反应终产物置于紫外-可见光分光光度计或半自动、全自动生化分析仪下,检测340nm处吸光度下降的速度,从而测算出甘油激酶活性大小,以上样品与试剂的混合比例体积为1/10至1/250,以上过程测定温度控制在20℃至50℃,反应时间控制在常规的6-8分钟,这种方法应用甘油激酶催化底物甘油和ATP生成甘油磷酸和ADP,再通过磷酸烯醇式丙酮酸与ADP反应生成ATP和丙酮酸,最后利用丙酮酸与还原型辅酶反应,氧化成NAD+,还原型辅酶在340nm处有吸收峰,因此从测定主波长340nm处吸光度下降的速度,可以推算出甘油激酶活性的大小,由此利用酶学方法,不易受到内、外源其他物质的干扰,酶方法简便、易于操作,酶反应的特异性促使测试结果准确,具有灵敏度高、特异性强的特点,酶反应都是在缓冲液条件下进行,没有污染环境问题、酶法测定方法不需要特殊、昂贵的仪器,测试成本低廉,因此可以保证具有较高的测试准确性,更便于推广应用。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1是本发明的甘油激酶活性测定方法的步骤流程图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
请参阅图1,本发明提供一种甘油激酶活性测定方法,包括如下步骤:
S1:将样品与主要由缓冲液、ATP、还原型辅酶Ⅰ、磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶、七水合硫酸镁、还原型谷胱甘肽及稳定剂组成的试剂依次混合;
S2:检测最终显色产物在波长为340nm处吸光度下降的速度,从而测算甘油激酶活性大小。
其中,将最终反应终产物置于紫外-可见光分光光度计或半自动、全自动生化分析仪下,检测340nm处吸光度下降的速度,从而测算出甘油激酶活性大小,以上样品与试剂的混合比例体积为1/10至1/250,以上过程测定温度控制在20℃至50℃,反应时间控制在常规的6-8分钟,这种方法应用甘油激酶催化底物甘油和ATP生成甘油磷酸和ADP,再通过磷酸烯醇式丙酮酸与ADP反应生成ATP和丙酮酸,最后利用丙酮酸与还原型辅酶反应,氧化成NAD+,还原型辅酶在340nm处有吸收峰,因此从测定主波长340nm处吸光度下降的速度,可以推算出甘油激酶活性的大小,由此利用酶学方法,不易受到内、外源其他物质的干扰,酶方法简便、易于操作,酶反应的特异性促使测试结果准确,具有灵敏度高、特异性强的特点,酶反应都是在缓冲液条件下进行,没有污染环境问题、酶法测定方法不需要特殊、昂贵的仪器,测试成本低廉,因此可以保证具有较高的测试准确性,更便于推广应用;
用以实现本发明方法的甘油激酶测定试剂盒可以是单试剂,包括:
也可以将以上单试剂配成如下双试剂,更有利于消除内源性干扰及试剂的污染:
试剂Ⅰ
试剂Ⅱ
缓冲液 40-200mmol/L
甘油 2-20mmol/L
稳定剂 10%-50%(总体积)
双试剂的配方不仅限于上述配方,其中试剂Ⅰ的成分,还原型辅酶Ⅰ(NADH)、乳酸脱氢酶及丙酮酸激酶等可以放在试剂Ⅱ,如此可以形成多种配方;
还可以将试剂配成如下三试剂,不但更加有利于消除内源性干扰及试剂的污染,还更有利于试剂的稳定;
试剂Ⅰ
试剂Ⅱ
试剂Ⅲ
缓冲液 40-200mmol/L
甘油 2-20mmol/L
稳定剂 10%-50%(总体积)
三试剂的配方不仅限于上述配方,其中试剂Ⅰ的成分,ATP、还原型辅酶Ⅰ(NADH)等可以单独或同时放在试剂Ⅱ或试剂Ⅲ中,其中试剂Ⅱ的成分,磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶等中任何单独或任二者可以放在试剂Ⅲ中,如此可以形成多种配方;
所述缓冲液的范围为6.0~11.0,缓冲液可以是三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液、Tris-盐酸/EDTA缓冲液、磷酸盐缓冲液、三乙醇胺缓冲液等中的一种,但不仅限于这些缓冲液;
此外,为了减少各试剂成分之间的交叉影响、保持试剂的稳定性,以便长期储存,以上单试剂、双试剂的试剂Ⅰ、试剂Ⅱ或三试剂的试剂Ⅰ、试剂Ⅱ、试剂Ⅲ中通常加入稳定剂,为总体积的10%-80%(或者10%-50%),稳定剂可以是乙二醇、丙三醇、甘油、聚糖、聚醇、硫酸铵、盐或腺苷二磷酸中的一种或一种以上;
将样品与主要由缓冲液、ATP、还原型辅酶Ⅰ、磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶、七水合硫酸镁、还原型谷胱甘肽及稳定剂组成的试剂按一定的操作步骤依次混合,使之发生如下反应:
本试剂盒的线性范围:回归方程为Y=bX+a,R2=0.9999;
分析灵敏度:最低检出限为0.1U/mg;
精密度评价:
批内变异系数:CV批内=2.5%;
批间变异系数:CV批间=5.8%;
准确度评价:回收率95%~105%;
稳定性评价:本试剂盒2~8℃保存一年后,重复精密度试验、准确度试验均能符合试剂盒要求(批内变异系数:CV批内>5%,批间变异系数:CV批间>15%,回收率在95%~105%之间)。
以上所揭露的仅为本申请一种或多种较佳实施例而已,不能以此来限定本申请之权利范围,本领域普通技术人员可以理解实现上述实施例的全部或部分流程,并依本申请权利要求所作的等同变化,仍属于本申请所涵盖的范围。
Claims (8)
1.一种甘油激酶活性测定试剂盒,其特征在于,按重量份计,其制备原料包括:
40-200mmol/L缓冲液、2-20mmol/L甘油、5-20mmol/LATP、1-5mmol/L还原型辅酶Ⅰ、2-5mmol/L磷酸烯醇式丙酮酸、10-20U/ml乳酸脱氢酶、5-10U/ml丙酮酸激酶、10-40mmol/L七水合硫酸镁、10-40mmol/L还原型谷胱甘肽和10%-50%稳定剂。
2.如权利要求1所述的甘油激酶活性测定试剂盒,其特征在于,
所述稳定剂为乙二醇、丙三醇、甘油、聚糖、聚醇、硫酸铵、盐或腺苷二磷酸中的至少一种。
3.如权利要求2所述的甘油激酶活性测定试剂盒,其特征在于,
所述缓冲液为三羟甲基氨基甲烷-盐酸缓冲液、Tris-盐酸/EDTA缓冲液、磷酸盐缓冲液、三乙醇胺缓冲液中的一种。
4.如权利要求3所述的甘油激酶活性测定试剂盒,其特征在于,
所述缓冲液的pH范围为6.0~11.0。
5.如权利要求4所述的甘油激酶活性测定试剂盒,其特征在于,
试剂配成单试剂、双试剂或三试剂。
6.一种甘油激酶活性测定方法,采用如权利要求5所述的甘油激酶活性测定试剂盒,其特征在于,包括如下步骤:
将样品与主要由缓冲液、ATP、还原型辅酶Ⅰ、磷酸烯醇式丙酮酸、乳酸脱氢酶、丙酮酸激酶、七水合硫酸镁、还原型谷胱甘肽及稳定剂组成的试剂依次混合;
检测最终显色产物在波长为340nm处吸光度下降的速度,从而测算甘油激酶活性大小。
7.如权利要求6所述的甘油激酶活性测定方法,其特征在于,
温度控制在20℃至50℃,反应时间控制在6-8分钟。
8.如权利要求7所述的甘油激酶活性测定方法,其特征在于,
被测样品与试剂的比例控制在1/10至1/250。
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