CN107356544B - 一种脂肪酶检测试剂盒 - Google Patents
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Abstract
本发明公开了一种脂肪酶检测试剂盒,该试剂盒由单试剂组成,该单试剂包括以下组分:缓冲体系浓度80mmol/L‑160mmol/L,PH=8.8;甘油三月桂酸酯100mmol/L‑300mmol/L;氧化型辅酶Ⅰ0.2mmol/L‑0.4mmol/L;甘油脱氢酶0.5KU/L‑3KU/L;防腐剂NaN30.6g/L‑1g/L;酶保护剂5g/L‑10g/L;展开剂0.4%‑1.6%;溶剂为去离子水。本发明的优点是无需样本前处理,可直接利用全自动生化分析仪进行大批量样本检测,操作简单、准确度高、重复性好、线性范围宽,适合临床应用推广。
Description
技术领域
本发明涉及生物试剂,具体涉及一种检测试剂盒,特别是指一种脂肪酶检测试剂盒。
背景技术
脂肪酶(Lipase,甘油酯水解酶)隶属于羧基酯水解酶类,能够逐步的将甘油三酯水解成甘油和脂肪酸。主要来源于胰腺,其次为胃及小肠,能水解多种含长链(8-18碳链)脂肪酸的甘油酯。脂肪酶合成于胰腺的腺泡组织,具有较高的器官特异性,胰腺炎发病早期,即可检测到血清脂肪酶浓度的升高,急性胰腺炎是一种临床常见、发病急、病情凶险、病死率高的急腹症,及时抽血检查血清脂肪酶,对诊断、治疗和预后有重要意义。早期检测血清脂肪酶能较好的区分急性和非急性胰腺炎,其血清表达水平和胰腺炎病情进展程度呈显著正相关,有较高的敏感性,是诊断胰腺炎病情的主要监测指标。
目前检测脂肪酶(LPS)的方法主要有PNPB法、pH-Stat法、滴定法及比浊法等。PNPB法、pH-Stat法、滴定法无法配合全自动生化分析仪,不适用于临床诊断;目前大部分LPS检测试剂盒均采用比浊法,该法的缺陷在于对底物的依赖性较大且反应的准确度不高、重复性较差,线性范围较窄。
因此,有必要对此进行改进。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供一种脂肪酶检测试剂盒,该检测试剂盒采用辅酶指示系统的连续监测法,无需样本前处理,可直接利用全自动生化分析仪进行大批量样本检测,操作简单、准确度高、重复性好、线性范围宽,适合临床应用推广。
为实现上述目的,本发明的技术方案是该试剂盒由单试剂组成,该单试剂包括以下组分:
缓冲体系浓度80 mmol/L-160 mmol/L,PH=8.6-9.0;
甘油三月桂酸酯 100 mmol/L-300 mmol/L
氧化型辅酶Ⅰ 0.2 mmol/L-0.4 mmol/L
甘油脱氢酶0.5KU/L-3KU/L
防腐剂NaN3 0.6 g/L-1 g/L
酶保护剂 5 g/L-10 g/L
展开剂,该展开剂为液体展开剂 0.4mL-1.6 mL/100mL;或该展开剂为固体展开剂0.4g-1.6g/100mL ;
溶剂为去离子水。
进一步设置是所述缓冲体系为柠檬酸-醋酸镁缓冲液、甘氨酸-醋酸镁缓冲液、咪唑-醋酸镁缓冲液、乙酸-醋酸镁缓冲液或硼酸-醋酸镁缓冲液。
进一步设置是所述的酶保护剂为海藻糖、蔗糖、甘露醇、果糖中的一种或多种组合。
进一步设置是所述的酶保护剂为海藻糖和蔗糖1:1质量比组合。
进一步设置是所述展开剂为吐温-20、吐温-80、脂肪醇聚氧乙烯(7)醚、月桂酸聚氧乙烯(9)酯、聚乙二醇6000、聚乙二醇8000、烷基酚聚氧乙烯(10)醚中的一种或多种组合。
进一步设置是所述展开剂为吐温-80和脂肪醇聚氧乙烯(7)醚1:3体积比组合。
本试剂盒采用辅酶指示系统的连续监测法,可以确定线性期并计算每分钟吸光度的变化(△A/min),根据此值再准确地计算酶活性,可以利用全自动生化分析仪进行检测;酶和底物反应的特异性好,检测结果准确度高。
本发明的试剂盒主要采用辅酶指示系统的连续监测法进行检测。其检测原理为:LPS催化分解甘油三月桂酸酯,生成甘油和脂肪酸。在氧化型辅酶Ⅰ(NAD+)的参与下,甘油脱氢酶(GDH)将甘油上的氢转至还原型辅酶Ⅰ(NADH),由于NADH在340 nm处有特征吸收,所以NADH的上升速率和LPS的活力成正比。反应式如下:
本发明涉及的脂肪酶检测试剂盒,无需样本前处理,可直接利用全自动生化分析仪进行大批量样本检测,操作简单、准确度高、重复性好、线性范围宽,适合临床应用推广。
有益效果:本发明采用的辅酶指示系统制备的脂肪酶(LPS)检测试剂盒,采用乙酸-醋酸镁缓冲液,不仅可以提高试剂的稳定性,且其中的Mg2+可作为LPS的激活剂而大大提高其准确度和灵敏度;同时加入展开剂,增强油水界面的表面张力,提高了试剂的反应度,增大了试剂可测线性范围。
下面结合说明书附图和具体实施方式对本发明做进一步介绍。
附图说明
图1是本发明实施例1的线性结果图;
图2是本发明实施例2的线性结果图;
图3是本发明实施例3的线性结果图。
具体实施方式
下面通过实施例对本发明进行具体的描述,只用于对本发明进行进一步说明,不能理解为对本发明保护范围的限定,该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。
实施例1
本发明提供的试剂盒为单一试剂,试剂的组成和成份如下:
乙酸-醋酸镁缓冲液80 mmol/L,PH=8.8,本实施例该PH值也可以为8.6或者8.9;
甘油三月桂酸酯 100 mmol/L
氧化型辅酶Ⅰ(NAD+) 0.2 mmol/L
甘油脱氢酶(GDH)0.5KU/L
防腐剂NaN3 0.6 g/L
酶保护剂海藻糖和蔗糖均2.5g/L
展开剂吐温-80(Tween-80) 0.1%和脂肪醇聚氧乙烯(7)醚 0.3%
实施例2
本发明提供的试剂盒为单一试剂,试剂的组成和成份如下:
乙酸-醋酸镁缓冲液120 mmol/L,PH=8.8
甘油三月桂酸酯 200 mmol/L
氧化型辅酶Ⅰ(NAD+) 0.3 mmol/L
甘油脱氢酶(GDH)1.8KU/L
防腐剂NaN3 0.8 g/L
酶保护剂海藻糖和蔗糖均3.5g/L
展开剂吐温-80(Tween-80) 0.25%和脂肪醇聚氧乙烯(7)醚 0.75%
实施例3
本发明提供的试剂盒为单一试剂,试剂的组成和成份如下:
乙酸-醋酸镁缓冲液160 mmol/L,PH=8.8
甘油三月桂酸酯 300 mmol/L
氧化型辅酶Ⅰ(NAD+) 0.4 mmol/L
甘油脱氢酶(GDH)3KU/L
防腐剂NaN3 1g/L
酶保护剂海藻糖和蔗糖均5g/L
展开剂吐温-80(Tween-80) 0.4%和脂肪醇聚氧乙烯(7)醚 1.2%
实施例4
将本发明实施例1-3试剂盒进行性能评价
1.准确度验证:
选用朗道低、高值质控品(1530,批号:1045UN;1532,批号:801UE),采用BS-420全自动生化分析仪进行检测,具体操作方法如表1 所述,采用单点定标法定标后,LPS测定值及空白吸光度值可从仪器上直接读取,为了减少偶然误差,重复测定三次取均值,计算测定值与靶值的相对偏差。
由以上结果可知,本发明的试剂盒测定均值位于靶值范围内,相对偏差明显<10%,说明本发明试剂盒准确度较高。
2.精密度实验:
选用临床常规样本,用本发明实施例1-3试剂盒重复测定20次,计算变异系数,结果见表3。
由以上结果可知,本发明试剂盒实施例1-3变异系数(CV)分别为0.96%、1.06%、0.84%,均明显<8%,说明本发明试剂盒具有良好的精密度。
3.线性范围检测:
制取脂肪酶浓度 350U/L的样本,分别按1、1/2、1/4、1/8、1/16、1/32、1/64稀释成7个不同浓度(Xi),用本发明试剂盒实施例1-3按照检测方法均测定3次取均值(Yi),以Xi为自变量,Yi为因变量求出线性回归方程,计算线性回归相关系数R2,将稀释浓度(xi)代入线性回归方程,计算yi的估算值Yi,及Yi,与yi的相对偏差。
从以上数据可知,本发明实施例1-3试剂盒线性相关系数分别为:0.9998、0.9999、0.9999,均大于0.9900,有非常好的线性,且所测线性范围较宽,可以满足临床需求。
综上性能评价可知,本发明实施例1-3试剂盒具有准确度高、重复性好、测定线性范围宽的优点,可配合全自动生化分析仪使用,在临床诊断胰腺炎病情方面有很好的应用价值。
应用实施例
本发明脂肪酶(LPS)检测试剂盒适用于各类全自动生化分析仪,现于全自动生化分析仪BS-420上的应用,其具体使用方法如下:
计算方法:样品中的LPS含量(U/L)=ΔAT/min×F
ΔAT/min:测定管平均每分钟相对空白管吸光度变化
注:TV:试剂样品总体积,SV为样品体积; 6.22:NAD+在340nm处的毫摩尔分子吸光系数;
1.0:比色皿光径(cm); 1000:U/mL到U/L的转换系数。
本发明所使用的质控品为朗道高、低值质控品;所测样品为不溶血血清。
Claims (2)
1.一种脂肪酶检测试剂盒,其特征在于:该试剂盒由单试剂组成,该单试剂包括以下组分:
缓冲体系 浓度160 mmol/L ;
甘油三月桂酸酯 300 mmol/L
氧化型辅酶ⅠNAD+ 0.4 mmol/L
甘油脱氢酶GDH 3KU/L
防腐剂NaN3 1g/L
酶保护剂 海藻糖和蔗糖 均5g/L
展开剂 吐温-80 0.4%和脂肪醇聚氧乙烯(7)醚 1.2%;
溶剂为去离子水;
所述缓冲体系为乙酸-醋酸镁缓冲液。
2.根据权利要求1所述的一种脂肪酶检测试剂盒,其特征在于:所述的缓冲体系的pH值为8.8。
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