CN105628930A - 一种灵敏度高的胶乳增强免疫比浊法肌钙蛋白i检测试剂 - Google Patents
一种灵敏度高的胶乳增强免疫比浊法肌钙蛋白i检测试剂 Download PDFInfo
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Abstract
本发明涉及肌钙蛋白I检测技术领域,特别涉及一种灵敏度高的肌钙蛋白I检测试剂,其中试剂中的主要成分为缓冲液、氯化锌、Thesit、EMULGEN-A90、氨基三乙酸(NTA)防腐剂,试剂R2的组分为:缓冲液、曲拉通-308、牛血清白蛋白(BSA)、防腐剂、TnI抗体包被胶乳颗粒等主要组成成分。其中Thesit、EMULGEN-A90、曲拉通-308三种表面活性剂的加入以及恰当的胶乳微球粒径大小的选择大大增加了反应的灵敏度,并且产品配置简单,价格低廉,非常适合大面积的临床推广。
Description
技术领域
本发明涉及一种灵敏度高的胶乳增强免疫比浊法肌钙蛋白I检测试剂,属于临床体外检测技术领域。
背景技术
肌钙蛋白,由T、C、I三亚基构成,和原肌球蛋白一起通过调节钙离子对横纹肌动蛋白ATP酶的活性来调节肌动蛋白和肌球蛋白相互作用。当心肌损伤后,心肌肌钙蛋白复合物释放到血液中,4-6小时后,开始在血液中升高,升高的肌钙蛋白I能在血液中保持很长时间6-10天。肌钙蛋白I具有高度心肌特异性和灵敏度,所以肌钙蛋白T已成为目前最理想的心肌梗死标志物。
患有各种冠状动脉疾患的病人必然会发生心肌细胞损伤.有些病人的临床表现可能不完全符合WHO关于AMI诊断标准(不稳定心绞痛就是其中之一),但却伴有某些心肌损伤 标志物(如cTnT等)升高,从而导致细胞内的组成成分渗漏入外周血循环.这使得心肌细胞损伤标志物的检测成为可能.cTnT和cTnI在AMI后(3~6h)血中浓度很快升高,和CK-MB(3~8h)相当或稍早,它们测定的特异性和灵敏度明显高于CK-MB.cTn具有相当长的诊断窗口期(cTnI7~9天,cTnT更长).cTn对急性胸痛病人(无论有无骨骼肌损伤)的诊断均优于CK-MB.研究表明:在对AMI的诊断方面cTnI和cTnT无显著差异,都能鉴别出CK-MB所不能检测出的心肌损伤.
心肌肌钙蛋白I测定开始于20世纪80年代中期,最早使用的是多克隆抗体检测的放射免疫比浊法,随后相继出现了运用单克隆抗体检测的各种不同的免疫法。目前检测的方法有肌钙蛋白I的测定主要有免疫学方法,检测方法则包括胶乳增强免疫比浊法,夹心免疫放射法、夹心酶联免疫法,化学发光以及电化学发光等相关方法,其中普通夹心免疫放射法、夹心酶联免疫法两种方法成本相对较低,操作相对较简单,但是若试验中所用的抗体为多克隆抗体,会交叉反应,且分析灵敏度较低。而化学发光和电化学发光这两种方法,准确度较好且反应的分析灵敏度高,能够快速准确的检测出结果,但是这两种方法需要特定的化学发光仪器,并且对实验室操作人员的要求相对较高,因此在临床中无法真正的普及使用,只有一些相对较大的实验室才具备相应的条件,而后期逐渐研发和使用的胶乳增强免疫比浊法操作简单,成本较低,能够较好的结合全自动生化分析仪器进行操作,方便快捷,但是本方法目前同样具有分析灵敏度低的缺陷,在临床使用过程中针对于低浓度的临床样本,容易出现零值,甚至出现负值,这给临床使用者带来很大不方便,因此根据本问题,作者在胶乳增强免疫比浊法检测血清肌钙蛋白I的基础之上进行优化,发明一种分析灵敏度高的胶乳增强免疫比浊法检测血清肌钙蛋白I。
发明内容
本发明的目的是提供一种用于检测血清肌钙蛋白I的试剂。
检测原理:
首先将特异性抗体结合于胶乳颗粒表面,样本与胶乳颗粒在缓冲液中混合,样本中的TnI与胶乳颗粒表面的抗体结合,使相邻的胶乳颗粒彼此交联,在505nm波长下检测溶液浊度变化,变化程度与样本中的TnI含量成正比:
一种准确度高的肌钙蛋白I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·············································100mmol/L,
氯化锌·············································0.1mmol/L-0.5mmol/L,
Thesit·············································1ml/L-3ml/L
EMULGEN-A90········································1ml/L-5ml/L,
氨基三乙酸(NTA)····································0.1g/L-1g/L,
防腐剂·············································0.5ml/L-1ml/L;
2)试剂R2的组分为:
缓冲液·············································100mmol/L,
曲拉通-308·········································1ml/L-5ml/L,
牛血清白蛋白(BSA)··································1g/L-5g/L,
防腐剂·············································0.5ml/L-1ml/L;
TnI抗体包被胶乳颗粒································适量
所述的肌钙蛋白I检测试剂,试剂R1中缓冲液为25℃,PH=7.6的咪唑缓冲液;
所述的肌钙蛋白I检测试剂,试剂R2中缓冲液为25℃,pH=7.6的咪唑缓冲液。
所述的肌钙蛋白I检测试剂,所述防腐剂为PC-300。
所述的肌钙蛋白I检测试剂,所述TnI抗体包被胶乳颗粒为羊抗人单抗体TnI抗体,抗体所包被的胶乳微球颗粒为50-55nm。
所述的肌钙蛋白I检测试剂来检测肌钙蛋白I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505nm。
所述的检测方法,R1试剂和R2试剂的比例为3:1。
本发明的创新处:
1)在试剂R1和R2中分别使用了咪唑缓冲液,咪唑缓冲液在反应中能够有较大的缓冲能力,对在保证缓冲能力的同时,且不会对反应体系产生负面影响;
2)在试剂的R1中加入Thesit和EMULGEN-A90两种表面活性剂,这两种表面活性剂的联合使用能有较好的提高试剂的乳化作用,能够较好的提高单抗体和肌钙蛋白I的结合,且能保证在反应过程中均衡稳定,保证反应曲线在仪器上的平滑;
3)在试剂R1中加入了氨基三乙酸(NTA),能够有效的螯合重金属离子,去除重金属离子对反应的干扰;
4)在R2中所选取的胶乳微球的颗粒粒径为直径相对较小的50-55nm胶乳颗粒,能够较好的增加反映的灵敏度;
5)在试剂R2中加入了曲拉通-308和牛血清白蛋白两种物质,能够有效的保护单抗体的活性和效价,且曲拉通-308能特异性的增加抗体和抗原的结合。
附图说明
图1为实施例2和化学发光相关性曲线图,
图2为实施例3和化学发光相关性曲线图。
图3为实施例1和化学发光相关性曲线图。
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
一种现有常见的肌钙蛋白I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1(R1):
甘氨酸缓冲液···················································PH7.6
氯化钠························································150mmol/L
曲拉通-100··················································1ml/L
试剂2(R2):
甘氨酸缓冲液··················································PH7.6
TnI抗体包被胶乳颗粒···········································适量
实施例2
一种准确度高的肌钙蛋白I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
试剂R1中含有
缓冲液·················································100mmol/L,
氯化锌·················································0.1mmol/L,
Thesit·················································1ml/L,
EMULGEN-A90············································1ml/L,
氨基三乙酸(NTA)········································0.1g/L,
防腐剂·················································0.5ml/L;
2)试剂R2的组分为:
缓冲液·················································100mmol/L,
曲拉通-308·············································1ml/L,
牛血清白蛋白(BSA)······································1g/L,
防腐剂·················································0.5ml/L;
TnI抗体包被胶乳颗粒····································适量
实施例3
本实施例描述的是一种重要组成部分含量增加的准确度高的肌钙蛋白I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做成成分
缓冲液··················································100mmol/L,
氯化锌··················································0.5mmol/L,
Thesit··················································3ml/L,
EMULGEN-A90·············································5ml/L,
氨基三乙酸(NTA)·········································1g/L,
防腐剂··················································1ml/L;
2)试剂R2的组分为:
缓冲液··················································100mmol/L,
曲拉通-308··············································5ml/L,
牛血清白蛋白(BSA)·······································5ml/L,
防腐剂··················································1ml/L;
TnI抗体包被胶乳颗粒·····································适量
3)检测使用方法:
本实施例描述的肌钙蛋白I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,利用速率法进行测定。分别将实施例1和实施例2的R1和R2按照3:1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,操作如表一:
表一实施例1试剂检测方法
计算:肌钙蛋白I含量(U/L)=(测定÷标准)×C标准。
4)相关性实验:利用实施例1、2和实施例3中的配方配制试剂,与化学发光方法检测血清肌钙蛋白I进行对照检测,同时检测了20个临床血清样本,检测结果如表二所示。并获得了3种试剂分别与化学发光实验检测结果的相关性曲线(如图1-图3所示),通过检测结果显示,实施例1试剂和实施例2以及实施例3与化学发光法检测血清的肌钙蛋白I相关系数分别为0.9739、0.9995、0.9990,说明了实施例2和实施例3中的试剂与化学发光试验的相关性要比实施例1与化学发光实验的相关性要好很多。
表二实施例1、实施例2、实施例3试剂与化学发光验检测血清对比检测结果
样本号 | 实施例2试剂(ug/L) | 实施例3试剂(ug/L) | 实施例1试剂(ug/L) | 化学发光实验(ug/L) |
1 | 1.61 | 1.56 | 1.2 | 1.69 |
2 | 0.75 | 0.72 | 0.86 | 0.73 |
3 | 0.43 | 0.52 | 0 | 0.45 |
4 | 0.02 | 0.01 | 0 | 0.01 |
5 | 0 | 0.01 | 0 | 0.01 |
6 | 0.21 | 0.24 | 0.16 | 0.25 |
7 | 0.63 | 0.63 | 0.59 | 0.69 |
8 | 7.53 | 7.39 | 7.33 | 8.03 |
9 | 10.02 | 10.69 | 8.69 | 10.55 |
10 | 0.12 | 0.09 | 0 | 0.1 |
11 | 0.36 | 0.42 | 0.21 | 0.39 |
12 | 0.29 | 0.31 | 0 | 0.27 |
13 | 0.31 | 0.29 | 0.91 | 0.3 |
14 | 0.01 | 0.01 | 0 | 0.024 --> |
15 | 0.05 | 0.04 | 0 | 0.06 |
16 | 0.71 | 0.71 | 0.79 | 0.69 |
17 | 9.68 | 9.58 | 5.69 | 9.75 |
18 | 4.35 | 4.29 | 4.38 | 4.28 |
19 | 1.55 | 1.55 | 1.39 | 1.58 |
20 | 1.11 | 1.19 | 0.98 | 1.19 |
与化学发光相关性 | 0.9995 | 0.9990 | 0.9739 | ----- |
4)灵敏度实验:
选取12个已知不同浓度的肌钙蛋白I样本(如表三),然后将十二个不同的样本分别使用本实施例的配方、实施例1和实施例2中的配方配置的试剂与化学发光法检测肌钙蛋白I检测方法,其中实施例1、2和本实施例的试剂借助全自动生化分析仪器,化学发光方法借助于化学发光仪器对不同浓度的同一肌钙蛋白I样本进行检测,检测结果如下:
表三12个不同低浓度梯度的肌钙蛋白四种方法检测结果对比情况
通过实验表明,实施例2和实施例3中的优化方案与化学发光法同时检测由高到低不同浓度的样本,检测结果相近,针对于非常低的样本同样可以检测出,而实施例1中的常规样本则无法检测出,说明本实施例和实施例2都具有良好的分析灵敏度。
Claims (9)
1.一种准确度高的肌钙蛋白I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:
一种准确度高的肌钙蛋白I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················································100mmol/L,
氯化锌················································0.1mmol/L,
Thesit················································1ml/L,
EMULGEN-A90···········································1ml/L,
氨基三乙酸(NTA)·······································0.1g/L,
防腐剂················································0.5ml/L;
2)试剂R2的组分为:
缓冲液················································100mmol/L,
曲拉通-308············································1ml/L,
牛血清白蛋白(BSA)·····································1g/L,
防腐剂················································0.5ml/L;
TnI抗体包被胶乳颗粒···································适量。
2.一种重要组成部分含量增加的准确度高的肌钙蛋白I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做成成分
缓冲液················································100mmol/L,
氯化锌················································0.5mmol/L,
Thesit················································3ml/L,
EMULGEN-A90···········································5ml/L,
氨基三乙酸(NTA)·······································1g/L,
防腐剂················································1ml/L;
2)试剂R2的组分为:
缓冲液················································100mmol/L,
曲拉通-308············································5ml/L,
牛血清白蛋白(BSA)·····································5ml/L,
防腐剂················································1ml/L;
TnI抗体包被胶乳颗粒···································适量。
3.根据权利要求1和权利要求2所述的肌钙蛋白I检测试剂,其特征在于试剂R1和R2中缓冲液均为25℃,pH为7.6的咪唑缓冲液。
4.根据权利要求1和权利要求2所述的肌钙蛋白I检测试剂,其特征在于所述表面活性剂为Thesit、EMULGEN-A90和曲拉通-308。
5.根据权利要求1和权利要求2所述的肌钙蛋白I检测试剂,其特征在于所述离子平衡剂为氯化锌。
6.根据权利要求1和权利要求2所述的肌钙蛋白I检测试剂,其特征在于所述重金属离子螯合剂为氨基三乙酸(NTA)。
7.根据权利要求1和权利要求2所述的肌钙蛋白I检测试剂,其特征在于所述胶乳微球的粒径为50-55nm,同时所包被的抗体为羊抗人TnI单抗。
8.一种使用权利要求1-7中任一项所述的肌钙蛋白I检测试剂来检测肌钙蛋白I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505nm。
9.根据权利要求8所述的检测方法,其特征在于R1试剂和R2试剂的比例为3:1。
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