CN111537740B - 一种森林脑炎病毒IgM抗体检测试剂盒及其应用 - Google Patents

一种森林脑炎病毒IgM抗体检测试剂盒及其应用 Download PDF

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CN111537740B
CN111537740B CN202010443526.2A CN202010443526A CN111537740B CN 111537740 B CN111537740 B CN 111537740B CN 202010443526 A CN202010443526 A CN 202010443526A CN 111537740 B CN111537740 B CN 111537740B
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常军亮
邹勇
孙宏亮
曹玉峰
张秀霞
唐剑光
吴月
韩慧利
李雨桐
严永男
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Abstract

本发明涉及一种森林脑炎病毒IgM抗体检测试剂盒及其应用，所述试剂盒包括酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂，所述的酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂中采用含有维生素C和乙二醇的稀释液作为稳定剂。本发明的试剂盒能够长期维持稳定和有效性，即使在0～4℃温度下避光保存200天以上的情况下能够维持60％以上(优选70％以上)的效价，对于临床使用具有重要意义。

Description

一种森林脑炎病毒IgM抗体检测试剂盒及其应用
技术领域
发明涉及生物检测领域，具体涉及一种医用检测用试剂盒，更具体地涉及一种森林脑炎病毒IgM抗体检测试剂盒及其应用。
背景技术
森林脑炎病毒又称蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)属于黄病毒属，森林脑炎病毒为球形颗粒，基因组为单股正链RNA，长约11kb，共编码3个结构蛋白即衣壳蛋白(C)、膜蛋白(M)、包膜蛋白(E)和7个非结构蛋白(NS1、NS2a、NS2b、NS3、NS4a、NS4b、NS5)，在3个结构蛋白中，E蛋白是病毒最重要的结构蛋白，其含有多种T细胞表位和B细胞表位，决定着病毒的组织嗜性，介导病毒与细胞膜受体的结合，诱导保护性的免疫反应。森林脑炎病毒至少有3个亚型，即西欧亚型、西伯利亚亚型和远东亚型。该病毒以侵袭中枢神经系统为主的方式引起急性传染病，该病病死率高，预后不好，约25-37％的患者会留下麻痹型后遗症。
在本申请人的在先申请CN109613249A中公开了一种森林脑炎病毒IgM抗体ELISA检测试剂盒及其制备方法。该发明申请所公开的试剂盒特别适用于国内森林脑炎病毒的早期辅助诊断，其具有特异性强、敏感性高、检测快速、操作方便、成本低廉等优点，适合临床推广。但是，申请人在后续的研究中发现，上述专利申请所公开的试剂盒即使是在低温环境下进行储存，仍然存在检测准确性显著下降的缺陷。鉴于上述情况，本申请拟对该问题进行改进。
发明内容
基于上述背景技术，本发明拟解决的技术问题在于提供一种储存稳定性显著提高的森林脑炎病毒IgM抗体检测试剂盒及其应用。为了实现本发明的发明目的，拟采用如下技术方案：
本发明一方面涉及一种森林脑炎病毒IgM抗体ELISA检测试剂盒，其特征在于：所述试剂盒包括酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂，所述的酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂中采用含有维生素C和乙二醇的稀释液作为稳定剂。
在本发明的一个优选实施方式中，所述的试剂盒还包括小鼠抗人IgM抗体(抗μ链)预处理酶标板、抗原试剂、浓缩洗液、显色液A液、显色液B液、终止液、阳性对照及阴性对照。
在本发明的一个优选实施方式中，所述抗原试剂为灭活的森林脑炎病毒，并非是任何一种重组森林脑炎病毒蛋白或其混合物。
在本发明的一个优选实施方式中，所采用的灭活森林脑炎病毒为我国流行的远东型森张株病毒。
本发明另一方面还涉及上述检测试剂盒在制备检测森林脑炎病毒的试剂盒中的应用。
本发明还涉及一种森林脑炎病毒森张株E蛋白的单克隆抗体试剂，其包括森林脑炎病毒森张株E蛋白的单克隆抗体、维生素C和乙二醇。
在本发明的一个优选实施方式中，所述维生素C的含量为0.05质量％～0.2质量％；所述乙二醇的含量为0.1质量％～5质量％。
在本发明的一个优选实施方式中，其特征在于所述试剂还含有作为缓冲成分的PBS。
在本发明的一个优选实施方式中，所述试剂在0～4℃温度下避光保存200天的情况下能够维持60％以上(优选70％以上)的效价。
有益效果
针对本发明特定的抗森林脑炎病毒特异性单克隆抗体，通过采用含有维生素C和乙二醇的稀释液，能够有效的提高本发明的抗森林脑炎病毒特异性单克隆抗体的稳定性。即使在0～4℃温度下避光保存6个月(优选12个月)以上的情况下能够维持60％以上(优选70％以上)的效价。
具体实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。在本发明中，如果没有特别说明，所述的百分比以及比例关系是指质量百分比或质量比。
实施例1
(1)按照CN109613249A说明书中实施例1和2的记载，制备获得抗森林脑炎病毒特异性单克隆抗体，经过测试，该单克隆抗体的中和效价为1:32倍。
(2)按下表所示的配方制备酶标试剂，并且检测其在长期储存过程中的稳定性。
组别 配方(稀释之后的单克隆抗体浓度均为1mg/ml
对照组1 含5％的牛血清蛋白的PBS为稀释液，
对照组2 含3％蔗糖+8％甘油的PBS为稀释液
对照组3 含0.1％明胶+8％甘油的PBS为稀释液
对照组4 含0.1％EDTA+1.5％山梨醇的PBS为稀释液
实验组1 含0.1％维生素C+1％乙二醇的PBS为稀释液
实验组2 含0.15％维生素C+0.5％乙二醇的PBS为稀释液
实验组3 含0.1％维生素C+2％乙二醇的PBS为稀释液
(3)将上述配方在0～4℃的温度下避光储存一段时间，然后用间接ELISA法测定上述配方中的单克隆抗体的效价，方案如下：(1)TBEV包被板的制备：将灭活的“森张”株纯化病毒用0.05mol/L，pH 9.6的包被液稀释成5μg/ml，包被酶标板，100μl/孔，4℃，20h；甩干后，每孔加入10％0.02mol/L，pH 7.2PBS稀释的牛血清白蛋白封闭，200μl/孔，4℃，16h；甩干，-20℃保存，备用；(2)单抗ELISA效价测定：分别采用上述表中的配方作为稀释液；NS-1骨髓瘤细胞培养上清为阴性对照，将稀释的单抗加到步骤(1)制备的TBEV包被板中，100μl/孔，37℃孵育1小时，甩干，PBST洗3遍；加入HRP标记的羊抗小鼠IgG酶结合物，1：8000稀释，100μl/孔，37℃孵育30分钟，甩干，PBST洗5遍；(3)加底物显色，加TMB A、B液显色，37℃孵育15分钟，OD450nm读数，cut-off＝2.1×阴性对照OD值作为判定标准。结果如下表所示。
Figure BDA0002504996980000041
基于上述实验结果可知，本发明的申请人尝试了本领域常见的单克隆抗体的稳定剂，其难以实现有效的稳定性，而采用本发明实施例中所使用的稳定剂，能够有效的提高本发明的抗森林脑炎病毒特异性单克隆抗体的稳定性。
以上描述了本发明优选实施方式，然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。

Claims (8)

1.一种森林脑炎病毒IgM抗体ELISA检测试剂盒，其特征在于：所述试剂盒包括酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂，所述的酶标森林脑炎病毒森张株E蛋白的单克隆抗体试剂中采用含有维生素C和乙二醇的稀释液作为稳定剂，所述维生素C的含量为0.05质量%~0.2质量%；所述乙二醇的含量为0.1质量%~5质量%。
2.根据权利要求1所述的检测试剂盒，其特征在于所述的试剂盒还包括小鼠抗人IgM抗体抗μ链预处理酶标板、抗原试剂、浓缩洗液、显色液A液、显色液B液、终止液、阳性对照及阴性对照。
3.根据权利要求2所述的检测试剂盒，其特征在于：所述抗原试剂为灭活的森林脑炎病毒，并非是任何一种重组森林脑炎病毒蛋白或其混合物。
4.根据权利要求3所述的检测试剂盒，其特征在于：所采用的灭活森林脑炎病毒为我国流行的远东型森张株病毒。
5.权利要求1~4任意一项所述的检测试剂盒在制备检测森林脑炎病毒的试剂盒中的应用。
6.一种森林脑炎病毒森张株E蛋白的单克隆抗体试剂，其包括森林脑炎病毒森张株E蛋白的单克隆抗体、维生素C和乙二醇，所述维生素C的含量为0.05质量%~0.2质量%；所述乙二醇的含量为0.1质量%~5质量%。
7.根据权利要求6所述的试剂，其特征在于所述试剂还含有作为缓冲成分的PBS。
8.根据权利要求6或7所述的试剂，其特征在于所述试剂在0~4℃温度下避光保存200天的情况下能够维持60%以上的效价。
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