CN116284354B - 检测重组人轮状病毒vp8抗原(vp8 p[4])的单克隆抗体及其应用 - Google Patents
检测重组人轮状病毒vp8抗原(vp8 p[4])的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种重组人轮状病毒VP8抗原(VP8 P[4])的单克隆抗体及其应用。所述的重组人轮状病毒VP8 P[4]抗原的单克隆抗体,其重链可变区(VH)氨基酸序列为如SEQ ID NO.1所示,轻链(VL+CL)氨基酸序列如SEQ ID NO.2所示。
Description
技术领域
本发明属于医药生物技术领域,具体的,涉及一种重组人轮状病毒VP8抗原(VP8 P[4])的单克隆抗体及其应用。
背景技术
轮状病毒是导致全世界范围内儿童、婴儿腹泻的最常见病原体,是引起严重脱水性腹泻的最常见原因,几乎每个孩子在5岁之前都被轮状病毒感染过,对经济造成巨大的损失。由于缺乏有效的治疗方式,由轮状病毒引发的疾病每年导致约21.5万名婴儿死亡,主要集中于低收入国家。
人轮状病毒属于呼肠孤病毒科轮状病毒属,为无包膜RNA病毒,颗粒直径约75nm,由三层二十面体蛋白衣壳组成。其基因组为包含11个节段的双链RNA,编码6个结构蛋白(VP1、VP2、VP3、VP4、VP6、VP7)和5个非结构蛋白(NSP1、NSP2、NSP3、NSP4、NSP5),內壳蛋白为VP6,核蛋白为VP1、VP2和VP3,最外层由两种蛋白形成,VP4和VP7,其中VP4形成刺突样结构。在轮状病毒感染细胞过程中,VP4蛋白经胰蛋白酶作用,裂解形成VP5*和VP8两个功能多肽片段。VP8蛋白主要参与受体识别,对病毒宿主范围和病毒感染具有重要作用。感染过程中宿主产生的VP8特异性单克隆抗体和抗血清,具有中和病毒的能力,可以阻断病毒与宿主细胞的结合和侵入。
根据VP8核苷酸序列差异,可以将A组轮状病毒分成不同的P基因型,其中P[8]最为常见,P[4]次之,P[6]主要分布在非洲地区。P[4]、P[6]和P[8]型的占比超过90%,所以基于VP8*抗原开发三价疫苗是目前全球轮状病毒候选疫苗的通用设计。
轮状病毒疫苗研发生产过程中对疫苗主要有效成分的质量监控非常重要。酶联免疫吸附试验(ELISA)具有灵敏、快速、耐受性强的优点,可以用于重组人轮状病毒VP8 P[4]抗原生产过程中的质量控制。
基于此,提出本发明。
发明内容
本发明首先涉及一种检测重组人轮状病毒VP8 P[4]抗原的单克隆抗体,其重链可变区(VH)氨基酸序列如SEQ ID NO.1所示:
SEQ ID NO.1:
轻链(VL+CL)氨基酸序列如SEQ ID NO.2所示,
SEQ ID NO.2:
优选的,所述的单克隆抗体为IgG型抗体,的Fc为鼠源Fc;
优选的,
所述的重组人轮状病毒VP8 P[8]抗原的氨基酸序列如SEQ ID NO.3所示:
SEQ ID NO.3:
其中包括:
前导序列:DKISDVSTIVPYIGPALN
连接肽序列:GSGSG
剩余为P[8]△VP8*序列。
所述的重组人轮状病毒VP8 P[6]抗原的氨基酸序列如SEQ ID NO.4所示:
其中包括:
前导序列:IDKISDVSTIVPYIGPALNI;
连接肽序列:GSGSG;
剩余为P[6]△VP8*序列。
所述的重组人轮状病毒VP8 P[4]抗原的氨基酸序列如SEQ ID NO.5所示:
其中包括:
前导序列:IDKISDVSTIVPYIGPALNI
连接肽序列:GSGSG
剩余P[4]△VP8*序列(64-223,GenBank:AB848998(Japan))。
本发明还涉及编码所述单克隆抗体的核酸片段。
本发明还涉及所述的单克隆抗体在制备检测试剂盒中的应用,所述的检测试剂盒为:检测重组人轮状病毒VP8 P[4]抗原的浓度的检测试剂盒。
优选的,所述的检测试剂盒为基于阻断ELISA检测原理的检测试剂盒。
一种检测重组人轮状病毒VP8 P[4]抗原的检测试剂盒,所述的试剂盒包括:
(1)检测有效量的所述单克隆抗体;
(2)必要的二抗、显色试剂、缓冲试剂;
所述单克隆抗体的重链可变区(VH)氨基酸序列为如SEQ ID NO.1所示,轻链(VL+CL)氨基酸序列如SEQ ID NO.2所示。
优选的,所述的单克隆抗体为IgG型抗体,Fc为鼠源Fc。
本发明的有益效果在于,
本发明所述的单克隆抗体208单抗,具有灵敏、快速、耐受性强的优点,用于重组人轮状病毒VP8 P[4]抗原生产过程中的质量控制,能够高效精准的定量疫苗中的抗原含量,对疫苗质量进行监控。
附图说明
图1、阻断ELISA实验流程图。
图2、208单抗检测人轮状病毒VP8 P[4]抗原的标准曲线。
具体实施方式
本发明所使用的主要试剂及其来源为:
208单抗(P4-1),其重链可变区(VH)氨基酸序列为:
SEQ ID NO.1:
其轻链(VH+CL)氨基酸序列为:
SEQ ID NO.2:
其他试剂若无特别说明,均为国产分析纯。
实施例1、阻断ELISA检测方法的建立
阻断ELISA检测方法原理:将供试品稀释至一定浓度后进行梯度稀释,再加入特异性抗体与抗原反应,将此反应液作为ELISA的一抗,与包被在固相载体上的抗原特异性结合,然后加入酶标二抗、显色液,最后加入终止液终止反应,通过测定特定波长吸光值进行定量分析,供试品吸光度值与其抗原浓度呈负相关,根据标准曲线计算供试品中抗原的浓度。阻断ELISA实验流程图见图1。
1.1、线性范围与抗体工作浓度的确定
(1)按照常规包被条件,将包被的208参比品(Y202001-02)稀释至4μg/ml进行包被(2-8℃包被16-24小时,包被液配方:Na2CO310.6g,加纯化水定容至1L,调pH至9.5-9.6)
(2)次日用1*PBST清洗掉未结合上的包被抗原,然后加入封闭液进行封闭(25℃封闭1小时),即制得包被了抗原的酶标板;
(3)使用时,依次加入系列稀释的208参比品、系列稀释的208单克隆抗体(P4-1)、系列稀释的辣根过氧化物酶标记的山羊抗小鼠单克隆抗体(IgG(H+L),25℃反应后加入辣根过氧化物酶显色体系的TMB显色液,最后用1M磷酸终止后读取特异性吸光值。
(4)经过试验,结果显示,我们最终得出酶标抗体最佳工作稀释倍数为5000倍;在329~2.57μg/ml范围内,吸光值与检测浓度(μg/ml)呈高度线性相关(R2>0.98)。
实施例2、阻断ELISA检测方法的验证
2.1、专属性验证
实验设计如下表所示:
说明:
201蛋白为重组人轮状病毒VP8 P[8]抗原,氨基酸序列如SEQ ID NO.3所示;
205蛋白为重组人轮状病毒VP8 P[6]抗原,氨基酸序列如SEQ ID NO.4所示;
208蛋白为重组人轮状病毒VP8 P[4]抗原,氨基酸序列如SEQ ID NO.5所示。
接受标准:各样品回收率80%~120%;其他抗原存在的情况下,不干扰供试品抗原的检测。
专属性验证结果
专属性验证结论:在201蛋白、205蛋白及201蛋白+205蛋白同时存在的情况下,依然可以准确检测出208蛋白的含量(回收率在可接受范围80%-120%内),即本检测方法专属性/特异性良好。专属性验证合格。
2.2、精密度(重复性)
实验设计:选取1批208蛋白,用稀释液分别稀释至高、中、低三个浓度分别为3.5倍、7倍、14倍,即在板内等体积稀释8个梯度进行检测,其余与标准曲线同法操作。
检测时,每个浓度重复测定3次。计算三次复测检测值CV值。
接受标准:供试品必须有至少3个稀释度在标准曲线线性范围内;同一批次供试品的三次复测检测值CV≤20%。
精密度(重复性)验证结果如下表:
精密度(重复性)结论:各浓度三次复测检测值之间的CV小于20%(分别为1.5%、4.0%、1.6%)。重复性验证合格。
2.3、精密度(中间精密度)
实验设计:另择一天两位实验员重复“重复性”实验,计算日间及人员差异。
接受标准:不同日期,不同人员,同一批次的供试品检测值CV≤20%。
精密度(中间精密度)验证结果如下表:
精密度(中间精密度)结论:不同日期、不同实验员对供试品的抗原检测结果CV小于20%(分别为6.2%、4.6%、9.6%)。中间精密度验证合格。
2.4、准确度
实验设计:选取1批208蛋白,用稀释液按表1进行稀释,然后进行加标,加标过程见表2,取C1、C2、C3、c1、c2、c3进行测定,计算回收率。
回收率(%)=(加标样品实测值-样品实测值)/加标理论值×100%
208蛋白稀释过程如下表
208蛋白准确度加标过程如下表
接受标准:回收率在80%~120%之间。
准确度验证结果如下表所示:
名称 | 加标理论值(μg/ml) | 加标实测值(μg/ml) | 回收率% |
208蛋白加标样品-高 | 164.6 | 376-400*0.5=176 | 107% |
208蛋白加标样品-中 | 82.3 | 196-203*0.5=94.5 | 115% |
208蛋白加标样品-低 | 41.2 | 97.64-101*0.5=47.14 | 114% |
准确度验证结论:各浓度加标回收率在80%~120%之间别(分别为107%、115%、114%),准确度验证合格。
2.5、标准曲线(线性和范围)
实验设计:总结精密度、准确度验证中的标准曲线数据,以得到标准曲线的线性及最佳检测范围。
接受标准:标准曲线(四参数拟合曲线)的相关系数均不小于0.98,各标准曲线的R2的CV≤10%;标准曲线最佳检测范围一致。
标准曲线(线性和范围)验证结果如下表:
标准曲线(线性和范围)验证结论:综合前几个验证可知,标准曲线线性良好,R2均大于0.99,R2的CV%小于1%,符合验证标准;标曲的线性范围一致,均为329~2.57μg/ml。208标准曲线如图2。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
Claims (7)
1. 一种检测重组人轮状病毒VP8 P[4]抗原的单克隆抗体,其特征在于,
其重链可变区(VH)氨基酸序列如SEQ ID NO.1 所示:
轻链(VL+CL)氨基酸序列如SEQ ID NO.2 所示;
所述的人轮状病毒VP8 P[4]抗原的氨基酸序列如SEQ ID NO.5 所示。
2. 根据权利要求1 所述的单克隆抗体,其特征在于,所述的单克隆抗体为IgG 型抗体,Fc为鼠源Fc。
3. 编码权利要求1 或2 所述单克隆抗体的核酸片段。
4. 权利要求1 或2 所述的单克隆抗体在制备检测试剂盒中的应用,所述的检测试剂盒为:检测重组人轮状病毒VP8 P[4]抗原的浓度的检测试剂盒。
5. 根据权利要求4 所述的应用,其特征在于,所述的检测试剂盒为基于阻断ELISA 检测原理的检测试剂盒。
6. 一种检测重组人轮状病毒VP8 P[4]抗原的检测试剂盒,所述的试剂盒包括:
(1)检测有效量的单克隆抗体;
(2)必要的二抗、显色试剂、缓冲试剂;
所述单克隆抗体的重链可变区(VH)氨基酸序列为如SEQ ID NO.1 所示,轻链(VL+CL)氨基酸序列如SEQ ID NO.2 所示;
所述的人轮状病毒VP8 P[4]抗原的氨基酸序列如SEQ ID NO.5 所示。
7. 根据权利要求6 所述的检测试剂盒,其特征在于,所述的单克隆抗体的Fc 为鼠源Fc。
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