CN117285620B - 抗aav9抗体及aav9滴度测定elisa试剂盒 - Google Patents
抗aav9抗体及aav9滴度测定elisa试剂盒 Download PDFInfo
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Abstract
本发明提供了一种高特异性结合AAV9病毒颗粒/AAV9载体的单克隆抗体,其不与变性解聚的病毒结合,且不与AAV2,5,8血清型结合。基于该抗体制备的快速AAV9滴度测定ELISA试剂盒具有1.03E+07 capsids/ml~6.5E+09 capsids/ml的线性范围,灵敏度达到1.03E+07 capsids/ml,且具有较好的精密度,可应用于基因治疗的AAV9载体滴度测定。其测定时间仅为1.5小时左右,操作时间缩短了约2倍。
Description
技术领域
本发明涉及抗体领域,具体地,涉及抗AAV9抗体及AAV9滴度测定ELISA试剂盒。
背景技术
腺相关病毒(AAV)作为基因治疗递送载体具有较高的安全性和临床价值, 已成为体内基因导入的主要平台之一。国际病毒学会将AAV划分为两个种属,其中AAV1-4和AAV6-13属于腺相关依赖性病毒A。AAV病毒粒子由60个VP亚基组成,组装衣壳的三个结构蛋白VP1:VP2:VP3的比例为1:1:10。VP1,2,3只在蛋白的N端序列上有差异,VP1,2都含有VP3的氨基酸序列,同时N端还有额外的其它氨基酸序列。AAV的序列和结构决定了不同血清型AAV与宿主细胞受体结合作用的差异,导致不同血清型的 AAV 对不同的组织和细胞感染效率不同,具有组织趋向性。 AAV可在心脏组织有效、持久、稳定、安全地表达外源基因,因而在心脏基因治疗策略中占据独特优势。AAV1、AAV2、AAV6、AAV8和AAV9型对心脏有较好的感染效率,其中AAV8和AAV9的感染效率较优于其他血清型,AAV9 是目前公认的高效特异性靶向心脏表达的病毒载体血清型。尽管 AAV8 的感染效果也不错,但是没有AAV9 的扩散性好。临床上靶向心肌的AAV血清型一般为1,6和9型,其中AAV1和AAV6均可有效靶向心肌和骨骼肌细胞,而AAV9则更加倾向于对心肌的感染。
对于治疗型AAV载体,准确的滴度测定是AAV基因治疗药物质控的重要组成部分,稳定可靠的滴度才能保证准确给药剂量。AAV的物理滴度一般是指基因组滴度或者衣壳滴度。基因组滴度常采用qPCR、Digital droplet PCR(ddPCR)检测,qPCR由于样品制备,引物设计,PCR效率等差异,不同批次不同实验室会产生实验偏差,ddPCR虽然能克服一些qPCR的局限,不同样品处理方法仍然会产生偏差。
而衣壳滴度主要是通过ELISA方法进行检测,ELISA方法具有可靠及重现性好的优点。对于应用于基因治疗的不同血清型的AAV载体,尤其是经过序列结构优化改造的AAV载体,都有需求开发出特异性抗体作为捕获和检测抗体,以ELISA方法测定AAV滴度。
发明内容
本发明旨在克服上述缺陷,提供一种特异性的抗AAV9抗体,以及包含所述抗体的快速AAV9滴度测定ELISA试剂盒。
本发明提供的一种抗AAV9抗体,其特征在于:
所述抗AAV9抗体包括重链可变区和轻链可变区;
其中,所述重链可变区的重链CDR包含SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3所示的序列;
所述轻链可变区的轻链CDR包含SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6所示的序列。
进一步地,本发明提供的一种抗AAV9抗体,其特征还在于:
所述重链可变区的CDR的氨基酸序列如SEQ ID NO.7所示。
进一步的本发明提供的一种抗AAV9抗体,其特征还在于:
所述抗AAV9抗体重链的氨基酸序列如SEQ ID NO.8所示。
进一步的本发明提供的一种抗AAV9抗体,其特征还在于:
所述轻链可变区的CDR的氨基酸序列如SEQ ID NO.9所示。
进一步的本发明提供的一种抗AAV9抗体,其特征还在于:
所述抗AAV9抗体轻链的氨基酸序列如SEQ ID NO.10所示。
进一步的,本发明提供的一种抗AAV9抗体的制备方法,其特征在于,包含如下步骤:
S1.将AAV9-GFP与佐剂混合后进行免疫;
S2.以脾脏细胞与对数生长期的骨髓瘤细胞融合,制备杂交瘤细胞;
S3.以AAV9-GFP和AAV2/9/8/5-GFP包板,间接ELISA法检测,筛选出只与AAV9-GFP结合而与其他血清型不结合的抗体细胞株;
S4.将扩大培养的杂交瘤单克隆细胞上清与AAV9-GFP结合,通过间接法ELISA,再次验证杂交瘤细胞株抗体与AAV9结合的特异性;
S5.将S4获取的杂交瘤细胞株培养至特定的细胞密度,送做杂交瘤单抗测序,基因合成编码信号肽,可变区及恒定区的抗体重链及轻链基因序列,分别构建进哺乳细胞表达载体,重组质粒转染Expi293哺乳细胞,分泌表达抗体,细胞表达上清经protein A 亲和纯化得到AAV9特异性重组抗体。
进一步的,本发明还提供了一种抗AAV9抗体在高特异性结合AAV9病毒颗粒/AAV9载体上的应用,其特征在于:
所述抗体不与变性解聚的AAV9结合。
进一步的,本发明还提供了一种抗AAV9抗体在高特异性结合AAV9病毒颗粒/AAV9载体上的应用,其特征在于:
所述抗体不与AAV2, 5 ,8血清型结合。
进一步的,本发明还提供了一种抗AAV9抗体在AAV9滴度测定ELISA试剂盒上的应用。
本发明的作用和效果:
本发明提供了一种高特异性结合AAV9病毒颗粒/AAV9载体的单克隆抗体,其不与变性解聚的VP1、VP2和/或VP3亚基结合,且不与AAV2, 5 ,8血清型结合。
基于该抗体制备的快速AAV9滴度测定ELISA试剂盒具有1.03E+07 capsids/ml~6.5E+09 capsids/ml的线性范围,灵敏度达到1.03E+07 capsids/ml,且具有较好的精密度,可应用于基因治疗的AAV9载体滴度测定。其测定时间仅为1.5小时左右,操作时间缩短了约2倍。
附图说明
图1、本实施例提供的35B重组抗体的特异性验证结果。
图2、本实施例提供的35B抗体的特异性验证结果。
图3、本实施例提供的快速法数据结果。
具体实施方式
本发明能够实施多种变更且能够具有各种实施例,因而要在附图中例示各特定实施例并对其进行说明。但这并不是要将本发明限定在特定的实施方式,而应当理解为包括落入本发明的思想以及技术范围的所有变更、等同物乃至替代物。
实施例1.抗AAV9抗体
抗AAV9抗体及AAV9滴度测定ELISA试剂盒,包括以下步骤:
1、小鼠免疫:
将AAV9-GFP(1E+13 vg/ml)1ul,以PBS 稀释100倍与等体积的氢氧化铝佐剂混合,混匀后以200μl/只剂量,选取6-8周雌性Balb/C小鼠进行皮下多点免疫。每次免疫间隔两周再以相同剂量和方式加强免疫,免疫四次以后进行血清效价检测,效价大于1:30000后进行骨髓瘤细胞SP2/0融合,于融合前三天用100μl AAV9-GFP(1E+13 vg/ml,取1ul用PBS 100倍稀释,不加佐剂)腹腔加强免疫一次。
2、杂交瘤细胞株的筛选及抗体亚型鉴定:
小鼠加强免疫3天后,取小鼠脾脏细胞与对数生长期的骨髓瘤细胞SP2/0通过PEG1500融合,制备杂交瘤细胞。10天之后,以AAV9-GFP和AAV2/8/5-GFP包板,间接ELISA法检测,筛选出只与AAV9-GFP结合而与其他血清型不结合的抗体细胞株,经有限稀释至单克隆状态后进行扩大培养和冻存。将扩大培养的杂交瘤单克隆细胞上清与AAV9-GFP结合,通过间接法ELISA,再次验证杂交瘤细胞株抗体与AAV9结合的特异性。
3、杂交瘤单抗测序及重组抗体表达纯化:
将35B杂交瘤细胞株培养至1E+07cell/ml细胞密度,送做杂交瘤单抗测序(苏州鸿讯生物科技股份有限公司)。基因合成编码信号肽,可变区及恒定区的抗体重链及轻链基因序列,分别构建进哺乳细胞表达载体,表达载体可选用pTT5、pCDNA3.1等任意哺乳细胞表达商业化载体。重组质粒转染Expi293哺乳细胞,分泌表达35B抗体,细胞表达上清经proteinA 亲和纯化得到AAV9特异性重组抗体35B。
该35B可用于抗体表征及ELISA试剂盒开发。
4. 35B重组抗体特异性验证
分别以1E+09 vg/ml浓度的AAV2/5/8/9-GFP包被微孔板,将重组35B 抗体以10ug/ml的初始浓度开始,3倍梯度稀释,每孔加样100μl,以山羊抗小 鼠Fc-HRP抗体0.5ug/ml做为二抗,显色反应终止后,在酶标仪上测定450nm吸 光度值。如图1所示,结果显示35B重组抗体只与AAV9载体反应,而不与AAV2/8/5这些常用于基因递送的AAV血清型载体反应,表明35B重组单抗具有良好的特异性。
5. 35B抗体与AAV9载体颗粒的结合
将35B抗体以5ug/ml的浓度包被在微孔板上,将AAV9-GFP(1E+13 vg/ml) 以PBST缓冲液1:1000倍稀释,取部分稀释好的样品95℃加热30分钟进行解聚变性,分别以稀释好的未变性和变性后的AAV9-GFP样品做为检测物,以生物素标记的358抗体5ug/ml做为检测抗体进行夹心法ELISA检测。 如图2所示,结果显示35B抗体只与未解聚的AAV9-GFP载体颗粒结合,识别AAV9颗粒的空间构象表位。不与变性解聚的AAV9 亚基结合。
KI372-A(重链)氨基酸序列:
SDVQLQESGPGLVKPSQSLSLTCSFTGYSSTIGYYWNWIRQFPGNKLEWMGYIRYDGINNYNPSLKNRI SITLDTSKNHVFLNLHSVTPEDTATYYCADYYDNGYFDVWGAGTTVTVSSKTTPPSVYPLAPGCGDTTGSSVTLGCL VKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK
KI373(轻链)氨基酸序列:
DIQMTQSPASLSVSVGETVTITCRASENIFSNLAWYQQKQGKSPQLLVYSATNLADGAPSRFSGSGSGT QYSLKINSLQSEDFGSYYCQHFWDTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
其中,加粗为CDR1,2,3,下划线为CDR区。
实施例2.AAV9滴度测定ELISA试剂盒
使用本发明制备的35B抗体分别开发了快速法和普通法AAV9滴度测定ELISA试剂盒,其具体的组成如下:
组分 | 标签 | 规格 |
包被反应板 | Anti-AAV9 Antibody Coated Plate | 96T/盒 |
标准品(6.5E+08capsids/ml) | AAV9 Standard(冻干粉) | 6.5E+08capsids /瓶×2 |
生物素标记抗体 | Biotinylated Anti-AAV9 Antibody | 30ul/管 |
HRP标记链霉亲和素 | Streptavidin-Enzyme Conjugate | 15ml/瓶 |
10X分析缓冲液 | Assay Diluent | 10ml/瓶 |
20X浓缩洗涤液 | 20X Wash Buffer | 15ml/瓶×2 |
TMB底物液 | TMB | 15ml/瓶 |
终止液 | Stop Solution | 10ml/瓶 |
具体检测方法如下:
(1)实验将试剂盒从冰箱中取出,置于室温环境下,平衡至室温。
(2)配制 1×洗液(PBST: PBS+0.05% Tween-20):将 20×浓缩洗液用纯水稀释至1×备用。
(3)配制 1×分析缓冲液(PBST+1% BSA):将 10×分析缓冲液用纯水稀释至 1×备用。
(4)配制 1×检测抗体:将检测抗体用 1×分析缓冲液稀释 500 倍备用。
(5)准备样本:细胞培养上清可直接加样或用细胞裂解液稀释后加样,如需使用细胞裂解液处理,裂解液需自行准备。
(6)配制标准品:取出标准品,加入 1ml 超纯水,室温溶解 20min,1000rpm 以上离心 30s,使液体集中于管底。取 200μl 标准品之后按 2 倍倍比依次将 6.5E+08capsids/ml 标准品稀释成 3.25E+08 capsids/ml、
1.63E+08capsids/ml、8.13E+07capsids/ml、4.06E+07capsids/ml、2.03E+07capsids/ml、1.02E+07capsids/ml共 7 个滴度,使用 1×分析缓冲液作为零点标准品,每个滴度标准品均应设置复孔。
(7)预洗:根据需要测试的量,取出 96 孔反应板,用洗板机洗板 1 次,每孔可加入 300μl 洗液, 洗涤后拍干,未使用的板条请尽快放入密封袋中并于 4-8℃保存。
(8)加样:分别将标准品和样本加入到 96 孔反应板中,每孔 100μl,37℃恒温振荡反应 20min。
(9)加 1×检测抗体:将上一步反应后的 96 孔板洗板 4 次,300μl/孔,拍干后加入检测抗体,每孔100μl,37℃恒温振荡反应 20min。
(10)加 HRP 标记物:将上一步反应后的 96 孔板洗板 4 次,300μl/孔,拍干后加入 HRP 标记物,每孔100μl,37℃恒温振荡反应 20min。
(11)显色读数:将上一步反应后的 96 孔板洗板 4 次,300μl/孔,拍干后加入显色液,100μl/孔,37℃避光显色 10min,加入终止液 50μl/孔,终止反应。立即使用酶标仪读取 450nm 波长 的 OD 值。
本发明的AAV9滴度测定试剂盒快速方便,1.5-2小时内便获得滴度测定结果,相较传统的3.5-4小时的检测时间,大大缩短了测定时间。
AAV9快速试剂盒精密度分析将 AAV9-GFP 颗 粒 用 1X 稀 释 缓 冲 液 稀 释成 2E+09capsids/ml 、 5E+08 capsids/ml、1.25E+08 capsids/ml高、中、低三个滴度,利用AAV9快速及普通ELISA试剂盒对线性范围内稀释的高、中、低三个滴度的AAV9-GFP颗粒连续检测20次,每天检测1次,连续检测20天,结果显示,批内和批间的CV%分别 为4.5%和3.5%,均在允许误差范围内(批内 CV%< 5%,批间CV%<5%),结果显示该试剂盒精密度高,重复性较好。
本试剂盒采用双抗体夹心酶联免疫吸附法测定样本中 AAV9 衣壳含量,将 AAV9单克隆抗体预包被于96 孔反应板中,并进行包装处理,保证其活性。加入校准品或待测样本后,标准品或样本中的 AAV9 衣壳会特异性结合到反应板上,再加入检测抗体和 HRP 标记物, 形成抗体-抗原-[检测抗体]-[HRP 标记物]复合物,通过洗涤操作,将多余的检测抗体及 HRP 标记物除去。加入显色液后,HRP 催化其显色,显色强度与样品中的 AAV9 衣壳滴度成正比。用终止液终止反应后,在酶标仪上读取 450nm 波长处的吸收值,即可计算出样品中的 AAV9 衣壳滴度。
以上虽然以实施例为中心进行了说明,但这只是例示而已,并不限定本发明,本领域普通技术人员清楚,在不逸出本实施例的本质特性的范围内能够进行以上并未例示的各种变形和应用。例如,实施例中所具体示出的各构成要素能够经变形而实施。而且,与这种变形和应用相关的各种不同点应当解释为包括在所附的权利要求书中所规定的本发明的范围。
Claims (8)
1.一种抗AAV9抗体,其特征在于:
所述抗AAV9抗体包括重链可变区和轻链可变区;
其中,所述重链可变区的重链CDR1为SEQ ID NO.1,CDR2为SEQ ID NO.2,CDR3为SEQ IDNO.3所示的序列;
所述轻链可变区的轻链CDR1为SEQ ID NO.4,CDR2为SEQ ID NO.5,CDR3为SEQ ID NO.6所示的序列。
2.如权利要求1所述的一种抗AAV9抗体,其特征在于:
所述重链可变区的氨基酸序列如SEQ ID NO.7所示。
3.如权利要求1所述的一种抗AAV9抗体,其特征在于:
所述抗AAV9抗体重链的氨基酸序列如SEQ ID NO.8所示。
4.如权利要求1所述的一种抗AAV9抗体,其特征在于:
所述轻链可变区的氨基酸序列如SEQ ID NO.9所示。
5.如权利要求1所述的一种抗AAV9抗体,其特征在于:
所述抗AAV9抗体轻链的氨基酸序列如SEQ ID NO.10所示。
6.如权利要求1-5任一所述的一种抗AAV9抗体在制备高特异性结合AAV9病毒颗粒/AAV9载体上的应用,其特征在于:
所述抗体不与变性解聚病毒结合。
7.如权利要求1-5任一所述的一种抗AAV9抗体在制备高特异性结合AAV9病毒颗粒/AAV9载体上的应用,其特征在于:
所述抗体不与AAV2, 5 ,8血清型结合。
8.如权利要求1-5任一所述的一种抗AAV9抗体在制备AAV9滴度测定ELISA试剂盒上的应用。
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