CN113817030A - 一种检测人新型冠状病毒的单克隆抗体及检测试剂盒 - Google Patents
一种检测人新型冠状病毒的单克隆抗体及检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一株杂交瘤细胞株及其单克隆抗体制备的检测试剂盒，本发明中所检测的SARS‑CoV‑2抗体是根据E蛋白，通过预测设计的特异性多肽，识别抗原表位如序列ID.NO.2所示。用该抗原免疫BALB/c小鼠，并使用体外细胞融合技术获得了产生特异抗体的杂交瘤细胞株6E9‑2，经鉴定该瘤细胞分泌的抗体为IgG1阳性。所述抗SARS‑CoV‑2‑E单克隆抗体杂交瘤细胞株6E9‑2，能够稳定分泌检测特异性强、灵敏度高的抗SARS‑CoV‑2的单克隆抗体，该抗体可用于制备诊断SARS‑CoV‑2的ELISA试剂盒。
Description
技术领域
本发明属于细胞技术领域，具体为一种杂交瘤细胞株及其单克隆抗体的应用
背景技术
新型冠状病毒肺炎(新冠肺炎)是新型冠状病毒(新冠病毒)感染导致的呼吸道传染病，潜伏期1到14天，主要表现是发热、乏力、干咳，少数病人伴有鼻塞、流涕、腹泻等症状。新冠肺炎重症化率和病死率较高，重症患者多在发病1周后出现呼吸困难和(或)低氧血症，严重患者快速进展至急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒、出凝血功能障碍等。流行病学调查显示，病患多可以追踪到与确诊病例有过近距离的密切接触情况。确诊病例需有病原学证据阳性结果，即病毒核酸检测。但是病毒核酸检测，受到样本和所选试剂等因素的限制，并且存在部分假阴性结果。
目前的研究证实不少新冠肺炎患者本身无症状，或仅有轻微症状，但是他们却能够将病毒传染给他人，核酸检测的阴性结果可能会导致疾病的蔓延。如何从人群中将这些隐性病例分辩出现，从而遵循“早诊断、早治疗、早隔离”的原则，成为了解决这一隐患的重要问题。
机体被病毒感染后，可通过体液免疫应答反应产生病毒抗原特异性抗体。新冠病毒抗体是人体感染新冠病毒后产生的抗体，其浓度水平随着病情进展而增加。因此，新冠病毒抗体检测有助于新冠肺炎的辅助诊断，能够最大程度的解决因为核酸检测漏诊的风险。
发明内容
本发明要解决的技术问题是提供一种SARS-CoV-2-E的抗原表位、单克隆抗体及其制备.
为解决上述技术问题，本发明采用以下技术方案：
1.一种SARS-CoV-2-E的抗原表位，序列表NO.1的氨基酸序列和NO.2 的多肽序列
2.一种识别SARS-CoV-2-E的抗原表位筛选得到的单克隆抗体
3.根据SARS-CoV-2-E的单抗筛选得到的一株杂交瘤细胞株，分类命名 Mus-6E9-2。
4.本杂交瘤细胞分泌的抗SARS-CoV-2-E蛋白的单克隆抗体6E9-2能与 SARS-CoV-2-E蛋白特异性结合。
5.单克隆抗体6E9-2亚型为IgG1。
6.单克隆抗体6E9-2可用于SARS-CoV-2的诊断鉴定。
7.针对SARS-CoV-2病毒的生物诊断方法包括ELISA试剂盒，试剂盒包含内容5所述的单克隆抗体6E9-2
8.杂交瘤细胞株的制备方法，用SARS-CoV-2-E作为抗原免疫BALB/c 小鼠；收集小鼠的脾细胞，将脾细胞与骨髓瘤细胞进行融合得到融合细胞；采用HAT培养基对所述融合细胞进行选择培养；使用 SARS-CoV-2-E蛋白抗原检测所述融合细胞的培养上清中的抗体，筛选得到一株稳定分泌单克隆抗体6E9-2的杂交瘤细胞6E9-2。
附图说明
图1：是基于6E9-2抗体建立的酶联免疫检测方法的标准曲线；
图2：6E9-2抗体对合成多肽检测结果
具体实施方式
为了是本专利的技术方案更加清晰，结合以下实施例对本发明作进一步说明
实施例1：SARS-CoV-2-E单克隆抗体的制备
1.SARS-CoV-2-E抗原表位的设计和制备，根据GenBank上的 SARS-CoV-2基因以及其相对应的蛋白序列，根据文献报道的其结构特征，通过免疫源性分析、亲疏水性及表面可及性分析，最终筛选采用 SARS-CoV-2核酸序列(序列表NO：1)的多肽序列(序列表NO：2)为抗原；送生工合成多肽。
2.免疫小鼠，初次免疫需将抗原多肽与弗氏完全佐剂按1∶1混匀500μl 乳化后，经后颈、左右大腿内侧间隙、左右腋下间隙各约80μl多点注射6-8周龄雌性Balb/c小鼠3只，每只小鼠抗原接种剂量为50μg，间隔两周后，抗原与弗氏不完全佐剂按1∶1混匀500μl乳化后颈、左右大腿内侧间隙、左右腋下间隙各约80μl接种，每只小鼠抗原接种剂量为50μg，间隔两周后，抗原与弗氏不完全佐剂按1∶1混匀500μl乳化后颈、左右大腿内侧间隙、左右腋下间隙各约80μl接种，每只小鼠抗原接种剂量为50μg。
3.抗体效价分析：将三次免疫小鼠及对照小鼠血液与离心管中室温静置4 小时，待凝血后，用离心机4℃，3000g离心5分钟，然后取上清-20℃储存备用；将抗原多肽以5μg/ml浓度用包被液稀释，每孔100μl加入ELISA板中，4度过夜备用；弃去含抗原液体，用包被液清洗ELISA 板三次，然后每孔加入130μl封闭液室温封闭1小时。将各组血清用 PBS稀释成不同梯度(以1∶50起步)备用；弃去封闭液，用PBS清洗 ELISA板3次。每孔加入100μl待检测稀释血清(使用已知抗体作为阳性对照)室温条件孵育一小时；弃去血清稀释液，用PBS清洗3次 ELISA板。然后每孔加入100μl HRP标记羊抗鼠IgG抗体(PBS稀释， 1∶2000)，室温孵育一小时；弃去二抗，用去离子水清洗10次ELISA 板，加入100μl TMB底物溶液，室温孵育10分钟，然后加入100μl 0.5M H2SO4避光终止反应；450nm下测OD值，分析抗体效价。以对照组小鼠血清作为阴性对照，以测定值与对照值得比≥2.1为阳性来判断免疫血清的效价。
4.养分巨噬细胞提取：取阴性对照小鼠处死后，预冷的不完全1640培养基注射到腹腔内冲洗，提取巨噬细胞，计数后，用1640完全培养基悬浮巨噬细胞，使其浓度在108/ml。向96孔板每孔加入100μl细胞悬浮液，37℃，5％CO2恒温箱培养过夜备用
5.细胞融合：融合前3天，取抗原多肽与等体积的PBS混匀后，以每只 50μg的量腹腔注射血清效价大于1∶105的待融合小鼠进行加强免疫；无菌取小鼠脾脏，制成脾细胞悬液，将10×107脾细胞与2×107SP2/0 细胞混合，200g室温离心10分钟，再用1640培养基(含双抗)洗涤、离心3遍，用200μl预热到的PBS悬浮离心管中细胞，然后加入200 μl预热的PEG1500轻轻混合，37℃水浴一分钟。然后缓慢加入20ml 预热到37℃的1640培养基(含双抗)终止反应。室温200g离心5分钟，弃去上清，用完全1640培养基将细胞稀释到原来的十分之一浓度备用。将先前铺好巨噬细胞的96孔板取出，弃去原本的培养基，然后加入稀释好的混合细胞液，每孔100μl，37℃，5％CO2恒温箱培养。此后每天对细胞进行镜检，并更换50μl新鲜的HAT 1640培养基(20％FBS，2mM谷氨酰胺，双抗，50μM巯基乙醇，1×HAT)。待细胞生长至铺满孔底一半面积以上，且不再出现死亡细胞残片，则判定该孔细胞皆为杂交瘤细胞，可进入筛选环节。
6.筛选分泌抗SARS-CoV-2-E的单克隆抗体的杂交瘤细胞：通过间接 ELISA法检测细胞培养上清，选择效价较高的杂交瘤细胞进行亚克隆化，使用有限稀释法连续克隆化2次，直至到100％细胞阳性率，最后获得稳定分泌抗SARS-CoV-2-E的单克隆抗体细胞株，标记为6E9-2；将克隆化后阳性率达100％的细胞扩增培养后液氮冻存；
7.腹水的制备和纯化：将筛选得到的杂交瘤细胞株6E9-2以每只2×106的细胞量注入液体石蜡预处理的8周龄的BALB/c雌性小鼠腹腔，随后饲养观察14天，小鼠腹部膨大时抽取腹水；采用亲和色谱法纯化单克隆抗体，以SDS-PAGE测定单克隆抗体的纯度。
实施例2：基于6E9-2单克隆抗体建立的酶联免疫检测方法
1.使用碳酸盐缓冲液(pH9.4，0.05M)吸附抗6E9-2单克隆抗体进行包被， 4℃孵育过夜后洗涤液洗板；
2.使用用5％牛血清白蛋白、0.05％Tween-20的磷酸盐缓冲液 (pH7.2，0.05M)对板子进行封闭处理；
3.用含1％牛血清白蛋白和0.05％Tween-20的磷酸盐缓冲液将标准样品 (合成多肽)、待测样品(血清)稀释后，加入到酶标板中，常温条件孵育45分钟，洗涤液洗板3次；
4.将用含1％牛血清白蛋白、0.05％Tween-20的磷酸盐缓冲液稀释的，经长链生物素(NHS-LC-biotin)标记的6E9-2单克隆抗体加入到酶标板中，常温条件孵育45分钟，然后用洗涤液洗板3次；
5.加入用含1％牛血清白蛋白、0.05％Tween-20的磷酸盐缓冲液稀释的亲和素标记的辣根过氧化物酶(HRP)，常温条件孵育45分钟，然后用洗涤液洗板；
6.加入TMB显色液，常温显色10分钟，加乙酸终止显色，然后分别于 OD450nm和OD630nm下测定吸光值，OD450减去OD630为校正后的吸光值。
7.生物素标记处理方法：将6E9-2单克隆抗体配成1mg/ml的溶液；用 DMSO将NHS-LC-biotin配成浓度为60mg/ml的溶液；取200ul 1mg/ml 的6E9-2单克隆抗体溶液，加入10ul 60mg/ml的NHS-LC-biotin溶液；混匀后在室温放置30分钟后，加入500mM ph 9.0的Tris溶液中止反应；最后加入大量pH7.4的1X PBS缓冲液，用排阻极限为30Kd的离心柱离心去除多余的生物素分子和进行缓冲液体系的平衡。
最后说明，以上实施例，仅用以说明本发明的技术方案。而不限于对本发明保护范围的限制，尽管实施例对本发明作了详细说明，所有对本发明的技术方案进行修改或者等同替换的发难，将不脱离本发明技术方案的实质保护范围。
序列表
DNA序列
多肽序列表
RTSFSCFRGILASYTSHPYCA。
Claims (9)
1.一种SARS-CoV-2的抗原表位，其特征在于：具有序列表NO.1的氨基酸序列和序列表NO.2的多肽序列。
2.根据权利要求1所述，其特征在于：“根据SARS-CoV-2-E的抗原表位筛选得到的单克隆抗体。
3.根据权利要求2所述，其特征在于：“根据SARS-CoV-2-E的单抗筛选得到的一株杂交瘤细胞株，分类命名Mus-6E9-2。
4.根据权利要求3所述，其特征在于：“本杂交瘤细胞是分泌的抗SARS-CoV-2-E蛋白的单克隆抗体6E9-2。
5.根据权利要求4所述，其特征在于：“单克隆抗体6E9-2，本抗体的特征在于：所述6E9-2能与SARS-CoV-2-E蛋白特异性结合。
6.根据权利要求5所述的单克隆抗体6E9-2，其特征在于：所述单克隆抗体6E9-2亚型为IgG1。
7.根据权利要求6所述其特征在于：“单克隆抗体6E9-2可用于在SARS-CoV-2的诊断鉴定中的应用。
8.根据权利要求7所述的应用，所述生物诊断特征在于：针对SARS-CoV-2病毒的生物检测，包括ELISA试剂盒。
9.根据权利要求8所述杂交瘤细胞株的制备方法，其特征在于：用SARS-CoV-2-E作为抗原免疫BALB/c小鼠；收集免疫后的小鼠的脾细胞，将脾细胞与骨髓瘤细胞进行融合得到融合细胞；采用HAT培养基对所述融合细胞进行选择培养；使用SARS-CoV-2-E蛋白抗原检测所述融合细胞的培养上清中的抗体，筛选得到一株稳定分泌单克隆抗体6E9-2的杂交瘤细胞6E9-2。
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