CN113969265A - 分泌IgG1型抗人补体C3d单抗杂交瘤细胞株及应用 - Google Patents
分泌IgG1型抗人补体C3d单抗杂交瘤细胞株及应用 Download PDFInfo
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Abstract
本发明公开一种分泌IgG1型抗人补体C3d单抗杂交瘤细胞株及用途，采用人补体C3d蛋白为抗原免疫BALB/c小鼠，经免疫、细胞融合、筛选、克隆，获得一株能稳定传代并分泌抗人补体C3d单抗的杂交瘤细胞株C3B1B1，为一种新的分泌IgG1型抗人补体C3d单抗的杂交瘤细胞株；同时还提供了该杂交瘤细胞株及其单抗的应用；所述杂交瘤细胞株分泌的抗人补体C3d单克隆抗体类型为IgG1型；将该细胞培养上清收集后浓缩，用C3d致敏细胞做试管凝集试验检测，浓缩液抗体效价可达1024。
Description
技术领域
本发明公开一种分泌IgG1型抗人补体C3d单抗杂交瘤细胞株，尤其是一种分泌IgG1型抗人补体C3d 单抗的杂交瘤细胞株、单克隆抗体，同时还提供了该细胞株的医用用途，属于生物技术领域。
背景技术
人补体C3是由α链(Mr120000)和β链(Mr75000)以二硫键连接而形成的二聚体。在C3转化酶的作用下，C3分子裂解成C3a和C3b。C3b受血清中I因子、H因子和蛋白酶的作用，又可逐级水解为iC3b、C3f、C3c、C3dg、C3d和C3g等片段，其中C3d是由C3dg裂解而来，是补体C3蛋白不能再被蛋白酶裂解的最小片段，始终与抗原共价结合。C3d蛋白的氨基酸序列位于C3蛋白的第996～1303位残基，由308个氨基酸组成，相对分子质量为35KDa。
人补体C3d在先天性和获得性免疫之间起桥梁作用，是补体活化的标志物。人体正常组织和血清中，补体C3d保持稳态，当机体出现异常时，补体系统被激活，导致机体组织和血清中补体C3d水平发生变化，所以机体内补体C3d水平可以用来衡量机体的健康状况和疾病的辅助诊断。有研究表明在感染性疾病、自身免疫性疾病、肾病和糖尿病及一些其他疾病中，机体内补体C3d水平显著高于正常水平。所以临床检测中补体C3d水平对某些疾病具有辅助诊断价值。
发明内容
为检测人补体C3d蛋白，本发明的目的在于提供一种分泌IgG1型抗人补体C3d 单抗的杂交瘤细胞株，本发明以人补体C3d蛋白为抗原通过杂交瘤技术制备了一株分泌IgG1型抗人补体C3d 单克隆抗体的杂交瘤细胞，将细胞培养上清浓缩后可作为人补体C3d检测试剂，应用于人补体C3d的检测中。
本发明提供了一种分泌IgG1型抗人补体C3d 单抗的杂交瘤细胞株，于2021年9月6日保藏于中国典型培养物保藏中心，地址：中国武汉.武汉大学，保藏号为CCTCC NO：C2021235，命名为杂交瘤细胞株C3B1B1。
本发明还提供了一种抗人补体C3d 单克隆抗体，所述抗人补体C3d 单克隆抗体由如上所述的分泌IgG1型抗人补体C3d 单克隆抗体的杂交瘤细胞株分泌产生。
所述抗人补体C3d 单克隆抗体类型为IgG1型；所述抗人补体C3d 单克隆抗体与人补体C3d发生特异性反应。
本发明进一步提供一种分泌IgG1型抗人补体C3d单抗的杂交瘤细胞株在制备人补体C3d检测试剂中的用途，包括步骤将待测样品与如上所述的抗人补体C3d单克隆抗体接触，以确定待测样本是否含人补体C3d。
本发明提供一种如上所述的抗人补体C3d单克隆抗体在人补体C3d检测上的应用。
本发明积极效果在于：
采用人补体C3d蛋白为抗原免疫BALB/c小鼠，经免疫、细胞融合、筛选、克隆，获得一株能稳定传代并分泌抗人补体C3d单抗的杂交瘤细胞株C3B1B1，为一种新的分泌IgG1型抗人补体C3d 单抗的杂交瘤细胞株；同时还提供了该杂交瘤细胞株及其单抗的应用；所述杂交瘤细胞株分泌的抗人补体C3d单克隆抗体类型为IgG1型；由本发明抗人补体C3d单克隆抗体能够与人补体C3d发生特异性反应。将该细胞培养上清收集后浓缩，用C3d致敏细胞做试管凝集试验检测，浓缩液抗体效价可达1024。
上述说明仅是本发明技术方案的概述，为了能够更清楚的了解本发明的技术手段，并可按照说明书的内容予以实施，以下用一些实例来详细说明。本发明的具体实施方式由以下实施例详细给出。
附图说明
图1为抗人补体C3d单克隆抗体试管凝集效价测定结果照片；
图2为抗人补体C3d单克隆抗体特异性鉴定结果照片；
图3为抗人补体C3d单克隆抗体Ig亚类鉴定结果图片；
图4为抗人补体C3d单克隆抗体蛋白免疫印迹结果图片；
图5为杂交瘤细胞株C3B1B1染色体核型分析结果照片；
图6为杂交瘤细胞株C3B1B1传代稳定性分析结果图片。
具体实施方式
以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
实施例1
杂交瘤细胞的获得
1. 免疫原的制备
将酵母菌用生理盐水稀释至浓度为2.4×108个/mL悬液；将酵母菌悬液煮沸30min，3000rpm离心5min，弃去上清，用生理盐水洗涤3次，每次3000rpm离心5min，弃去上清；采集新鲜AB型人全血，3500rpm离心15min，分离血清；将以上酵母菌裂解物重悬于等体积 AB型人血清中，混匀，20℃恒温孵育30min，取出后用生理盐水洗涤3次，3000rpm离心5min，弃上清，最后用10mL生理盐水重悬，-20℃分装保存；
2. 动物免疫与试血
（1）用免疫原免疫4周龄，体重约20g的BALB/c小鼠，免疫原剂量为500µl/只，皮下多点注射；
（2）每间隔一周免疫一次；
（3）三次免疫后，尾静脉加强免疫，抗原剂量为100µl/只小鼠，3天后眼眶静脉采血100μL/只，3500rpm离心15min，分离血清，进行血清效价检测；小鼠血清试管凝集效价大于200进行细胞融合试验；
3. 细胞融合
（1）取NS-1细胞与上述小鼠脾细胞；
（2）融合前5-7天，对进行扩大培养，调整细胞状态，使其达到对数生长期，细胞活率在95％以上；将对数生长期的NS-1细胞从培养箱中取出，轻轻晃动细胞，收集细胞悬液于50mL离心管，1000rpm离心10min，弃上清，再用10mL 1640培养液重悬细胞，备用；
（3）提取试血合格的Balb/c小鼠脾脏细胞按1:5~1:8的比例混匀，1000rpm离心15min，弃上清液；
（4）使用50％的PEG4000作为融合剂进行融合，融合2min，加入50mL1640培养液终止反应，800rpm离心5min，弃上清液；
（5）细胞沉淀用HAT培养液重悬，将细胞均匀加入96孔培养板中，置于37℃、5％CO2培养箱内培养；
4 .杂交瘤细胞的筛选及克隆
（1）制备致敏细胞；
① C3d致敏细胞：取9.24%蔗糖溶液38mL，与2mL新鲜无抗凝剂全血混匀，轻轻震荡，在37℃水浴中孵育15min，用缓冲盐水洗涤3次，2000rpm离心3min，弃上清，将洗涤后的压积细胞，分别1:4（或1:1）加入0.1%胰酶，轻轻震荡，在37℃水浴中孵育10min-30min；用缓冲盐水洗涤3次，2000rpm离心3min，加入细胞保存液于2-8℃保存；
②C4b致敏细胞：取100mL10%的蔗糖溶液，加入120～150mg Na2EDTA，使其溶解后，取20mL加入2mL新鲜全血，轻轻震荡在37℃水浴中孵育15min，用缓冲盐水洗涤3次，2000rpm离心3min，弃上清，加入细胞保存液于2-8℃保存；
③ C4d致敏细胞：将洗涤后的压积C4b致敏细胞，分别1:4（或1:1）加入0.1%胰酶，轻轻震荡，在37℃水浴中孵育10min-30min；用缓冲盐水洗涤3次，2000rpm离心3min，加入细胞保存液于2-8℃保存；
（2）筛选及克隆化
① 融合细胞在培养箱培养5~7天后，细胞克隆约上百个细胞进行筛选；
②取杂交瘤细胞上清液至96孔板中，150µl/孔。将待测样本各取40μl分别加到U型板中，每个待测样本加3个孔；将三种C3致敏细胞用生理盐水配制成0.3%悬液，将C3d、C4b、C4d致敏细胞分别加到待测样本的3个孔中，40μl/孔；用板式振荡器将加完抗体和细胞的U型板混匀，300g离心30s，再用板式振荡器震荡，观察是否有凝集反应；
③筛选出与C3d细胞发生凝集，与C4b和C4d细胞不发生凝集的细胞进行克隆化和培养；
获得一株能稳定分泌抗人补体C3d单克隆抗体的杂交瘤细胞株C3B1B1；经多次传代和多次冻融复苏后，细胞株均能良好生长，并能稳定分泌抗体；扩大培养后，细胞冻存，置于液氮中保存。
实施例2
抗人补体C3d单克隆抗体的制备
1. 将实施例1获得的细胞株C3B1B1使用10%完全1640培养液扩大培养；
2. 收集细胞培养上清，使用中空纤维柱将细胞培养上清浓缩，除菌后得到抗人补体C3d单克隆抗体浓缩液，盐水试管法凝集效价可达1024；
3. 根据生产需求，将浓缩的抗人补体C3d单克隆抗体适当稀释，根据需求加入抗体稳定剂，即可作为抗人补体C3d单克隆抗体试剂，用来进行人补体C3d的鉴定或作为抗人球蛋白检测卡（抗IgG、抗C3d）或抗人球蛋白检测卡（抗IgG＋C3d）等试剂盒的原料。
实施例3
抗人补体C3d单克隆抗体效价测定
采用盐水试管法，测定实施例2制备抗人补体C3d单克隆抗体与C3d致敏细胞反应的效价；
（1）取试管一列，每管中加0.2mL生理盐水，在第2管中加入抗C3d单克隆抗体浓缩液0.2mL，用移液器依次进行倍比稀释，从稀释后的最后一管中移去0.2mL；
（2）向每一试管中加入2% C3d致敏细胞悬液0.2mL。置室温孵育5min，立即1000rpm离心1分钟；取出试管，观察并记录结果；
（3）对照应没有凝集，以待测抗体的最大稀释度仍能出现肉眼可见红细胞凝集，作为抗体的效价。
实验结果如图1所示，实施例2制备的抗人补体C3d单克隆抗体试管凝集效价为1024。
实施例4
抗人补体C3d单克隆抗体特异性鉴定
采用盐水试管法对实施例2制备抗人补体C3d单克隆抗体进行特异性鉴定，具体步骤如下：
（1）取C3d、C4b、C4d致敏细胞和3人份A1、B、O细胞用生理盐水洗涤3次，2000rpm离心5min，去除上清，用生理盐水配成2%的红细胞悬液；
（2）将试管排成2列，每列6支，做好标记，第一列试管各加待检样品0.1mL，然后在各试管中分别加入2%的A1、B、O、C3d、C4b、C4d细胞悬液0.1mL；第二列试管做红细胞悬液对照，即将待检样品改为生理氯化钠溶液，然后在各试管中分别加入上述2%红细胞悬液0.1mL，置室温反应15分钟，1000rpm离心1分钟，轻摇试管，肉眼观察有无凝集；
（3）红细胞悬液对照组无凝集的前提下，观察并记录实施例2制备的抗人补体C3d单克隆抗体与相应红细胞的凝集结果。反之如对照组有凝集反应则试验不成立，需重复试验；
（4）结果如图2所示，抗C3d单克隆抗体与C3d致敏细胞发生凝集反应，与C4b、C4d、A1、B、O细胞不发生凝集反应，未发生溶血和其他不易分辨现象。
实施例5
小鼠单克隆抗体Ig亚类鉴定，采用商品化试剂盒（Mouse Ig Isotyping INSTANTELISA，厂家：eBioscience）对实施例2制备抗人补体C3d单克隆抗体Ig亚类分型进行鉴定；实验结果如图3所示，抗C3d单克隆抗体为IgG1型。
实施例6
抗人补体C3d蛋白单克隆抗体蛋白免疫印迹试验
对实施例2制备抗人补体C3d单克隆抗体进行Western Blot试验，具体步骤如下：
（1）凝胶配制
配置5%浓缩胶，10%分离胶；
（2）样品处理
对实施例2制备抗人补体C3d单克隆抗体10μL，加入10μL还原型loading buffer，再加入80μL生理盐水，置于1.5mL EP管内，100℃加热10min；
（3）SDS-PAGE电泳
电泳上样，处理后的抗人补体C3d抗体上样10μL/孔，蛋白Marker上样量为5µL；恒压电泳，初始电压为80V，进入分离胶时调至120V，当溴酚蓝迁移胶底处停止电泳；
（4）转膜
电泳结束后取出按照蛋白大小切割凝胶，再按凝胶大小剪切PVDF膜；PVDF膜置于甲醇浸泡2 min后放入纯化水清洗两次；按照滤纸、凝胶、PVDF膜、滤纸的顺序放置在转移槽内，200mA恒流转膜1h；
（5）封闭
转膜完毕，将PVDF膜置于预先配置好的5%BSA（PBST配制）封闭液中，37℃封闭1 h；封闭完毕，用PBST洗涤5次，每次5 min；
（6）孵育二抗
加入HRP标记羊抗鼠IgG（1∶3000稀释）10mL，37℃孵育1 h。用PBST洗涤5次，每次5min；
（7）显色
使用DAB辣根过氧化物酶显色试剂盒，按说明书操作进行显色；
（8）对实施例2制备抗人补体C3d单克隆抗体进行Western Blot试验，结果如图4所示，可见抗人补体C3d单克隆抗体重链及轻链，重链分子量约50KD，轻链分子量约25KD，鉴定此抗体为鼠IgG。
实施例7
杂交瘤细胞株染色体核型分析
（1）将实施例1获得杂交瘤细胞株C3B1B1在传代培养48小时后，加秋水仙素，使秋水仙素最终浓度为0.8μg/mL，继续37℃培养4～6小时。将以上细胞1000rpm离心10min，弃去上清液；
（2）向细胞沉淀加入8mL 0.5%的氯化钾低渗液，用吸管吹打均匀，置37℃水浴30min；1000rpm离心10min，弃去上清液；
（3）向细胞沉淀缓慢加入5mL固定液（甲醇：乙酸=3：1）并混匀，37℃水浴固定20min，1000rpm离心10min，弃上清；
（4）向细胞沉淀中加入5mL固定液，1000rpm离心5min，弃上清，留下少许固定液，轻轻混匀细胞；
（5）用移液器吸取细胞滴于预冷的载玻片上，在火焰上经过几次，自然干燥；用Giemsa染液染色20min，用流水冲洗干净后，自然干燥，用显微镜观察染色体数，挑选染色体分散好、中期相多、形态规则的盖玻片封存并拍照记录结果；
（6）杂交瘤细胞的染色体数目接近两种亲本细胞的染色体数目之和，即小鼠脾细胞的染色体数目（40条）与小鼠骨髓瘤细胞NS-1的染色体数目（54～64）之和；
（7）实施例1获得杂交瘤细胞株C3B1B1染色体数目结果如图5所示，染色体数在94～103条之间。
实施例8
杂交瘤细胞株C3B1B1传代稳定性检测
（1）将实施例1获得杂交瘤细胞株C3B1B1扩大培养至细胞培养瓶；
（2）待细胞处于对数生长期，对细胞进行计数，取样稀释到4×105个/ml，接种于一个新的细胞培养瓶中，培养液为10%FBS完全1640培养基；
将方瓶放置二氧化碳培养箱中培养7天后，对细胞培养上清液进行实施例3所述抗人补体C3d单克隆抗体效价测定；
（3）实施例1获得杂交瘤细胞株C3B1B1传代稳定性检测结果如图6所示，杂交瘤细胞株C3B1B1能够稳定传代。
Claims (3)
1.一种分泌IgG1型抗人补体C3d单抗的杂交瘤细胞株，其特征在于：保藏于中国典型培养物保藏中心，保藏日为2021年9月6日，保藏号为CCTCC NO:C2021235；命名为杂交瘤细胞株C3B1B1。
2.如权利要求1所述的一种分泌IgG1型抗人补体C3d单抗的杂交瘤细胞株在制备人补体C3d检测试剂中的用途。
3.一种抗人补体C3d单克隆抗体，其特征在于：由权利要求1所述的分泌IgG1型抗人补体C3d型单抗的杂交瘤细胞株C3B1B1分泌产生；
所述的一种抗人补体C3d单克隆抗体类型为IgG1型；所述抗人补体C3d单克隆抗体与人补体C3d发生特异性反应。
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