CN110358739B - 一株分泌抗酚酞单克隆抗体的杂交瘤细胞株js及其应用 - Google Patents
一株分泌抗酚酞单克隆抗体的杂交瘤细胞株js及其应用 Download PDFInfo
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Abstract
一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS及其应用,属于食品安全免疫检测领域。本发明一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏日期2019年3月7日,保藏编号CGMCC No.17394。本发明将酚酞的完全抗原与等量弗氏佐剂混合乳化,通过颈背部皮下多点注射免疫BALB/c小鼠。再经过间接竞争酶联免疫法筛选细胞并三次亚克隆,最终得到一株单克隆抗体杂交瘤细胞株。此细胞株分泌的单克隆抗体,对酚酞具有较好的特异性和检测灵敏度(IC50值为15.4 ng/mL),可实现对保健食品中酚酞残留量的检测,为食品中酚酞残留的免疫检测提供了原料,具有实际应用价值。
Description
技术领域
本发明涉及一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS及其应用,属于食品安全免疫检测领域。
背景技术
酚酞(Phenolphthalein)是一种弱的有机酸,在常温下为白色或微带黄色的细小晶体。其有多重用途,酚酞具有在酸性溶液中呈无色,在碱液或碱金属碳酸盐溶液中呈红色,在化学实验中常作为酸碱滴定的指示剂。在有机合成中主要用于合成塑料特别是二氮杂萘酮聚芳醚酮类聚合物。
而酚酞在医用为接触性泻药,其刺激结肠黏膜增强并组织肠内液体吸收,将水和电解质在结肠蓄积产生缓泻作用而促进排便。在临床上作为***日益提高,肥胖也成了很多人面临的健康问题,越来越多的人选择购买中药减肥类保健产品来辅助减肥。但由于中药成分显效慢,很多不法商家在商品内添加酚酞等化学药物,以达到快速见效的目的。消费者在不知情的情况下长期服用添加酚酞的减肥保健食品将会带来严重的不良反应,可能会诱发结肠炎、流产大出血、甚至结肠癌等疾病。
目前,减肥类保健食品中酚酞基本采用拉曼光谱、高效液相色谱、薄层色谱法、液-质谱串联以及分光光度计法等方法检测。这些检测方法均有检测成本高、耗时较长及检测人员专业素质要求高等缺点。另外,还有报道利用酚酞在酸性和中性溶液为无色在pH>10的碱性环境中呈粉红色醌式结构这一显色原理制备酚酞检测卡来检测,该方法虽然操作简单,便于携带但具有准确度差的缺点,并且不可以进行定量分析。而基于酶联免疫分析方法(ELISA)建立的一种酚酞免疫学检测方法具有简单、灵敏、快捷和高通量的优点。此方法的一个重要前提即需筛选出针对酚酞的高特异性单克隆单体。
发明内容
本发明的目的是提供一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS及其应用,由该细胞株制备的抗体对酚酞具有较好特异性和检测灵敏度,可以用来建立酚酞的免疫学检测方法。
本发明的技术方案,一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCC No.17394。
酚酞单克隆抗体,它由所述保藏编号为CGMCC No.17394的分泌抗酚酞单克隆抗体的杂交瘤细胞株JS分泌产生。
所述酚酞单克隆抗体的应用,用于食品安全检测中酚酞残留的分析检测。
本发明提供的酚酞单克隆抗体杂交瘤细胞株JS的制备基本步骤为:
(1)半抗原结构:
(2)完全抗原酚酞-KLH的制备:称取3.55mg酚酞(酚酞与钥孔血蓝蛋白KLH摩尔比为9000:1),17.6mg N,N'-羰基二咪唑CDI,溶解于300μL N,N-二甲基甲酰胺DMF中,37℃下搅拌反应1h,称为A液;取6mg KLH,用2mL 0.01M硼酸盐缓冲溶液BB, pH=8.6溶解,称为B液,再逐滴将A液缓慢加入到B液中,室温反应36h;然后用0.01M PBS溶液透析,除去未反应的小分子半抗原,得到完全抗原酚酞-KLH,并通过紫外吸收扫描方法进行鉴定。
(3)小鼠的免疫:将完全抗原酚酞-KLH与等量弗氏佐剂混合乳化后,对BALB/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫用完全弗氏佐剂,剂量为100μg/只;多次加强免疫用不完全弗氏佐剂且剂量减半即为50μg/只;冲刺免疫不用佐剂,直接用生理盐水稀释后腹腔注射,剂量再减半即为25μg/只。首次免疫与第二次加强免疫之间间隔一个月,多次加强免疫之间间隔21天,冲刺免疫与最后一次加强免疫之间间隔18-21天。通过间接竞争酶联免疫法(ic-ELISA)观测小鼠免疫效果即检测小鼠血清的效价和抑制。
(4)细胞融合与细胞株建立:通过聚乙二醇(PEG 4000)法将小鼠脾细胞和小鼠骨髓瘤细胞进行融合,采用选择性培养基(HAT培养基)筛选出杂交瘤细胞,并用HT培养基进行细胞培养。融合一周后利用ic-ELISA法检测阳性细胞孔,并进一步利用ic-ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对抑制较好的阳性细胞孔进行亚克隆,一周后再次检测、挑孔、亚克隆。按上述方法进行三次亚克隆后获得酚酞的高分泌特异抗体的单克隆杂交瘤细胞株JS。
(5)杂交瘤细胞株性质的鉴定:通过ic-ELISA测定灵敏度和特异性。
本发明的有益效果:本发明提供的细胞株JS分泌的单克隆抗体,对酚酞具有较好的特异性和检测灵敏度(IC50值为15.4ng/mL),可实现对保健食品中酚酞残留量的检测,为食品中酚酞残留的免疫检测提供了原料,具有实际应用价值。
生物材料样品保藏:一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCC No.17394。
附图说明
图1 单克隆抗体JS对酚酞的标准抑制曲线。
具体实施方式
本发明下面的实施例仅作为本发明内容的进一步说明,不能作为本发明的限定内容或范围。下面通过实施例对本发明作进一步说明。
本发明通过将完全抗原酚酞-KLH免疫小鼠,通过细胞融合,HAT选择性培养基培养,通过ic-ELISA筛选细胞上清,最终得到了针对酚酞具有高分泌特异性抗体的杂交瘤细胞株JS。
实施例1 杂交瘤细胞株JS的制备
(1)完全抗原的合成:称取3.55mg 酚酞(酚酞与钥孔血蓝蛋白KLH摩尔比为9000:1),17.6mg N,N'-羰基二咪唑(CDI),溶解于300μL N,N-二甲基甲酰胺(DMF)中,37℃下搅拌反应1h (称为A液);取6mg KLH,用2mL 0.01M硼酸盐缓冲溶液(BB, pH=8.6)溶解(称为B液),再逐滴将A液缓慢加入到B液中,室温反应36h;然后用0.01M PBS溶液透析,除去未反应的小分子半抗原,得到完全抗原酚酞-KLH,并通过紫外吸收扫描方法进行鉴定。
(2)动物免疫:将酚酞完全抗原与等量弗氏佐剂混合乳化后,对BALB/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫用完全弗氏佐剂,剂量为100μg/只;多次加强免疫用不完全弗氏佐剂且剂量减半即为50μg/只;冲刺免疫不用佐剂,直接用生理盐水稀释后腹腔注射,剂量再减半即为25μg/只。首次免疫与第二次加强免疫之间间隔一个月,多次加强免疫之间间隔21天,冲刺免疫与最后一次加强免疫之间间隔18-21天。通过间接竞争酶联免疫法(ic-ELISA)观测小鼠免疫效果即检测小鼠血清的效价和抑制。
(3)细胞融合:在冲刺免疫三天后,按照常规PEG(聚乙二醇,分子量为4000)方法进行细胞融合,具体步骤如下:
a、小鼠摘眼球取血,颈椎脱臼法处死小鼠后,立即放入 75% 酒精中消毒,浸泡 5min左右,无菌操作取出小鼠的脾脏,用注射器胶头适度研磨并通过200目细胞筛网得到脾细胞悬液,收集,离心(1200rpm,8 min),用RPMI-1640(无酚红)培养基洗涤脾细胞三次,最后一次离心后,将脾细胞稀释到一定体积,计数,备用;
b、收集SP2/0细胞: 融合前 7-10 天,将 SP2/0 瘤细胞用含10% FBS(胎牛血清)RPMI-1640(无酚红) 培养基在5% CO2培养箱中培养。 融合前要求SP2/0瘤细胞数量达到(1-4)*107,保证融合前SP2/0瘤细胞处于对数生长期。融合时,收集瘤细胞,悬浮于RPMI-1640(无酚红)基础培养液中,进行细胞计数;
c、融合过程7min: 第1min,将1mL的PEG 4000 由慢到快滴加到细胞中;第2min,静置。第3min 和第4min,在1min内滴加1mL RPMI-1640(无酚红)培养基;第5min 和第6min,在1min内滴加2mL RPMI-1640(无酚红) 培养基;第7min,每10s 滴加1mL 的 RPMI-1640(无酚红) 培养基。然后37℃温浴5 min。 离心(800 rpm,10 min),弃上清,细胞轻轻敲散,并向其内加入含20%胎牛血清,2% 50×HAT的RPMI-1640(无酚红)选择性培养基(HAT培养基),按照200μL/ 孔加到 96 孔细胞板,置于37℃,5% CO2培养箱中培养。
(4)细胞筛选与细胞株建立:在细胞融合后的第3天用HAT培养基对融合细胞进行半换液;第5天用含20% 胎牛血清,1%的100×HT的RPMI-1640(无酚红)过渡培养液(HT培养基)进行全换液;第7天取细胞上清进行筛选。筛选分两步:第一步先用ic-ELISA法筛选出阳性细胞孔,第二步选用酚酞为标准品,用ic-ELISA法对阳性细胞进行抑制效果测定。选择对酚酞标准品有较好抑制的细胞孔,采用有限稀释法进行亚克隆,七天后用同样的方法进行检测。按上述方法进行三次亚克隆,最终获得酚酞单克隆抗体细胞株JS。
(5)单克隆抗体的制备:取8-10周龄BALB/c小鼠,每只小鼠腹腔注射无菌石蜡油1mL;7天后每只小鼠腹腔注射1×106 酚酞杂交瘤细胞,从第七天开始收集腹水,将腹水通过辛酸-饱和硫酸铵法进行抗体纯化。在偏酸条件下,正辛酸可以沉淀腹水中除IgG免疫球蛋白外的其他杂蛋白,然后离心,弃沉淀;再用等量饱和度的硫酸铵溶液沉淀 IgG型的单克隆抗体,离心,弃上清,用0.01 M PBS溶液(pH7.4)溶解后,透析脱盐,最终得到纯化后的单克隆抗体置于-20℃保存。
(6)单克隆抗体的鉴定:
6.1包被:将包被原酚酞-BSA用0.05M pH9.6 碳酸盐缓冲液从1μg/mL开始3倍比稀释,100μL/孔,37℃反应2h;
6.2洗涤:将板内溶液倾去,并用洗涤液洗涤3次,每次3min;
6.3封闭:拍干后,加入200μL/孔封闭液,37℃反应2h。洗涤后烘干备用;
6.4加样:将抗血清从1:1000开始倍比稀释,并加入到各稀释度的包被孔中,100μL/孔,37℃反应30min;充分洗涤后,加入1:3000稀释的HRP-羊抗鼠IgG,100μL/孔,37℃反应30min;
6.5显色:将酶标板取出,充分洗涤后,每孔加入100μL的TMB显色液,37℃避光反应15min;
6.6终止和测定:每孔加入50μL终止液以终止反应,然后用酶标仪测定各孔的OD450值。
用ic-ELISA测定单克隆抗体酚酞的IC50为:15.4ng/mL,说明对酚酞有很好的灵敏度,可用于酚酞免疫分析检测。
溶液的配置:
碳酸盐缓冲液(CBS):称取Na2CO3 1.59 g,NaHCO3 2.93 g,分别溶于少量双蒸水后混合,加双蒸水至约800mL混匀,调pH值至9.6,加双蒸水定容至1000mL,4℃贮存备用。
磷酸盐缓冲液(PBS):8.00g NaCl,0.2g KCl,0.2g KH2PO4,2.9g Na2HPO4·12 H2O,溶于800mL纯水中,用NaOH或HCl调pH到7.2~7.4,定容至1000mL;
PBST:含0.05 % 吐温20的PBS;
TMB显色液:A液:Na2HPO4 .12H2O 18.43g,柠檬酸 9.33g,纯水定容至1000mL;B液:60mg TMB 溶于100mL乙二醇中。A、B液按体积比5:1混合即为TMB显色液,现用现混。
综上所述仅为本发明的较佳实施例而已,并非用来限定本发明的实施范围。即凡依本发明申请范围的内容所作的等效变化与修饰,都应为本发明的技术范畴。
Claims (3)
1.一株分泌抗酚酞单克隆抗体的杂交瘤细胞株JS,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCC No.17394。
2.酚酞单克隆抗体,其特征在于:它由权利要求1所述保藏编号为CGMCC No.17394的分泌抗酚酞单克隆抗体的杂交瘤细胞株JS分泌产生。
3.权利要求2所述酚酞单克隆抗体的应用,其特征在于:用于食品安全检测中酚酞残留的分析检测。
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