CN110205303A - 一株利福平单克隆抗体杂交瘤细胞株nlc及其应用 - Google Patents
一株利福平单克隆抗体杂交瘤细胞株nlc及其应用 Download PDFInfo
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Abstract
本发明涉及一株利福平单克隆抗体杂交瘤细胞株NLC及其应用,属于食品安全免疫检测领域。本发明细胞株NLC,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,保藏编号CGMCC No.17391。将利福平完全抗原与等量弗氏佐剂混合乳化,通过免疫BALB/c小鼠。首次免疫用完全弗氏佐剂,多次加强免疫用不完全弗氏佐剂,最后一次用利福平完全抗原冲刺免疫(腹腔注射)。将高效价,低IC50小鼠的脾细胞,通过PEG方法与小鼠骨髓瘤细胞融合;再经过间接竞争酶联免疫法筛选并三次亚克隆,最终得到杂交瘤细胞株NLC。此细胞株分泌的单克隆抗体,对利福平具有较好的特异性和检测灵敏度,可实现对水产品中利福平残留量的检测,具有实际应用价值。
Description
技术领域
本发明涉及一株利福平单克隆抗体杂交瘤细胞株NLC及其应用,属于食品安全免疫检测领域。
背景技术
利福平(Rifampicin,RFP)又名甲哌利福霉素,是一种半合成利福霉素类抗生素,为广谱抗生素,对结核杆菌有高度抗菌活性,临床主要用于抗结核治疗,也是一种水产专用的渔药。利福平适用于海水养殖鱼、虾、蟹类等因弧菌、球菌引起的出血、腹水、溃烂、肠炎、白便;对海参因细菌感染导致的化皮,肿嘴;在鱼、虾、蟹、贝类育苗期间,对发光菌、弧菌等具有强力抑杀作用。利福平虽未批准用于水产动物,但在实际水产养殖中使用广泛,在人体内蓄积对健康产生危害,其耐药性也有转移扩散到人体的风险,对消费者的健康存在很大的隐患。有报道指出利福平可致人肝功能、肾功能损害以及各种皮疹等疾病。
目前国内外对水产品中使用的利福平没有特定的限量标准。我国在《2018年兽药残留标准制修订项目的通知》中提出了制定《水产品中利福平残留量的测定LC-MS-MS》标准。因此,为保障水产品质量安全和消费者身体健康,促进水产养殖业的健康、可持续发展,有必要对水产品中的利福平残留进行检测和控制。目前对利福平的检测主要是仪器检测,尤其以高效液相色谱法为主。然而这些仪器分析方法的前处理复杂,需要消耗大量人力物力,不适用于大量样品的快速检测;因此为了维护消费者的利益,建立一种针对利福平的高效、快速的检测方法势在必行。
酶联免疫法(ELISA)是一种极为简便、快速的检测方法,可实现大量样品的快速检测,且检测时对样本的纯度要求不高。因此,建立高效的免疫学检测方法很有必要,而建立此方法的一个重要前提即需筛选出针对利福平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利福平单克隆抗体杂交瘤细胞株NLC,由该细胞株制备的抗体对利福平具有较好特异性和检测灵敏度,可以用来建立利福平的免疫学检测方法。
本发明的技术方案,一株利福平单克隆抗体杂交瘤细胞株NLC,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CGMCCNo.17391。
利福平单克隆抗体,它由所述保藏编号为CGMCC No.17391的利福平单克隆抗体杂交瘤细胞株NLC分泌产生。
所述利福平单克隆抗体的应用,用于食品安全检测中利福平残留的分析检测。
本发明提供的利福平单克隆抗体杂交瘤细胞株NLC的制备基本步骤为:
(1)完全抗原RFP-CDI-KLH的制备:称取11.0mgRFP(利福平与血蓝蛋白(KLH)摩尔比为6000:1)和30.3mg N,N'-羰基二咪唑(CDI),溶解于600 μL无水1,4-二氧六环中,48-52℃左右活化6-8h,加入100μL水终止反应,称为A液;取10 mg KLH,加入等体积pH=8.6、0.01M碳酸盐缓冲溶液CB,称为B液;再逐滴将A液缓慢加入到B液中,室温反应24h;即得偶联物RFP-CDI-KLH混合液;然后用0.01M PBS(pH=7.2)溶液透析三天,除去未反应的小分子半抗原,得到完全抗原RFP-CDI-KLH,并通过紫外吸收扫描方法进行鉴定;
(2)小鼠的免疫:将利福平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利福平的高分泌特异抗体的利福平单克隆抗体杂交瘤细胞株NLC;
(4)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NLC分泌的单克隆抗体,对利福平具有较好的特异性和检测灵敏度(IC50值为9.9ng/mL),可实现对水产品中利福平残留量的检测,具有实际应用价值。
生物材料样品保藏:一株利福平单克隆抗体杂交瘤细胞株NLC,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,简称CGMCC,地址为: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,保藏编号为CGMCC No.17391。
附图说明
图1 NLC单克隆抗体的抑制标准曲线。
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。
本发明通过利福平完全抗原免疫小鼠,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,最终得到了针对利福平具有高分泌特异性抗体的杂交瘤细胞株。
实施例1 杂交瘤细胞株NLC的制备
(1)完全抗原RFP-CDI-KLH的制备:称取11.0mgRFP(利福平与血蓝蛋白(KLH)摩尔比为6000:1)和30.3mg CDI,溶解于600 μL无水1,4-二氧六环中, 50℃左右活化6-8h,加入100μL水终止反应,称为A液;取10 mg KLH,加入等体积pH=8.6、0.01M碳酸盐缓冲溶液CB,称为B液;再逐滴将A液缓慢加入到B液中,室温反应24h;即得偶联物RFP-CDI-KLH混合液;然后用0.01M PBS(pH=7.2)溶液透析三天,除去未反应的小分子半抗原,得到完全抗原RFP-CDI-KLH,并通过紫外吸收扫描方法进行鉴定;
(2)动物免疫:将利福平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 5 min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,8min),用RPMI-1640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 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培养基;第5min 和第6min,在1min内滴加2mL RPMI-1640 培养基;第7min,每10s 滴加1mL 的 RPMI-1640 培养基。然后37℃温浴5min。 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,第二步选用利福平为标准品,用ic-ELISA法对阳性细胞进行抑制效果测定。选择对利福平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,最终获得利福平单克隆抗体细胞株NLC;
(5)单克隆抗体的制备与鉴定: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 利福平杂交瘤细胞,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;
5.1包被:将包被原RFP-CDI-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;
5.2洗涤:将板内溶液倾去,并用洗涤液洗涤3次,每次3min;
5.3封闭:拍干后,加入200μL/孔封闭液,37℃反应2h。洗涤后烘干备用;
5.4加样:将纯化后的单克隆抗体从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;
5.5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;
5.6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OD 450值。
用ic-ELISA测定单克隆抗体利福平的IC50为:9.9ng/mL,说明对利福平有很好的灵敏度,可用于利福平免疫分析检测。
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:60mgTMB 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。
Claims (4)
1.一株利福平单克隆抗体杂交瘤细胞株NLC,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.17391。
2.利福平单克隆抗体,其特征在于:它由权利要求1所述保藏编号为CGMCC No.17391的利福平单克隆抗体杂交瘤细胞株NLC分泌产生。
3.权利要求2所述利福平单克隆抗体的应用,其特征在于:用于食品安全检测中利福平残留的分析检测。
4.利福平单克隆抗体杂交瘤细胞株NLC免疫用完全抗原的制备方法,其特征是步骤如下:称取11.0mg RFP,使得利福平RFP与血蓝蛋白KLH摩尔比为6000:1,和30.3mg CDI,溶解于600μL无水1,4-二氧六环中,48-52℃活化6-8h,加入100μL水终止反应,称为A液;取10mgKLH,加入等体积pH=8.6、0.01M碳酸盐缓冲溶液CB,称为B液;再逐滴将A液缓慢加入到B液中,室温反应24h,即得偶联物RFP-CDI-KLH混合液;然后用pH=7.2、0.01M的PBS溶液透析三天,除去未反应的小分子半抗原,得到完全抗原RFP-CDI-KLH,并通过紫外吸收扫描方法进行鉴定。
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