CN108998424A - 一株马兜铃酸a单克隆抗体杂交瘤细胞株及其应用 - Google Patents
一株马兜铃酸a单克隆抗体杂交瘤细胞株及其应用 Download PDFInfo
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Abstract
一株马兜铃酸A单克隆抗体杂交瘤细胞株及其应用,属于食品安全免疫检测领域。本发明包括一株马兜铃酸A单克隆抗体杂交瘤细胞株SS0709,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.14688,保藏日期为2017年9月5日。马兜铃酸A单克隆抗体,其由所述保藏编号为CGMCC No.14688,马兜铃酸A单克隆抗体杂交瘤细胞株SS0709分泌产生。所述马兜铃酸A单克隆抗体的应用,用于中药材中马兜铃酸A分析检测。本发明细胞株分泌的单克隆抗体,对马兜铃酸A具有较好的特异性和检测灵敏度(IC50值为4ng/mL),可实现对中药材中马兜铃酸A检测,为中药材中马兜铃酸A的免疫检测提供了原料,具有实际应用价值。
Description
技术领域
本发明涉及一株马兜铃酸A单克隆抗体杂交瘤细胞株及其应用,属于食品安全免疫检测领域。
背景技术
马兜铃酸(Aristolochic acids,简称AAs),被称为马兜铃总酸、增噬力酸或木通甲素,是一类硝基菲羧酸,马兜铃酸类化合物是马兜铃科马兜铃属植物特有的一类硝基菲类化合物,包括马兜铃酸A、B、C和D等。近年来, 马兜铃酸的肾毒性已引起广泛关注,各国纷纷对含马兜铃酸的中药采取管制及限制措施。2017年10月27日,世界卫生组织国际癌症研究机构公布的致癌物清单初步整理参考,马兜铃酸、含马兜铃酸的植物在一类致癌物清单中。研究表明, 马兜铃酸类化合物的毒性主要由马兜铃酸A或其代谢物马兜铃内酰胺所致。因此, 在检测马兜铃酸时,常选择马兜铃酸A为指标性成分。
马兜铃科药用植物在我国广泛使用,由于马兜铃科植物许多名称混淆,为确保临床用药的安全有效,对于疑含有马兜铃酸类物质的药用植物及制剂必须进行严格检测,制订限量用药标准。目前,检测马兜铃酸A的方法主要有薄层色谱法、紫外分光光度法、高效液相色谱法及酶联免疫分析法等。薄层色谱法和紫外分光光度法灵敏度较低;高效液相色谱法(HPLC)灵敏度较高、测定准确,是目前测定马兜铃酸A最常用的方法,但仪器设备昂贵,对实验人员专业技能要求高。酶联免疫分析法具有灵敏、准确、前处理简单、适合大通量筛选等优点,在医学诊断、食品安全检测、环境污染物监测等领域已得到广泛应用。本发明制备了马兜铃酸A的单克隆抗体, 建立了马兜铃酸A的酶联免疫分析技术。
发明内容
本发明的目的是提供一株马兜铃酸A单克隆抗体杂交瘤细胞株SS0709及其应用,由该细胞株制备的抗体对马兜铃酸A具有较好特异性和检测灵敏度,可以用来建立马兜铃酸A的免疫学检测方法。
本发明的技术方案,一株马兜铃酸A单克隆抗体杂交瘤细胞株SS0709,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.14688,保藏日期为2017年9月5日。
马兜铃酸A单克隆抗体,其由所述保藏编号为CGMCC No.14688,马兜铃酸A单克隆抗体杂交瘤细胞株SS0709分泌产生。
所述马兜铃酸A单克隆抗体的应用,用于中药材中马兜铃酸A分析检测。
杂交瘤细胞株的制备方法如下:
(1)半抗原的制备:
原药 Mol. Wt: 341.272
由于AAs化学结构式中含有活泼基团(羧基),因此本发明将原药直接作为半抗原。
本发明采用碳二亚胺(EDC)将AAs半抗原的羧基与载体蛋白上的氨基偶联以制备完全抗原。
(2)完全抗原AAs-EDC-BSA的制备:称取马兜铃酸A 1.4 mg,加入500μL DMF溶解,充分溶解后向其中加入EDC 2.5mg和NHS 1.6mg,并于30℃搅拌6h,作为A液。称取10mg的BSA,加入5mL CB溶解,作为B液。将A液滴加入B液中,偶联12h,混合液用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AAs-EDC-BSA,并通过紫外吸收扫描方法进行鉴定;
(3)小鼠免疫:将AAs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)。首次免疫采用马兜铃酸A的完全抗原与完全弗氏佐剂混合,剂量为100μg/只;多次加强免疫采用马兜铃酸A的完全抗原与不完全弗氏佐剂混合,剂量为50μg/只;最后一次用马兜铃酸A完全抗原与生理盐水混合冲刺免疫。采用腹腔注射,剂量为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AAs的高分泌特异抗体的单克隆杂交瘤细胞株SS0709;
(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SS0709分泌的单克隆抗体,对AAs具有较好的特异性和检测灵敏度(IC50值为4ng/mL),可实现对中药材中AAs的检测,为中药材中AAs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:一株马兜铃酸A单克隆抗体杂交瘤细胞株SS0709,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,保藏日期2017年9月5日,保藏编号为CGMCCNo.14688,分类命名为单克隆细胞株。
附图说明
图1是马兜铃酸A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。
本发明通过小鼠免疫,细胞融合,ic-ELISA筛选,杂交瘤细胞有限稀释亚克隆,最终得到了针对马兜铃酸A的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SS0709的制备
(1)半抗原:马兜铃酸A化学结构式中含活泼基团(-COOH),即可直接作为半抗原。
(2)完全抗原的制备:称取马兜铃酸A 1.4 mg,加入500μL DMF溶解,充分溶解后向其加入EDC 2.5mg和NHS 1.6mg,并于30℃搅拌6h,作为A液。称取10mg的BSA,加入5mL CB溶解,作为B液。将A液滴加入B液中,偶联12h,混合液用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AAs-EDC-BSA,并通过紫外吸收扫描方法进行鉴定;
(3)小鼠免疫:将AAs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;冲刺免疫直接用生理盐水稀释后腹腔注射,减半为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:在冲刺免疫三天后,按照常规PEG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,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。37℃温浴5min后离心(800rpm,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。
(5)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,第二步选用马兜铃酸A为标准品,用ic-ELISA法对阳性细胞进行抑制效果测定。选择对马兜铃酸A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,最终获得马兜铃酸A单克隆抗体细胞株SS0709。
(6)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 硫酸多粘菌素B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。
6.1包被:将包被原AAs-EDC-OVA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OD 450值。
用ic-ELISA测定单克隆抗体马兜铃酸A的IC50为:4ng/mL,说明对马兜铃酸A有很好的灵敏度,可用于马兜铃酸A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液体积比按5:1混合即为TMB显色液,现用现混。
Claims (4)
1.一株马兜铃酸A单克隆抗体杂交瘤细胞株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.14688,保藏日期为2017年9月5日。
2.马兜铃酸A单克隆抗体,其特征在于:其由权利要求1所述保藏编号为CGMCCNo.14688,马兜铃酸A单克隆抗体杂交瘤细胞株SS0709分泌产生。
3.权利要求2所述马兜铃酸A单克隆抗体的应用,其特征在于:用于中药材中马兜铃酸A分析检测。
4.权利要求1所述杂交瘤细胞株所用完全抗原的制备方法,其特征在于步骤如下:完全抗原AAs-EDC-BSA的制备:称取马兜铃酸A 1.4 mg,加入500μL DMF溶解,充分溶解后向其中加入EDC 2.5mg和NHS 1.6mg,并于30℃搅拌6h,作为A液;称取10mg的BSA,加入5mL CB溶解,作为B液;将A液滴加入B液中,偶联12h,混合液用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AAs-EDC-BSA,并通过紫外吸收扫描方法进行鉴定。
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CN112881701A (zh) * | 2021-01-19 | 2021-06-01 | 南昌大学 | 一种用于检测中药中马兜铃酸a和b的试纸条及其制备方法 |
CN114262367A (zh) * | 2021-12-23 | 2022-04-01 | 天津科技大学 | 一种马兜铃酸特异结合多肽及应用 |
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