CN110117575B - 一株嘧霉胺单克隆抗体杂交瘤细胞株hfg及其应用 - Google Patents
一株嘧霉胺单克隆抗体杂交瘤细胞株hfg及其应用 Download PDFInfo
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Abstract
一株嘧霉胺单克隆抗体杂交瘤细胞株HFG及其应用,属于食品安全免疫检测技术领域。本发明嘧霉胺单克隆抗体杂交瘤细胞株HFG,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.17389。本发明通过将嘧霉胺完全抗原免疫小鼠,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,最终得到了针对嘧霉胺具有高分泌特异性抗体的杂交瘤细胞株HFG。此细胞株分泌的单克隆抗体,对嘧霉胺具有较好的特异性和检测灵敏度,IC50值为2.3ng/mL,可用来建立嘧霉胺的免疫学检测方法,实现对水果、蔬菜中嘧霉胺残留量的检测,为食品中嘧霉胺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
本发明涉及一株嘧霉胺单克隆抗体杂交瘤细胞株HFG及其应用,属于食品安全免疫检测技术领域。
背景技术
在蔬菜及水果的生长期、收获期及收获后的储存期需要喷洒杀菌剂来抑制病原真菌的生长,预防植物的真菌疾病,防止果实腐烂。嘧霉胺属于苯胺嘧啶类杀菌剂,能抑制真菌分泌水解酶,从而抑制真菌对感染组织的降解和消化,是广谱杀菌剂,能有效地控制果实、蔬菜和观赏植物上的灰霉病、叶痂及其他采后病害。比如,在葡萄园中常施用嘧霉胺来抑制霉菌和灰霉病。与此同时,杀菌剂嘧霉胺在果实上的残留问题也亟待解决。
有研究表明自然环境中的杀菌剂会影响两栖类的变态生长,致畸。嘧霉胺在哺乳动物体内的毒性较低,但有研究表明嘧霉胺在小鼠、大鼠、狗及水生生物中具有潜在的致癌毒性。因此,开发针对于食品及环境中的嘧霉胺安全快速高效的检测方法对食品安全有着重要的意义。
目前,嘧霉胺残留物最常用气相色谱(GC),液相色谱(LC),高效液相色谱(HPLC),串联质谱联用(MS-MS)进行监测。近年来也发展了新型的检测方法,如:电化学传感器及金纳米粒子(AuNPs)支持的表面增强拉曼散射(SERS)。然而这些方法需要昂贵的仪器,专业的人员,且耗时。因此,针对嘧霉胺,开发一种廉价、简单、快速安全的检测方法是很有必要的。
近几年来,越来越多的化学残留物和污染物通过生物分析技术被检测出来,竞争性酶联免疫吸附试验(ELISA)是最常用的免疫化学方法之一,它操作简单,高灵敏度,便携性,可承受的成本和高样品通量,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嘧霉胺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嘧霉胺单克隆抗体杂交瘤细胞株,由该细胞株制备的抗体对嘧霉胺具有较好特异性和检测灵敏度,可以用来建立嘧霉胺的免疫学检测方法。
本发明的技术方案,一株嘧霉胺单克隆抗体杂交瘤细胞株HFG,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.17389。
嘧霉胺单克隆抗体,它由所述保藏编号为CGMCC No.17389的嘧霉胺单克隆抗体杂交瘤细胞株HFG分泌产生。
所述嘧霉胺单克隆抗体的应用,用于食品安全检测中嘧霉胺残留的分析检测。
本发明提供的嘧霉胺单克隆抗体杂交瘤细胞株HFG的制备步骤为:
(1)嘧霉胺结构:
(2)嘧霉胺半抗原的制备:
将2-氯-4,6-二甲基嘧啶(713mg,5mmol),4-氨基苯甲酸(685.7mg,5mmol)的二恶烷(20mL)溶液在75℃下搅拌30小时。 冷却至室温后,将其减压干燥,用甲酸(0.5ml)溶解残余物,缓慢加入水使其沉淀,用冷水反复洗涤沉淀物并真空干燥。 最后,进行HPLC,得到粉红色粉末状的嘧霉胺衍生物(78mg),即嘧霉胺半抗原;
(3)完全抗原嘧霉胺-BSA的制备:
称取1mg 嘧霉胺半抗原(嘧霉胺半抗原与牛血清白蛋白(BSA)摩尔比为60:1),2.3mg N-羟基琥珀酰亚胺(NHS),溶解于300μL N,N-二甲基甲酰胺(DMF)中,室温搅拌反应10min;再称取1.45mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),用100μL DMF充分溶解后,加入到嘧霉胺半抗原溶液中,室温搅拌反应6-8 h(称为A液)。取10mg BSA,用2mL0.01M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,室温反应过夜;然后用0.01M PBS溶液透析,除去未反应的小分子半抗原,得到完全抗原嘧霉胺-BSA,并通过紫外吸收扫描方法进行鉴定;
(4)小鼠的免疫:将嘧霉胺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;
(5)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嘧霉胺的高分泌特异抗体的单克隆杂交瘤细胞株HFG;
(6)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HFG分泌的单克隆抗体,对嘧霉胺具有较好的特异性和检测灵敏度(IC50值为2.3ng/mL),可以用来建立嘧霉胺的免疫学检测方法,实现对水果、蔬菜嘧霉胺残留量的检测,为食品中嘧霉胺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:一株嘧霉胺单克隆抗体杂交瘤细胞株HFG,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.17389。
附图说明
图1是HFG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。
本发明通过将嘧霉胺完全抗原免疫小鼠,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,最终得到了针对嘧霉胺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HFG的制备
(1)完全抗原的合成:称取1mg嘧霉胺半抗原,2.3mg N-羟基琥珀酰亚胺(NHS),溶解于300μL N,N-二甲基甲酰胺(DMF)中,室温搅拌反应10min;再称取1.45mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),用100μL DMF充分溶解后,加入到嘧霉胺半抗原溶液中,室温搅拌反应6-8 h(称为A液)。取10mg BSA(嘧霉胺与牛血清白蛋白(BSA)摩尔比为60:1),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,室温反应过夜;然后用0.01M PBS溶液透析,除去未反应的小分子半抗原,得到完全抗原嘧霉胺-BSA,并通过紫外吸收扫描方法进行鉴定;
(2)动物免疫:将嘧霉胺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,第二步选用嘧霉胺为标准品,用ic-ELISA法对阳性细胞进行抑制效果测定。选择对嘧霉胺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,最终获得嘧霉胺单克隆抗体细胞株HFG;
(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 嘧霉胺杂交瘤细胞,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:将包被原嘧霉胺-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;
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;
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OD450值。
用ic-ELISA测定单克隆抗体嘧霉胺的IC50为:2.3ng/mL,说明对嘧霉胺有很好的灵敏度,可用于嘧霉胺免疫分析检测。
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。
Claims (4)
1.一株嘧霉胺单克隆抗体杂交瘤细胞株HFG,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. 17389。
2.嘧霉胺单克隆抗体,其特征在于:它由权利要求1所述保藏编号为CGMCC No.17389的嘧霉胺单克隆抗体杂交瘤细胞株HFG分泌产生。
3.权利要求2所述嘧霉胺单克隆抗体的应用,其特征在于:用于食品安全检测中嘧霉胺残留的分析检测。
4.嘧霉胺单克隆抗体杂交瘤细胞株HFG所采用的抗原的制备方法,其特征是步骤如下:
(1)半抗原的制备:将2-氯-4,6-二甲基嘧啶5mmol,4-氨基苯甲酸5mmol的二恶烷20mL溶液在75℃下搅拌30h;冷却至室温后,将其减压干燥,用甲酸0.5mL溶解残余物,缓慢加入水使其沉淀,用冷水反复洗涤沉淀物并真空干燥;最后,进行HPLC,得到粉红色粉末状的嘧霉胺衍生物,即嘧霉胺半抗原;
(2)完全抗原嘧霉胺-BSA的制备:称取1mg嘧霉胺半抗原,2.3mg N-羟基琥珀酰亚胺NHS,溶解于300μL N,N-二甲基甲酰胺DMF中,室温搅拌反应10min;再称取1.45mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC,用100μL DMF充分溶解后,加入到嘧霉胺半抗原溶液中,室温搅拌反应6-8 h,作为A液;取10mg BSA,使嘧霉胺半抗原与牛血清白蛋白BSA摩尔比为60:1,用2mL 0.01M pH=8.6硼酸盐缓冲溶液BB溶解,作为B液;再逐滴将A液缓慢加入到B液中,室温反应过夜;然后用0.01M PBS溶液透析,除去未反应的小分子半抗原,得到完全抗原嘧霉胺-BSA,并通过紫外吸收扫描方法进行鉴定。
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