CN113684187A - 一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株及其制备方法和应用 - Google Patents
一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株，命名为单克隆细胞株LKS，于2021年05月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22324。本发明的杂交瘤细胞株可以高效且稳定地分泌氟啶草酮单克隆抗体，应用于氟啶草酮的免疫分析检测时具有较好的灵敏度和特异性，IC50值为0.86ng/mL，对氟啶草酮类似物交叉率小于10％。
Description
技术领域
本发明涉及免疫检测技术领域，尤其涉及一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株及其制备方法和应用。
背景技术
氟啶草酮(Fluridone)属于吡咯烷酮类除草剂，可用于小麦、水稻、玉米、棉花、牧场和非耕地等，用于防除一年生禾本科杂草，阔叶杂草及某些多年杂草，还适用于某些对草甘膦产生抗性的杂草，是一种内吸传导型除草剂，可被植物的根吸收并传导至叶片，其作用机理是抑制番茄红素脱氢酶，导致植物体内的类胡萝卜素生物合成减少，从而引起叶绿素损耗，最终抑制光合作用造成植物死亡。目前，有关氟啶草酮在动植物中的残留有较多的研究。美国已经规定了其在奶、蛋、棉籽等30多种农产品中的残留限量，因此，研究建立氟啶草酮的残留检测方法具有重要意义。
对于氟啶草酮残留的检测，常采用仪器分析方法。但这些方法存在样品前处理复杂和检测时间长等问题，不适用于大量样品的快速检测，因此，有必要建立一种针对氟啶草酮的高效、快速的检测方法。
酶联免疫法(ELISA)是一种极为高效、敏感、快速的检测方法，检测时对样本的前处理简单、纯化步骤少、分析容量大、检测成本低而且操作简便，适用于大量样本的现场快速检测，因此在农药残留分析中得到了广泛应用。而使用酶联免疫法检测氟啶草酮的前提是得到对氟啶草酮具有高特异性和高灵敏度的单克隆抗体，因此，找到一种制备对氟啶草酮具有高特异性和高灵敏度的单克隆抗体的方法十分关键。
发明内容
为解决上述技术问题，本发明提供了一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株及其制备方法，并应用该菌株及其产生的单克隆抗体进行氟啶草酮的检测。
本发明的第一个目的是提供一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株，命名为单克隆细胞株LKS，于2021年05月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22324。
本发明的第二个目的是提供上述杂交瘤细胞株的制备方法，包括以下步骤：
S1：将氟啶草酮完全抗原制备成弗氏佐剂与不完全弗氏佐剂，后将弗氏佐剂注射入免疫动物体内，首次免疫采用完全弗氏佐剂，加强免疫采用不完全弗氏佐剂；
其中，氟啶草酮完全抗原由氟啶草酮半抗原制备得到，氟啶草酮半抗原的结构如式I所示；
S2：对经过上述免疫过程的免疫动物进行采血，检测免疫动物的血清免疫效价和免疫抑制能力，筛选出血清中氟啶草酮抗体含量高的免疫动物；
S3：将筛选出的免疫动物用不完全弗氏佐剂进行加强免疫，然后采用不含弗氏佐剂的氟啶草酮完全抗原进行冲刺免疫；
S4：将冲刺免疫后的免疫动物的脾细胞和骨髓瘤细胞融合并进行培养，检测阳性细胞孔并测定阳性细胞孔的抑制效果，对有最好抑制效果的阳性细胞孔进行亚克隆，得到分泌氟啶草酮单克隆抗体的杂交瘤细胞株；
进一步地，氟啶草酮完全抗原由氟啶草酮半抗原活化后与钥孔血蓝蛋白或牛血清蛋白偶联得到，氟啶草酮完全抗原的结构如式II所示：
进一步地，使用N-羟基琥珀酰亚胺活化氟啶草酮半抗原。
进一步地，整个免疫过程包括1次首次免疫、4-5次加强免疫和1次冲刺免疫。
进一步地，首次免疫与加强免疫之间间隔28-30天，加强免疫之间间隔20-22天，加强免疫与冲刺免疫之间间隔18-21天。
进一步地，首次免疫的剂量为95-105μg/30g体重，加强免疫的剂量为45-55μg/30g体重，冲刺免疫的剂量为20-30μg/30g体重。
进一步地，在步骤S1中，弗氏佐剂通过背部皮下注射入免疫动物体内。
进一步地，在步骤S2中，第3次免疫过程结束后第7天进行采血。
进一步地，在步骤S3中，通过腹腔注射进行冲刺免疫。
进一步地，在步骤S4中，冲刺免疫结束3天后进行细胞融合。
进一步地，在步骤S4中，融合的细胞在RPMI-1640培养基上进行培养。
本发明的第三个目的是提供一种氟啶草酮单克隆抗体，由保藏编号为CGMCCNo.22324的杂交瘤细胞株分泌得到。
进一步地，向免疫动物腹腔注射石蜡油，再腹腔注射保藏编号为CGMCC No.22324的杂交瘤细胞株，注射后收集腹水，将腹水纯化，得到上述氟啶草酮单克隆抗体。
本发明的第四个目的是提供上述杂交瘤细胞株或氟啶草酮单克隆抗体在检测氟啶草酮中的应用，尤其是应用于食品安全检测中氟啶草酮残留的分析检测。
一种包括上述杂交瘤细胞株和/或氟啶草酮单克隆抗体的氟啶草酮检测试剂盒。
进一步地，氟啶草酮检测试剂盒中还包括氟啶草酮包被原，氟啶草酮包被原由氟啶草酮半抗原活化后与鸡卵白蛋白偶联得到。
借由上述方案，本发明至少具有以下优点：
本发明的杂交瘤细胞株通过新合成的氟啶草酮半抗原和氟啶草酮完全抗原配合合理的免疫过程得到，该菌株可以高效且稳定地分泌氟啶草酮单克隆抗体，应用于氟啶草酮的免疫分析检测时具有较好的灵敏度(IC50值为0.86ng/mL)和特异性(对氟啶草酮类似物交叉率小于10％)。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
生物材料保藏
单克隆细胞株LKS，所述单克隆细胞株LKS已于2021年05月13日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.22324，保藏地址为北京市朝阳区北辰西路1号院3号。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为本发明的氟啶草酮单克隆抗体对氟啶草酮的抑制标准曲线。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中涉及的培养基如下：
RPMI-1640培养基(mg/L)：L-精氨酸290、L-门冬酰胺50、L-门冬氨酸20、L-胱氨酸二盐酸盐65.15、L-谷氨酸20、甘氨酸10、L-组氨酸15、L-羟脯氨酸20、L-异亮氨酸50、L-亮氨酸50、L-赖氨酸盐酸盐40、L-甲硫氨酸15、L-苯丙氨酸15、L-脯氨酸20、L-丝氨酸30、L-苏氨酸20、L-色氨酸5、L-酪氨酸23.19、L-缬氨酸20、对氨基苯甲酸1、硝酸钙100、无水硫酸镁48.84、无水磷酸二氢钠676.13、氯化钾400、氯化钠6000、葡萄糖2000、还原谷胱甘肽1、酚红5、L-谷氨酰胺300、生物素0.2、D-泛酸钙0.25、叶酸1、i-肌醇35、烟酰胺1、氯化胆碱3、盐酸吡哆醇1、核黄素0.2、盐酸硫胺素1、维生素B12 0.005、碳酸氢钠2000。
下述实施例中涉及的试剂如下：
碳酸盐缓冲液(CBS)：称取Na2CO3 1.59g，NaHCO3 2.93g，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 Na2HPO4·12H2O，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；
抗体稀释液：PBS加入0.1％明胶。
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。
下述实施例中涉及的检测方法如下：
氟啶草酮抑制率检测方法：通过棋盘试验选择ic-ELISA中最合适的抗原和抗体浓度。用碳酸盐缓冲液(CBS)将抗原稀释至0.03，0.1，0.3和1μg/mL，并用抗体稀释液将抗体稀释至0.03，0.1，0.3和1μg/mL。选择最佳工作点后，将氟啶草酮标准品稀释0，0.04，0.12，0.37，1.11,3.33，10和30ng/mL等浓度，按照ic-ELISA操作步骤，最后用OriginPro 8.5做图(结果如图1所示)，获得氟啶草酮标准抑制曲线，计算IC50。
实施例1氟啶草酮半抗原的合成
将172mg氟啶草酮类似物3-(2-羟基苯基)-1-甲基-5-(3-(三氟甲基)苯基)-4(1H)-吡啶酮溶于10mL丙酮中，加入123mg 6-溴己酸乙酯、83mg无水碳酸钾、8mg碘化钾回流48h，过滤混合物并将丙酮蒸发，将残留物溶解在6.7mL乙醇和3.3mL 1M NaOH中，并回流过夜，然后用2.5M的HCl将溶液酸化至pH<2，并用乙酸乙酯萃取三次。得到的氟啶草酮半抗原的结构如下：
实施例2氟啶草酮完全抗原的合成
称取2.75mg氟啶草酮半抗原(FRD-COOH)，1.5mg 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2.76mg 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FRD-COOH溶液中，室温搅拌反应4-6h(称为A液)。取6mg KLH，用0.01M碳酸盐缓冲液(CBS)稀释至3mg/mL(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，得到氟啶草酮完全抗原(FRD-COOH-KLH)，并通过紫外吸收扫描方法进行鉴定。
实施例3氟啶草酮包被原的合成
将2.17mg氟啶草酮半抗原(FRD-COOH)、1.87mgN-羟基琥珀酰亚胺(NHS)溶解于300μL无水N,N-二甲基甲酰胺(DMF)中，室温搅拌反应10min，得到氟啶草酮半抗原(FRD-COOH)溶液；将3.16mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶解于100μL无水DMF后，加入到FRD-COOH溶液中，室温搅拌进行反应4-6h，得到A液；将6mg鸡卵白蛋白(OVA)用1mL浓度为0.01mmol/L的碳酸盐缓冲液(CBS)稀释，得到B液；将逐滴将A液缓慢加入到B液中进行反应，得到反应液；用PBS溶液透析反应液，除去未反应的小分子半抗原，得到氟啶草酮包被原(FRD-COOH-OVA)。
实施例4分泌氟啶草酮单克隆抗体的杂交瘤细胞株的制备
1、动物免疫的获得：将氟啶草酮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/30g；多次加强免疫用不完全弗氏佐剂且剂量减半即为50μg/30g；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/30g；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20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；
2、细胞融合：在冲刺免疫3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、断尾取血，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，用注射器的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3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，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 1500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(800rpm，8min)，弃上清，重悬入含20％胎牛血清、2％的50×HAT的RPMI-1640筛选培养液中，按照200μL/孔加到96孔细胞板，置于37℃、5％CO2培养箱中培养；
3、细胞筛选与细胞株建立：在细胞融合的第3天对融合细胞进行RPMI-1640筛选培养液半换液，第5天进行用含20％胎牛血清，1％的100×HT的RPMI-1640过渡培养液进行全换液，在第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，第二步选用氟啶草酮为标准品，用ic-ELISA法对阳性细胞进行抑制效果测定；
选择对氟啶草酮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，最终获得氟啶草酮单克隆抗体细胞株。
实施例5氟啶草酮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氟啶草酮杂交瘤细胞，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0.01M的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。
使用间接竞争ELISA，测得氟啶草酮单克隆抗体的IC50值为0.86ng/mL、对氟啶草酮类似物交叉率小于10％，说明对氟啶草酮有很好的灵敏度和特异性，可用于氟啶草酮免疫分析检测。其中，交叉率＝(氟啶草酮的IC50/类似物的IC50)×100％)，氟啶草酮类似物的IC50值见下表。
表1氟啶草酮类似物的IC50值
实施例6氟啶草酮单克隆抗体的应用
将实施例5制备的单克隆抗体应用于氟啶草酮的ELISA添加回收试验，具体步骤如下：
(1)将用碳酸盐缓冲液(CBS)稀释好的浓度为0.3μg/mL的包被原包被96孔酶标板，每孔100μL，37℃包被2h后，用PBST洗液洗板三次，每次每孔200μL，每次3min，拍干；
(2)用含0.2％明胶的CBS进行封闭，每孔200μL，37℃封闭2h，用PBST洗液洗板三次，每次每孔200μL，每次3min，拍干；
(3)用磷酸盐缓冲液(PBS)分别配置0，0.04，0.12，0.37，1.11,3.33，10和30ng/mL的氟啶草酮标准溶液，将标准溶液以及待检测样品提取液，分别加入到已经封闭好的酶标板中，每孔50μL，每个样品重复3个孔，再每孔加入50μL以1:32000稀释的抗氟啶草酮单克隆抗体，37℃反应0.5h后，洗板拍干；
(4)每孔加入100μL用含0.1％明胶的PBS以1:3000稀释的HRP标记的羊抗鼠IgG二抗，37℃反应0.5h后，洗板拍干；
(5)每孔加入100μL的TMB显色液，37℃显色15min后，每孔加入50μL 2M的H2SO4终止液，450nm测吸光值。
氟啶草酮单克隆抗体对氟啶草酮的抑制标准曲线如图1所示，可见用ic-ELISA测定氟啶草酮单克隆抗体的IC50值为0.86ng/mL，说明该抗体对氟啶草酮有较好的灵敏度，可用于氟啶草酮的免疫分析检测。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种分泌氟啶草酮单克隆抗体的杂交瘤细胞株，其特征在于：所述杂交瘤细胞株命名为单克隆细胞株LKS，于2021年05月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22324。
2.权利要求1所述的杂交瘤细胞株的制备方法，其特征在于，包括以下步骤：
S1：将氟啶草酮完全抗原制备成弗氏佐剂与不完全弗氏佐剂，后将所述弗氏佐剂注射入免疫动物体内，首次免疫采用完全弗氏佐剂，加强免疫采用不完全弗氏佐剂；
其中，所述氟啶草酮完全抗原由氟啶草酮半抗原制备得到，所述氟啶草酮半抗原的结构如式I所示；
S2：对经过上述免疫过程的免疫动物进行采血，检测免疫动物的血清免疫效价和免疫抑制能力，筛选出血清中氟啶草酮抗体含量高的免疫动物；
S3：将筛选出的免疫动物用不完全弗氏佐剂进行加强免疫，然后采用不含弗氏佐剂的氟啶草酮完全抗原进行冲刺免疫；
S4：将冲刺免疫后的免疫动物的脾细胞和骨髓瘤细胞融合并进行培养，检测阳性细胞孔并测定阳性细胞孔的抑制效果，对有最好抑制效果的阳性细胞孔进行亚克隆，得到所述分泌氟啶草酮单克隆抗体的杂交瘤细胞株；
3.根据权利要求2所述的制备方法，其特征在于，所述氟啶草酮完全抗原由所述氟啶草酮半抗原活化后与钥孔血蓝蛋白或牛血清蛋白偶联得到；所述氟啶草酮完全抗原的结构如式II所示：
4.根据权利要求2所述的制备方法，其特征在于：整个免疫过程包括1次首次免疫、4-5次加强免疫和1次冲刺免疫。
5.根据权利要求4所述的制备方法，其特征在于：首次免疫与加强免疫之间间隔28-30天，加强免疫之间间隔20-22天，加强免疫与冲刺免疫之间间隔18-21天。
6.根据权利要求4所述的制备方法，其特征在于：首次免疫的剂量为95-105μg/30g体重，加强免疫的剂量为45-55μg/30g体重，冲刺免疫的剂量为20-30μg/30g体重。
7.一种氟啶草酮单克隆抗体，其特征在于，所述氟啶草酮单克隆抗体由权利要求1所述的杂交瘤细胞株分泌得到。
8.权利要求1所述的杂交瘤细胞株或权利要求7所述的氟啶草酮单克隆抗体在检测氟啶草酮中的应用。
9.一种氟啶草酮检测试剂盒，其特征在于：所述氟啶草酮检测试剂盒中包括权利要求1所述的杂交瘤细胞株和/或权利要求7所述的氟啶草酮单克隆抗体。
10.根据权利要求9所述的氟啶草酮检测试剂盒，其特征在于：所述氟啶草酮检测试剂盒中还包括氟啶草酮包被原，所述氟啶草酮包被原由氟啶草酮半抗原活化后与鸡卵白蛋白偶联得到。
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