CN116376847B - 一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株及其应用 - Google Patents
一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株及其应用 Download PDFInfo
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Abstract
本发明公开了一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株及其应用，属于免疫检测领域。本发明分泌噁唑菌酮单克隆抗体的杂交瘤细胞株，已于2023年02月08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.45462。本发明获得的噁唑菌酮单克隆抗体细胞株可以用于免疫分析检测，其对噁唑菌酮有较好的检测灵敏度和特异性，IC50值为1.31ng/mL、对噁唑菌酮类似物交叉率小于1％，可用于制备噁唑菌酮的免疫检测产品，为噁唑菌酮的残留检测提供高效的检测方法及手段。
Description
技术领域
本发明属于免疫检测技术领域，尤其涉及一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株及其应用。
背景技术
噁唑菌酮是一种新型保护性杀菌剂，主要通过阻断泛醌醇酶，抑制病原菌的孢子释放，起到杀菌的作用，用于防治各种霜霉病，黑斑病和晚疫病。目前，我国已引进使用噁唑菌酮，用于防治农作物尤其是马铃薯、葡萄和谷物的病虫害，但是不恰当的使用会导致其在农作物的残留过量进而损害消费者的身体健康。因此，针对噁唑菌酮的残留检测，有必要去建立一个高效快速的检测方法。
对于噁唑菌酮农药残留的检测，常采用高效液相色谱法、气相色谱法、气相或液相色谱质谱联用法。但这些方法存在样品前处理复杂和检测时间长等不足，不适用于大量样品的快速检测，为了维护广大消费者的利益，有必要建立一种针对噁唑菌酮的简单、快速且灵敏的检测方法。
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噁唑菌酮的前提是得到对噁唑菌酮具有高特异性和高灵敏度的单克隆抗体，因此，找到一种制备对噁唑菌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，本发明提供了一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株，该杂交瘤细胞株分泌的噁唑菌酮单克隆抗体对噁唑菌酮具有较好的特异性和检测灵敏度(IC50值为1.31ng/mL)，可以用于建立噁唑菌酮的免疫学检测方法，检测食品中噁唑菌酮的残留。
本发明的第一个目的是提供一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株于2023年02月08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CGMCC No.45462。
进一步地，杂交瘤细胞株通过如下结构所示的噁唑菌酮半抗原制备的完全抗原免疫动物得到：
进一步地，所述完全抗原由噁唑菌酮半抗原与载体蛋白偶联得到。
进一步地，所述载体蛋白包括但不限于牛血清蛋白BSA、卵清蛋白OVA、钥孔血蓝蛋白KLH等。
本发明的第二个目的是提供上述分泌噁唑菌酮单克隆抗体的杂交瘤细胞株在噁唑菌酮检测中的应用。
本发明的第三个目的是提供一种噁唑菌酮单克隆抗体，所述噁唑菌酮单克隆抗体由上述杂交瘤细胞株分泌得到。
本发明的第四个目的是提供上述噁唑菌酮单克隆抗体在噁唑菌酮检测中的应用。
本发明的第五个目的是提供一种噁唑菌酮检测产品，所述噁唑菌酮检测产品中包括上述噁唑菌酮单克隆抗体。当然，检测产品可制备成试剂盒、试剂、试纸条等任何形式。
进一步地，上述噁唑菌酮检测产品可用于食品安全检测中噁唑菌酮的残留分析检测。
进一步地，所述噁唑菌酮检测产品中还包括噁唑菌酮包被原。
进一步地，所述噁唑菌酮包被原由噁唑菌酮半抗原活化后与鸡卵白蛋白偶联得到。
本发明的上述技术方案相比现有技术具有以下优点：
本发明提供了一种对噁唑菌酮具有较高亲和力、较好的特异性以及检测灵敏度的单克隆抗体杂交瘤细胞株，噁唑菌酮单克隆抗体对噁唑菌酮的IC50为1.31ng/mL，对噁唑菌酮类似物交叉率小于1％，可以用来建立噁唑菌酮的酶联免疫检测方法，为间接竞争ELISA试剂盒以及胶体金试纸条的研发推广奠定了基础，有助于实现对噁唑菌酮残留量的检测，为食品中噁唑菌酮残留的免疫检测提供了免疫检测方法和原料，具有实际应用价值。
生物材料保藏
单克隆细胞株SFA，所述单克隆细胞株SFA已于2023年02月08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CGMCCNo.45462，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，其中
图1为本发明噁唑菌酮单克隆抗体对噁唑菌酮的标准抑制曲线。
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 KH2PO4，2.9gNa2HPO4·12H2O，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：
噁唑菌酮抑制率检测方法：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，将噁唑菌酮标准品稀释0，0.03，0.1，0.3，0.9,2.7，8.1和24.3ng/mL等浓度，按照ic-ELISA操作步骤，最后用OriginPro 8.5做图，获得噁唑菌酮标准抑制曲线，计算IC50。
实施例1噁唑菌酮半抗原的合成
由于噁唑菌酮小分子不具有免疫原性，不能刺激小鼠产生免疫应答，进而产生抗体，因此需通过蛋白连接技术将噁唑菌酮偶联到蛋白上，使其获得免疫原性；蛋白偶联技术中常用的活泼基团有氨基，羧基，羟基，巯基等，鉴于噁唑菌酮分子结构中不含有氨基，羧基，羟基，因此需要在其结构上衍生出羧基。
本发明衍生后的噁唑菌酮半抗原结构如下：
衍生过程包括如下：
取噁唑菌酮100mg于三角烧瓶中，用5mL吡啶溶解，再加入112mg丁二酸酐，70℃水浴磁力搅拌反应4h，使用氮气将反应液吹干，沉淀物溶解于2mL三氯甲烷溶液中，等体积超纯水萃取三次。收集有机相，氮气吹干，沉淀物即为衍生产物。
实施例2噁唑菌酮完全抗原的合成
噁唑菌酮完全抗原，简称为FAM-COOH-BSA，分子式如下：
称取6.5mg噁唑菌酮半抗原(FAM-COOH)，4.2mg 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5.8mg 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FAM-COOH溶液中，室温搅拌反应4-6h(称为A液)。取6mgBSA，用0.01M碳酸盐缓冲液(CBS)稀释至2mg/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0.01MPBS溶液透析，除去未反应的小分子半抗原，得到完全抗原FAM-COOH-BSA，并通过紫外吸收扫描方法进行鉴定。
实施例3噁唑菌酮包被原的合成
将5.6mg噁唑菌酮半抗原(FPF-COOH)、3.4mgN-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，得到噁唑菌酮半抗原(FAM-COOH)溶液；将4.6mg 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FAM-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3mL浓度为0.01M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，得到包被原(FAM-COOH-OVA)。
实施例4分泌噁唑菌酮单克隆抗体的杂交瘤细胞株的制备
1、动物免疫的获得：将噁唑菌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/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；
2、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、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，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，第二步选用噁唑菌酮为标准品，用ic-ELISA法对阳性细胞进行抑制效果测定；
选择对噁唑菌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，最终获得噁唑菌酮单克隆抗体细胞株SFA。
实施例5噁唑菌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噁唑菌酮杂交瘤细胞，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，测得噁唑菌酮单克隆抗体的IC50值为1.31ng/mL、对噁唑菌酮类似物交叉率小于1％，说明对噁唑菌酮有很好的灵敏度，可用于噁唑菌酮免疫分析检测(交叉率＝(噁唑菌酮的IC50/类似物的IC50)×100％)。
验证了上述单克隆抗体对噁霉灵、恶霜灵、咪唑菌酮等类似物的IC50及交叉反应率，具体如下表所示。
表1噁唑菌酮单克隆抗体对噁霉灵、恶霜灵、咪唑菌酮的IC50及交叉反应率
| IC50(ng/mL) | 交叉反应率(％) | |
| 噁唑菌酮 | 1.31 | 100 |
| 噁霉灵 | >500 | <1 |
| 恶霜灵 | >500 | <1 |
| 咪唑菌酮 | >500 | <1 |
实施例6噁唑菌酮单克隆抗体的应用
将杂交瘤细胞株通过体内腹水制备的单克隆抗体应用于噁唑菌酮的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的噁唑菌酮标准溶液，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稀释的抗噁唑菌酮单克隆抗体，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；
噁唑菌酮单克隆抗体对噁唑菌酮的抑制标准曲线如图1所示，用ic-ELISA测定噁唑菌酮单克隆抗体的IC50值为1.31ng/mL，说明该抗体对噁唑菌酮有较好的灵敏度，可用于噁唑菌酮的免疫分析检测。
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 (6)
1.一株分泌噁唑菌酮单克隆抗体的杂交瘤细胞株，其特征在于：所述杂交瘤细胞株于2023年02月08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CGMCCNo.45462。
2.一种噁唑菌酮单克隆抗体，其特征在于：所述噁唑菌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。
3.权利要求2所述的噁唑菌酮单克隆抗体在检测噁唑菌酮中的应用。
4.一种噁唑菌酮检测产品，其特征在于：所述噁唑菌酮检测产品中包括权利要求2所述的噁唑菌酮单克隆抗体。
5.根据权利要求4所述的噁唑菌酮检测产品，其特征在于：所述噁唑菌酮检测产品中还包括噁唑菌酮包被原。
6.权利要求4或5所述的噁唑菌酮检测产品在食品安全检测中的应用。
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