CN114181910B - 一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株dc 1f5及其应用 - Google Patents
一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株dc 1f5及其应用 Download PDFInfo
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Abstract
本发明公开了属于食品安全免疫检测领域的一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5及其应用。该分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5，保藏编号为CGMCC NO.45014，分泌产生的脱落酸单克隆抗体具有较好亲和力和较高灵敏度，对脱落酸的50％抑制浓度IC50为0.49ng/mL，可用于制备脱落酸的免疫检测试剂盒以及胶体金试纸条，建立脱落酸酶联免疫检测方法，或建立胶体金免疫层析试纸条快速检测方法，为动物源性食品中脱落酸的检测提供有力的检测方法及手段。
Description
技术领域
本发明属于食品安全免疫检测领域，具体涉及一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5及其应用。
背景技术
脱落酸(C15H20O4)，是一种重要的植物激素，影响植物的重要生理作用，可引起叶芽、枝芽的休眠，干枯叶子的脱落，抑制细胞的分裂分化，调控植物体内细胞中的基因序列的表达等生理作用。脱落酸在植物体内的分布主要集中于干枯的叶片，茎、根、种子等部位。脱落酸与生长素、乙烯、赤霉素、细胞分裂素并称为植物的五大激素。脱落酸可以抑制细胞的分裂与伸长，延缓种子的发育，生长，可用于种子保存。脱落酸可以刺激乙烯的生成，从而可以催熟果实。脱落酸在作物生产和植物栽培种有着广阔的发展前景，给人类带来巨大的经济和社会效益，引起了人们极大的关注。
脱落酸的常规检测方法包括高效液相色谱串联质谱，超高效液相色谱，超高效液相色谱-串联质谱和气相色谱串联质谱等。这些方法在不同程度上存在一些缺陷，如耗时，仪器昂贵和大量的样品预处理程序等。因此，这些方法不适合现场进行脱落酸的高通量检测分析。因此需要分析系统，这意味着这些方法在现场分析中的应用受到限制。
免疫分析方法具有低成本、高通量、高灵敏、对技术人员相对要求低等特点，因此适用于大量样品的快速筛查。而使用免疫分析方法对脱落酸进行检测的前提是得到对脱落酸具有高特异性和高灵敏度的单克隆抗体，因此，找到一种制备对脱落酸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
为了解决现有技术中的不足，本发明的目的在于提供一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5及其应用。
本发明第一方面提供一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5，其保藏编号为CGMCC NO.45014。
本发明第二方面提供一种抗脱落酸单克隆抗体，由杂交瘤细胞株DC 1F5分泌产生。
本发明第三方面提供所述杂交瘤细胞株DC 1F5或所述抗脱落酸单克隆抗体在检测脱落酸中的应用、在制备脱落酸检测试剂盒中的应用或在制备脱落酸检测胶体金试纸条中的应用。
本发明第四方面提供一种脱落酸检测试剂盒，包含所述杂交瘤细胞株DC 1F5或所述抗脱落酸单克隆抗体。
进一步地，所述脱落酸检测试剂盒还包括脱落酸包被原。
优选地，所述脱落酸包被原由脱落酸与鸡卵白蛋白偶联得到。
本发明第五方面提供一种脱落酸检测胶体金试纸条，包含所述杂交瘤细胞株DC1F5或所述抗脱落酸单克隆抗体。
进一步地，所述脱落酸检测胶体金试纸条还包括脱落酸与载体蛋白偶联得到的包被原。
进一步地，所述载体蛋白为鸡卵白蛋白。
本发明的上述技术方案中，分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5的制备方法，包含如下步骤：
(1)免疫原的制备与鉴定：以脱落酸为原料，通过活化酯法与蛋白载体的氨基相连，反应结束后，通过透析分离完全抗原和未偶联的小分子半抗原，完全抗原通过紫外吸收扫描方法鉴定；
(2)小鼠的免疫：选取BALB/c小鼠进行免疫，将免疫原与弗氏佐剂乳化完全后，通过皮下多点注射免疫小鼠，首次免疫采用弗氏完全佐剂，加强免疫使用弗氏不完全佐剂，冲刺免疫不用佐剂，与生理盐水混合均匀后直接进行腹腔注射，冲刺免疫时免疫剂量为前一次免疫剂量的一半；各次免疫间隔为三周；免疫过程包含1次首次免疫、4次加强免疫和1次冲刺免疫；第三次免疫后，间隔一周采血检测血清效价和抑制；选择抑制最好的小鼠，在五免后18天冲刺免疫，准备融合。
(3)细胞融合与细胞株建立：通过聚乙二醇(PEG 2000)法，使小鼠脾细胞和小鼠骨髓瘤细胞融合，通过HAT培养基培养，利用间接ELISA检测阳性细胞孔，并进一步利用间接竞争ELISA法测定阳性细胞孔的抑制效果，通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆，最终筛选获得杂交瘤细胞株DC 1F5。
本发明的有益效果为：
本发明通过将脱落酸完全抗原免疫小鼠，通过细胞融合，HAT选择性培养基培养，通过间接ELISA和间接竞争ELISA筛选细胞上清，最终获得分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5，该杂交瘤细胞株分泌产生的脱落酸单克隆抗体具有较好亲和力、较高的特异性和较高灵敏度，对脱落酸的50％抑制浓度IC50为0.49ng/mL，可用于制备脱落酸的免疫检测试剂盒以及胶体金试纸条，建立脱落酸酶联免疫检测方法，或建立胶体金免疫层析试纸条快速检测方法，为动物源性食品中脱落酸的检测提供有力的检测方法及手段。
附图说明
图1为脱落酸单克隆抗体对脱落酸的标准抑制曲线。
保藏说明
分泌抗脱落酸单克隆抗体杂交瘤细胞株DC 1F5，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，分类命名单克隆细胞株，保藏编号CGMCC No.45014，保藏日期2021年12月16日。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
本发明实施例中涉及的溶液，其配置如下：
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，0.24g KH2PO4，3.62g 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％Tween20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1：5混合即为TMB显色液，现用现混。
本发明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。
实施例1：脱落酸完全抗原的合成
取4.5mg的脱落酸，再加入5.0mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和3.7mg NHS(N-羟基琥珀酰亚胺)，使用DMF(N,N-二甲基甲酰胺)溶解，室温搅拌，活化6h；另取15mg BSA(牛血清蛋白)溶解于3mL、0.05M、pH9.6的CBS(碳酸盐缓冲溶液)溶液中；将上述活化液逐滴加入BSA溶液中，室温搅拌反应过夜后，取出免疫原PBS透析3天，得到脱落酸完全抗原，-20℃分装保存。
脱落酸具有如下式(a)所示化学结构式，脱落酸完全抗原具有如下式(b)所示化学结构式：
实施例2：抗脱落酸单克隆抗体杂交瘤细胞株的制备
1、动物免疫：选择健康的6～8周龄的BALB/c小鼠进行免疫。取脱落酸完全抗原(1mg/mL)与等量弗氏佐剂乳化均匀后，通过皮下多点注射免疫BALB/c小鼠。首次免疫采用弗氏完全佐剂，首次免疫剂量为每只100μL；加强免疫使用弗氏不完全佐剂，加强免疫剂量为每只50μL；冲刺免疫不用佐剂，与生理盐水混合均匀后直接进行腹腔注射，冲刺免疫时免疫剂量为前一次免疫剂量的一半；各次免疫间隔为三周；免疫过程包含1次首次免疫、4次加强免疫和1次冲刺免疫。第三次免疫后，间隔一周采血检测血清效价和抑制；选择抑制最好的小鼠，在五免后18天冲刺免疫，准备融合。
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2000)方法进行细胞融合，具体步骤如下：
(1)无菌取小鼠脾脏，研磨并通过200目细胞筛网得到脾细胞悬液，并进行细胞计数；
(2)收集SP2/0细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
(3)将脾细胞和SP2/0细胞按照2～10:1的计数比例混合，离心后用PEG融合，时间1min，之后按照从慢到快，加入RPMI-1640基础培养液，离心后悬浮于含20％胎牛血清、2％的50×HAT的RPMI-1640筛选培养液中，加到96孔细胞培养板，置于37℃、5％CO2的培养箱中培养。
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：第一步先用间接ELISA筛选出阳性细胞孔，第二步选用脱落酸为标准品，用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对脱落酸有较好抑制的细胞孔，采用有限稀释法进行亚克隆，用同样的方法进行检测。重复三次，获得细胞株DC 1F5。
实施例3：抗脱落酸单克隆抗体的制备与鉴定
取8～10周龄BALB/c小鼠，每只小鼠腹腔注射石蜡油1mL；7天后每只小鼠腹腔注射1×106杂交瘤细胞，从第7天开始收集腹水，将腹水通过辛酸-饱和硫酸铵法纯化，获得的单抗置于-20℃保存。
使用小鼠单抗亚型鉴定试剂盒对腹水纯化获得的单克隆抗体进行免疫球蛋白亚型鉴定，其亚型为IgG2b型，具体如表1所示。
表1.脱落酸单克隆抗体的亚型鉴定
| 抗体亚型亚类 | OD值 |
| IgA | 0.133 |
| IgG1 | 0.113 |
| IgG2a | 0.233 |
| IgG2b | 2.022 |
| IgG3 | 0.106 |
| IgM | 0.073 |
使用间接竞争ELISA法，测定单克隆抗体对的IC50为0.49ng/mL，并验证了其对赤霉素、萘乙酸、吲哚乙酸的IC50及交叉反应率，交叉反应率＝(脱落酸的IC50/类似物的IC50)×100％。具体如表2所示。
表2.脱落酸单克隆抗体对脱落酸、赤霉素、萘乙酸、吲哚乙酸的IC50及交叉反应率
| IC50(ng/mL) | 交叉反应率 | |
| 脱落酸 | 0.49 | 100％ |
| 赤霉素 | ＞500 | ＜5％ |
| 萘乙酸 | ＞500 | ＜5％ |
| 吲哚乙酸 | ＞500 | ＜5％ |
实施例4：抗脱落酸单克隆抗体的应用
将杂交瘤细胞株DC 1F5通过体内腹水制备的单克隆抗体应用于脱落酸ELISA添加回收试验，具体步骤如下：
(1)用碳酸盐缓冲液(CBS)稀释好的0.1μg/mL的脱落酸包被原包被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。脱落酸包被原由脱落酸与鸡卵白蛋白偶联得到。
(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0，0.02，0.05，0.1，0.2，0.5，1，2μg/L的脱落酸标准溶液，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 1：16000稀释的抗脱落酸单克隆抗体，37℃反应半小时后，洗板拍干。
(4)每孔加入100μL用含0.1％明胶的PBS 1：3000稀释的HRP标记的羊抗鼠IgG二抗，37℃反应半小时后，洗板拍干。
(5)每孔加入100μL TMB显色液，37℃显色15min后，每孔加入50μL 2M H2SO4终止液，450nm测吸光值。
脱落酸单克隆抗体对脱落酸的标准抑制曲线如图1所示，对脱落酸的50％抑制浓度IC50为0.49ng/mL，说明该抗体对脱落酸有较好的灵敏度。
(6)添加回收及样品前处理：取新鲜或回温(冷藏保存)的牛奶5g，添加三个不同剂量的脱落酸标准品，分别为5ng、10ng、20ng。将其置于50mL离心管中，缓慢滴入50％氢氧化钾溶液1mL，在旋涡混合器上充分振荡，缓慢滴入乙酸乙酯20mL，在旋涡混合器上振荡10min，然后放入离心器中以3000r/min离心5min。移取4mL上清液于另一支离心管中，氮气吹干，加入1mL含有10％甲醇的PBS复溶，取50μL用于检测。采用间接竞争ELISA进行添加回收试验，其回收率分别为91.2％、101.5％、95.6％。
显然，上述实施例仅仅是为清楚地说明所作的举例，而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的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸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 (9)
1.一株分泌抗脱落酸单克隆抗体的杂交瘤细胞株DC 1F5，其特征在于，所述杂交瘤细胞株DC 1F5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，保藏编号为CGMCC NO.45014，保藏日期2021年12月16日。
2.一种抗脱落酸单克隆抗体，其特征在于，由权利要求1所述的杂交瘤细胞株DC 1F5分泌产生。
3.权利要求1所述杂交瘤细胞株DC 1F5或权利要求2所述抗脱落酸单克隆抗体在检测脱落酸中的应用、在制备脱落酸检测试剂盒中的应用或在制备脱落酸检测胶体金试纸条中的应用。
4.一种脱落酸检测试剂盒，其特征在于，包含权利要求1所述杂交瘤细胞株DC 1F5或权利要求2所述抗脱落酸单克隆抗体。
5.根据权利要求4所述的脱落酸检测试剂盒，其特征在于，所述脱落酸检测试剂盒还包括脱落酸包被原。
6.根据权利要求5所述的脱落酸检测试剂盒，其特征在于，所述脱落酸包被原由脱落酸与鸡卵白蛋白偶联得到。
7.一种脱落酸检测胶体金试纸条，其特征在于，包含权利要求2所述抗脱落酸单克隆抗体。
8.根据权利要求7所述的脱落酸检测胶体金试纸条，其特征在于，所述脱落酸检测胶体金试纸条还包括脱落酸与载体蛋白偶联得到的包被原。
9.根据权利要求8所述的脱落酸检测胶体金试纸条，其特征在于，所述载体蛋白为鸡卵白蛋白。
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