CN118724904A - 一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用 - Google Patents
一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用 Download PDFInfo
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- CN118724904A CN118724904A CN202410591919.6A CN202410591919A CN118724904A CN 118724904 A CN118724904 A CN 118724904A CN 202410591919 A CN202410591919 A CN 202410591919A CN 118724904 A CN118724904 A CN 118724904A
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Abstract
本发明涉及一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用，属于食品安全免疫检测领域。该分泌苦参碱单克隆抗体的杂交瘤细胞株BONT，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，保藏日期为2024年04月18日，保藏编号为CGMCC No.45902。该细胞株分泌产生的苦参碱单克隆抗体具有较好亲和力、较高的特异性和较高灵敏度(IC50值为0.39ng/mL)，可用于制备苦参碱的免疫检测试剂盒以及胶体金试纸条，建立苦参碱的免疫学检测方法，为食品中苦参碱残留的检测提供有力的检测方法。
Description
技术领域
本发明涉及食品安全免疫检测领域，尤其是指一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用。
背景技术
苦参碱(matrine)是由豆科植物苦参的根、茎、果实经乙醇等有机溶剂提取的一种生物碱，属于喹诺里西啶类的衍生物羽扇豆生物碱类。苦参碱具有多种生物活性，包括免疫调节、抗炎、抗菌、抗肿瘤等。它能够促进巨噬细胞吞噬和增强T淋巴细胞增殖，从而提高机体免疫力以对抗感染和疾病。此外，苦参碱还是一种天然的植物杀虫剂，能够防治蔬菜上菜青虫、菜蚜、瓜蚜、螟虫、瓢虫、果树上的天慕毛虫、刺蛾、蝽象等，粮食作物上粘虫、小麦吸浆虫和蝗虫等多种害虫。苦参碱对害虫具有触杀、胃毒作用，施用后可麻痹害虫的神经中枢系统，导致害虫窒息死亡。苦参碱作用的靶标大，能抑制抗药性的产生，对已经产生抗性的害虫仍有很强的活性。研究表明，长期摄入残留苦参碱的食品会引发腹痛、恶心、呕吐等胃肠道不适症状，出现头晕、胸闷、呼吸困难等情况，甚至可能会引起溶血反应，损害肝肾功能。
目前，针对苦参碱的检测方法主要为仪器检测，常用的有液相色谱法和液相色谱串联质谱法。仪器检测方法具有很高的灵敏度和特异性，存在一些缺点，例如需要彻底的样品净化，高溶剂消耗，昂贵的设备以及熟练的技术人员。因此，需要一种快速，简单的分析苦参碱残留的检测方法。
酶联免疫法(ELISA)是一种高效、灵敏的检测方法，检测时样本前处理过程简单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，在农药残留分析中得到广泛应用。酶联免疫法检测的前提是得到对目标检测物具有高特异性和高灵敏度的单克隆抗体，因此，制备对苦参碱具有高特异性和高灵敏度的单克隆抗体的方法十分关键。发明人尝试通过杂交瘤细胞制备抗苦参碱单克隆抗体，但在制备能分泌抗苦参碱单克隆抗体的杂交瘤细胞株的过程中，如何制备半抗原和完全抗原、如何使小鼠产生强免疫，还需进一步的研究；如何使得制备出的杂交瘤细胞株能够成功分泌出抗苦参碱单克隆抗体，还需进一步的研究；如何使得分泌出的单克隆抗体特异性强、灵敏度高，也还需进一步的研究。
发明内容
为解决上述技术问题，本发明提供了一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用。
本发明通过以下技术方案实现：
本发明第一个目的是提供一种苦参碱半抗原，所述苦参碱半抗原的结构式为：
本发明第二个目的是提供所述的苦参碱半抗原的制备方法，包括以下步骤：
将槐果碱与4-氨基丁酸甲酯盐酸盐在碱性溶液中混合反应，得到苦参碱半抗原。所述碱性溶液为氢氧化钠；所述反应的条件为80℃下搅拌16h。
本发明第二个目的是提供一种苦参碱人工抗原，由所述的苦参碱半抗原与载体蛋白偶联得到。
在本发明的一些实施例中，所述载体蛋白选自钥鸡卵白蛋白、孔血蓝蛋白、牛血清白蛋白、人血清白蛋白、乳铁蛋白、辣根过氧化物酶、甲状腺球蛋白、免疫球蛋白和激素中的至少一种。
本发明第三个目的是提供一株分泌苦参碱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，分类命名为单克隆细胞株，保藏日期为2024年04月18日，保藏编号为CGMCC No.45902。
本发明第四个目的是提供一种苦参碱单克隆抗体，由所述的杂交瘤细胞株分泌产生。
本发明第五个目的是提供一种组合物，所述组合物中含有所述的杂交瘤细胞株和/或所述的苦参碱单克隆抗体。
本发明第六个目的是提供一种苦参碱免疫检测试剂盒，所述苦参碱免疫检测试剂盒包含所述杂交瘤细胞株或所述苦参碱单克隆抗体。
在本发明的一些实施例中，所述苦参碱免疫检测试剂盒还包含偶联有信号物质的苦参碱或所述的苦参碱半抗原。
在本发明的一些实施例中，所述苦参碱免疫检测试剂盒还包含酶标板、苦参碱包被抗原、苦参碱标准液、酶标记二抗和底物反应液。
在本发明的一些实施例中，所述苦参碱包被抗原具有如下所示结构：
本发明第七个目的是提供一种苦参碱检测胶体金试纸条，所述胶体金试纸条含有所述的杂交瘤细胞株、所述的苦参碱单克隆抗体和所述的组合物中的至少一种。
在本发明的一些实施例中，所述苦参碱检测胶体金试纸条，包括样品垫、胶体金结合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上依次设有检测线和质控线，所述胶体金结合垫上包被所述苦参碱单克隆抗体。所述检测线由苦参碱包被抗原印制得到。所述苦参碱包被抗原具有如下所示结构：
本发明第八个目的是提供所述的杂交瘤细胞株、所述的苦参碱单克隆抗体、所述的组合物、所述的苦参碱免疫检测试剂盒或所述的苦参碱检测胶体金试纸条在检测苦参碱中的应用。
本发明分泌苦参碱单克隆抗体的杂交瘤细胞株的制备方法，包含如下步骤：
(1)设计并制备苦参碱半抗原；
(2)制备苦参碱完全抗原，将获得的苦参碱完全抗原配制弗氏佐剂与不完全弗氏佐剂；
(3)将得到的弗氏佐剂通过背部皮下注射，注射进入BALB/c小鼠体内进行多次免疫，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；
(4)将经过上述免疫过程的小鼠进行采血，通过间接ELISA检测小鼠血清免疫效价和免疫抑制能力，筛选出血清中苦参碱抗体含量高的获得免疫的小鼠；
(5)将筛选出的小鼠用不完全弗氏佐剂再进行最后一次加强免疫，然后通过腹腔注射进行冲刺免疫，冲刺免疫采用不含弗氏佐剂的苦参碱完全抗原进行；
(6)将进行冲刺免疫后的BALB/c小鼠的脾细胞和骨髓瘤细胞融合，将融合的细胞通过培养基培养，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亚克隆，最终筛选出获得能分泌苦参碱单克隆抗体的杂交瘤细胞株。
在本发明的上述制备方法中，所述步骤(3)、(5)中的首次免疫与加强免疫之间间隔一个月，加强免疫之间间隔21天，加强免疫与冲刺免疫之间间隔18～21天。
在本发明的上述制备方法中，所述步骤(3)、(5)中的首次免疫剂量为100μg/只，加强免疫剂量为50μg/只，冲刺免疫剂量为25μg/只。
在本发明的上述制备方法中，所述步骤3、5中的免疫过程，包含1次首次免疫、4次加强免疫和1次冲刺免疫；
在本发明的上述制备方法中，所述步骤4中的采血为第3次免疫过程结束后第7天进行采血。
在本发明的上述制备方法中，所述步骤6中的细胞融合是在冲刺免疫结束3天后进行。
在本发明的上述制备方法中，所述步骤6中的细胞融合是通过聚乙二醇(PEG4000)法进行的。
在本发明的上述制备方法中，所述步骤6中的培养基为RPMI-1640培养基。
在本发明的上述制备方法中，所述步骤6中的亚克隆次数为3次。
本发明的有益效果为：
本发明提供了一种苦参碱半抗原、杂交瘤细胞株、抗体及其应用。本发明提供了分泌苦参碱单克隆抗体的杂交瘤细胞株4F12，该杂交瘤细胞株分泌产生的苦参碱单克隆抗体具有较好亲和力、较高的特异性和较高灵敏度(IC50值为0.39ng/mL)，可用于制备苦参碱的免疫检测试剂盒以及胶体金试纸条，建立苦参碱的免疫学检测方法，为食品中苦参碱残留的检测提供有力的检测方法。
生物材料保藏：
一株分泌苦参碱单克隆抗体的杂交瘤细胞株BONT，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，保藏日期为2024年04月18日，保藏编号为CGMCCNo.45902。
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是本发明中苦参碱单克隆抗体的标准抑制曲线。
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。
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B120.005、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Na2CO31.59 g，NaHCO32.93 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 KH2PO4，2.9gNa2HPO4·12H2O，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：含0.1％明胶的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：
苦参碱抑制率检测方法：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0.01μg/mL，0.03μg/mL，0.1μg/mL和0.3μg/mL，并用抗体稀释液将抗体稀释至0.01μg/mL，0.03μg/mL，0.1μg/mL和0.3μg/mL。选择最佳工作点后，将苦参碱标准品均稀释为8个浓度(0ng/mL，0.019ng/mL，0.056ng/mL，0.167ng/mL，0.5ng/mL，1.5ng/mL，4.5ng/mL，13.5ng/mL)，按照ic-ELISA操作步骤，最后用Origin2021作图(结果如图1所示)，获得苦参碱标准抑制曲线，计算IC50。
实施例1：苦参碱半抗原制备。
由于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小分子偶联到蛋白上，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，为了得到能够特异性识别苦参碱的单克隆抗体，因此需要设计并衍生一个好的半抗原，具体衍生步骤如下所示。
称取槐果碱(500.0mg，2.0mmol),4-氨基丁酸甲酯盐酸盐(467.0mg,3.0mmol)溶于10mL水中，随后向混合液中滴加5mLNaOH(162.0mg,4.1mmol)水溶液，80℃下搅拌16h。反应结束后，真空浓缩混合物，通过高效液相色谱纯化得到白色固体化合物(苦参碱半抗原)。
实施例2：苦参碱完全抗原的合成。
称取4.70mg半抗原，4.63mg N-羟基琥珀酰亚胺(NHS)，溶解于300μLN,N-二甲基甲酰胺(DMF)中，室温搅拌反应10min；再称取7.69mg 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，加入到半抗原溶液中，室温搅拌反应6-8h(称为A液)。取10mg BSA，用0.01M磷酸盐缓冲液(PBS)稀释至5mg/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，得到完全抗原(如下式)，并通过紫外吸收扫描方法进行鉴定。
实施例3：苦参碱包被抗原的合成。
包被原合成具体步骤为：将2.33mg半抗原、2.30mgN-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，得到半抗原溶液；将3.81mg 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，加入到半抗原溶液中，室温搅拌进行反应6-8h，得到A液；将10mg鸡卵白蛋白(OVA)用2mL浓度为0.01mol/L的磷酸盐缓冲液(PBS)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(如下式)。
实施例4：分泌苦参碱单克隆抗体的杂交瘤细胞株的制备。
1、动物免疫的获得
将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由慢到快滴加到细胞中；第2min，静置；第3min和第4min，在1min内滴加1mLRPMI-1640培养基；第5min和第6min，在1min内滴加2mLRPMI-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，第二步选用苦参碱为标准品，用ic-ELISA法对阳性细胞进行抑制效果测定。
选择对苦参碱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，最终获得能够分泌识别苦参碱单克隆抗体的细胞株。
实施例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(pH 7.4)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，测得单克隆抗体对苦参碱的的IC50为0.39ng/mL，并验证其对其它类似物的交叉反应率，具体如表1所示，说明对苦参碱有很好的灵敏度，可用于苦参碱免疫分析检测。
表1苦参碱单克隆抗体4F12交叉反应率测定
实施例6：苦参碱单克隆抗体的应用。
将杂交瘤细胞株4F12通过体内诱生腹水制备的单克隆抗体应用于苦参碱的ELISA检测试验，具体步骤如下：
(1)将用碳酸盐缓冲液(CBS)稀释好的浓度为0.1μg/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)分别配置系列梯度的标准溶液，将标准溶液分别加入到已经封闭好的酶标板中，每孔50μL，做三个平行，再每孔加入50μL稀释至0.03μg/mL的单克隆抗体，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50μL2 M的H2SO4终止液，450nm测吸光值；
用Origin2021作图，获得苦参碱标准抑制曲线(结果如图1所示)，苦参碱单克隆抗体对苦参碱的IC50为0.39ng/mL，说明对苦参碱有很好的灵敏度，可用于苦参碱免疫分析检测。
实施例7：苦参碱免疫检测试剂盒。
本实施例提供一种苦参碱免疫检测试剂盒，其包含实施例5制备的苦参碱单克隆抗体4F12、酶标板、苦参碱包被抗原、苦参碱标准液、HRP标记的羊抗鼠IgG二抗和TMB显色液。
免疫检测试剂盒检测苦参碱的原理为：利用间接竞争ELISA法检测待测样本中苦参碱的含量。酶标板微孔内预先包被苦参碱的包被抗原，加入苦参碱标准液或待测样品、苦参碱单克隆抗体、HRP标记的羊抗鼠IgG二抗和TMB显色液，制作苦参碱的标准抑制曲线，根据苦参碱标准抑制曲线和待检测样品的吸光度值，确定待检测样品中的苦参碱含量。采用本领域常用方法进行操作即可实现苦参碱残留的检测。
实施例8：苦参碱检测胶体金试纸条。
本实施例提供一种胶体金试纸条，其包括样品垫、胶体金结合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上依次设有检测线和质控线，所述胶体金结合垫上包被实施例5制备的苦参碱单克隆抗体。所述检测线由苦参碱包被抗原印制得到。所述质控线由羊抗鼠IgG二抗印制得到。胶体金试纸条的组装方式采用本领域常用方式即可。
胶体金试纸条检测苦参碱的原理为：利用间接竞争法原理检测待测样品中是否含有苦参碱。如果待测样品中含有苦参碱，则检测线不显色，质控线显色。如果待测样品中不含有苦参碱，则检测线和质控线均显色。采用本领域常用方法进行操作即可实现苦参碱的检测。
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)
1.一种苦参碱半抗原，其特征在于，所述苦参碱半抗原的结构式为：
2.权利要求1所述的苦参碱半抗原的制备方法，其特征在于，包括以下步骤：
将槐果碱与4-氨基丁酸甲酯盐酸盐在碱性溶液中混合反应，得到苦参碱半抗原。
3.一种苦参碱人工抗原，其特征在于，由权利要求1所述的苦参碱半抗原与载体蛋白偶联得到。
4.根据权利要求3所述的苦参碱人工抗原，其特征在于，所述载体蛋白选自钥鸡卵白蛋白、孔血蓝蛋白、牛血清白蛋白、人血清白蛋白、乳铁蛋白、辣根过氧化物酶、甲状腺球蛋白、免疫球蛋白和激素中的至少一种。
5.一株分泌苦参碱单克隆抗体的杂交瘤细胞株，其特征在于，由权利要求1所述苦参碱半抗原通过合成人工抗原，免疫小鼠得到；所述杂交瘤细胞株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，保藏日期为2024年04月18日，保藏编号为CGMCC No.45902。
6.一种苦参碱单克隆抗体，其特征在于，由权利要求5所述的杂交瘤细胞株分泌产生。
7.一种组合物，其特征在于，所述组合物中含有权利要求5所述的杂交瘤细胞株和/或权利要求6所述的苦参碱单克隆抗体。
8.一种苦参碱免疫检测试剂盒，其特征在于，所述苦参碱免疫检测试剂盒包含权利要求5所述杂交瘤细胞株或权利要求6所述苦参碱单克隆抗体。
9.一种苦参碱检测胶体金试纸条，其特征在于，所述胶体金试纸条含有权利要求5所述的杂交瘤细胞株、权利要求6所述的苦参碱单克隆抗体和权利要求7所述的组合物中的至少一种。
10.权利要求5所述的杂交瘤细胞株、权利要求6所述的苦参碱单克隆抗体、权利要求7所述的组合物、权利要求8所述的苦参碱免疫检测试剂盒或权利要求9所述的苦参碱检测胶体金试纸条在检测苦参碱中的应用。
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