CN114752568A - 呋塞米单克隆抗体、杂交瘤细胞株及应用 - Google Patents
呋塞米单克隆抗体、杂交瘤细胞株及应用 Download PDFInfo
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- CN114752568A CN114752568A CN202210083958.6A CN202210083958A CN114752568A CN 114752568 A CN114752568 A CN 114752568A CN 202210083958 A CN202210083958 A CN 202210083958A CN 114752568 A CN114752568 A CN 114752568A
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- furosemide
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- hybridoma cell
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Abstract
本发明提供呋塞米单克隆抗体、杂交瘤细胞株及应用，属于食品安全免疫检测领域。杂交瘤细胞株的保藏编号为：CGMCC No.45023。本发明先合成呋塞米完全抗原，使用弗氏佐剂混合乳化，注射免疫BALB/c小鼠。筛选出高效价，低IC50的小鼠脾细胞，通过PEG法与小鼠骨髓瘤细胞融合，选择性培养基，筛选出两种细胞融合后的杂交瘤细胞；再经过间接竞争酶联免疫法筛选细胞并多次亚克隆，得到单克隆抗体杂交瘤细胞株。此细胞株分泌的单克隆抗体，对呋塞米具有较好的检测灵敏度，对呋塞米的50％抑制浓度IC50为0.49ng/mL，用于免疫检测试剂盒以及胶体金试纸条，为保健食品中添加呋塞米的检测提供有力的检测手段。
Description
技术领域
本发明属于食品安全免疫检测领域，具体涉及呋塞米单克隆抗体、杂交瘤细 胞株及应用。
背景技术
呋塞米又名呋喃苯胺酸，利尿灵，利尿磺胺，化学名称是2-[(2-呋喃甲基)氨 基]-5-(氨磺酰基)-4-氯苯甲酸。具有利尿、扩张血管的药理作用。临床上用于治 疗心脏性水肿、肾性水肿、肝硬化腹水、机能障碍或血管障碍所引起的周围性水 肿，并可促使上部尿道结石的排出，该药利尿作用强而短，为强效利尿药，特别 是对其他利尿药无效的病例；可用于治急性肺水肿，脑水肿，急性肾功能衰竭和 高血压等疾病；配合补液该品可促进毒物排泄。随着人们的审美观念的改变和爱 美之心，减肥药的需求量和使用量急剧增大，也使含有呋塞米的减肥药的使用趋 势上升，引发了很多的健康和社会问题，因此，迫切需要设计一种准确度高、可 操作性强的检测分析保健食品中呋塞米含量的方法。
呋塞米的常规检测方法包括紫外分光光度法，高效液相色谱法，电化学方法 超快速液相色谱-串联质谱法法等。这些方法在不同程度上存在一些缺陷：耗时， 昂贵的仪器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高通量 分析地呋塞米的检测。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。而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。
发明内容
为解决相关技术中存在的问题，本发明提供一种分泌呋塞米单克隆抗体的杂 交瘤细胞株及其应用，由该细胞株制备的单克隆抗体对呋塞米具有较好的亲和力 和检测灵敏度，可以用来建立呋塞米酶联免疫检测方法，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。
一方面，本发明提供一株分泌呋塞米单克隆抗体的杂交瘤细胞株，已于2021年12月16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，保藏名称为 单克隆细胞株DIC，保藏编号CGMCC No.45023，保藏地址为北京市朝阳区北辰 西路1号院3号。
本发明还提供一种呋塞米单克隆抗体，其是由保藏编号为CGMCC No.45023 的分泌呋塞米单克隆抗体的杂交瘤细胞株分泌产生。
又一方面，提供一种呋塞米单克隆抗体的制备方法，包括：取BALB/c小鼠， 腹腔注射石蜡油，再腹腔注射保藏编号为CGMCC No.45023的杂交瘤细胞株， 注射后收集腹水，将腹水纯化，将获得的呋塞米单克隆抗体低温保存。
又一方面，提供一种所述呋塞米单克隆抗体的应用，用于食品中呋塞米残留 的检测。
又一方面，提供一种分泌呋塞米单克隆抗体的杂交瘤细胞株的应用，用于制 备呋塞米单克隆抗体
再一方面，提供一种分泌呋塞米单克隆抗体的杂交瘤细胞株的应用，应用于 检测呋塞米。
进一步地，应用于食品中呋塞米残留的检测。
另一方面，提供一种试剂盒，含有所述呋塞米单克隆抗体。
又一方面，所述试剂盒应用于食品中呋塞米残留的检测。
又一方面，提供一种胶体金试纸条，含有所述的呋塞米单克隆抗体。
又一方面，所述胶体金试纸条应用于食品中呋塞米残留的检测。
本发明提供的细胞株DIC的制备基本步骤为：
(1)免疫原的制备与鉴定：以呋塞米为原料，通过活化酯法蛋白载体的氨 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6～8周龄的BALB/c小鼠进行免疫。将免疫原与福 氏佐剂乳化完全后，通过皮下多点注射免疫小鼠，首次免疫采用福氏完全佐剂， 加强免疫使用福氏不完全佐剂，冲刺免疫时免疫剂量为前一次免疫剂量的一半， 与生理盐水混合均匀后直接进行腹腔注射；各次免疫间隔为三周。第三次免疫后， 间隔一周采血检测血清效价和抑制；
(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 DIC；
(4)杂交瘤细胞株性质的鉴定：采用小鼠单抗Ig类/亚类鉴定用酶标二抗套 装测定；IC50值、交叉反应率和亲和力的测定通过ELISA法。
与现有技术相比，本发明至少具有以下有益效果：
本发明本发明提供的杂交瘤细胞株分泌的单克隆抗体，对呋塞米有较好的检 测灵敏度和亲和力；可实现对食品中呋塞米残留量的检测，为建立快速、简便、 价廉、灵敏、特异的呋塞米检测方法提供了新手段；本发明提供的合成呋塞米免 疫原的方法，合成步骤更加简化，有效，为今后人们的研究提供了合成免疫原的 思路与方法。
生物材料保藏
一株分泌呋塞米单克隆抗体的杂交瘤细胞株，分类命名为：单克隆细胞株 DIC，保藏单位为：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：为北京市朝阳区北辰西路1号院3号，保藏编号为：CGMCC No.45023，保 藏日期为：2021年12月16日。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面 的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1(a)是呋塞米半抗原的化学结构式，图1(b)是呋塞米完全抗原的化 学结构式。
图2是呋塞米单克隆抗体的标准抑制曲线。
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。
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 Na2HPO4·12 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：
呋塞米的抑制率检测方法：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0.01，0.03，0.1和0.3μg/mL， 并用抗体稀释液将抗体稀释至0.01，0.03，0.1和0.3μg/mL。选择最佳工作点后， 将呋塞米标准品稀释为7个浓度(0.125，0.25，0.5，1，2，4，8ng/mL)，按 照ic-ELISA操作步骤，最后用OriginPro 8.5做图(结果如图2所示)，获得呋 塞米的标准抑制曲线，计算IC50。
实施例1呋塞米人工抗原的制备：
呋塞米完全抗原的合成：取4.5mg的呋塞米，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，得到呋塞米半抗原； 另取15mg BSA(牛血清蛋白)溶解于3mL、0.05M、pH9.6的CB(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8h后，然后 用0.01MPBS透析3天，除去未反应的小分子半抗原，得到呋塞米完全抗原，-20℃ 分装保存。
实施例2：分泌呋塞米单克隆抗体杂交瘤细胞株的制备
2.1动物免疫的获得
选择健康的6～8周龄的Balb/C小鼠进行免疫。将麦角乙二胺免疫原与等量 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100ug/只；多次加强免疫用不完全弗 氏佐剂且剂量减半即为50ug/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25ug/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。
2.2细胞融合与筛选
在冲击免疫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、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，并离心(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％C02培养箱中培养。
2.3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，第二步选用呋塞米为标准品，用ic-ELISA法对阳性细胞进行抑 制效果测定。选择对呋塞米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，最终获得分 泌呋塞米单克隆抗体细胞株DIC。
实施例3：呋塞米单克隆抗体的制备
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。
实施例4：呋塞米单克隆抗体的应用
将杂交瘤细胞株DIC通过体内腹水制备的单克隆抗体应用于呋塞米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的呋塞米包被作为包被原 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0，0.02，0.05，0.1，0.2，0.5，1， 2μg/L的呋塞米标准溶液。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 稀释的呋塞米单克隆抗体，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；
(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 三个不同剂量的呋塞米标准品，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％。
实施例5灵敏度及特异性
表1利用ic-ELISA测定了所制备的呋塞米的单克隆抗体的效价和抑制，数 据表明，当抗体抑制率≥50％时，效价在2.0以上，表现了该抗体具有较好的亲 和力。
表1呋塞米的单克隆抗体的效价和抑制
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2所示。
表2呋塞米单克隆抗体的亚型鉴定
使用间接竞争ELISA法，测定单克隆抗体对呋塞米的IC50为0.49ng/mL， 并验证了其对地克珠利等的IC50及交叉反应率，具体如表3所示。
表3呋塞米单克隆抗体对呋塞米、螺内酯、坎利酮、安非他酮的IC50及交叉反应率
| IC<sub>50</sub>(ng/mL) | 交叉反应率 | |
| 呋塞米 | 0.49 | 100％ |
| 螺内酯 | ＞500 | ＜5％ |
| 坎利酮 | ＞500 | ＜5％ |
| 安非他酮 | ＞500 | ＜5％ |
实施例6：呋塞米免疫检测试剂盒
本实施例提供一种呋塞米免疫检测试剂盒，其包含实施例3制备的呋塞米单 克隆抗体、酶标板、呋塞米包被抗原、呋塞米标准液、HRP标记的羊抗鼠IgG 二抗和TMB显色液。
呋塞米免疫检测试剂盒检测呋塞米的原理为：采用间接竞争ELISA法检测 待测样本中呋塞米的含量。酶标板微孔内预先包被呋塞米包被抗原，加入呋塞米 标准液或待测样品、呋塞米单克隆抗体、HRP标记的羊抗鼠IgG二抗和TMB显 色液，制作呋塞米标准抑制曲线，根据呋塞米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呋塞米含量。采用本领域常用方法进行操作即可实现 呋塞米的检测。
实施例7：呋塞米检测胶体金试纸条
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制备的呋塞米单克隆抗体。所述检测线由呋塞米包被抗原印制 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。
呋塞米检测胶体金试纸条检测呋塞米的原理为：利用间接竞争法原理检测待 测样品中是否含有呋塞米。如果待测样品中含有呋塞米，则检测线不显色，质控 线显色。如果待测样品中不含有呋塞米，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呋塞米的检测。
通过以上实施例可以看出，本发明关于呋塞米的人工抗原的合成步骤简洁， 有效，可有效用于免疫分析中，为后续的的研究分析提供了方便的途径，提供的 细胞株DIC分泌的单克隆抗体，对具呋塞米有较好的特异性和检测灵敏度(IC50为0.49ng/mL)，可实现对保健品等食品中呋塞米残留量的检测，为建立快速、 简便、价廉、灵敏、特异的呋塞米检测方法提供了新手段。
以上仅以较佳实施例对本发明的技术方案进行介绍，但是对于本领域的一般 技术人员，依据本发明实施例的思想，应能在具体实施方式上及应用范围上进行 改变，故而，综上所述，本说明书内容不应该理解为本发明的限制，凡在本发明 的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本发明的权 利要求范围之内。
Claims (11)
1.一株分泌呋塞米单克隆抗体的杂交瘤细胞株，已于2021年12月16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，保藏名称为单克隆细胞株DIC，保藏编号为CGMCCNo.45023，保藏地址为北京市朝阳区北辰西路1号院3号。
2.一种呋塞米单克隆抗体，其特征在于，由权利要求1所述保藏编号为CGMCC No.45023的分泌呋塞米单克隆抗体的杂交瘤细胞株分泌产生。
3.一种如权利要求2所述的呋塞米单克隆抗体的制备方法，其特征在于，包括：取BALB/c小鼠，腹腔注射石蜡油，再腹腔注射保藏编号为CGMCC No.45023的杂交瘤细胞株，注射后收集腹水，将腹水纯化，将获得的呋塞米单克隆抗体低温保存。
4.如权利要求2所述呋塞米单克隆抗体的应用，其特征在于，用于食品中呋塞米残留的检测。
5.如权利要求1所述的分泌呋塞米单克隆抗体的杂交瘤细胞株的应用，其特征在于，用于制备呋塞米单克隆抗体。
6.如权利要求1所述的分泌呋塞米单克隆抗体的杂交瘤细胞株的应用，其特征在于，应用于检测呋塞米。
7.如权利要求6所述的应用，其特征在于，应用于食品中呋塞米残留的检测。
8.一种试剂盒，其特征在于，含有权利要求2所述的呋塞米单克隆抗体。
9.权利要求8所述的试剂盒应用于食品中呋塞米残留的检测。
10.一种胶体金试纸条，其特征在于，含有权利要求2所述的呋塞米单克隆抗体。
11.权利要求10所述的胶体金试纸条应用于食品中呋塞米残留的检测。
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