CN116286669B - 一株分泌托拉塞米单克隆抗体的杂交瘤细胞株及其应用 - Google Patents
一株分泌托拉塞米单克隆抗体的杂交瘤细胞株及其应用 Download PDFInfo
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Classifications
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Abstract
本发明公开了一株分泌托拉塞米单克隆抗体的杂交瘤细胞株及其应用,属于食品安全免疫检测领域。本发明合成了托拉塞米半抗原,制备托拉塞米完全抗原,并将它与等量弗氏佐剂混合乳化完全,通过背部皮下注射免疫BALB/c小鼠,首次免疫用弗氏完全佐剂,多次加强免疫用弗氏不完全佐剂,最后一次用托拉塞米完全抗原冲刺免疫。取高效价、低IC50小鼠脾细胞,通过PEG方法与骨髓瘤细胞融合,经过间接竞争酶联免疫法的筛选和三次亚克隆,得到杂交瘤细胞株。此细胞株分泌的单克隆抗体,对托拉塞米具有较好的特异性和检测灵敏度(IC50为0.8ng/mL)。本发明成果可用于建立托拉塞米含量的免疫检测方法,具有实际应用价值。
Description
技术领域
本发明涉及食品安全免疫检测技术领域,尤其是指一株分泌托拉塞米单克隆抗体的杂交瘤细胞株及其应用。
背景技术
托拉塞米(torasemide)属于吡啶磺酰脲类利尿剂,分子式为C16H20N4O3S,相对分子质量348.4,是一种白色结晶固体,两性物质,为白色结晶固体,两性物质,在酸碱环境中溶解度高于中性条件。原料可制成片剂、胶囊和注射剂。托拉塞米是市场上新出现的Na+-K+-2Cl-共转运抑制剂,其药理作用和药物代谢均优于呋塞米,于1993年在德国上市,次年在美国上市。但一些研究表明,托拉塞米比呋塞米有更多的并发症。常见的不良反应有头痛、头晕、厌食、肌肉痉挛、恶心呕吐、高血糖、高尿酸血症、便秘和腹泻以及过敏反应,长期大量使用导致水电解质失衡以及视力缺失,因此被列入2021年保健食品风险监测项目。
有关托拉塞米药物残留在动物组织中的检测方法,国内外已有少量报道,主要是用荧光光度法、气相色谱法、气相色谱-串联质谱法、高效液相色谱法、液相色谱-串联质谱法超高效液相色谱-高分辨质谱法等,检测样品主要为果蔬、茶叶、地表水和饮用水。仪器检测方法可进行定量分析并具有较低的检测限,但是通常需要昂贵的仪器和复杂的操作,前处理及检测时间长,严重制约了这些检测方法的推广。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。本发明的目的在于提供一种对托拉塞米具有较高亲和力和检测灵敏度的单克隆抗体杂交瘤细胞株的制备方法。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。
发明内容
为解决上述技术问题,本发明提供了一株分泌托拉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托拉塞米具有较好的亲和力和灵敏度,可以用来建立托拉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。
本发明的第一个目的在于提供一株杂交瘤细胞株,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,简称CGMCC,保藏编号CGMCCNo.45459,保藏日期2023年02月08日,分类命名:单克隆细胞株。
本发明的第二个目的在于提供托拉塞米半抗原,用于制备所述的杂交瘤细胞株,所述托拉塞米半抗原结构式:
本发明的第三个目的在于提供一种托拉塞米单克隆抗体,由所述的杂交瘤细胞株分泌得到。
本发明的第四个目的在于提供一种组合物,所述组合物包括,所述组合物中含有所述的杂交瘤细胞株或所述的托拉塞米单克隆抗体。
本发明的第五个目的在于提供一种试剂盒,所述试剂盒中含有所述的杂交瘤细胞株、所述的托拉塞米单克隆抗体或所述的组合物。
在本发明的一个实施例中,还包括酶标板、托拉塞米包被抗原、托拉塞米标准液、酶标记二抗和底物反应液。
本发明的第六个目的在于提供一种胶体金试纸条,所述胶体金试纸条含有所述杂交瘤细胞株、所述的托拉塞米单克隆抗体或所述组合物。
在本发明的一个实施例中,所述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托拉塞米单克隆抗体。
本发明的第七个目的在于提供一种芯片,所述芯片含有所述杂交瘤细胞株、所述的托拉塞米单克隆抗体、所述组合物。
本发明的第八个目的在于提供所述的杂交瘤细胞株、所述的托拉塞米单克隆抗体、所述的组合物、所述的试剂盒、所述的胶体金试纸条或所述的芯片在检测托拉塞米中的应用。
本发明提供的细胞株的制备基本步骤为:
(1)半抗原的合成:通过选择在远离磺酰脲基的苯环上引入羧基,保持托拉塞米原有的分子结构。
(2)免疫原的制备与鉴定:以托拉塞米衍生物为原料,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;(具体见图1)。
(3)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;
(4)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;
(5)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。
本发明的上述技术方案相比现有技术具有以下优点:
(1)、本发明获得的托拉塞米单克隆抗体,对托拉塞米有较好的检测灵敏度和亲和力。
(2)、一种新的合成托拉塞米半抗原和免疫原的方法,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。
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是免疫原的紫外吸收光谱表征。
图2是托拉塞米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。
本发明通过将托拉塞米完全抗原免疫小鼠,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,最终得到了对托拉塞米有较好亲和力和灵敏度的单克隆抗体杂交瘤细胞株。
实施例1:托拉塞米单克隆抗体杂交瘤细胞株的制备
1、托拉塞米半抗原的合成:其合成路线如下:
在100mL三颈烧瓶中分别加入5mL乙腈、500mg 4-氯吡啶-3-磺胺和528mg三乙胺,不断搅拌,缓慢滴加221mg异氰酸异丙酯,25℃搅拌2h,真空浓缩,用C18色谱柱(乙腈:水(0.1%碳酸氢铵)=5%~40%)对混合物进行纯化,得到化合物1。称取600mg化合物1溶于6mL的醋酸溶液,向其中加入311mg 3-氨基苯甲酸,25℃下搅拌5h。减压下去除醋酸,残渣经高效液相色谱法纯化得到白色固体粉末,即为托拉塞米半抗原。
2、完全抗原的合成:取5.2mg上述托拉塞米半抗原,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、pH 9.6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,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
托拉塞米包被原的制备方法:取4.6mg上述托拉塞米半抗原,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10mg OVA(鸡卵白蛋白)溶解于3mL、0.05M、pH 9.6的CB(碳酸盐缓冲溶液)溶液中;将上述活化液逐滴加入OVA溶液中,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
通过紫外可见分光光度法对所合成的完全抗原在波长500nm~200nm范围内处进行扫描,表征完全抗原合成是否成功。实验结果见图1。
3、动物免疫:选择健康的6~8周龄的BALB/c小鼠进行免疫。取托拉塞米完全抗原(1mg/mL)与等量福氏佐剂乳化均匀后,通过皮下多点注射免疫BALB/c小鼠,每只100μL。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;选择抑制最好的小鼠,在五免后18天冲刺免疫,准备融合。
4、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:
(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。
5、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,第二步选用托拉塞米为标准品,用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对托拉塞米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。
6、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.托拉塞米单克隆抗体的亚型鉴定
使用间接竞争ELISA法,测定单克隆抗体对托拉塞米的IC50为0.80ng/mL(结果见图2),并验证了其对他达拉非等的IC50及交叉反应率,具体如表2所示。
表2托拉塞米单克隆抗体对托拉塞米、氯磺丙脲、甲苯磺丁脲等的IC50及交叉反应率
由表2可知,托拉塞米是一种含有磺酰脲基的利尿剂,对其他含有磺酰脲基的化合物测定交叉,说明托拉塞米抗体具有良好的特异性,对托拉塞米结构类似物没有识别能力,CR均<1%,因此应用托拉塞米建立的ELISA方法具有优异的特异性,能够满足实际检测需求。
7、抗体应用:
(一)、将杂交瘤细胞株通过体内腹水制备的单克隆抗体应用于托拉塞米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的托拉塞米包被原包被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、0.02μg/L、0.05μg/L、0.1μg/L、0.2μg/L、0.5μg/L、1μg/L、2μg/L的托拉塞米标准溶液。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稀释的抗托拉塞米单克隆抗体,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100μLTMB显色液,37℃显色15min后,每孔加入50μL2 M H2SO4终止液,450nm测吸光值;
(6)添加回收及样品前处理:取新鲜的黄瓜样品5g,添加三个不同剂量的托拉塞米标准品,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90.2%、103.1%、99.3%。
(二)、样品的分析
(1)、溶液的配置:
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,0.24g KH2PO4,3.62gNa2HPO4·12H2O,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。
(2)、选择保健片剂和胶囊样本,根据托拉塞米抗体的灵敏度,在阴性样本中添加4个浓度:0、2、5、10ng/g,样本经提取后,ELISA检测液稀释20倍后检测。实验结果如表3所示,ELISA对保健片剂和胶囊中托拉塞米的回收率为98.9%~108.0%,变异系数小于7.7%;准确性良好,满足要求,可以用于保健食品中托拉塞米的定量分析。
表3保健片剂和胶囊中托拉塞米的添加回收(n=3)
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 (9)
1.一株杂交瘤细胞株,其特征在于,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CGMCC No.45459,保藏日期2023年02月08日,分类命名:单克隆细胞株。
2.一种托拉塞米单克隆抗体,其特征在于,由权利要求1所述的杂交瘤细胞株分泌得到。
3.一种组合物,其特征在于,所述组合物包括,所述组合物中含有权利要求1所述的杂交瘤细胞株或权利要求2所述的托拉塞米单克隆抗体。
4.一种试剂盒,其特征在于,所述试剂盒中含有权利要求1所述的杂交瘤细胞株、权利要求2所述的托拉塞米单克隆抗体或权利要求3所述的组合物。
5.根据权利要求4所述试剂盒,其特征在于,还包括酶标板、托拉塞米包被抗原、托拉塞米标准液、酶标记二抗和底物反应液。
6.一种胶体金试纸条,其特征在于,所述胶体金试纸条含有权利要求2所述的托拉塞米单克隆抗体。
7.根据权利要求6所述的胶体金试纸条,其特征在于,所述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权利要求2所述托拉塞米单克隆抗体。
8.一种芯片,其特征在于,所述芯片含有权利要求1所述杂交瘤细胞株、权利要求2所述的托拉塞米单克隆抗体、权利要求3所述组合物。
9.权利要求2所述的托拉塞米单克隆抗体、权利要求3所述的组合物、权利要求4或5所述的试剂盒、权利要求6或7所述的胶体金试纸条或权利要求8所述的芯片在检测托拉塞米中的应用。
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