CN113897338B - 一株分泌2,4-d单克隆抗体的杂交瘤细胞株及其应用 - Google Patents
一株分泌2,4-d单克隆抗体的杂交瘤细胞株及其应用 Download PDFInfo
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Abstract
一株分泌2,4‑D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2,4‑D完全抗原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。首次免疫用完全弗氏佐剂,多次加强免疫用不完全弗氏佐剂,最后一次用2,4‑D完全抗原冲击免疫。取高效价低IC50小鼠的脾细胞,通过PEG方法与小鼠骨髓瘤细胞融合,经过间接竞争酶联免疫法筛选和三次亚克隆,得到一株杂交瘤细胞株。此细胞株分泌的单克隆抗体,对2,4‑D具有较好的特异性和检测灵敏度,IC50值为1.7ng/mL,为食品中2,4‑D残留的免疫检测提供了原料,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领域,尤其是涉及一株分泌2,4-D单克隆抗体的杂交瘤细胞株及其应用。
背景技术
2,4-D(2,4-二氯苯氧乙酸),是人工合成、类似于生长素的一种植物生长调节剂、除草剂,主要作为有机除草剂在农业中使用。它能扰乱植物体内激素平衡,广泛应用于水稻、小麦、高粱、甘蔗、玉米等单子叶作物的生产中,防治阔叶杂草。另外,2,4-D还可以作为水果和蔬菜的防腐剂,延长其保质期。由于其成本低,2,4-D的使用逐年增加。因此,对人和环境的危害也在增加。喷洒在土壤表面的除草剂渗入土壤,污染地下水和地表水。同时,它很容易通过皮肤和呼吸进入人体,导致中毒。
为了有效监督监测食品中使用2,4-D的情况,需要寻找一种特异性好,灵敏度高的测定方法,而目前相关技术中,检测方法如薄层层析法、气相色谱法、液相色谱法等,分离纯化过程冗长,灵敏度低,加上食品中干扰物多,难以获得准确结果。因此建立快速简便的2,4-D检测方法具有重要意义。建立高效的免疫学检测方法,筛选高特异性的单克隆抗体是重要前提。
发明内容
本发明的目的是提供一株2,4-D单克隆抗体杂交瘤细胞株,由该细胞株制备的抗体对2,4-D具有较好特异性和检测灵敏度,可以用来建立2,4-D的免疫学检测方法。
本发明的技术方案,一株2,4-D单克隆抗体杂交瘤细胞株,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,保藏编号为CGMCC No.22332。
本发明的另一目的是提供2,4-D单克隆抗体,其由所述保藏编号为CGMCCNo.22332的杂交瘤细胞株分泌产生。
本发明的又一目的是提供2,4-D单克隆抗体的制备方法,由2,4-D原药与蛋白的偶联物免疫小鼠后获得。
本发明的再一目的是提供一种所述2,4-D单克隆抗体的应用,用于食品安全检测中2,4-D残留的分析检测。
本发明第五个目的是提供一种试剂盒,该试剂盒包含所述的2,4-D单克隆抗体。
本发明第六个目的是提供一种试剂盒,应用在检测2,4-D含量中,进一步地,用于食品安全检测中2,4-D的分析检测。
本发明提供的分泌2,4-D单克隆抗体的杂交瘤细胞株的制备基本步骤为:
(1)完全抗原的制备:
a、免疫原2,4-D-BSA的制备:2,4-D药物的化学结构中具有羧基官能团,可直接作为半抗原与蛋白进行偶联,具体操作如下:称取1mg 2,4-D原药(2,4-D原药和牛血清白蛋白(BSA)摩尔比为30:1),1.56mg 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2.6mg 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2,4-D原药溶液中,室温搅拌反应6-8h(称为A液)。取牛血清白蛋白BSA 10mg,加入3mL硼酸缓冲溶液(称为B液),在室温条件,逐滴将A液加入到B液中,室温反应过夜,即得偶联物2,4-D-BSA混合液,通过透析分离完全抗原和未偶联的小分子半抗原;
b、包被原2,4-DB-OVA的制备:2,4-DB与2,4-D相比,具有更长的间隔臂,药物的化学结构中也具有羧基官能团,可直接与蛋白进行偶联,具体操作如下:称取3.3mg 2,4-DB,1-乙基碳二亚胺盐酸盐7.6mg,N-羟基琥珀酰亚胺4.6mg,用400μL无水N,N-二甲基甲酰胺溶解(称为A液),室温搅拌反4-5h。称取10mg鸡卵白蛋白OVA,溶解于4mL硼酸缓冲溶液中,在室温条件下,逐滴将A液加入到B液中,室温反应过夜,即得偶联物2,4-DB-OVA混合液,通过透析分离完全抗原和未偶联的小分子半抗原。
(2)小鼠的免疫:2,4-D-BSA完全抗原与等量弗氏佐剂混合乳化后,通过背部皮下注射免疫BALB/c小鼠。首次免疫用完全弗氏佐剂,多次加强免疫用不完全弗氏佐剂。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。最后一次用2,4-D-BSA完全抗原(不含佐剂)冲击免疫;通过间接竞争酶联免疫法(ic-ELISA)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(PEG4000)法将小鼠脾细胞和小鼠骨髓瘤细胞融合,通过HAT培养基培养,利用间接竞争酶联免疫法(ic-ELISA)检测阳性细胞孔,并进一步利用ic-ELISA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,最终筛选获得分泌2,4-D单克隆抗体的杂交瘤细胞株;
本发明的有益效果:本发明所制备的杂交瘤细胞株能够有效分泌2,4-D的单克隆抗体,对2,4-D具有较好的特异性和检测灵敏度,IC50值为1.7ng/mL,可实现对水、果蔬、谷物中2,4-D残留量的检测,为食品中2,4-D残留的免疫检测提供了原料,具有实际应用价值。
生物材料样品保藏:一株分泌2,4-D单克隆抗体的杂交瘤细胞株,已于2021年05月13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XSMS,保藏编号为CGMCC No.22332,保藏地址为:北京市朝阳区北辰西路1号院3号。
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为2,4-D单克隆抗体对2,4-D的抑制标准曲线。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
本发明通过将2,4-D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ic-ELISA筛选细胞上清,最终得到了对2,4-D有较好特异性和灵敏度的单克隆抗体杂交瘤细胞株。
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1:5混合即为TMB
显色液,现用现混。
实施例1杂交瘤细胞株的制备:
(1)完全抗原的合成:
a、免疫原2,4-D-BSA的制备:2,4-D药物的化学结构中具有羧基官能团,可直接与蛋白进行偶联,具体操作如下:称取1mg 2,4-D原药(2,4-D原药和牛血清白蛋白(BSA)摩尔比为30:1),1.56mg 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2.6mg 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2,4-D原药溶液中,室温搅拌反应6-8h(称为A液),取牛血清白蛋白BSA 10mg,加入3mL硼酸缓冲溶液(称为B液),在室温条件,逐滴将A液加入到B液中,室温反应过夜,即得偶联物2,4-D-BSA混合液,通过透析分离完全抗原和未偶联的小分子半抗原,得到免疫原2,4-D-BSA;
b、包被原2,4-DB-OVA的制备:2,4-DB与2,4-D相比,具有更长的间隔臂,药物的化学结构中也具有羧基官能团,可直接与蛋白进行偶联,具体操作如下:称取3.3mg2,4-DB,1-乙基碳二亚胺盐酸盐7.6mg,N-羟基琥珀酰亚胺4.6mg,用400μL无水N,N-二甲基甲酰胺溶解(称为A液),室温搅拌反4-5h。称取10mg鸡卵白蛋白OVA,溶解于4mL硼酸缓冲溶液中,在室温条件下,逐滴将A液加入到B液中,室温反应过夜,即得偶联物2,4-DB-OVA混合液,通过透析分离完全抗原和未偶联的小分子半抗原,得到包被原2,4-DB-OVA。
(2)动物免疫:选择健康的6~8周龄的BALB/c小鼠进行免疫。取2,4-D完全抗原与等量弗氏佐剂混合乳化后,通过背部皮下注射免疫BALB/c小鼠。第一次免疫用完全弗氏佐剂,之后都用不完全弗氏佐剂。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。第三次免疫后7天采血,使用ic-ELISA测定小鼠血清效价和抑制,选择效价高抑制好的小鼠,在第五次免疫后21天冲击免疫,腹腔注射,要求冲免剂量减半且不含任何佐剂。
(3)细胞融合:在冲击免疫三天后,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、摘眼球取血,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。
(4)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筛选出阳性细胞孔,第二步选用2,4-D为标准品,用ic-ELISA对阳性细胞进行抑制效果测定。选择对2,4-D标准品均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,获得分泌2,4-D单克隆抗体的杂交瘤细胞株。
实施例2单克隆抗体的制备、鉴定及应用:
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-硫酸铵法纯化。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 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。
(1)包被:将包被原2,4-DB-OVA用0.05M pH9.6碳酸盐缓冲液从1μg/mL开始倍比稀释,100μL/孔,37℃反应2h。
(2)洗涤:将板内溶液倾去,并用洗涤液洗涤3次,每次3min.
(3)封闭:拍干后,加入200μL/孔封闭液,37℃反应2h。洗涤后烘干备用。
(4)加样:将抗血清从1:1000开始倍比稀释,并加入到各稀释度的包被孔中,100μL/孔,37℃反应30min;充分洗涤后,加入1:3000稀释的HRP-羊抗鼠IgG,100μL/孔,37℃反应30min。
(5)显色:将酶标板取出,充分洗涤后,每孔加入100μL的TMB显色液,37℃避光反应15min.
(6)终止和测定:每孔加入50μL终止液以终止反应,然后用酶标仪测定各孔的OD450值。
用ic-ELISA测定单克隆抗体对2,4-D检测的IC50为1.7ng/mL。
表1还列出了用ic-ELISA测定该单克隆抗体检测2,4-D结构类似物的IC50和CR值。从表1可以看出,该单克隆抗体与其他与2,4-D结构相似的植物生长调节剂没有交叉反应,说明获得的单克隆抗体对2,4-D具有很好的特异性,可以用于实际样品的检测。
表1.单克隆抗体与2,4-D结构类似物的交叉反应
综上,本发明所制备的杂交瘤细胞株能够有效分泌2,4-D的单克隆抗体,对2,4-D具有很好的特异性和检测灵敏度,IC50值为1.7ng/mL,可实现对水、果蔬、谷物中2,4-D残留量的检测,为食品中2,4-D残留的免疫检测提供了原料,具有实际应用价值。
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 (8)
1.一株分泌2,4-D单克隆抗体的杂交瘤细胞株,已于2021年05月13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XSMS,保藏编号CGMCCNo.22332,保藏地址为北京市朝阳区北辰西路1号院3号。
2.一种2,4-D单克隆抗体,其特征在于,它由权利要求1所述保藏编号为CGMCCNo.22332的杂交瘤细胞株分泌产生。
3.如权利要求2所述的2,4-D单克隆抗体的制备方法,其特征在于,由权利要求1所述保藏编号为CGMCC No.22332的杂交瘤细胞株分泌产生。
4.权利要求2所述2,4-D单克隆抗体的应用,其特征在于,用于食品安全检测中2,4-D残留的分析检测。
5.一种试剂盒,其特征在于,含有权利要求2所述的2,4-D单克隆抗体。
6.权利要求5所述试剂盒在检测2,4-D含量的应用。
7.一种根据权利要求5所述的试剂盒,其特征在于,所述试剂盒用于食品安全检测中2,4-D的分析检测。
8.一种根据权利要求6所述试剂盒在检测2,4-D含量的应用,其特征在于,用于食品安全检测中2,4-D的分析检测。
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