抗拟除虫菊酯类农药广谱性单克隆抗体的研制及鉴定

文孟棠 刘媛 闫帅 张霄 王恒 刘贤金
摘要制备了抗拟除虫菊酯类农药(Pyrethroids)的广谱性单克隆抗体,并鉴定其免疫学特性;以间苯氧基苯甲酸(PBA)为半抗原,用活性酯法将其与牛血清蛋白(BSA)偶联制得人工抗原PBABSA免疫Balb/c小鼠,5次免疫后选择效价最高、对PBA识别能力最强的小鼠取脾细胞与SP2/0骨髓瘤细胞在PEG1500作用下融合,经间接ELISA筛选及有限稀释法进行亚克隆,分离阳性细胞株,腹水诱导法大量制备单克隆抗体,并用Protein G亲和柱纯化,间接ELISA法测定抗体效价、亚型、亲和力常数及对拟除虫菊酯类农药的作用;UV结果显示,PBABSA成功偶联,获得1株稳定分泌抗拟除虫菊酯类农药单克隆抗体的杂交瘤细胞株4H11,其培养腹水抗体效价为1∶6.5×106,其抗体亚类为IgG1,对PBA的亲和力常数为2.5×10 L/mol,对PBA的IC50为208.9 μg/L,检出限为21 μg/L,对高效氯氰菊酯、氟氰戊菊酯、氰戊菊酯和氯氰菊酯的IC50分别为1.01, 2.15, 3.16和3.67 μg/L。
[KH*3/4D][HTH]关键词拟除虫菊酯类农药;广谱特异性;单克隆抗体;酶联免疫吸附法
[HK][FQ(32,X,DY-W][CD15]20140123收稿;20140512接受
本文系国家自然科学基金(No.31201535)、江苏省社会发展项目(No.BE2012750)、江苏省农业自主创新基金(No.CX(12)5042)、948项目(No.2011Z46)资助
* Email:jaasliu@jaas.ac.cn
1引言
拟除虫菊酯(Pyrethroids)是一类杀虫谱广、高效、低毒、残留少的合成杀虫剂,被广泛应用于农业领域中的害虫防治。但其残留期较长,对某些非靶标生物毒性很高;通过食物链进入到机体后对哺乳动物的生殖、免疫和心血管有毒副作用 \[1,2\],联合国粮农组织(FAO)和世界卫生组织(WHO)已对它们在蔬菜和水果等上的残留做了严格限量\[3\],我国也对不同拟除虫菊酯类农药做了相应的最大残留限量\[4\]。目前,定量测定拟除虫菊酯的方法主要为气相色谱法、高效液相色谱法及气相色谱质谱联用\[5\],但其检测仪器昂贵,技术要求高,且样品需经过复杂的前处理,不能满足我国农产品质量监控工作中大量样品快速初筛的要求\[6\]。目前,已有针对拟除虫菊酯类广谱性农药抗体的报告,但多采用多克隆抗体制备技术。 Bonwick等\[7\] 将PBA和氯苯氧乙酸(CPA)分别与载体蛋白偶联合成免疫原,制得的多克隆抗体对PBA抑制中浓度(IC50)为100 mg/L,检出限为10 mg/L; 文献\[8,9\]分别对I和Ⅱ型拟除虫菊酯类农药进行了ELISA免疫分析,得到了不同类型菊酯类农药的检出限;骆爱兰等\[10\]以PBA为半抗原,制备出拟除虫菊酯类农药的广谱性多克隆抗体,能识别氯菊酯、甲氰菊酯、三氟氯菊酯、溴氰菊酯,其IC50约4 mg/L。
相对而言,单抗纯度高、专一性强、重复性好、能持续地大量生产,克服了多克隆抗体的非均一性和数量有限性。针对单种拟除虫菊酯类农药的单克隆抗体已有报告,如Kong等\[11\]研制出特异性针对溴氰菊酯的单克隆抗体,其IC50达17 μg/L;Chen等制备出一个高灵敏度的拟除虫菊酯类农药广谱性单克隆抗体,IC50在1.7~298.5 μg/L范围内\[12\],但其半抗原需重新合成复杂结构;徐敦明等\[13\]以商品化的PBA为半抗原(不需任何化学修饰)制备拟除虫菊酯类农药的单克隆抗体,对溴氰菊酯、甲体氯氰菊酯、甲氰菊酯、氰戊菊酯均有特异性识别,IC50分别为0.79,0.74,0.63和0.8 mg/L。现虽有以PBA为半抗原直接制备的单克隆抗体,但是本研究所制备的单克隆抗体识别的拟除虫菊酯类农药的种类与之前文献报道不同,能识别高效氯氰菊酯、氟氰戊菊酯、氯氰菊酯和氰戊菊酯,为多种拟除虫菊酯类农药的快速检测方法奠定了基础。
2实验部分
2.1仪器与试剂
Multiscan ascent酶标仪(Thermo),酶标板(Corning,96孔),细胞培养板(Corning),恒温培养箱(Napco);倒置显微镜(Olympus)。
高效氯氰菊酯标准品、甲氰菊酯标准品、氟氰戊菊酯标准品、氰戊菊酯标准品、氯氰菊酯标准品、氯菊酯标准品、溴氰菊酯标准品、三氟氯氰菊酯标准品(农业部农药检定所提供);RPMI1640培养液、HAT选择培养液,HT选择培养液(Gibco产品);8氮杂鸟嘌呤、50%PEG1500、弗氏完全佐剂(FCA)和弗氏不完全佐剂(FIA)、小鼠单克隆抗体分型试剂、卵清蛋白(OVA)、牛血清蛋白(BSA)、间苯氧基苯甲酸(PBA),均购于Sigma公司;辣根过氧化物酶标记羊抗鼠IgG(武汉博士德公司); 其它试剂均为国产分析纯。
实验动物:Balb/c雌性小鼠(购于扬州大学比较医学中心)。
2.2实验方法
2.2.1人工抗原的合成和鉴定以PBA为半抗原,参照文献分别用活性酯法\[14\]和混合酸酐法\[15\]将其分别与BSA和OVA偶联合成免疫抗原和包被抗原,用紫外分光光度计扫描鉴定。
2.2.2动物免疫及小鼠血清效价的测定将PBABSA与佐剂等体积混合乳化,对6~8周龄的Balb/c雌性小鼠进行腹腔注射免疫,免疫剂量为100 μg/只。首免用弗氏完全佐剂,之后每2周用弗式不完全佐剂加强免疫。融合前3天进行冲击免疫,免疫剂量为200 μg/只。
采用间接ELISA法测血清效价:PBAOVA包被,包被浓度2 mg/L,包被量100 μL/孔,4 ℃过夜包被,PBST 洗板3 次(下同);用1% OVA封闭,200 μL/孔,37 ℃温育1 h,洗板;加小鼠血清,100 μL/孔,用PBS倍比稀释,设阴性对照(NC)和空白对照(BC), 37 ℃温育1 h,洗板;加GaMIgGHRP,100 μL/孔,37 ℃温育1 h,洗板;加酶底物TMB 显色液100 μL/孔,显色15 min;每孔加50 μL 2 mol/L H2SO4终止反应,用酶标仪读OD450 nm值;结果判断: 待测孔OD450 nm值≥NC OD450 nm 值的2.1 倍(P/N≥2.1),判为阳性。
用间接竞争 ELISA法测定单克隆抗体对不同浓度PBA的灵敏度:将上述测定效价的间接ELISA法步骤中加血清一步改为加50 μL抗体和50 μL不同浓度的PBA标准品溶液,其余各步相同。结果判断:读数与PBA浓度呈明显负相关者即表示小鼠产生了特异性针对PBA的抗体。
2.2.3杂交瘤细胞株的建立(1)细胞融合按常规操作方法进行细胞融合。融合后的细胞在
37 ℃、 5% CO2恒温培养箱中选择培养。第1~7天用HAT培养基,第7~14天换用HT培养基,14天后换用完全1640培养基, 观察杂交瘤细胞的生长状况。(2)阳性杂交瘤细胞的筛选及亚克隆融合7~10天后,对杂交瘤细胞的上清液用间接ELISA进行检测。以融合前小鼠血清为阳性对照,饲养细胞的培养上清液为阴性对照。选择OD450 nm值高、对PBA特异性抑制率最高的阳性杂交瘤细胞孔用有限稀释法\[16\]进行亚克隆。(3)单克隆抗体的大量制备及纯化采用体内诱生腹水法制备单克隆抗体\[17\],将收集的腹水离心除去细胞和其它沉淀物,收集上清液,
Symbolm@@ 70 ℃冻存备用。
采用亲和层析柱的方法纯化腹水抗体(用饱和硫酸铵沉淀法作为对比),用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE电泳)鉴定纯化效果。
2.2.4单克隆抗体免疫学特性的鉴定采用间接 ELISA 法测定单克隆抗体效价。采用间接竞争 ELISA法测定单克隆抗体对PBA的灵敏度。以PBA标准品浓度的对数为横坐标,以抑制百分率为纵坐标作图。
3结果与讨论
3.1免疫抗原和包被抗原的紫外鉴定
能否将小分子半抗原和蛋白成功偶联是制备单克隆抗体的关键,直接关系到动物免疫后能否产生针对目标物的高质量抗体\[19\]。结果表明,与BSA、PBA相比, PBABSA的吸收曲线发生明显改变,在 PBA的最大吸收峰处296 nm的吸光值明显增加,最高吸收峰有一定偏移,推测免疫抗原PBABSA偶联成功;同样PBAOVA也成功偶联(图 1)。经计算得其偶联比分别为25∶1和12∶1。
3.4单克隆抗体免疫学特性的鉴定
3.4.1单克隆抗体纯度的鉴定由SDSPAGE电泳结果(图4)可知,饱和硫酸铵沉淀法得到的抗体纯度不高; Protein G亲和纯化后得到的抗体重、轻链分子量约为55和23 kDa, 与预期大小一致,说明抗体纯化效果较好。
3.4.2单克隆抗体效价和灵敏度的测定将纯化后的4H11对PBA进行竞争抑制ELISA实验,得到其线性回归方程I=41.214lgC+78.08, R2=0.994;计算IC50为208.9μg/L。表明纯化后的单抗4H11效价与灵敏度均有增加(表1和图5)。
3.4.3单克隆抗体亚型的鉴定通过小鼠单克隆抗体分型试剂盒测得4H11阳性杂交瘤细胞株诱生腹水产生的抗体为lgG1型。
3.5单克隆抗体对不同拟除虫菊酯类农药敏感性测定
由表2可见,4H11单克隆抗体对高效氯氰菊酯、氟氰戊菊酯、氯氰菊酯和氰戊菊酯的灵敏度相对较高,对甲氰菊酯标准品的识别度相对减弱,对三氟氯氰菊酯和溴氰菊酯的灵敏度较低在10 mg/L的标准品浓度能到达30%的抑制率,不能识别氯菊酯。
由结构式可见,PBA含有拟除虫菊酯类农药的一个共性结构基团:苯醚基团,以此结构作为半抗原制备的抗体不仅可以特异性识别PBA,而且可以含有此基团的多种拟除虫菊酯类农药。但是抗体对被测物的化学结构极为敏感,其结构上的微小变化都可引起抗体对其的识别度的极大变化\[20\]。同时,此抗体可高灵敏识别高效氯氰菊酯、氟氰戊菊酯、氯氰菊酯和氰戊菊酯属Ⅱ型菊酯,对属于Ⅰ型菊酯的氯菊酯不能识别,说明氰基对于抗体识别具有很大影响,这文献\[21\]一致;另外,键和电子云也可影响抗原抗体的结合紧密度\[22\]。但抗体对同是Ⅱ型菊酯且包含一个氰基的溴氰菊酯的识别灵敏度很低,溴原子可能是抑制基团,说明不同的基团对于抗体结合的影响程度不同\[7\],具体原因还有待进一步研究。同时,ELISA条件还有改进之处,灵敏度仍然有提升空间。
4结论
本研究成功合成了 PBABSA人工免疫抗原,通过细胞融合技术获得1 株高亲和力的杂交瘤细胞株4H11,经多次传代、冻存与复苏,杂交瘤分泌抗体稳定。本研究获得的4H11腹水型单克隆抗体具有效价高、敏感性和广谱性强的特点,能同时灵敏地识别高效氯氰菊酯、氟氰戊菊酯、氯氰菊酯、氰戊菊酯,对甲氰菊酯、溴氰菊酯和三氟氯氰也有一定的识别作用,相对于同样以PBA为半抗原制备拟除虫菊酯类农药多抗,对PBA的IC50从3.86 mg/L提升到0.21 mg/L,且对拟除虫菊酯农药的灵敏度也有增强。本方法适用于拟除虫菊酯类农药残留现场快速检测。
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AbstractThe objective of this study is to generate broad spectrum monoclonal antibody(mAb) against a group of pyrethroid insecticides and to identify its immunological characteristics. The generic hapten 3phenoxybenzoic acid (PBA) was conjugated to carrier protein BSA by activated ester method. Balb/c mice were immunized with PBABSA. The titer of polyclonal antibody (pAb) was detected by indirect enzymelinked immunosorbent assay (ELISA) after five times immunization. The mouse with high titer and sensitivity was selected for cell fusing. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect noncompetitive ELISA based on the coating antigen PBAovoalbumin (PBAOVA). Highsensitivity and highspecificity mAb was prepared after subcloning using limiting dilution method. Purified mAb was obtained after purified by saturated ammonia sulfate precipitation and protein G affinity column. The immunological characteristics of mAb such as titer, antibody subtypes, affinity constant and the sensitivity to pyrethroid insecticides were characterized by indirect ELISA; The results of UV spectroscopy and SDSPAGE showed that PBABSA artificial antigen was synthesized successfully. A hybridoma cell line (4H11) secreting anti pyrethroid mAb was established. The titre of ascites was up to 1: 6.5×106, and the mAb was IgG1 subtype. The affinity constant of the mAb to PBA was about 2.5×107 L/mol, with a IC50 value of 208.83 μg/L and a detection limit of 21.23 μg/L to PBA. Simultaneously, betacypermethrin, flucythrinate, cypermethrin and fenvalerate were sensitively recognized by the mAb with the IC50 of 1.01, 2.15, 3.16 and 3.67μg/L, respectively.
KeywordsPyrethroid pesticides; Broad specificity; Monoclonal antibody; Enzymelinked immunosorbent assay
22Nathalie D B, Pichon V, Hennion M C. J. Chromatogr. A, 2003, 999(1): 3-15
AbstractThe objective of this study is to generate broad spectrum monoclonal antibody(mAb) against a group of pyrethroid insecticides and to identify its immunological characteristics. The generic hapten 3phenoxybenzoic acid (PBA) was conjugated to carrier protein BSA by activated ester method. Balb/c mice were immunized with PBABSA. The titer of polyclonal antibody (pAb) was detected by indirect enzymelinked immunosorbent assay (ELISA) after five times immunization. The mouse with high titer and sensitivity was selected for cell fusing. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect noncompetitive ELISA based on the coating antigen PBAovoalbumin (PBAOVA). Highsensitivity and highspecificity mAb was prepared after subcloning using limiting dilution method. Purified mAb was obtained after purified by saturated ammonia sulfate precipitation and protein G affinity column. The immunological characteristics of mAb such as titer, antibody subtypes, affinity constant and the sensitivity to pyrethroid insecticides were characterized by indirect ELISA; The results of UV spectroscopy and SDSPAGE showed that PBABSA artificial antigen was synthesized successfully. A hybridoma cell line (4H11) secreting anti pyrethroid mAb was established. The titre of ascites was up to 1: 6.5×106, and the mAb was IgG1 subtype. The affinity constant of the mAb to PBA was about 2.5×107 L/mol, with a IC50 value of 208.83 μg/L and a detection limit of 21.23 μg/L to PBA. Simultaneously, betacypermethrin, flucythrinate, cypermethrin and fenvalerate were sensitively recognized by the mAb with the IC50 of 1.01, 2.15, 3.16 and 3.67μg/L, respectively.
KeywordsPyrethroid pesticides; Broad specificity; Monoclonal antibody; Enzymelinked immunosorbent assay
22Nathalie D B, Pichon V, Hennion M C. J. Chromatogr. A, 2003, 999(1): 3-15
AbstractThe objective of this study is to generate broad spectrum monoclonal antibody(mAb) against a group of pyrethroid insecticides and to identify its immunological characteristics. The generic hapten 3phenoxybenzoic acid (PBA) was conjugated to carrier protein BSA by activated ester method. Balb/c mice were immunized with PBABSA. The titer of polyclonal antibody (pAb) was detected by indirect enzymelinked immunosorbent assay (ELISA) after five times immunization. The mouse with high titer and sensitivity was selected for cell fusing. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect noncompetitive ELISA based on the coating antigen PBAovoalbumin (PBAOVA). Highsensitivity and highspecificity mAb was prepared after subcloning using limiting dilution method. Purified mAb was obtained after purified by saturated ammonia sulfate precipitation and protein G affinity column. The immunological characteristics of mAb such as titer, antibody subtypes, affinity constant and the sensitivity to pyrethroid insecticides were characterized by indirect ELISA; The results of UV spectroscopy and SDSPAGE showed that PBABSA artificial antigen was synthesized successfully. A hybridoma cell line (4H11) secreting anti pyrethroid mAb was established. The titre of ascites was up to 1: 6.5×106, and the mAb was IgG1 subtype. The affinity constant of the mAb to PBA was about 2.5×107 L/mol, with a IC50 value of 208.83 μg/L and a detection limit of 21.23 μg/L to PBA. Simultaneously, betacypermethrin, flucythrinate, cypermethrin and fenvalerate were sensitively recognized by the mAb with the IC50 of 1.01, 2.15, 3.16 and 3.67μg/L, respectively.
KeywordsPyrethroid pesticides; Broad specificity; Monoclonal antibody; Enzymelinked immunosorbent assay
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