溴氰菊酯农药残留免疫检测技术研究
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摘要
本研究利用实验室自主合成的人工免疫抗原,免疫BALB/c小鼠获得了特异性较高的多克隆抗体;进一步将小鼠脾细胞与SP2/0细胞融合,制得杂交瘤细胞,使用半固体HAT培养基筛选,获得四株阳性杂交瘤细胞;对四株细胞腹水单克隆抗体的特性进行鉴定,均具有较强的特异性,为酶联免疫和免疫亲和技术检测溴氰菊酯奠定了基础。主要的研究内容及结果如下:
1、自主设计合成了两种溴氰菊酯半抗原结构,即分别引入氨基活性位点的Hp-1和引入羧基活性位点的Hp-2,通过长程免疫小鼠,制备出对溴氰菊酯具有较高灵敏度的两种多克隆抗体。用小鼠免疫制备的多克隆抗体最高效价均可达64000,且抗体的灵敏度分别为0.117μg/mL和0.160μg/mL。
2、优化了融合细胞的培养方法,缩短了细胞克隆的时间。采用半固体培养基有效缩短了培养周期,减少了细胞污染的概率。采用该细胞培养条件培养新融合的细胞,采用间接非竞争性ELISA初筛和间接竞争性ELISA终筛相结合,最终从14株细胞中得到了四株能稳定分泌抗溴氰菊酯抗体的杂交瘤细胞株,分别命名为2B12,2C1,2F1和3D4。
3、对这四株细胞分泌的抗体特性作了初步鉴定,经ELISA鉴定四株单克隆抗体2B12、2F1、3D4三株细胞均为IgG1型,2C1为IgG2b型。在单抗的SDS—PAGE电泳实验中,2B12、2C1、2F1和3D4重链的分子量均为50 KD左右,轻链分子量为25 KD左右。用ELISA检测腹水上清的效价,2B12,2C1,2F1,3D4的腹水效价可达1:5.12×105～1:1.024×106,四株单抗2B12,2C1,2F1,3D4对溴氰菊酯的亲和力大小分别为7.161×108 L/moL,2.05×108 L/moL,7.0×108 L/moL,6.7×108 L/moL。在特异性实验中,四株细胞均对I型拟除虫菊酯类农药没有交叉反应,与II型拟除虫菊酯的交叉反应随着竞争物与溴氰菊酯的类似程度的升高而升高,但交叉反应率均低于35%。
4、通过优化ELISA检测条件建立标准曲线,得到该方法的IC50值为17.0±3.3μg/L,最低检测限为1.2±1.3μg/L,工作范围为0.501μg/L～499.8μg/L。在添加回收试验中,对来自环境中的水样进行了过滤处理后用甲醇PBS以5倍的体积进行稀释,分别添加10到1000μg/mL的溴氰菊酯标准品,测得回收率范围在89±2.5%～107.9±6.3%。该试验结果证明,所建立的ELISA体系适用于对实际样品进行快速、高灵敏度的检测。
Antiserum was produced by immuning Balb/c mice with the artificial antigen systhesized in the laboratory. Then hybridoma cells were produced by fusion of mice spleen cells with sp2/0 cells, and four hybridoma cell lines were screened from the semi-solid culture medium which contained HAT. The monoclonal antibody in ascites of the four cell lines were characterized and the results showed that all antibodies had very high specificity, which provided a foundation for the detection of Deltamethrin by ELISA and immuno-affinity technology.
1. The immune response could be induced effectively by the artificial antigen, and the highest titers of the antiserum was achieved 1:64000,as the cross-reactivity was 0.117μg/mL and 0.160μg/mL,respectively.
2. The culture method of fusion cell was optimized, and the survival rate of hybridoma cells was highly improved . By comparison of liquid culture medium, the semi-solid medium was adopted and the clone cycle was shortened with decreased polluting rate of the hybridomas.
3. Four strains of stable secreting monoclonal antibodies against deltamethrin hybridoma cell lines were established by combination of indirect non-competitive and indirect competitive ELISA screening, which were named 2B12, 2C1, 2F1 and 3D4, respectively. Three monoclonal antibodies 2B12,2F1,3D4 are all IgG1 type and 2C1 is IgG2b type characterized by ELISA. The molecular weight of three mcAbs were determined by SDS-PAGE electrophoresis, 2B12,2C1,2F1,3D4 heavy chains were all 50KD and light chain are all 25KD. Ascites titers of four cell lines all reached to 1:5.12×105～1:1.024×106. The affinity constants of four monoclonal antibodies 2B12, 2C1, 2F1 and 3D4 were 7.161×108 L/moL, 2.05×108 L/moL, 7.0×108 L/moL, 6.7×108 L/moL, respectively. In the experiment of specificity, the cross reaction of the four antibodies with type I pyrethroids no cross reaction was found with deltamethrin, and the cross-reactivity with type II pyrethroids was enhanced as the structure being close to the deltamethrin, but the hightest CR was no more than 35%.
4. A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies (2B12) was developed for the quantitative detection of deltamethrin. The IC50 for deltamethrin was 17.0±3.3μg/L, and the lower detection limit was 1.2±1.3μg/L, The working range was assigned to a concentration 0.501μg/L～499.8μg/L. To reduce the analysis time, the river water samples were pretreated with filter paper to clean-up. River water samples fortified with deltamethrin were analyzed according to this method. 89±2.5%～107.9±6.3% recoveries were observed as the the spike levels ranging from 10 to 1000μg/L. These results suggested that the developed ELISA could be used for the rapid and sensitive determination of deltamethrin in agro-food and environment.
1、自主设计合成了两种溴氰菊酯半抗原结构,即分别引入氨基活性位点的Hp-1和引入羧基活性位点的Hp-2,通过长程免疫小鼠,制备出对溴氰菊酯具有较高灵敏度的两种多克隆抗体。用小鼠免疫制备的多克隆抗体最高效价均可达64000,且抗体的灵敏度分别为0.117μg/mL和0.160μg/mL。
2、优化了融合细胞的培养方法,缩短了细胞克隆的时间。采用半固体培养基有效缩短了培养周期,减少了细胞污染的概率。采用该细胞培养条件培养新融合的细胞,采用间接非竞争性ELISA初筛和间接竞争性ELISA终筛相结合,最终从14株细胞中得到了四株能稳定分泌抗溴氰菊酯抗体的杂交瘤细胞株,分别命名为2B12,2C1,2F1和3D4。
3、对这四株细胞分泌的抗体特性作了初步鉴定,经ELISA鉴定四株单克隆抗体2B12、2F1、3D4三株细胞均为IgG1型,2C1为IgG2b型。在单抗的SDS—PAGE电泳实验中,2B12、2C1、2F1和3D4重链的分子量均为50 KD左右,轻链分子量为25 KD左右。用ELISA检测腹水上清的效价,2B12,2C1,2F1,3D4的腹水效价可达1:5.12×105～1:1.024×106,四株单抗2B12,2C1,2F1,3D4对溴氰菊酯的亲和力大小分别为7.161×108 L/moL,2.05×108 L/moL,7.0×108 L/moL,6.7×108 L/moL。在特异性实验中,四株细胞均对I型拟除虫菊酯类农药没有交叉反应,与II型拟除虫菊酯的交叉反应随着竞争物与溴氰菊酯的类似程度的升高而升高,但交叉反应率均低于35%。
4、通过优化ELISA检测条件建立标准曲线,得到该方法的IC50值为17.0±3.3μg/L,最低检测限为1.2±1.3μg/L,工作范围为0.501μg/L～499.8μg/L。在添加回收试验中,对来自环境中的水样进行了过滤处理后用甲醇PBS以5倍的体积进行稀释,分别添加10到1000μg/mL的溴氰菊酯标准品,测得回收率范围在89±2.5%～107.9±6.3%。该试验结果证明,所建立的ELISA体系适用于对实际样品进行快速、高灵敏度的检测。
Antiserum was produced by immuning Balb/c mice with the artificial antigen systhesized in the laboratory. Then hybridoma cells were produced by fusion of mice spleen cells with sp2/0 cells, and four hybridoma cell lines were screened from the semi-solid culture medium which contained HAT. The monoclonal antibody in ascites of the four cell lines were characterized and the results showed that all antibodies had very high specificity, which provided a foundation for the detection of Deltamethrin by ELISA and immuno-affinity technology.
1. The immune response could be induced effectively by the artificial antigen, and the highest titers of the antiserum was achieved 1:64000,as the cross-reactivity was 0.117μg/mL and 0.160μg/mL,respectively.
2. The culture method of fusion cell was optimized, and the survival rate of hybridoma cells was highly improved . By comparison of liquid culture medium, the semi-solid medium was adopted and the clone cycle was shortened with decreased polluting rate of the hybridomas.
3. Four strains of stable secreting monoclonal antibodies against deltamethrin hybridoma cell lines were established by combination of indirect non-competitive and indirect competitive ELISA screening, which were named 2B12, 2C1, 2F1 and 3D4, respectively. Three monoclonal antibodies 2B12,2F1,3D4 are all IgG1 type and 2C1 is IgG2b type characterized by ELISA. The molecular weight of three mcAbs were determined by SDS-PAGE electrophoresis, 2B12,2C1,2F1,3D4 heavy chains were all 50KD and light chain are all 25KD. Ascites titers of four cell lines all reached to 1:5.12×105～1:1.024×106. The affinity constants of four monoclonal antibodies 2B12, 2C1, 2F1 and 3D4 were 7.161×108 L/moL, 2.05×108 L/moL, 7.0×108 L/moL, 6.7×108 L/moL, respectively. In the experiment of specificity, the cross reaction of the four antibodies with type I pyrethroids no cross reaction was found with deltamethrin, and the cross-reactivity with type II pyrethroids was enhanced as the structure being close to the deltamethrin, but the hightest CR was no more than 35%.
4. A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies (2B12) was developed for the quantitative detection of deltamethrin. The IC50 for deltamethrin was 17.0±3.3μg/L, and the lower detection limit was 1.2±1.3μg/L, The working range was assigned to a concentration 0.501μg/L～499.8μg/L. To reduce the analysis time, the river water samples were pretreated with filter paper to clean-up. River water samples fortified with deltamethrin were analyzed according to this method. 89±2.5%～107.9±6.3% recoveries were observed as the the spike levels ranging from 10 to 1000μg/L. These results suggested that the developed ELISA could be used for the rapid and sensitive determination of deltamethrin in agro-food and environment.
引文
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