抗头孢噻呋多克隆抗体和抗氯霉素多克隆抗体制备的研究
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摘要
头孢噻呋作为第一个动物专用的头孢菌素类抗生素,由于其在兽医临床上的日益广泛使用,其在动物性食品中的残留也逐渐引起人们的高度关注,快速检测头孢噻呋在动物性食品中的残留成为畜牧生产实践中亟待解决的问题。氯霉素是第一个人工合成的广谱抗生素,因其效高价廉,在我国畜牧业中得到广泛应用,成为畜禽疾病防治的重要药物。随着氯霉素的广泛应用及研究的深入,发现其有许多严重的副作用,因此,必须控制氯霉素在动物性食品中的残留,建立一种快速检测氯霉素残留的方法。
本研究采用免疫学技术,成功制备抗头孢噻呋多克隆抗体和抗氯霉素多克隆抗体,并初步建立了检测头孢噻呋和氯霉素在动物性食品中残留的ELISA方法,为其单克隆抗体的制备和试剂盒的开发打下了良好的基础。主要研究内容如下:
1成功制备了抗-CEF多克隆抗体
1.1采用直接法将头孢噻呋(CEF)与牛血清蛋白(BSA)和卵清蛋白(OVA)分别合成免疫抗原(CEF-BSA)和检测抗原(CEF-OVA),用紫外(UV)、SDS聚丙烯酰胺凝胶电泳法进行鉴定,并用正交分解法优化了抗原合成过程。
1.2用CEF-BSA免疫Balb/c小鼠得到抗CEF多克隆抗体,间接ELISA检测抗血清的效价为1:135000,间接竞争ELISA测定CEF的标准曲线,线性范围为100 ng/mL-1000 ng/mL,线性方程是y=-2.6x+206.0,相关系数(r)为0.98,IC_(50)是206.0 ng/mL,检测限为49.1 ng/mL,以200 ng/mL-800 ng/mL浓度添加时,牛奶中的回收率为79.2%-94.5%,变异系数为5.1%-13.4%。
2成功制备了抗-CAP多克隆抗体
2.1将氯霉素(CAP)和琥珀酸酐合成半抗原琥珀酰氯霉素(CAP-HS),然后在碳化二亚胺(EDC)作用下与牛血清蛋白(BSA)和卵清蛋白(OVA)分别合成免疫抗原(CAP-BSA)和检测抗原(CAP-OVA),用紫外(UV)、SDS聚丙烯酰胺凝胶电泳法进行鉴定,并用正交分解法优化了抗原合成过程。
2.2用CAP-BSA免疫家兔和Balb/c小鼠都得到抗CAP多克隆抗体,间接ELISA检测抗血清的效价为1:162000,间接竞争ELISA测定CAP的标准曲线,线性范围为10 ng/mL-100 ng/mL,线性方程是y=-4.3x+32.4,相关系数(r)为0.98,IC_(50)是32.4ng/mL,检测限为0.94ng/mL,以20ng/mL-100ng/mL浓度添加时,鸡蛋中的回收率为79.4%-98.5%,变异系数为0.5%-4.3%。
Ceftiofur(CEF) is cephalosporin used on veterinarian clinic at first,because it is used extensively day by day in veterinarian,its residue in animal food has already aroused great concern from people gradually.How to detect the CEF residues quickly has became the problem resolved urgently.Chloramphenicol(CAP) is a highly-effective broad-spectrum antibiotics which is widely used in stockbreeding. But it has been found that CAP residues in animal tissues and/or animal products has toxic effects to human body.Therefore,it is necessary to work out an analytical method to monitoring CAP residues.
In the paper,the technique of molecular immunology was used to the preparation of the anti-CEF polyclonal antibody and the anti-CAP polyclonal antibody,The two above polyclonal antibody establish the foundation for the preparation of their monoclonal antibody and the exploitation of their ELISA Kit.The main content and results of the study are descripted as follow:
1 The anti-ceftiofur polyclonal antibody was prepared successfully.
1.1 The chemical crosslinking was used to conjugate ceftiofur(CEF) to BSA and obtained artificial immunogen CEF-BSA,the coating antigen CEF-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CEF-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.
1.2 Balb/c mice was immunized with CEF-BSA,the titre of polyclonal antibody(pAb) was 135000 by indirect ELISA,The linear range of calibration curves to detect CEF was 100 ng/mL-1000 ng/mL,The curve equation is y=-2.6x+206.0,coefficient correlation r=0.98,its IC_(50) is 206.0 ng/mL and the limit of detection was 49.1 ng/mL, The recoveries ranged from 79.2%to 94.5%for detecting CEF residues in milk and the coefficients of variation was 5.1%to 13.4%.
2 The anti- chloramphenicol polyclonal antibody was prepared successfully.
2.1 The active group carboxyl was introduced to Chloramphenicol(CAP) and formed CAP half succinaate(CAP-HS).The carbodiimide(EDC) was used to conjugate CAP-HS to BSA and obtained artificial immunogen CAP-HS-BSA,the coating antigen CAP-HS-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CAP-HS-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.
2.2 Rabbits and Balb/c mice were immunized with CAP-HS-BSA,the titre of polyclonal antibody(pAb) was 162000 by indirect ELISA,The linear range of calibration curves to detect CAP was 10 ng/mL-100 ng/mL,The curve equation is y=-4.3x+32.4,coefficient correlation r=0.98,its IC_(50) is 32.4 ng/mL and the limit of detection was 0.94 ng/mL,The recoveries ranged from79.4%to 98.5%for detecting CAP residues in eggs and the coefficients of variation was 0.5%to 4.3%.
本研究采用免疫学技术,成功制备抗头孢噻呋多克隆抗体和抗氯霉素多克隆抗体,并初步建立了检测头孢噻呋和氯霉素在动物性食品中残留的ELISA方法,为其单克隆抗体的制备和试剂盒的开发打下了良好的基础。主要研究内容如下:
1成功制备了抗-CEF多克隆抗体
1.1采用直接法将头孢噻呋(CEF)与牛血清蛋白(BSA)和卵清蛋白(OVA)分别合成免疫抗原(CEF-BSA)和检测抗原(CEF-OVA),用紫外(UV)、SDS聚丙烯酰胺凝胶电泳法进行鉴定,并用正交分解法优化了抗原合成过程。
1.2用CEF-BSA免疫Balb/c小鼠得到抗CEF多克隆抗体,间接ELISA检测抗血清的效价为1:135000,间接竞争ELISA测定CEF的标准曲线,线性范围为100 ng/mL-1000 ng/mL,线性方程是y=-2.6x+206.0,相关系数(r)为0.98,IC_(50)是206.0 ng/mL,检测限为49.1 ng/mL,以200 ng/mL-800 ng/mL浓度添加时,牛奶中的回收率为79.2%-94.5%,变异系数为5.1%-13.4%。
2成功制备了抗-CAP多克隆抗体
2.1将氯霉素(CAP)和琥珀酸酐合成半抗原琥珀酰氯霉素(CAP-HS),然后在碳化二亚胺(EDC)作用下与牛血清蛋白(BSA)和卵清蛋白(OVA)分别合成免疫抗原(CAP-BSA)和检测抗原(CAP-OVA),用紫外(UV)、SDS聚丙烯酰胺凝胶电泳法进行鉴定,并用正交分解法优化了抗原合成过程。
2.2用CAP-BSA免疫家兔和Balb/c小鼠都得到抗CAP多克隆抗体,间接ELISA检测抗血清的效价为1:162000,间接竞争ELISA测定CAP的标准曲线,线性范围为10 ng/mL-100 ng/mL,线性方程是y=-4.3x+32.4,相关系数(r)为0.98,IC_(50)是32.4ng/mL,检测限为0.94ng/mL,以20ng/mL-100ng/mL浓度添加时,鸡蛋中的回收率为79.4%-98.5%,变异系数为0.5%-4.3%。
Ceftiofur(CEF) is cephalosporin used on veterinarian clinic at first,because it is used extensively day by day in veterinarian,its residue in animal food has already aroused great concern from people gradually.How to detect the CEF residues quickly has became the problem resolved urgently.Chloramphenicol(CAP) is a highly-effective broad-spectrum antibiotics which is widely used in stockbreeding. But it has been found that CAP residues in animal tissues and/or animal products has toxic effects to human body.Therefore,it is necessary to work out an analytical method to monitoring CAP residues.
In the paper,the technique of molecular immunology was used to the preparation of the anti-CEF polyclonal antibody and the anti-CAP polyclonal antibody,The two above polyclonal antibody establish the foundation for the preparation of their monoclonal antibody and the exploitation of their ELISA Kit.The main content and results of the study are descripted as follow:
1 The anti-ceftiofur polyclonal antibody was prepared successfully.
1.1 The chemical crosslinking was used to conjugate ceftiofur(CEF) to BSA and obtained artificial immunogen CEF-BSA,the coating antigen CEF-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CEF-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.
1.2 Balb/c mice was immunized with CEF-BSA,the titre of polyclonal antibody(pAb) was 135000 by indirect ELISA,The linear range of calibration curves to detect CEF was 100 ng/mL-1000 ng/mL,The curve equation is y=-2.6x+206.0,coefficient correlation r=0.98,its IC_(50) is 206.0 ng/mL and the limit of detection was 49.1 ng/mL, The recoveries ranged from 79.2%to 94.5%for detecting CEF residues in milk and the coefficients of variation was 5.1%to 13.4%.
2 The anti- chloramphenicol polyclonal antibody was prepared successfully.
2.1 The active group carboxyl was introduced to Chloramphenicol(CAP) and formed CAP half succinaate(CAP-HS).The carbodiimide(EDC) was used to conjugate CAP-HS to BSA and obtained artificial immunogen CAP-HS-BSA,the coating antigen CAP-HS-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CAP-HS-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.
2.2 Rabbits and Balb/c mice were immunized with CAP-HS-BSA,the titre of polyclonal antibody(pAb) was 162000 by indirect ELISA,The linear range of calibration curves to detect CAP was 10 ng/mL-100 ng/mL,The curve equation is y=-4.3x+32.4,coefficient correlation r=0.98,its IC_(50) is 32.4 ng/mL and the limit of detection was 0.94 ng/mL,The recoveries ranged from79.4%to 98.5%for detecting CAP residues in eggs and the coefficients of variation was 0.5%to 4.3%.
引文
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