莫能菌素多克隆抗体制备及应用
摘要
本文合成莫能菌素结合抗原，制备高特异性抗体，改变包被原的结构，以提高酶联免疫吸附测定(enzyme-linkedimmunosorbentassay，ELISA)灵敏度，并在成功研制多克隆抗体基础上，初步进行了应用。旨在建立一种简便、可信、快速、灵敏的莫能菌素残留分析方法，以对组织中莫能菌素残留进行监控，保证畜禽产品的质量。
1．莫能菌素抗原制备
生产中制备的莫能菌素以钠盐形式存在，没有直接与选用的载体蛋白质(BSA、OVA)偶联的基团，因此不能直接用于人工抗原的合成，必须加以修饰形成含羧基的物质才能合成人工抗原。本试验采用琥珀酸酐法合成莫能菌素-琥珀酸酐衍生物(MON-HS)，并通过薄层层析进行鉴定。采用混合酸酐法将莫能菌素-琥珀酸酐(MON-HS)与载体蛋白(BSA，OVA)共价偶联，合成免疫抗原(Mon-HS-OVA)，合成的免疫原通过SDS-PAGE电泳法进行鉴定，并通过蛋白染色法和香草醛法测得MON与BSA的结合比为14.7：1。
2．抗莫能菌素多克隆抗体制备
用合成的Mon-HS-BSA免疫新西兰大白兔，多次免疫后取血清，经间接ELISA法测定抗体效价为1：25600(2号)。以MON浓度的对数为横坐标，抑制率为纵坐标，作标准曲线，用这种方法确定的I_(50)为32.4ng／ml，以90％抑制率为其灵敏度，灵敏度为2.75ng／ml。为检验抗体的特异性，用盐霉素，海南霉素，磺胺间甲氧嘧啶等3种药物作为竞争抗原，做ELISA，结果显示与这3种药物没有交又反应。
3．抗莫能菌素多克隆抗体应用
为了验证实际检测效果，通过样品给药实验，证实了可以用ELISA法测定鸡蛋中的MON。
This paper includes three parts of research, synthesis of the antigen and development of the polyclonal antibodies against MON and application for the determination of MON residues in eggs, the results was shown as follows:
1. Development of the antigen against MON
In production Monensin is existed as Monensin sodium salt. However, Monensin sodium salt has not any group which are directly coupled with protein conjugations such as BSA,OVA. So it can not synthesize immugen directly and must be modified to the substance contained of carboxyl to produce antibody. We applied succinate acid anhydride method for transformation to monensin-succinate acid anhydride derivative. The materials of the transformation were appraised by thin layer chromatography. The result of appraising is approved successfully.
The Mon-HS was coupled with protein (BSA, OVA) by the mixed acid anhydride reaction method. The conjugation included the immunogen(Mon-HS-BSA) and the coating antigen(Mon-HS-OVA). The synthesized antigens were appraised by SDS-PAGE. The protein-hapten conjugate rate was 14.7:1(Mon-HS-BSA). 2. Development of the polyclonal antibodies against MON
High titer polyclonal antibodies against Monensin were produced from rabbits immunized with the synthesized immunogen conjugates Mon-HS-BSA. Through the indirect competitive enzyme-linked immunosorbent assay(icELISA).The final titer of antibodies were 1:25600(No.2). Calibration graphs were prepared by plotting log (MON concentration) against percentage binding. The percentage binding was calculated from the absorbance obtained in the absence (Bo) and the presence (B) of MON in standards and samples as follows: binding= B/Bo. The I_(50) of this assay was established to be 32.4ng /ml, and the sensitivity of detection was 2.75ng/ml. To test the speciality of antibodies, SAL and HAN and SMM were used in the competitive ELISA as the competitive antigen. The result proved that the antibodies shows no cross-eaction to the other three antibiotics.
1．莫能菌素抗原制备
生产中制备的莫能菌素以钠盐形式存在，没有直接与选用的载体蛋白质(BSA、OVA)偶联的基团，因此不能直接用于人工抗原的合成，必须加以修饰形成含羧基的物质才能合成人工抗原。本试验采用琥珀酸酐法合成莫能菌素-琥珀酸酐衍生物(MON-HS)，并通过薄层层析进行鉴定。采用混合酸酐法将莫能菌素-琥珀酸酐(MON-HS)与载体蛋白(BSA，OVA)共价偶联，合成免疫抗原(Mon-HS-OVA)，合成的免疫原通过SDS-PAGE电泳法进行鉴定，并通过蛋白染色法和香草醛法测得MON与BSA的结合比为14.7：1。
2．抗莫能菌素多克隆抗体制备
用合成的Mon-HS-BSA免疫新西兰大白兔，多次免疫后取血清，经间接ELISA法测定抗体效价为1：25600(2号)。以MON浓度的对数为横坐标，抑制率为纵坐标，作标准曲线，用这种方法确定的I_(50)为32.4ng／ml，以90％抑制率为其灵敏度，灵敏度为2.75ng／ml。为检验抗体的特异性，用盐霉素，海南霉素，磺胺间甲氧嘧啶等3种药物作为竞争抗原，做ELISA，结果显示与这3种药物没有交又反应。
3．抗莫能菌素多克隆抗体应用
为了验证实际检测效果，通过样品给药实验，证实了可以用ELISA法测定鸡蛋中的MON。
This paper includes three parts of research, synthesis of the antigen and development of the polyclonal antibodies against MON and application for the determination of MON residues in eggs, the results was shown as follows:
1. Development of the antigen against MON
In production Monensin is existed as Monensin sodium salt. However, Monensin sodium salt has not any group which are directly coupled with protein conjugations such as BSA,OVA. So it can not synthesize immugen directly and must be modified to the substance contained of carboxyl to produce antibody. We applied succinate acid anhydride method for transformation to monensin-succinate acid anhydride derivative. The materials of the transformation were appraised by thin layer chromatography. The result of appraising is approved successfully.
The Mon-HS was coupled with protein (BSA, OVA) by the mixed acid anhydride reaction method. The conjugation included the immunogen(Mon-HS-BSA) and the coating antigen(Mon-HS-OVA). The synthesized antigens were appraised by SDS-PAGE. The protein-hapten conjugate rate was 14.7:1(Mon-HS-BSA). 2. Development of the polyclonal antibodies against MON
High titer polyclonal antibodies against Monensin were produced from rabbits immunized with the synthesized immunogen conjugates Mon-HS-BSA. Through the indirect competitive enzyme-linked immunosorbent assay(icELISA).The final titer of antibodies were 1:25600(No.2). Calibration graphs were prepared by plotting log (MON concentration) against percentage binding. The percentage binding was calculated from the absorbance obtained in the absence (Bo) and the presence (B) of MON in standards and samples as follows: binding= B/Bo. The I_(50) of this assay was established to be 32.4ng /ml, and the sensitivity of detection was 2.75ng/ml. To test the speciality of antibodies, SAL and HAN and SMM were used in the competitive ELISA as the competitive antigen. The result proved that the antibodies shows no cross-eaction to the other three antibiotics.
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