脱氧雪腐镰刀烯醇（呕吐毒素）酶标抗原的合成及免疫抗体的制备

Preparation of enzyme-labled antigen andantiserum for deoxynivalenol(vomiting toxin)

利用琥珀酸酐法合成了脱氧雪腐镰刀烯醇-琥珀酸酐衍生物(3-HS-DON),并用DCC法将该衍生物与牛血清白蛋白(BSA)偶联得到的结合物(DON-HS-BSA)作为免疫抗原免疫新西兰大白兔,获得效价为1∶10000的特异性抗脱氧雪腐镰刀烯醇(DON)的抗体。同时,用EDC法将3-HS-DON与辣根过氧化物酶(HRP)偶联得到酶标抗原(DON-HS-HRP)。通过圆二色光谱(CD)看到偶联后蛋白二级结构发生了改变,酶活稍有下降;用分光光度法测得酶标抗原酶活损失<5%,偶联物中DON与HRP的结合比为5.29。说明:该酶偶联物及制备的抗体可作为酶联免疫吸附法(ELISA)或免疫传感器法测试脱氧雪腐镰刀烯醇时所需的酶标抗原及抗体。

A succinyl derivation of deoxynivalenol（3-HS-DON）was prepared using the method of mixed anhydride reaction .The derivation was conjugated to BSA using the reagent DCC.Polyclonal antibody to DON was prepared from rabbits immunized with DON-HS-BSA conjugate.High titer（1∶10000）multiclonal anti-DON antisera were obtained.Meanwhile the derivation 3-HS-DON was also conjugated to horseradish peroxidase（HRP）using the reagent EDC.The molar incorporation ration of 3-HS-DON into HRP in the purified conjugation product was determined by ultraviolet spectrophotometry.By circular dichroism（CD）of study the change of secondary structure and activity before and after the reaction by circular dichroism（CD）.The results showed that the lose of HRP activity was less than 5% and the molar incorporation ration of 3-HS-DON into HRP reached 5.29.Thus the enzyme-labled antigen（DON-HS- HRP）and the polyclonal antibody can be used for the determination of deoxynivalenol by ELISA or immunosensor.