Preparation and Identification of Zearalenone Complete Antigen
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摘要: 为了降低基层检测部门检测谷物中玉米赤霉烯酮（ZEN）的成本,研制国产化检测试剂盒非常必要,而试剂盒的研制离不开完全抗原的支持。本试验的主要内容是将ZEN肟化,生成玉米赤霉烯酮肟（ZENO）,再用DCC-NHS活性酯法将ZENO与牛血清白蛋白（BSA）、鸡卵白蛋白（OVA）偶联合成ZEN完全抗原,最后透析将未偶联的小分子ZEN除去,得到的即为完全抗原。包被原ZEN-BSA浓度为1.62 mg·mL-1,免疫原ZEN-OVA浓度为1.33mg·mL-1。采用UV法、SDS-PAGE法对完全抗原进行鉴定,同时建立间接ELISA方法验证包被原ZEN-BSA。结果表明:采用活性酯法合成的ZEN完全抗原在319nm处出现新的吸收峰,且SDS-PAGE图表明ZEN-BSA分子量集中在60～70kDa,ZEN-OVA分子量集中在40～50kDa,不同于BSA和OVA分子量,但相差不大。根据紫外扫描图计算得到ZEN-BSA结合比为7.0∶1,ZEN-OVA结合比为6.1∶1。根据免疫学方法可知,OD450nm值随着毒素浓度的增加而减小。本试验利用3种方法对ZEN完全抗原进行鉴定,证明ZEN完全抗原偶联成功。Abstract: In order to reduce the cost of detecting zearalenone（ZEN）in grains by the basic-level testing department,it is necessary to develop a domestically produced testing kit,and the development of the kit need the support of complete antigen.The main content of this experiment is to oximize ZEN to produce zearalenone oxime（ZENO）,and then combine ZENO with bovine serum albumin（BSA）and chicken ovalbumin（OVA）by the DCC-NHS active ester method.ZEN complete antigen.Finally,dialysis removes the unconjugated small molecule ZEN,and the result is complete antigen.The original coating ZEN-BSA concentration was1.62 mg·mL-1,and the immunogen ZEN-OVA concentration was 1.33 mg·mL-1.The UV method and SDSPAGE method were used to identify the complete antigen.At the same time,an indirect ELISA method was established to verify the original coating ZEN-BSA.The results showed that the ZEN complete antigen synthesized by the active ester method had a new absorption peak at 319 nm,and the SDS-PAGE chart showed that the molecular weight of ZEN-BSA was concentrated at 60-70 kDa,and the molecular weight of ZEN-OVA was concentrated at 40-50 kDa.Different from the molecular weight of BSA and OVA but not much different.The ZEN-BSA binding ratio was 7.0∶1 and the ZEN-OVA binding ratio was 6.1∶1 calculated according to the UV scan.According to the immunological method chart,the OD450 nmvalue decreased with the increase of the toxin concentration.This test uses three methods to identify the ZEN complete antigen,which proves that the ZEN complete antigen coupling is successful.
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Keywords:
- zearalenone /
- antigen synthesis /
- enzyme-linked immunosorbent assay /
- toxin detection
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