玉米赤霉烯酮结构类似物在免疫亲和柱柱容量实时监测中的应用

Application of zearalenone structural analogues in real-time monitoring of column capacity in immunoaffinity columns

中文关键词:  玉米赤霉烯酮  免疫亲和柱  高效液相色谱法  柱容量  实时监测

英文关键词:zearalenone  immunoaffinity column  high performance liquid chromatography  column capacity  real-time monitoring

中文摘要:

      目的 探索一种可以在检测过程中实时监测玉米赤霉烯酮(zearalenone, ZEN)免疫亲和柱柱容量的方法。方法 将两种ZEN的天然结构类似物: α-玉米赤霉烯醇(α-zearalanol, α-ZEL)、β-玉米赤霉烯醇(β-zearalanol, β-ZEL)与戊二酸酐酯化反应合成了ZEN的两种人工结构类似物(α-ZEL-G、β-ZEL-G), 样品纯化后, 通过荧光、紫外、高分辨率质谱法对其进行了鉴定; 利用免疫亲和柱的抗体建立了竞争酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)曲线, 通过半抑制浓度(half maximal inhibitory concentration, IC50)比较它们与抗体之间的亲和力。分别将α-ZEL-G和β-ZEL-G作为柱容量示踪物, 与不同浓度的ZEN一起加入免疫亲和柱结合, 探索建立一种免疫亲和柱柱容量实时监测的方法。结果 ZEN、α-ZEL-G和β-ZEL-G的竞争ELISA曲线的IC50分别为: 2.0、1.3和10.0 ng/mL, 表明α-ZEL-G与抗体的亲和力略高于ZEN, 而β-ZEL-G与抗体的亲和力则明显低于ZEN与抗体的亲和力。实验结果表明: α-ZEL-G在不同柱容量的免疫亲和柱中会影响ZEN的回收率, 而β-ZEL-G的加入量在柱容量50%时, 不同浓度的ZEN的回收率大于80%。结论 与抗体亲和力更低的β-ZEL-G更适合用于柱容量实时监测, 在实际样品的检测中显示出良好的应用效果。

英文摘要:

      Objective To explore a method that can monitor the immunoaffinity column capacity of zearalenone (ZEN) in real time during detection. Methods The 2 kinds of artificial structural analogues of ZEN (α-ZEL-G and β-ZEL-G) were synthesized by esterification of α-zearalanol (α-ZEL) and β-zearalanol (β-ZEL) with glutaric anhydride in this study. The products were purified and identified by fluorescence, ultraviolet and high-resolution mass spectrometry. A competitive enzyme-linked immunosorbent assay (ELISA) curve was established using the same antibody with immunoaffinity columns, and their affinity with the antibody was compared by half maximal inhibitory concentration (IC50). α-ZEL-G and β-ZEL-G were used as column capacity tracers and added to the immunoaffinity column together with different concentrations of ZEN to explore a method for real-time monitoring of immunoaffinity column capacity. Results The IC50 of the competitive ELISA curves of ZEN, α-ZEL-G and β-ZEL-G were 2.0, 1.3 and 10.0 ng/mL, respectively, indicating that the affinity of α-ZEL-G with antibody was slightly higher than that of ZEN, while the affinity of β-ZEL-G with antibody was significantly lower than that of ZEN with antibody. The experimental results showed that: α-ZEL-G in immunoaffinity columns with different column sizes would affect the recovery rates of ZEN. Whereas the recovery rates of ZEN with different concentration were more than 80% while the adding concentrations of β-ZEL-G was 50% of the column capacity. Conclusion β-ZEL-G with lower antibody affinity is more suitable for real-time monitoring of column volume. It shows good application results in the test of actual samples.