Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism
Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism
复制标题
开发基于单克隆的 ic-ELISA 测定动物组织中的吉他霉素并模拟研究其分子识别机制
DOI：
10.1016/j.foodchem.2021.129465
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发表时间：
2021-07-08
期刊：
影响因子：
8.8
通讯作者：
Peng, Dapeng
中科院分区：
文献类型：
--
作者：
Li, Long;Hou, Ren;Peng, Dapeng
为了监测吉他霉素(KIT)的残留，制备了抗KIT的单抗，通过对最敏感的抗体KA/2A9的优化，获得了对KIT最敏感的抗体KA/2A9的半数抑制浓度(IC50)为5.7+/-1.4mU/L。KIT在不同动物组织中的检出限范围为22.47~29.32mU g/kg，强化组织的回收率为70%~120%，变异系数小于20%。在此基础上，首次制备了试剂盒特异性单链抗体Ka/2A9/3。同源模建和分子对接结果表明，KA/2A9/3scFv的关键氨基酸是TYR-92(CDRL3)、SER-93(CDRL3)、ASP-155(CDRH1)和GLY-226(CDRH3)，其中氢键是主要作用力。然后，虚拟突变提供了一种进化KA/2A9/3单链抗体的方法。这些结果有助于了解抗KIT单链抗体的抗原抗体结合机制，并为抗KIT单链抗体的体外亲和力成熟提供有效信息。
To monitor the residue of kitasamycin (KIT), a monoclonal antibody against KIT was prepared, and a 50% inhibition concentration (IC50) of 5.7 +/- 1.4 mu g/L was achieved with the most sensitive antibody, KA/2A9, by optimizing ELISA conditions. The LODs for KIT in different animal tissues ranged from 22.47 mu g/kg to 29.32 mu g/kg, and the recoveries of the fortified tissues were 70% similar to 120% with coefficients of variation below 20%. Then, KIT-specific scFv KA/2A9/3 was prepared for the first time. Homologous modeling and molecular docking results indicated that the key amino acids of KA/2A9/3 scFv are TYR-92 (CDRL3), SER-93 (CDRL3), ASP-155 (CDRH1) and GLY-226 (CDRH3), and the hydrogen bond is the main force. And then, virtual mutation provides a method to evolve KA/2A9/3 scFv antibodies. These results contribute to comprehending the antigen-antibody binding mechanism and provide effective information for in vitro affinity maturation of anti-KIT scFv.