旋光异构毒氟磷抗体制备及其手性识别性能研究

Dufulin is a novel virus inhibitor which was developed and synthesized by the Center for Research and Development of Fine Chemicals of Guizhou University. In our research, the Polyclonal and monoclonal antibodies of the anti-Dufulin were obtained and the competitive inhibition enzyme-linked immunosorbent assay (ELISA) were established. Chrial antibodies were prepared and the difference among their ability of chiral recognition were preliminary investigated. The ELISA method were established for the quantitative detection of Dufulin after the reaction conditions were optimized. To explore the chiral recognition of the antibody and the structure-activity relationship between antibody and antigen through the data of cross-reaction. Three PcAb were prepared and ELISA detection methods were established. Chrial antibodies were preapared and the difference among their ability of chiral recognition were preliminary investigated. The ELISA data of cross-reactivity provide support to construct the quantitative structure activity relationship (QSAR) between antibody and antigen.

本项目以自主创制品种新型抗植物病毒剂毒氟磷为研究对象，合成制备毒氟磷外消旋体和两个旋光异构体半抗原和完全抗原，进而制备分析毒氟磷的多克隆抗体和单克隆抗体。通过条件优化，建立间接或直接竞争酶联免疫吸附（ELISA）检测方法。通过抗体和毒氟磷外消旋体和两个异构体之间的交叉反应，考察抗体的手性识别差异性。通过毒氟磷类似结构化合物及其旋光异构体的交叉反应数据，探讨抗原抗体构效关系，考察抗原抗体反应机制。预期建立基于单克隆抗体的毒氟磷的ELISA检测方法；在外消旋体和异构体多克隆抗体检测方法的基础上，构建抗原抗体定量构效关系模型。

项目以毒氟磷及其类似物为研究对象，通过杂交瘤技术建立抗毒氟磷及其类似物的高特异性杂交瘤细胞系，通过体内诱生腹水制备单克隆抗体，开发了检测毒氟磷及其类似物的酶联免疫分析方法，并对取得如下创新性结果和主要结论：.1. 根据毒氟磷及其类似物的分子结构，利用2-甲酰苯氧乙酸、氨基酸合成得到了分别含有3个亚甲基和5个亚甲基的带有羧基的半抗原DHF和DHS；从硝基取代的膦酸酯通过还原反应得到了带有氨基的半抗原DHN。.2. 通过碳二亚胺法将含有羧基的衍生物偶联到载体蛋白KLH和BSA上，戊二醛法将含有氨基的半抗原偶联到蛋白上，成功合成了人工抗原。Balb/c小鼠的三免血清ELISA实验结果表明，具有碳链间隔臂的人工抗原DHF-KLH和DHS-KLH具有良好的免疫原性和反应原性，为单克隆抗体的制备提供了理想材料。.3. 通过对Balb/c小鼠五免血清的间接ELISA试验和间接竞争ELISA试验。选择免疫效果好的抗原和小鼠，按照实验室常规单克隆抗体制备与鉴定方法，获得了一株生长稳定、分泌抗体效价高的杂交瘤细胞株DHS3A2，并进行了鉴定。.4. 在制备的单克隆抗体mAb-DHS3A2的基础上，通过对固相载体、有机溶剂、pH值、离子强度、封闭物种类、点板时间、工作浓度等因素的筛选比较，优化了毒氟磷间接竞争(ic-ELISA)和直接竞争ELISA(dc-ELISA)的方法和条件，并对两种方法的性能进行了比较。.5. 建立了土壤、烟草、番茄和中草药太子参中毒氟磷残留的ic-ELISA分析方法，分析了基质对ELISA检测结果的影响。结果表明，所建立的ic-ELISA方法与HPLC方法相关性良好，可以满足毒氟磷在这几种基质中残留分析的要求。.6. 采用ic-ELISA方法分析了不同水体和土壤中的毒氟磷的降解。建立的ELISA方法适用于环境水样和土壤中痕量毒氟磷含量的测定。降解试验结果表明，毒氟磷为易降解农药，以毒氟磷为有效成分的农药在合理施用后不易造成环境污染。.7. 采用2D-,3D-QSAR进行计算机分子模拟计算。