半抗原驱动有机磷农药抗体分离可变区分子进化及其非竞争免疫分析基础

Generally,small hapten pollutants with low molecular weight are not considered amenable to sandwich enzyme-linked immunosorbent assay (ELISA) due to their difficulty of simultaneous recognition by two antibodies. Therefore,the immunoassays for small haptens are always based on a competitive model. However, the competitive immunoassay model has several disadvantages such as lower sensitivity and specificity, high background than sandwich ELISA. So,the competitive assay model has become one of the bottlenecks limiting sensitivity for small hapten pollutants in immunoassay. Previously, a single antibody variable region (scFv) directed against O,O-diethyl phosphorothioate (DPE), a group of organophosphorus pesticides,was obtained in our group. In this project, with DPE as target, based on "hapten-driven Fv stabilization mechanism', we intend to develop a novel noncompetive immunoassay model by through molecular envolution of separated antibody variable region VH and VL chains, namely, split fragment variable(spFv). The contents include three aspects (1)Analysis of molecular recognition between DPE and recombinant scFv based on bioinformatics. The three-dimensional model of scFv is obtained by homology modeling and the amino acid residues responsible for binding DPE in complementarity determining region (CDR) of anti-DPE scFv are determined through molecular docking. (2)Establisment of hapten-driven envolution system of spFv based on ribosome display. The randomized VH (or VL) fragments CDR of scFv is displayed by ribosome in vitro and mixed with the target DPE, before being applied to VL(or VH) , which is immobilized on microtiterplates and subjected to rapid selection by panning, futher to obtain specific spFv showed significant binding toward DPE.(3)Development of noncompetetive immunassay for DPE based on bifunctional VH or VL and evaluation of the assay effects. The soluble spFv fragments selected are expressed as VH/VL-alkaline phosphatase or VH/VL-biotin fusion protein in Escherichia coli, purified, and subjected to a noncompetitive "open-sandwich" ELISA assay for DPE. The affinitiy, sensitivity, working range and measurement time will be evaluated by established assay method. The results from our project will be useful and meaningful for improving the immunoassay technology for all the other small hapten pollutants.

"竞争法检测模式"是当前限制小分子污染物免疫分析灵敏度的技术瓶颈之一。针对此，以二乙氧基硫代磷酸酯类(DPE)有机磷农药为代表性研究对象，在前期获得特异性单链抗体工作基础上，以定向进化抗体分离可变区、构建非竞争检测新模式为目标，重点开展3 方面研究：1）基于生物信息学的半抗原DPE与重组抗体分子识别特征；2）半抗原驱动的抗体分离可变区分子进化系统；3）功能化分离可变区对DPE"夹心"分析基础。通过同源模建，分子对接，解析抗体与药物分子结合结构特征，重点进化与药物分子直接作用的热点氨基酸残基；利用抗原存在可使抗体重链、轻链结合稳定性增强这一机制，借助核糖体展示，以半抗原驱动，筛选亲和力成熟抗体分离可变区，并与功能性蛋白融合表达，以此探索有机磷农药非竞争免疫分析新模式。本研究方法对其他小分子污染物的研究具有借鉴性，研究成果将对提升食品小分子污染物免疫分析水平及深化免疫分析理论基础均有重要意义。

针对小分子污染物免疫分析中"竞争法”检测模式本身灵敏度有限的技术瓶颈，本项目以二乙氧基硫代磷酸酯类有机磷农药（DPPs）为代表性研究对象，开展了基于半抗原驱动抗体可变区的非竞争检测新模式研究。得到的结果如下：. 基于生物信息学，建立了一种抗体可变区筛选分析的新方法（授权专利201110420334.0），以此得到了针对半抗原DPPs的单链抗体基因片段，进而对抗体结构进行了三维模建以及与药物分子的分子对接，结合分子对接打分和19种有机磷农药与抗体的IC50之间的线性相关性分析以及单链抗体定点突变实验，明确抗体活性口袋当中与药物结合的特异氨基酸CDR-L3的Trp92、Thr93、Thr94和Arg96，CDR-H2的Gln168、Ala170，CDR-H3的leu212、Tyr213起主要识别作用，特别是关键氨基酸位点Arg96，直接影响到抗原抗体结合的亲和力。同时，对VH/VL之间的结合能进行计算，并对比药物存在前后结合能的大小，发现获得的一对VH和VL片段在有DPPs存在时，结合能达到1.4477×104kJ/mol，远高于单纯VH和VL之间的结合力-4.50×10-1kJ/mol，以此筛选获得有效抗体可变区VH、VL片段。进而通过载体构建、转化大肠杆菌并诱导表达，分别得到20kDa大小的VL-Flag标签融合表达蛋白和70kDa大小的VH-碱性磷酸酶AP融合表达蛋白，实现抗体可变区的双功能化。最后，以获得双功能化的抗体可变区VL-Flag和VH-AP建立乙基对硫磷农药OS-ELISA非竞争免疫分析方法，该方法的检测限为 1ng/mL，IC50 分别为 6.75 ng/mL，线性范围为1.22~37.26 ng/mL，与基于有机磷单链抗体建立的竞争ELISA方法相比（IC50为26.7 ng/mL），本项目建立的非竞争模式比竞争模式灵敏度提高4倍。. 在本项目资助下，在J AGR FOOD CHEM等杂志上发表论文12篇，其中SCI收录8篇，EI收录3篇，其他1篇；授权专利1项；培养研究生6人，其中博士生1名。本项目相关成果作为部分内容获得2016年中国植保学会科技一等奖1项。