麻痹性贝类毒素多特异性抗体识别特性及高灵敏比率荧光免疫分析研究

Paralytic shellfish poisoning toxins (PSPts) is one of the key toxins which pose a serious threat on the safety of edible marine products and prosperous development of our national marine products trade. Currently, immunoassays for PSPts are mainly capable of analyzing one or a few PSPts simultaneously since antibodies exhibit high affinity to one or a few PSPts, but show low binding affinity to other PSPts, which results in high false negative rate. To address these problem, in this project, novel general hapten against PSPts will be designed to induce broad-specific antibodies against PSPts by computer-assisted molecular modeling. Then, intensive investigation of the recognition mechanism between antibody and PSPts will be performed to provide guiding theory for improving hapten designing, thus inducing broad-specificity improved antibodies against PSPts. Meanwhile, taking advantage of the high sensitivity and precision of ratiometric florescent analysis technology, some nano-materials based ratiometric fluorescent sensing system highly sensitively responding to enzymatic reactions and enzyme-functionized immuno-nanoprobe will be constructed by virtue of nanotechnology and biological labeling strategy. During this process, exploration of fluorescence energy transfer mechanism, optimization of working conditions for these ratiometric fluorescent sensing system and systematic investigation of the design, preparation of immuno-nanoprobe. Finally, a rapid, sensitive, stable and economical ratiometric fluorescent immunoassay for visual detection of PSPts will be developed. It is anticipated that this project will not only enhance the monitoring of PSPts in edible marine products and offshore sea environment, but also providing a technology platform for rapid detection of other chemical poisons residues in foods.

麻痹性贝类毒素（PSPts）是影响海洋水产品食用安全及进出口贸易的重要毒素，当前其免疫分析存在检测对象少、灵敏度不足等问题。本项目拟借助计算机分子模拟创新PSPts通用半抗原设计，制备广谱特异性抗体并深入研究抗体识别特性，进而指导半抗原改进设计，最终获得广谱特异性显著提高的通用抗体。同时利用纳米材料和生物标记技术，构筑多种对酶促反应高灵敏响应的比率荧光传感体系及酶功能化免疫纳米探针以放大检测信号，研究基于不同构筑方式的比率荧光传感体系的工作机制及其传感性能，系统对功能化纳米探针的设计、制备、及信号放大规律进行研究。发展快速、灵敏、稳定的PSPts多残留比率荧光免疫分析方法。本研究将提升对食用海产品中PSPts的监测与防控能力及为食品中其他化学有害物残留快速检测提供技术借鉴有重要意义。

项目以麻痹性贝类毒素（PSPts）为研究对象，开展PSPts广谱识别抗体制备，抗体识别特性及高灵敏比率荧光免疫分析分析方法研究等内容研究，取得的主要研究成果如下：（1）借助计算机模拟优选PSPts中的C1/C2与STX为半抗原，制备了两株可分别识别四种PSPts的广谱性多克隆抗体（anti-C1/C2与anti-STX）。其中anti-STX对PSPts的亲和力更高，对STX、dcSTX、GTX2/3、NEO的半抑制浓度（IC50）分别达到0.16 ng/mL、0.28 ng/mL、0.57 ng/mL、5.2 ng/mL。（2）构建一种基于纳米酶-天然酶级联催化、邻苯二胺（OPD）为单一荧光信号前驱体，对碱性磷酸酶（ALP）高灵敏响应的比率荧光传感体系，与基于ALP免疫分析体系结合，建立了一种高灵敏检测贝类样品中PSPts的比率荧光免疫分析方法，检测限（LOD）达到0.8 ppb，较传统ELISA方法提升6.25 倍。（3）针对天然蛋白酶稳定性不足，以稳定性更好、成本更低的纳米酶代替天然辣根过氧化物酶（HRP）标记抗体构建免疫探针，建立了一种天冬氨酸掺杂普鲁士蓝纳米酶介导的PSPts比率荧光分析方法， LOD达到2.75 ppb，满足国际限量标准。（4）发表论8篇，其中SCI论文5篇，EI论文2篇，核心期刊1篇，申请专利1件，授权1件；（5）项目执行期间，项目负责人由讲师晋升为副教授，培养毕业研究生2名，读研究生2名。