硝基呋喃代谢物多价半抗原的抗体诱导及抗体与代谢物原型识别机制研究

The safety of antibiotics metabolites degraded in animal has raised public concerns. A lot of these metabolites are with low molecular weight (<150 Da); therefore the direct determination of these metabolites without derivatization using immunoassays is still unavailable. Previously, the hapten of 3-amino-2-oxazolidone (AOZ, 102Da), the main metabolite of furazolidone, was conjugated to the two amido of lysine to obtain a divalent hapten and then coupled to carrier protein as new immunogen. Antibodies specific to AOZ was obtained by using this new immunogen, while it failed to obtain specific antibodies to AOZ using the traditional immunogen (direct coupling AOZ hapten to carrier protein). Based on this interesting result, in this project, we intend to synthesize haptens against nitrofuran with multiple level using lysine as basal unit. The antibodies production ability induced by the multiple haptens will be studied. The relationship model between the multiple haptens and the antibodies affinity will be constructed. Based on the obtained antibodies, the specificity and recognition mechanism between the antibodies and nitrofuran metabolites will be studied. Finally, immunoassays without derivatization to determine nitrofuran metabolites will be developed. The results of this project will provide a new strategy on the production of antibodies with high affinity for haptens with super-low molecule weight.

抗生素在动物体内代谢产物的安全性已引起人们的广泛关注。多数代谢产物的分子量很低（一般＜150 Da），国际上直接针对其代谢物原型的免疫检测尚未取得突破。本课题组前期制备了呋喃唑酮（硝基呋喃类药物中的一种）代谢产物3-氨基-2-恶唑烷酮（AOZ，102 Da）的2价半抗原，与载体蛋白偶联后可以刺激动物产生特异性免疫应答。本项目拟在此基础上，合成硝基呋喃类药物代谢物的多价半抗原，探讨其诱导产生特异性识别代谢物抗体的能力，建立不同价数半抗原与抗体亲和力的关系模型，在获得抗体的基础上研究抗体与代谢物的反应特性及其识别机制，最终建立检测对象无需衍生化（即代谢物原型）的免疫检测方法。本项目的实施不但为直接检测硝基呋喃代谢产物原型提供新方法，而且为建立其他超低分子量有害物的免疫检测方法提供理论与技术基础，具有重要的意义。

本项目重点以硝基呋喃代谢物AMOZ和AOZ为对象，针对其分子量小、结构简单等特点，引入赖氨酸树状分子作为载体，研究其提高半抗原免疫原性的能力，主要的结果如下：（1）合成了系列半抗原，并以赖氨酸为基础单元合成了从2价到8价的树状分子，制备树状半抗原和树状抗原，通过动物免疫及抗血清鉴定，研究了不同价数半抗原的抗体诱导能力及其抗体活性，建立半抗原价数与抗体亲和力的关系模型。结果发现4价树状分子具有显著提高半抗原免疫原性的效果，与传统载体蛋白相比，其更有利于刺激动物免疫应答产生抗体，并基于此制备了特异性识别AMOZ的抗体；（2）制备了AMOZ特异性单克隆抗体，并建立了直接检测AMOZ的无需衍生化的免疫分析方法，半抑制浓度IC50值为20.7ng/mL，方法的检出限IC10为0.95ng/mL，检测范围为IC20~IC80：2.92ng/mL ~145.2ng/mL；同时建立了AMOZ和AOZ荧光偏振、量子点标记免疫层析试纸条等多种免疫分析方法，并进行实际应用；（3）利用噬菌体展示技术筛选制备了AMOZ单链抗体，经鉴定与亲本单克隆抗体具有相似性能，利用同源模建、分子动力学和分子对接等技术建立了AMOZ单链抗体与半抗原的识别模型，明确了Ser32、Tyr33、Arg99、Asp100、Tyr102、Tyr220、Asp221等七个氨基酸残基为关键识别氨基酸残基，后续定向进化仍在研究中。本项目结果为建立其他超低分子量有害物的免疫检测方法提供理论与技术支撑。在项目资助下，发表了SCI论文6篇，EI论文1篇；获得授权发明专利2项；项目组2名成员晋升高级职称，已培养博士毕业生1名，硕士毕业生3名，本科毕业生8名；在读博士研究生1名。