新型抗A0Z/AMOZ双特异性单链抗体的构建及其多残留同时免疫分析方法研究

Furazolidone and furaltadone are nitrofuran antibacterial agents,they were once widely used in livestock and poultry breeding and aquaculture industry.The two kinds of agents have been banned for use as a veterinary medicine in food animals in many countries because these parent drug and theirs metabolite exhibit high toxic,carcinogenic,and mutagenic effects.However furazolidone and furaltadone are still illegally used in some countries because they are cheap and effective.Thus,it is necessary to continuously monitor for the misuse of furazolidone and furaltadone.Several AOZ derivative immunogen will be synthesized and used for immunizeing animal in this project.The against AOZ derivatives monoclonal antibody hybridoma cell will be obtained by hybridoma technique.The gene of scFv antibody against AOZ derivative will be selected and obtained by phage display technique on the basis of the hybridoma cell prepared in this project.The gene of scFv antibody against AMOZ derivative had been obtained in our previous research work.The two scFv genes will be ligated by SOE-PCR technique and the anti-AOZ/AMOZ bispecific single-chain antibody gene will be constructed and expressed and purified. Several kinds of AOZ/AMOZ multiresidue immunoassay new methods based on the bispecific single-chain antibody will be studied and established in this project.This project will provide a kind of new and low-cost and highly specific recombinant antybody for the study of AOZ/AMOZ multiresidue immunoassay and will provide new research idea and gateway for the immunoassay methods establishing of veterinary drugs multiresidue and will provide scientific basis for development of commercial test kit of veterinary drugs multiresidue with recombinant antybody in future.

呋喃唑酮和呋喃它酮是硝基呋喃类抗生素，在畜禽水产养殖业中曾被广泛应用，因其原药及代谢物具有很大毒性和致畸致癌致突变作用，已禁止使用，由于价格低疗效好，目前非法使用现象仍很严重，有必要加强监控。本项目拟合成多种呋喃唑酮代谢物（AOZ）衍生物免疫原，免疫动物后采用杂交瘤技术筛选抗AOZ衍生物单克隆抗体杂交瘤细胞，从此杂交瘤细胞出发，采用噬菌体抗体库技术构建筛选抗AOZ衍生物单链抗体基因，通过重叠延伸PCR技术将其和前期工作已获得的抗呋喃它酮代谢物(AMOZ)衍生物单链抗体基因连接，构建抗AOZ/AMOZ双特异性单链抗体基因并表达纯化，研究建立基于此双特异性单链抗体的AOZ/AMOZ多残留同时免疫分析新方法。本项目将为A0Z/AMOZ多残留免疫检测提供新型低成本高特异的重组抗体，为兽药多残留免疫检测方法建立提供新的研究思路和途径，为今后利用重组抗体开发兽药多残留免疫检测商品化试剂盒提供科学依据。

背景：呋喃唑酮和呋喃它酮是硝基呋喃类抗生素，在畜禽水产养殖业中曾被广泛应用，因其原药及代谢物具有很大毒性和致畸致癌致突变作用，已禁止使用，由于价格低疗效好，目前非法使用现象仍很严重，有必要加强监控。. 主要研究内容：AOZ衍生物免疫抗原和包被抗原的合成和鉴定；动物免疫和抗AOZ衍生物单克隆抗体阳性杂交瘤细胞筛选; 抗AOZ衍生物单链抗体构建和制备; 抗AOZ/AMOZ双特异性单链抗体构建和制备; 基于双特异性单链抗体的AOZ/AMOZ多残留同时免疫分析方法研究。. 重要结果和关键数据：①合成并鉴定了9种计划书所设计的AOZ衍生物半抗原和10种文献未报道过且计划书内没有设计的新型AOZ衍生物半抗原，得到了一系列鉴定结果正确的AOZ衍生物完全免疫抗原（19种）和包被抗原（20种）。②小鼠免疫实验发现大部分AOZ衍生物免疫抗原都能得到特异性多克隆抗体，只是特异性有较大的差别。③选取免疫效果较好的免疫小鼠作为后续细胞融合试验的对象，采用杂交瘤技术筛选得到了一株抗AOZ衍生物单抗的阳性细胞株1D2，制备得到的腹水单抗效价为1:50万（间接ELISA），特异性IC50为31.56 ng/mL（icELISA），单抗亚型重链是IgG2b型，轻链是Kappa型。④从抗AOZ衍生物单抗阳性杂交瘤细胞出发，构建制备得到的抗AOZ衍生物单链抗体特异性IC50为40.23 ng/mL（dcELISA）。⑤构建制备得到的抗AOZ/AMOZ双特异单链抗体特异性IC50为75.16 ng/mL（呋喃唑酮 ）+36.78 ng/mL（呋喃它酮）（dcELISA）。⑥利用本项目制备得到的双特异性单链抗体建立了AOZ/AMOZ多残留同时直接竞争酶联免疫分析（dcELISA）和化学发光免疫分析（dcCLEIA）两种方法。利用建立的这些方法对鱼肉、虾肉、鸡肉等动物性食品中AOZ/AMOZ多残留进行了同时免疫检测并用液相色谱-质谱进行了确证。. 科学意义：本项目为AOZ/AMOZ多残留同时免疫检测提供了一种新型的特异性较好、成本较低的基因工程双特异抗体，为兽药多残留同时免疫检测多特异重组抗体制备和分析方法建立提供了新思路，为今后利用重组抗体开发兽药多残留免疫检测商品化试剂盒提供了科学依据，具有很好的应用前景。