本项目针对基因工程抗体亲和力偏低，在痕量农药残留检测领域实际应用困难的突出问题。拟借鉴亲和力成熟技术的高效抗体重组、改性优势，发挥时间分辨荧光免疫分析高灵敏与多组分分析特点，完成以下工作：①对已筛选到的苏云金杆菌杀虫蛋白Cry1Ac和伊维菌素（IVM）单链抗体基因，采用错配PCR、定点突变等方法，分别构建2种大容量亲和力成熟库。②采用磁珠液相筛选和游离抗原洗脱等优化筛选技术，分离鉴定出20-30种Cry1Ac、IVM成熟抗体株。比较不同成熟策略效率，分析蛋白及小分子农药特异性抗体在成熟过程中结构修饰差异，预测成熟关键表位。③选择亲和力显著提高或具广谱识别能力的抗体株，经可溶性表达后，建立3-4种高灵敏或广谱Cry1Ac、IVM及其家族同系物的时间分辨荧光免疫分析方法。为农药基因工程抗体亲和力成熟技术做出探索，并建立高灵敏度的农药残留检测方法。

Due to its relative lower affinities, engineering antibodies usually have some outstanding problems in practical applications of detecting trace amounts of pesticides. This project intends to use affinity maturation and time-resolved fluoroimmunoassay to resolve these problems.①Two large-capacity affinity maturation libraries are separately constructed for single chain variable fragments(scFv)of Bacillus thuringiensis (Bt) Cry1Ac toxin and ivermectin (IVM) by error PCR and site-directed mutagenesis. ②20-30 positive clones against Cry1Ac and IVM are isolated from affinity maturation libraries by using magnetic beads and free antigen elution. The efficiency of these strategies and the different structural modification requirements of protein or hapten specific antibodies are discussed. The key epitopes of affinity maturation are also predicted. ③The clones which have significantly improvement in affinities or specificities are identified and expressed in soluble formats. Base on these antibodies,3-4 high sensitive or broad specific time-resolved fluoroimmunoassay are established for Cry1Ac, IVM or their analogues. This project will make an exploration in affinity maturation of engineering antibodies and their use in detecting trace amounts of pesticides.