酶联免疫吸附法快速检测畜禽产品中西马特罗药物残留

为建立高效的西马特罗(Cimaterol, CIM)检测方法,试验用以重氮化法合成的西马特罗完全抗原免疫新西兰大白兔,提取兔脾细胞总RNA,反转录合成cDNA第一链,设计兼并引物,扩增兔源抗体重链可变区(VH)和轻链可变区(VL),通过45 bp的连接肽(Linker)连接成VH-Linker-VL并大量扩增；利用噬菌体表面展示技术将单链抗体(ScFv)片段与噬菌体展示载体pCANTAB-5e连接,电转化至大肠杆菌TG1感受态细胞,得到兔源噬菌体ScFv抗体库。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)判断是否偶联成功,用紫外扫描(UV)及基质辅助激光解析串联飞行时间质谱(MALDI-TOF-MS)测定人工抗原的偶联比。将富集的抗体库侵染对数期大肠杆菌进行抗体库滴度测定合成抗体库的质量进行鉴定,对抗体库进行三轮淘选,取最优的CIM-ScFv进行氨基酸序列分析。结果表明：CIM-BSA及CIM-OVA偶联比分别为8:1及10:1,CIM-BSA免疫原免疫新西兰大白兔,间接ELISA测得免疫血清校价为1:32000,免疫血清半数抑制浓度为9.77 ng/mL, 曲线的拟合度为0.997。抗西马特罗兔源噬菌体ScFv免疫库的插入率为92%,库容为1×10_¹⁰ pfu,多样性为83.3%,经过4轮淘选,获得一株与CIM有结合活性的噬菌体ScFv菌。以上结果表明西马特罗完全抗原的合成效果良好,可用于建立西马特罗检测方法,兔源噬菌体抗体库为快速获得CIM单链抗体奠定基础,为西马特罗快速检测提供新思路。

Rapid detection of cimaderol residues in livestock and poultry products by enzyme linked immunosorbent assay

In order to establish an efficient CIM detection method, basing on the synthesis of cimaterol complete antigen by diazotization. Immunizing the New Zealand white rabbits and extracting the total RNA of rabbit splenocytes was extractedtotal. Merge primers are designed to amplify the heavy chain variable region (VH) and light chain variable region (VL) of rabbit antibody, which are connected into VH linker VL through linker. Then amplifying in large quantities. connect them to VH-Linker-VL through a 45 bp linker peptide (Linker) and amplify them in large quantities.Using the single-chain antibody fragment (ScFv) which links to the phage display vector pCANTAB-5e by phage surface display technology, and electroporated into E. coli TG1 Chemical Competent Cells to obtain a rabbit-derived phage ScFv antibody library. The polyacrylamide gel electrophoresis (SDS-PAGE) is used to determine whether or not the success of coupling. Thus, the coupling ratio of artificial antigens is determined by ultraviolet scanning (UV) and matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDI-TOF-MS). The enriched antibody library is infected with log-phase Escherichia coli, and the quality of the synthesized antibody library is determined by antibody library titer. The antibody library is subjected to three rounds of elimination, and the optimal CIM-ScFv is selected for amino acid sequence analysis. The results show that the coupling ratios of CIM-BSA and CIM-OVA are 8:1 and 10:1 respectively. The CIM-BSA immunogen is immunized with New Zealand white rabbits. The immune serum tilter is measured by indirect ELISA was 1:32000, and the half of the immune serum is inhibited. The concentration is 9.77 ng/mL, and the fitting degree of the curve is 0.997. The insertion rate of the anti-cimaterol rabbit-derived phage ScFv immune is 92%, the capacity is 1×10_¹⁰ pfu, and the diversity is 83.3%. After 4 rounds of elimination, a phage ScFv strain with binding activity to CIM is obtained as a result. The above results show that the synthesis effect of the complete antigen of CIM is good, and it can be used to establish the detection method of cimaterol. The rabbit-derived phage antibody capacity lays the foundation for the rapid acquisition of CIM single-chain antibody, and provides new ideas for the rapid detection of CIM.