可替宁单克隆抗体的制备及其在免疫学检测中的应用

目的: 研制可替宁单克隆抗体（单抗），并建立检测人体尿液样本中可替宁的免疫检测方法。方法: 利用人工合成的可替宁-牛血清白蛋白（COT-BSA）免疫BALB/c小鼠，通过细胞融合技术筛选一种对可替宁具有高特异性和敏感性的单抗，并将该单抗用于建立间接竞争ELISA（ic-ELISA）和胶体金免疫层析试纸，检测人体尿液中可替宁的含量。结果: 所研制的可替宁单抗经ic-ELISA鉴定，半数有效抑制浓度为21 ng/mL，经胶体金免疫层析试纸鉴定，截断值为100 ng/mL。所建立的ic-ELISA方法检测限为0.1 ng/mL，回收率为99.41%~117.98%，批间和批内变异系数分别小于等于15.31%和15.07%。所建立的胶体金免疫层析试纸稳定性和重复性检测准确率均为100%。结论: 以制备的可替宁单抗为基础开发的ic-ELISA和胶体金免疫层析试纸可快速、可靠地检测人体尿液中可替宁含量，可作为环境烟草烟雾在人体内暴露程度的有效检测方法。

Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection

Objective: To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples. Methods: BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine. Results: The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC₅₀) value of 21 ng/mL; and it was also identified by colloidal gold immunochromatographic strip assay with a cut-off value of 100 ng/mL. For ic-ELISA, the range of detection was 0-100 ng/mL with a minimal limit of 0.1 ng/mL; the recovery of assay was 99.41%-117.98%, and the intra-assay and inter-assay coefficient variations were not higher than 15.31%and 15.07%, respectively. For colloidal gold immunochromatographic strip assay, the accuracy of stability and repeatability both were 100%. Conclusion: The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.