【摘要】 目的制备泰乐菌素(Tylosin)单克隆抗体,并建立泰乐菌素竞争ELISA检测方法。方法用羰基二咪唑将半抗原泰乐菌素分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,制备泰乐菌素免疫抗原Tylosin-BSA和检测抗原Tylosin-OVA,用紫外光谱扫描检测Tylosin-BSA偶联物,辣根过氧化物酶标记Tylosin-OVA偶联物。采用杂交瘤技术,制备特异性抗泰乐菌素单克隆抗体,建立泰乐菌素竞争ELISA检测方法。结果筛选出2株稳定分泌抗泰乐菌素单抗的杂交瘤细胞株3E4和2G10,2G10分泌的单抗腹水效价为6×10-4,抗体类型为IgG1型,轻链为κ链,在酸、碱及热条件下均较稳定。选择该单抗建立了泰乐菌素竞争ELISA检测方法。该方法灵敏度达50ng/ml,标准曲线线性关系良好(r=0.9827),且特异性、稳定性较好,准确性、精密性较高。结论已成功制备了泰乐菌素单克隆抗体,并建立了竞争ELISA检测方法,可用于动物源性食品中残留泰乐菌素含量的检测。更多还原

【Abstract】 Objective To prepare the monoclonal antibody(McAb)against Tylosin for development of a competitive ELISA. Methods Tylosin was coupled with carrier protein bovine serum albumin(BSA)and ovalbumin(OVA)by N, N’-carbonyldiimida- zole (CDI)to prepare complete antigens Tylosin-BSA and Tylosin-OVA respectively. Tylosin-BSA was identified by ultraviolet scan- ing, and Tylosin-OVA was labeled with horse radish peroxidase (HRP). The McAb specific to Tylosin was prepared by hybridoma technique to develop a competitive ELISA for Tylosin. Results Two hybridoma cell strains secreting McAb against Tylosin stably, i.e. 3E4 and 2G10, were screened. The McAb secreted by strain 2G10, at a titer of 6 × 10-4 in ascites, was IgG1 with κ chain and was acid-, alkaline- and heat-stable. The competitive ELISA developed with McAb 2G10 reached a sensitivity of 50 ng / ml, and showed high specificity, stability, accuracy and precision. The determination result by the developed ELISA showed good linear rela- tionship to Tylosin concentration within a range of 50 ~ 800 ng / ml (r = 0.9827). Conclusion The McAb against Tylosin was suc- cessfully prepared, and a competitive ELISA for residual Tylosin content in the foods from animal origins was developed.更多还原