利用特异性抗体建立金黄色葡萄球菌肠毒素B快速检测方法

Establishment of Rapid Detection Methods for Staphylococ⁃cal enterotoxin B Using Specific Antibodies

目的:利用特异性靶向金黄色葡萄球菌肠毒素B(SEB)的抗体建立2种体外快速检测SEB的方法。方法:通过间接ELISA法及生物膜干涉技术测定3株抗体(LXY8、LXY9和LXY10)与SEB的结合活性;通过竞争ELISA法鉴定3株抗体的表位交叉情况;利用配对抗体建立双夹心ELISA检测法和胶体金试纸条层析技术检测SEB。结果:3株抗体与SEB均具有较高的亲和力(LXY8:KD=5.25×10^-10 mol/L;LXY9:KD=5.18×10^-9 mol/L;LXY10:KD=8.93×10^-10 mol/L);经间接ELISA法获得生物素标记的抗体与SEB结合的最低饱和浓度为0.25μg/mL,以该浓度作为抗原表位竞争的浓度,竞争ELISA结果表明3株抗体与SEB结合的表位不交叉;根据双夹心配伍实验的结果,获得LXY8与LXY10的最佳配对,SEB的最低检测限为240 pg/mL,而基于LXY8与LXY10配对的胶体金检测试纸条能够在5~10 min内完成样品的检测,且检测灵敏度约为2 ng/mL。结论:基于双夹心酶联免疫吸附技术建立了一种特异、灵敏的体外检测SEB的方法,利用胶体金层析技术开发了SEB快速检测的胶体金检测试纸条,能够实现SEB现场快速高效检测。

Objective:To establish two different methods for staphylococcal enterotoxin B(SEB)rapid detection uses the specific SEB-targeted pairwise antibody.Methods:The binding activity of three strains of antibodies(LXY8,LXY9 and LXY10)with SEB was measured by biofilm interference technology.The epitope of the three antibodies were mapped by indirect ELISA assays and competitive ELISA assays.Results:Three antibodies have high affinity with SEB(LXY8:KD=5.25×10^-10 mol/L;LXY9:KD=5.18×10^-9 mol/L;LXY10:KD=8.93×10^-10 mol/L).It is 0.25μg/mL that the minimum saturation concentration of biotin-labeled antibody binding to SEB obtained by indi⁃rect ELISA assays.The results of competitive ELISA showed that the epitopes of the three antibodies and SEB did not cross by using this concentration as the optimal concentration for epitope competition.According to the re⁃sults of the double-sandwich compatibility experiment,we obtained the best pairing of LXY8 and LXY10.The low⁃est detection limit of SEB is 240 pg/mL,and the lowest detection limit of SEB of the colloidal gold detection test strips based on the pairing of LXY8 and LXY10 is 2 ng/mL that can be detected within 5~10 min.Conclusion:Based on the double-sandwich enzyme-linked immunosorbent technology,a specific and sensitive method for in vi⁃tro detection of SEB was established.On the other hand,colloidal gold chromatography technology was used to de⁃velop colloidal gold detection test strips for rapid detection of SEB to achieve rapid and efficient on-site detec⁃tion.