应用双抗体夹心ELISA检测产志贺毒素大肠杆菌Ⅱ型志贺毒素

Establishment of a double-antibody sandwich ELISA for the detection of shiga toxin type Ⅱ in shiga toxin-producing Escherichia coli infection

目的：构建双抗体夹心ELISA检测Ⅱ型志贺毒素（ StxⅡ）,以利于产志贺毒素大肠杆菌（ STEC）感染的临床快速诊断。方法应用业已制备的StxⅡ特异性杂交瘤细胞株,筛选、鉴定最佳抗体配对,构建双抗体夹心ELISA检测体系,对16株STEC临床分离株培养上清中StxⅡ进行检测,并对该体系的特异性和敏感性进行评价。结果从获得的多株单抗分子中,成功筛选出最佳配对抗体S2D8和S2C6,构建基于S2D8/S2C6的双抗体夹心ELISA检测体系,该体系对StxⅡ纯品的检测底限是4 ng/ml,并成功从STEC培养上清中检测出StxⅡ及其变种,而与StxⅠ不结合。其检测效能与商业化胶体金试剂一致,显示出良好的敏感性和特异性。结论 S2 D8/S2 C6单抗的双抗体夹心法ELISA检测试剂的制备为基于毒素分子作为靶分子的STEC 免疫诊断、治疗研究提供基础资料。

Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ （ StxⅡ） in shiga toxin-producing Escherichia coli （ STEC） infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡ and its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.