I型志贺毒素时间分辨荧光免疫分析方法的建立

Establishment of time-resolved fluorescence immunoassay for detection of Shiga toxin type I

目的构建基于时间分辨荧光免疫分析技术(TRFIA)的高灵敏性、高特异性的I型志贺毒素(StxI)双抗体夹心免疫检测法,用于产志贺毒素大肠杆菌(STEC)感染的临床快速诊断。方法采用两株针对StxI不同表位的单克隆抗体2F6-F8和8E7-E6,构建双抗体夹心TRFIA,并评价该方法的灵敏度、特异性以及稳定性;以PCR检测stxI基因结果为金标准,以TRFIA、ELISA、Duopath~?Verotoxins免疫胶体金检测试剂盒,检测54例StxI毒素粗提物,比较灵敏度、特异性、阳性预测值、阴性预测值和与PCR法的一致性。结果建立的以StxI为靶向分子的TRFIA,可以在2h内完成对StxI检测,检测最大稀释倍数为1:1 000的StxI阳性菌株毒素粗提物样本,灵敏度比ELISA提高了约2个数量级。Stx I的TRFIA与StxII型无交叉反应,检测结果在1个月内仍保持较高稳定性。与PCR方法相比,TRFIA灵敏度(93.8%)高于ELISA(82.4%)和Duopath~?Verotoxins免疫胶体金(57.1%),差异有统计学意义(P<0.05);其特异性(100.0%)、阳性预测值(100.0%)、阴性预测值(97.4%)均大于等于ELISA(分别为97.2%、93.3%、92.1%)和Duopath~?Verotoxins免疫胶体金(分别为100.0%、100.0%、87.0%);TRFIA检测结果与PCR一致性(Kappa=0.955)高于ELISA(Kappa=0.809)法和Duopath~?Verotoxins免疫胶体金(Kappa=0.664)。结论成功构建StxI-TRFIA快速检测方法检测样品中StxI毒素,为TRFIA在毒素检测方面的推广应用提供了依据。

Objective To establish a highly sensitive and specific double-antibody sandwich immunoassay based on time-resolved fluorescence immunoassay(TRFIA)for Shiga toxin I(StxI);to facilitate the rapid diagnosis of Shiga toxin-producing Escherichia coli(STEC)infection.Methods Two monoclonal antibodies against StxI,named as 2 F6-F8 and 8 E7-E6 were used for establishing StxI double antibody sandwich TRFIA.Its sensitivity,specificity and stability were then evaluated,PCR detection for genes encoding StxI was used as the standard reference for comparison.StxI in the culture supernatants of 54 clinical isolated STEC strains were analyzed by StxI-TRFIA,StxI-ELISA and Duopath~?Verotoxins test,the sensitivity,specificity,positive predictive value,negative predictive value and consistency of above methods were compared with those by PCR analysis.Results A TRFIA was developed for the detection of StxI and its variants in STEC strains within 2 h.The crude toxin extract samples of StxI positive strains with the maximum dilution ratio of 1:1 000 could be detected,resulting an increment of approximately 100-fold sensitivity compared to ELISA.TRFIA of StxI did not cross-react with StxII,and the results remained stable within 1 month.Compared with the PCR method,the sensitivity of TRFIA(93.8%)was higher than that of ELISA(82.4%)and Duopath~?Verotoxins immunocolloidal gold chromatography(57.1%)(P<0.05);the specificity(100.0%),positive predictive value(100.0%)and negative predictive value(97.4%)were higher than that of ELISA(97.2%,93.3%and 92.1%,respectively)and Duopath~?Verotoxins immunocolloidal gold chromatography(100.0%,100.0%and 87.0%,respectively);the consistency of TRFIA and PCR(Kappa=0.955)was higher than ELISA(Kappa=0.809)and Duopath~?Verotoxins immunocolloidal gold chromatography(Kappa=0.664).Conclusion A rapid detection method for StxI toxin in samples by StxI-TRFIA has been successfully established.It provides a practical basis for the popularization and application of TRFIA in toxin detection.