产志贺毒素大肠杆菌志贺毒素Ⅰ型双抗体夹心ELISA检测方法的建立

Establishment of double- antibody sandwich enzyme- linked immunosorbent assay for detection of shiga toxin type Ⅰ in shiga toxin- producing Escherichia coli

为建立双抗体夹心ELISA法检测产志贺毒素大肠杆菌(STEC)Ⅰ型志贺毒素(Stx1),利用重组Ⅰ型志贺毒素A亚单位(Stx1A)免疫小鼠,制备单克隆抗体,从中筛选出非竞争性的2株单抗,构建双抗体夹心法ELISA检测方法,并对试验样品中的志贺毒素进行检测。结果显示:共获得4株针对Stx1A的单克隆抗体,分别为1H2-G7、2H1-C12、8E7-E6和2F6-F8。当8E7-E6作为包被抗体,而2F6-F8作为检测抗体时,其检测毒素的OD值最高。对样品中的Stx进行检测,表明该方法只有效识别Stx1,对Stx2不识别。结论:成功建立了用于检测Stx1的双抗体夹心ELISA法,为临床上快速诊断STEC感染奠定了基础。

To establish double- antibody sandwich enzyme- linked immunosorbent assay（ ELISA） for detection of shiga toxin type 1（ Stx1） of shiga toxin- producing Escherichia coli（ STEC）,the recombinant shiga toxin type I A subunit（ Stx1A） was used to immunize mice to develop monoclonal antibodies（ MAbs）,and two un- competitive MAbs were screened to set up double- antibody sandwich ELISA.This method was further evaluated by detecting Stx1 from supernatants of STEC cultures. Four strains of hybridomas which can secrete MAbs against Stx1 A,namely 1H2- G7,2H1- C12,8E7- E6 and 2F6- F8,were obtained. The highest OD value could be obtained using 8E7- E6 as coating antibody and 2F6- F8 as detection antibody in double- antibody sandwich ELISA. The ELISA method could detect Stx1 but not Stx2. It suggests that a double- antibody sandwich ELISA for detecting Stx1 was successfully established,which provides a foundation for the clinical diagnosis of STEC infection.