耐药蛋白NDM-1的双抗体夹心ELISA检测方法研究

Detection of the antimicrobial-resistance protein NDM-1 by double antibody sandwich ELISA

旨在建立一种检测细菌中新德里金属β-内酰胺酶(NDM)耐药蛋白双抗体夹心ELISA方法。采用化学合成的方法获得优化后的NDM-1蛋白基因序列,构建表达载体pET-28a-NDM-1(G29-R270),并在大肠杆菌中进行IPTG诱导表达,用SDS-PAGE和Western blot验证重组蛋白。以纯化的NDM-1蛋白作为免疫原,免疫BALB/c小鼠,获得了2株杂交瘤细胞株,分别命名为3H5和4G1,分别作为捕获抗体和检测抗体,通过对包被条件、包被抗体和检测抗体浓度等进行优化,建立双抗体夹心ELISA方法,并对所建立方法的敏感性、特异性等进行了评价。结果表明,经15℃条件下诱导16 h后,SDS-PAGE和Western blot显示目的条带清晰,大小正确。经优化后的双抗体夹心ELISA方法中,捕获抗体和检测抗体的稀释度分别为1∶2 000和1∶5 000,使用0.05 mol/L碳酸盐缓冲液(pH值9.6),4℃过夜包被。该方法对NDM-1阳性大肠杆菌和肺炎克雷伯菌的最低检出浓度为1×10^(5)CFU/mL,NDM-1阳性鲍曼不动杆菌和铜绿假单胞菌的最低检出浓度为1×10^(6)CFU/mL。该方法检测NDM-1、NDM-4、NDM-5和NDM-9,与4种阳性对照菌有明显的交叉,但与大肠杆菌ATCC 25922、大肠杆菌ATCC 35150、鲍曼不动杆菌ATCC 19606、肺炎克雷伯菌ATCC 10031和铜绿假单胞菌ATCC 9027均无交叉反应。板内变异系数小于6%,板间变异系数小于10%,说明本方法具有良好的重复性。综上所述,本研究建立了一种高特异性和高灵敏的双抗体夹心ELISA方法,为耐碳青霉烯类细菌中NDM-1耐药蛋白检测提供了一种技术手段。

This study was to establish a sandwich ELISA method for detecting the NDM-1 resistant protein in bacteria. The optimized NDM-1 protein gene sequence was obtained by chemical synthesis, the expression vector pET-28 a-NDM-1(G29-R270) was constructed, the expression vector was induced by IPTG in BL-21(DE3), and the recombinant protein was verified by SDS-PAGE and Western blot. The purified NDM-1 protein was used as an immunogen to immunize Balb/c mice, and two hybridoma cell lines were obtained and were named 3 H5 and 4 G1, respectively. They were used as the capture antibody and the detection antibody, respectively. Then, the coating conditions and the concentrations of the antibody and detection antibodies were optimized, the double-antibody sandwich ELISA method was established. Next, the sensitivity and specificity of the established method were evaluated. The results showed that after 16 hours of induction at 15 ℃, SDS-PAGE and Western blot both showed that the target band was clear and the size was correct. In the optimized double-antibody sandwich ELISA method, the dilutions of capture antibody and the detection antibody were 1∶2 000 and 1∶5 000, respectively, using 0.05 mol/L carbonate buffer(pH=9.6), and by coating overnight at 4 ℃. The minimum detectable concentration of this method for NDM-1 positive Escherichia coli(E. coli) and Klebsiella pneumonia was 1×10^(5)CFU/mL, and the minimum detectable concentration of NDM-1 positive Acinetobacter baumannii and Pseudomonas aeruginosa was 1×10^(6)CFU/mL. With this method, obvious cross-overs were observed in detecting the four positive controls of NDM-1, NDM-4, NDM-5 and NDM-9;but there was no cross reaction with E. coli ATCC 25922, E.