Establishment And Utilization Of Detection Method Of New Delhi Metallo-β-lactamase

ObjectivesUsing New Delhi Metallo-β-lactamase-1 (NDM-1 prokaryotic expression vector induced expression of NDM-1 and 6×His tag fusion protein was purified by protein NDM-1 affinity chromatography. Using the in vivo culture method, the hybridoma cell lines 3F8 secreting anti NDM-1 monoclonal antibody to BALB/c mice abdominal cavity, preparation of anti NDM-1 rat monoclonal antibody. Based on the McAb and colloidal gold marked technology, according to the principle of competitive inhibition, the gold labeled immune spot detection method of NDM-1 was established and applied to the detection of NDM-1 in bacterial culture.Methods1. After been induced by IPTG, the NDM-1-6×His fusion protein was expressed in E.coli(DE3) at a high level, and was purified by affinity chromatography and identified by SDS-PAGE. Enzymetic kinetics test was carried out to identify the activity of recombinant NDM-16×His protein.2. Preparation and Purification of McAb against NDM-1,The in vivo culture method, will be able to hybridoma cell line 3F8 secreting monoclonal antibody against NDM-1 were stable in BALB/c mice. Thirty-six milliliter of ascites were produced by six BLAB/c mice after injecting 106 cell per mouse. Affinity chromatography method was used in the purification process, and4.50 mg purified McAb can be obtained from 5 ml affinity chromatography column each time. ELISA method was used to analyze the immune activity of antibody.3. Preparation of colloidal gold and immune gold probeAccording to the sodium citrate reduction method, colloidal gold with a diameter of 20 nm was successfully prepared. Based on that, the optimal condition under McAb against NDM-1, which was conjugated to colloidal gold was established. The ideal pH value is 7.4, and the idea concentration is 8μg McAb against NDM-1,/mL colloidal gold. The immune activity of gold probe was analysed with ELISA.Results1. NDM-1-6×His fusion protein was expressed and purified, and the purified fusion protein was proved to have high enzyme activity by enzymatic kinetic experiments.2. Development of NDM-1 dot immunogold filtration assayDot immunogold filtration assay (DIGFA) was developed for the detection of NDM-1 based on the competitive inhibition principle. In this system, with gold labeled anti NDM-1 monoclonal antibody as the analysis probe, complete antigen NDM-1 acted as thecoating antigen, goat anti-mouse IgG was immobilized on the membrane acting as thecontrol dot. When the concentration of the coating antigen was 6ng/ml, immunogold probe was in its stock solution, the sensitivity for NDM-1 detection was 6ng/ml by visual observation. The detection time was about 10 min.ConclusionsThis research has obtained the recombinant Escherichia coli high expression strains and preparation of mice anti NDM-1 monoclonal antibody.Based on the McAb and colloidal gold labeling technology, according to the principle of competitive inhibition,the gold labeled immune spot detection method of NDM-1 was established and its initial application in the detection of NDM-1 in bacterial culture...