Development A Colloidal Gold Immunochromatographic Strip For Detection Of Erwinia Amylovory

Erwinia amylovory is a serious pathogenic bacterium in North America that can infect Rosaceae to cause pear fire blight.It spreads quickly and cause huge economic losses in many regions.Because of its largely hosts,more than 220 species are encroached.It is hard to remove the Ea.pathogen after damaged,growth and reproduction affected.So far there is no definite occurrence of pear fire blight in China,but the probability of introduction of Ea.is greatly increased.Therefore,it is very urgent to development a rapid and effective diagnostic method for detection of Ea.Scholars have put forward many effective methods,but the existing problems are:cumbersome operation,expensive,professional,etc.Do not suit for the rapid detection of field.Therefore,it is urgent to establish efficient detection method in field.The paper takes Ea.as the research object to establish a colloidal gold immune chromatographic test strip method for detection it.The main research results are as follows:(1)The Monoclonal Antibody and Polyclonal Antibody preparation:The Ea.imm unogen with adjuvant into BALB/c mice intraperitoneal,then ELISA method to detect the serum antibody titer,and select the titer has higher before strengthening immune mice.Preparation of immune spleen cells,by using hybridoma technology make it fus ed with myeloma cells.After a series of fused cells subcloned,6 cell lines strain stabl y secreting anti-Ea.were obtained,named as 1H1?3B7?5A3?5B6?6A4?6E12,all identified to be subclass antibodies IgG2b.The antiserum was obtained by beating asc ites in mice,after using antibody affinity column to purified it.Certified,the three m Ab 1H1,5A3,6E12 had good specificity could distinguish Ea.form Ep.specifically,the titer all reach 128000,provide materials for the establishment of immunochromato graphic strips.Polyclonal antibody:the immunogen of Ea.was injected into New Zealand rabbits,and polyclonal antibody was obtained through the purified column of the immunized anti-serum.(2)Establishment of immunochromatographic method:Using the trisodium citrate r educe of hypochlorous acid to prepare colloidal gold particles,the monoclonal antibod y 6E12 was labeled it as capture antibody,polyclonal antibody 0550 and goat anti-mo use IgG were fixed on Nitrocellulose Membranes as detection and control area antibod y.Assembled the colloidal gold immune chromatography test paper for detection Ea.The experimental condition was optimized--the best pH of colloidal gold labeled of antibody is 8.0;the best antibody concentration to stabilize colloidal gold was 110?g/mL;the concentration of control line and test line antibody was 0.3 mg/mL and 3.3 mg/mL;the binding pad was sealed with 2%skimmed milk powder.Specific experi ment showed that the colloidal gold strip had no positive reaction with Ep.Sensitivity experiment showed that the minimum detection limit of the strip was 1×105 CFU/m L.Stability experiment showed that the colloidal gold strip has good repeatability and stability.At the same time,300 samples of test strips were sampled,the accuracy rate was 98%,and the whole operation could be completed within 10 minutes.