Development Of A Colloidal Gold Immunochromatography Test Strip For Detection Of Erwinia Amylovora Based On Monoclonal Antibody

Pear fire blight is a bacterial disease caused by Erwinia amylovora(EA).It mainly harms the Rosaceae.With the characteristics of heavy damage,rapid transmission,and difficulty in removing the pathogen after the plant is infested.It seriously affects the growth and reproduction of plants and is one of the most important plant quarantine pests in China.Pear fire blight occured in many places in the world,such as the United States,Germany,France and Canada,and various countries pay more attention to the disease.For the areas where the fire blight pathogen has not yet been discovered,the most important content is to prevent the introduction and occurrence of the disease,and do a good job in the inspection and quarantine of the pear fire blight.Therefore,it is very important to establish an accurate,sensitive,efficient,and suitable method for large-scale field detection of EA.My laboratory has established a colloidal gold immunochromatographic test strip detection method using polyclonal antibody 0550 as the capture antibody and monoclonal antibody 6E12 as the gold-labeled antibody.This method is accurate,sensitive and efficient,It has the advantages of portability and is suitable for large-scale and rapid detection in the field.However,due to the uncertainty in the production of polyclonal antibodies,the development and application of test strips are limited by polyclonal antibodies,and there is also instability.Therefore,based on these problems and on the basis of the preliminary work in the laboratory,this study took EA as the research object,aimed to screen out a new monoclonal antibody against EA as the capture antibody,compared with the original gold.The antibody 6E12 was used for pairing recognition,and develop of a test strip for colloidal gold immunochromatography of EA based on monoclonal antibody.which overcomes the problems caused by the instability of polyclonal antibody production in the preparation of the test strips.It will has a wider field application prospects.This research mainly includes the following contents:(1)Antibody preparation:The single colony of EA was injected into the experimental mice.And choose the mice of higher titers as a contributor to splenocytes,Fusion of spleen cells and myeloma cells,four cell lines which could stably secrete monoclonal antibodies against EA were successfully screened,respectively.These are 2C2,3E11,4D10 and 6B10.The subtypes of the antibodies secreted by the four cell lines selected this time and the cell line 6E12 stored in the laboratory were determined,and it was found that the antibody subtypes secreted by the cells 3E11,4D10,and 6E12 were Ig G2b,and the antibodies secreted by the cells 2C2 and 6B10 was Ig G2a.Preparing a large number of monoclonal antibodies by in vitro culture method,and antibody affinity column is used to purify the obtained antibodies,which provides materials for subsequent experimental research.(2)The establishment of a colloidal gold immunochromatographic method based on monoclonal antibodies:the antibodies secreted by the four strains of cells selected this time and the antibodies secreted by the cells 6E12 stored in the laboratory are paired to identify each other,After a lot of experiments,it was found that the combination of 3E11 and 6E12have a good recognition capabilities.Therefore,in this study,the monoclonal antibody 6E12against EA was used as the gold-labeled antibody(recognition antibody),and the newly screened monoclonal antibody 3E11 against the EA was used as the detection line antibody(capture antibody).Anti-mouse Ig G was respectively coated on nitrocellulose membrane(NC membrane)to construct a monoclonal antibody-based method for the detection of EA colloidal gold immunochromatographic test strips,and optimize the relevant working conditions.According to the sensitivity test,it find that the detection limit of the test strip for EA is 1×10~6 CFU/m L;the specific experimental test did not find that the test strip has a cross-reaction with the Erwinia pyrifoliae(EP).And through a large number of repeated experiments,the test strip has high repeatability and stability,the entire reaction process can be completed within 5 minutes,and with the reaction time increasing,the experimental phenomenon will not change significantly.It could be widely used in field testing.