Preparation Of Monoclone Antibodies Against Tomato Zanote Spot Virus(TZSV)and Development Of Colloidal Gold Immunochromatographic Assay For Detection Of TZSV

Tomato zanote spot virus is a new species in the genus of tomato spotted wilt virus,host plant reported has 166 species from 34 families,which makes plants grow exceptions in some or all of the serious impediment to the growth of plants,affecting the quality and reduce production.In Yunnan province,it has caused serious harm to the local tomato,pepper production.The current detection methods need special equipment and professional staff,it is difficult to meet the need of rapid and on-site detection.Therefore,it is of important significance to develop a simple,efficient,rapid detection methods in plant disease diagnosis,disease epidemic law analysis,prediction and disease prevention and control and other aspects.In this study,the monoclonal antibodies against tomato zanote spot virus were producted,and a colloidal gold immunochromatographic assay for detection of tomato zanote spot virus was successfully developed,based on the monoclonal antibodies and colloidal gold immunochromatographic technology,this method has the advantages of simple operation,short reaction time,the specificity,sensitivity,basically meet the detection requirement.Tomato zonate spot virus nucleocapsid protein was achieved by prokaryotic expression,and immunize BALB/c female mice after purification and dialysis deionized as antigen.After third immunization,the serum titer was detected by ELISA.The mouse that owned the highest titer was chosen for boosted immune.Using hybridoma technique,spleen cells from the chosen mouse and myeloma cells sp2/0 wsa fused by PEG]500.Then the clone anti-TZSV,after subcloning two or three times,three positive hybridoma cell lines which can stably secrete the anti-TZSV monoclonal antibodies were selected,named 3A2,5D2,5F7.they were all identified to be subclass antibodies IgM k.ELISA titers of the acsites were 128000,64000,64000,respectively.Dot-ELIS A showed that the three mAb of acsites could distinguish TZSV form TSWV specifically.In western blot,mAb3A2,5D2 and 5F7 all could react with Tomato zonate spot virus,and produce specific bands.Using the euglobulin precipitation method,gold chloride was reduced with sodim citrate,which was coupled with the purified monoantibody.By monoclonal antibody pairing test,3A2 was choosed to as the gold labeled antibody,and select 5D2 as coating antibody in test line.In this experiment,the final choice that pH 7.5 of colloidal gold were labeled to 150ug/ml of purified antibody 3A2,Using Sigma Goat anti-mouse IgG,its’ concentration diluted 10 times,as coating protein on control line of nitrocellulose membrane.After optimization concentration antibody coupled with gold chloride,reaction condition,assembled into a strip.The specificity and sensitivity analysis of test strip showed that the test preparation within 5～10min,can specifically recognize the tomato zonate spot virus,no cross reaction with CMV,TMV,SMV,TSWV,INSV,the sensitivity of strip also meet the detection requirements.has a potential application prospect in the prevention of tomato zonate spot virus dissemination.