Preparation Of Monoclonal Antibody Against Dimethorphine And Study On Immunological Detection Methods

Objective:Prepare dimethomorph monoclonal antibodies with high sensitivity,good specificity and strong affinity,and establish immunological detection and analysis methods to provide technical support for the rapid detection of dimethomorph residues in agricultural products.Methods:Spleen cells from immunized mice were fused with SP2/0 myeloma cells,and hybridoma cells were selected by HAT medium.The positive cells were screened from hybridoma cells by ELISA method,and the cells were subcloned by the limiting dilution method until the cells secreted antibodies stably,the cells were expanded,cul-tured,frozen,and ascites-type monoclonal antibodies were prepared.After purification of ascites,the specificity,sensitivity,affinity,titer and subtype of the antibody are iden-tified.Based on the obtained antibodies,the detection methods of dimethomorph ic-ELISA and colloidal gold immunochromatography strip were established,and the methods were preliminary evaluated.Results:(1)Ten hybridoma cell lines that stably secrete anti-dimethomorph monoclo-nal antibodies were screened,and the sensitivity of the antibody(IC₅₀)was between 5-251 ng/m L;All antibodies showed good specificity to dimethomorph and did not cross-react to the nine selected substances that might interfere with it;The subtype identifi-cation results show that the light chains of the 10 antibodies are all Kappa(?)chains,and 8 of them are heavy chains of Ig G1.The other two strains are Ig G2a and Ig G2b subtypes.(2)A dimethomorph ic-ELISA method was established by using 25E6 antibody.Under the optimal conditions,the minimum detection limits(LOD)of this method were 12.5ng/m L,IC₅₀ 50 ng/m L,and the linear range was 12.5?250 ng/m L.The method was accurate and the recovery rate was in line with the standard of addition.The established IC-ELISA method and LC-MS method were used to determine the content of diphenyl morpholine in real cucumber samples.The results of the two methods were highly cor-related(R²=0.9563),which further indicated that the established IC-ELISA method had high accuracy and reliability.(3)A colloidal gold immunochromatographic method for the detection of Dimetho-morph was established by using antibody(14E12).Under the optimal conditions,the line elimination effect was obvious when the concentration of dimethomorph was 125ng/m L,and the color development of the test line was obviously weakened when the concentration was 31.2ng/m L,which could be determined as weak positive.The test results of colloidal gold strip for real cucumber samples are in good agreement with the LC-MS results,which indicates that the prepared strip has high reliability and accuracy.Conclusion:10 strains of dimethomorph monoclonal antibody hybridoma cells were screened,and dimethomorph ic-ELISA and colloidal gold immunochromatographic de-tection methods were initially established,which provided a basis for the development of rapid detection products of Dimethomorph pesticide residues in agricultural products.