Production of a Monoclonal Antibody against Eugenol and Development of Colloidal Test Strip Detection Method
In order to prepare highly specific and highly sensitive monoclonal antibodies against eugenol,a colloidal gold immunochromatography was developed for the detection of eugenol.The study used nucleophilic substitution method to synthesize eugenol semi-antigen(Eugenol-COOH),identified by mass spectrometry(HRMS);eugenol immunoantigen(Eugenol-BSA)and encapsulated antigen(Eugenol-OVA)were prepared by carbodiimide method,identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)and ultraviolet scanning;immunoantigen Eugenol-BSA was used to immunize mice.The mice were immunized with the antigen Eugenol-BSA,and after cell fusion,screening and cloning,a hybridoma cell line(3D9)that could stably secrete anti-eugenol monoclonal antibody was finally obtained,and the ascites was prepared,and the ascites potency was determined by the indirect competition ELISA(icELISA)method.The monoclonal antibodies were identified,and the detection method of colloidal gold immunochromatographic test strips was established through optimization,and the sensitivity and stability of the test strips were evaluated.The Eugenol-BSA conjugation ratio was about 16∶1,and the Eugenol-OVA conjugation ratio was about 10∶1,and the complete antigen conjugation was successful.The antibody type and subclass of the 3D9 cell line were all IgG1,and the light chain was κ chain.The titer of the monoclonal antibody was 1×10-6,the linear regression equation between the concentration of eugenol and the inhibition rate was y=9.6711nx+10.539(R2=0.998),and the half maximal inhibitory concentration(IC50)was 59.2 ng·mL-1,which indicated that the prepared anti-eugenol monoclonal antibody had high detection sensitivity.The optimal pH of colloidal gold labeling was 9,and the addition amount of 0.2 mol·L-1 K2CO3 was 12 μL,the optimal amount of labeled antibody was 7 μg·mL-1,the concentration of T-line disappearance of colloidal gold test strips was 1.8 μg·mL-1,and the maked eye detection limit for this parameter was 0.3 μg·mL-1.This study counld provide an effective detection method for the on-site detection of eugenol.
eugenolmonoclonal antibodyartificial antigencolloidal gold test strip
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维普
