Development Of Neomycin Detection By Elisa

We established Enzyme-Linked Immunosorbnent Assay (ELISA) for neomycin detection. And also, we further detected residues in food samples sensitively and specificly. Based on the successful synthesis of neomysin antigen, we developed and characterized its polyclonal antibody, and also carried on the preliminary inquisition to the monoclonal antibody preparation.Neomycin is a broad-spectrum antibacterial antibiotics which was separated from streptomyces fradiae, and showed a significant antimicrobial effect to Gram-negative bacterias and most Gram-positive bacterias. It has been widely used in our country in agriculture, forest and husbandry, and has caused great concern of the growing NEO residues. In order to rapidly and effectively detect and monitor NEO residues from animal-originated foods, it was successfully established in this experiment by firstly synthesizing neomycin antigen, preparing the polyclonal antibody (pAb) and monoclonal antibody (mAb) against the neomycin, and establishing the ELISA methods for the detection of NEO residues.Neomycin antigen was synthesized by the method of glutaraldehyde and immunized New Zealand rabbits, and ultimately we obtained the anti-neomycin antibody. We established an indirect competitive ELISA (ic-ELISA) method to detect neomycin antibody and got the antiserum titer of 512000. Also, we optimized the coating concentration, coating time, competition time, pH and other experimental conditions which would affect the ELISA analysis. In the optimal experimental conditions, we re-established the standard inhibition curve of neomycin. We got good results with the detection limit below 0.01ng/ml, IC50 of 0.965 ng/mL and the linear range of 0.03～29.5 ng/ml. The results also showed the optimal concentration dilutions of coating antigen and antibody of 1:2000 and 1:16000, respectively., In this section ,we applied four aminoglycoside drugs like gentamicin, streptomycin, dihydrostreptomycin and kanamycin in the indirect competition ELISA as the competition antigen to test the specificity of antibody. And no cross-reactions of the neomycin antibody took were displayed with other aminoglycodides. We also determined the specificity of neomycin antiserum with IC50 4.88 ng/mL by one-step ELISA. Compared with ic-ELISA, the results of one-step ELISA were almost the same and could save 40 minutes, which was much convenient to the fast and on-line detection.Based on the successful synthesis of neomycin immunogen, we immunized BALB/C mise and prepared the polyclonal antibody. Then, we carried on the cytomixis using the hybridoma technology with the cytomixis rate of 95.57 % and positive rate of 38.96 %. Positive sera were screened using ic-ELISA method. Finally, after four times clone, we gained two highly sensitive and specific monoclonal antibodys which were named 2C6-F4-G10 and 3D4-F5-C3.