Synthesis And Identification Of Neomycin Artificial Antigen, Preparation Of Neomycin Antiserum And Development Of An Enzyme-linked Immunosorbent Assay For The Detection Of Neomycin

Neomycin, an aminoglycoside antibiotics, is widely used in veterinary medicine to treat bacterial infections in animals. It is classified as a broda-spectrum antibiotics due to its growth inhibition of Gram-positive bacteria and Gram-negative bacteria. However, despite its impressive clinical successes, neomycin is potentially ototoxic and nephrotoxic to human and animals; thus monitoring of its residues in foods is essential for the maintenance of public health.Therefore, simple and reliable analytical methods are required to monitor neomycin residue levels in foods. Enzyme-linked immunosorbent assay (ELISA) is relatively sensitive and simple for the detection of antibiotics. In the category of the aminoglycosides, no reasearch about ELISA has been reported at home. In this study, infrared spectrum was used to establish the analysis method of neomycin artificial antigen and an enzyme-linked immunosorbent assay using neomycin polyclonal antibodies was developed for the detection of neomycin elementarily. The main results were as following:1. Infrared spectrum was used to establish the analysis method of neomycin artificial antigen.2. On the basis of infrared spectrum, the synthesis conditions of neomycin artifical antigen were optimized. The temperature of reaction was 25℃and the concentration of EDC was 8.3 mg/mL.3. Neomycin was linked to Bovine Serum albumin(BSA) and keyhole limpethemocyanin(KLH) respectively by EDC coupling which was optimized. Themolar ratio of neomycin to BSA and KLH was 3:1 and 10:1 respectively.4. Four New Zealand White rabbits were subcutaneously immunized with conjugates (BSA-NEO and KLH-NEO) and the antiserum was obtained. For the determination of the antibody titer, indirect ELISA was used. The two rabbits immunized with BSA-NEO both developed good antibody titers using KLH-NEO as the coating conjugate, the highest titer was 1.28×10~5. One rabbit immunized with KLH-NEO developed good titer using BSA-NEO as the coating conjugate, but the other one failed.5. The conditions of direct competitive ELISA were optimized. The optimal dilution ratio of antibodies and HRP-NEO were 1:1000 and 1:500 respectively. The optimal reaction time was 60mim. Based on direct competitive ELISA, the standard curve was established and the regression equation of the crave was y = -16.475x + 113.76, R~2 = 0.9962. The average coefficient of variation was 3.82% and the lowest detection limit was 27.67 ng/mL.6. The conditions of indirect competitive ELISA were optimized. The concentration of antigen, antibody and the secondary antibody conjugate were determinated by chessboard format. The optimal dilution of antigen, antibody and the secondary antibody conjugate were 1:400, 1:2000 and 1:2000 respectively. Based on indirect competitive ELISA, the standard curve was established and the regression equation of the crave was y = -11.35x + 109.36, R~2 = 0.9945. The average coefficient of variation was 1.71% and the lowest detection limit was 51.29ng/mL.7. Indirect competitive ELISA was used in a competitive format with gentamicin, kanamycin, ampicillin, erythromycin and tetracycline. Because gentamincin, kanamycin have similar structure with neomycin, the cross-reaction rate of gentamincin and kanamycin with antibody were respectively 2.04% and 0.02%. While the cross-reaction rates of other antibiotics with antibody were below 0.01%. The results showed that the polyclonal antibodies of neomycin could detect neomycin specially. The detected samples recovery rate ranged from 132.5% to 246.5%.