Preliminary Study Of Immunology Assays For Rapid Quantitative Detection Of Amantadine

Amantadine(AMD) is an antiviral drug, which is used to kill flu virus for human.AMD is a prohibitive veterinary medicine. However, in recent years, AMD was illegally large-scale and excessly using in animal feeding. It not only increases viral resistance, but also threats human health. In this study, an indirect competition enzyme linked immunosorbent assay(ELISA) and immune colloidal gold technique(GICT) for rapid quantitative detection of AMD were initially established. Actual samples were tested and compared with UPLC-MS/MS to evaluate the accuracy of the methods. The results showed that the methods had high accuracy, simple operation, and high throughout. The two methods are suitable for rapid detection of AMD in the actual sample.Firstly, BSA and OVA were conjugated with AMD by glutaraldehyde method.The UV-vis spectrum of the AMD, carrier protein and AMD-BSA was determined.The results showed that the conjugates had characteristic absorption peak of carrier protein and AMD. It proved that AMD-OVA and AMD-BSA were successfully synthesized. AMD-OVA was immunized with Balb/c mice to prepare anti-AMD polyclonal antibody. The results of indirect competition ELISA showed that the inhibition rate of the polyclonal antibody of AMD was less than 10%.In this study, an indirect competition ELISA was established for rapid quantitative detection of AMD. AMD-BSA concentration, monoclonal antibody concentration, coating buffer, blocking buffer, pH, and competition reaction time were optimized. The optimal results were as follow: the concentration of AMD-BSA was 6.7 ?g/mL, the concentration of monoclonal antibody was 5.5 ?g/mL, coating buffer was 0.01 mol/L PBS, blocking buffer was 0.5% skim milk, pH was 7.4, and competition time was 40 min. Under the optimal conditions, the IC50 and limit of detection(LOD) of the ELISA were 11.93 ng/mL and 1.18 ng/mL, respectively. And there was a good linear ranged from 2.5 to 100 ng/mL. The ELISA had 27.12% of cross-reactivity with rimantadine.A GICT was also established for rapid quantitative detection of AMD. Antibody was conjugated to colloidal gold by electrostatic adsorption. The AMD-BSA conjugate and goat anti-mouse antibody were spotted on the nitrocellulose(NC)membrane at the test line(T-line) and control line(C-line), respectively. AMD-BSA concentration of T-line, goat anti-mouse antibody concentration of C-line, coupling pH, labeled amount, amount of colloidal gold probes, and detection time were optimized. The optimal results were as follow: the concentration of AMD-BSA on T-line was 1.5 mg/mL, the concentration of goat anti-mouse antibody on C-line was1.0 mg/mL, coupling pH was 6.0,amount of labeled antibody was 4 ?g/mL,amount of colloidal gold probes was 1.6 ?L, and detection time was 15 min. Under the optimal conditions, the IC50 and limit of detection(LOD) of the GICT were 8.44ng/mL and 1.80 ng/mL, respectively. And there was a good linear ranged from 2.5 to25 ng/mL. The GICT had 11.53% of cross-reactivity with rimantadine.Indirect competition ELISA was compared with GICT. ELISA and GICT had little difference in sensitivity. The recovery and linear range of ELISA was better than that of GICT. However, the detection time of GICT was 15 min while the detection time of ELISA was 2 h. The two methods were confirmed to be accuracy by comparing with UPLC-MS/MS.