Production Of Monoclone Antibody And Colloidal Gold Immunchromatographic Test Strip Against Norfloxacin

Norfloxacin?NOR?is one of Quinolone drugs,which was widely used in the treatment of various infectious diseases of animals,livestock products.However,NOR residues in livestock products cause serious damage to people's health.Immunological analysis method is based on antigen-antibody reaction,with simple operation,strong specificity,high sensitivity,and can quickly detect a variety of samples.Therefore,in this study immunological analysis method was used to prepare anti-NOR monoclonal antibody to detect NOR in samples.The small molecule antigen NOR was conjugated with BSA and OVA proteins by EDC method.Then NOR-BSA conjugate and NOR-OVA can be obtained.Three female mice were immunized by NOR-BSA conjugate.And the hybridoma cell lines were obtained by cell fusing technology.Positive cells were screened by indirect ELISA and indirect competitive ELISA.Finally,three hybridoma cell lines named7C4,11G7 and 8G3 were screened out.The titer of cell culture supernatant and the ascites titer after purification of three hybridoma cell lines were 1:512,1:1024,1:1024and 1:128000,1:128000,1:128000 respectively.The subtypes of three cells were IgG2b,IgG2b,IgG1.The competition standard curve was developed by indirect ELISA,the half maximal inhibitory concentration(IC₅₀)was 31.225 ng,the detection of linear range was 5.011 ng⁶30.95 ng,and the detection limit was 2.75 ng.The affinity constant Kaff was 4.6×10⁸.There have low cross-reactivity between ciprofloxacin?27.22%?,enrofloxacin?11.29%?,orbifloxacin?11.03%?,ofloxacin?9.73%?,danofloxacin?7.38%?,sarafloxacin?2.57%?,difloxacin?0.61%?antimicrobial agents.We use one monoclonal antibody to prepare colloidal gold strip.100 mL acide chloraurique was restored by 1.8 mL 1%sodium citrate to prepare colloidal gold,colloidal gold has maximum absorbance at 520 nm by uv scan,and particle size was18.41 nm.pH effect of the preferred labeled colloidal gold and the optimum amount of labeled protein were studied,so the best colloidal gold pH was added 5?L K₂CO₃to colloidal gold,and the optimum amount of the antibody was 6?g.When the gold-labeled antibody was diluted three times may has the best color.To assemble the colloidal gold strip,we use Sarturius CN95 as NC membrane,the test line was coated with a concentration of 0.4 mg/mL of anti-norfloxacin monoclonal antibody,and the control line was coated with a concentration of 0.6 mg/mL of goat anti-mouse secondary antibody.The sensitivity of prepared colloidal gold strip was 30 ng/mL.When tested samples with NOR standard substance,the NOR in milk and fish was 50ng/mL could be checked out.