Establishment And Primary Applycation Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Norfloxation

Norfloxacin(NFLX) was conjugated with bovine serum albumin(BSA) and human serum albumin(HSA), separatedly with EDC method to form artificial antigens. Mice inoculated with different immunogens above produced antibodies against norfloxacin. The conjugants were identified by UV spectrum method. The rate of conjugation were 60:1 (NFLX-BSA) and 60:1(NFLX-HSA). The results of the indirect Enzyme-linked Immunosorbent Assay(ELISA) showed the titers of the conjugatants(NFLX-BSA and NFLX -HSA) were above 1:6400.Indirect Enzyme-linked Immunosorbent Assay(ELISA) method was developed to detect antibodies against norfloxacin. The optimum condition of the ELISA are set as follows: 1ug/mL of NFLX -HSA(norfloxacin -human serum albumin) conjugation used as a coated antigen dissolved in carbonate buffer solution(CBS,pH 9.6,0.05mol/L), 0.5 % glutin in PBS(pH7.4,0.01mol/L) as a blocking agent post-coating, horse radish heroxidase-labeled sheep anti-mouse IgG(HRP-anti IgG) diluted by PBS(pH 7.4,0.1mol/L) in 1:1000 as a secondary antibody, and TMB (3,3',5,5'-tetramethyl benzidine dihydrochloride) as a sbustrate.The specificity and repetitiveness of the ELISA was primely proved by the blocking test. The ELISA was conducted to detect the antisera of mouse and presented good results.One clone which can stably secrete the McAbs against norfloxacin. was obtained after fuse and named cell strain 3C2. The ELISA titers of the McAbs in cell supernatant and ascite were 1:51200 and 1:1638400, respectively. The rate of cross reactivity with other antibiotics were below 0.01%. The cell strain 3C2 also can stably secrete the McAbs after twenty-five setial passages and freezing ang recicfing three times in six months.Indirect competitive enzyme-linked immunosorbent assay(ciELISA) was developed by the McAbs in ascite of the cell strain 3C2. The optimum conditions in some critical steps of the ELISA were set as follows: the optimal concentrations of the coating antigen and the McAbs against norfloxacin were 1μg/mL and 1:1 ×10~5. Quantitation of the norfloxacin was linear from 10μg/mL to 10ng/mL and the limit of detection(LDM) was 10ng/mL. The regression equation was y = 0.3091-0.2246x, R~2 =0.8759.The ciELISA established in the persent study should be futher consummanted to suit producing rapid diagnostic slip by combining gold-labeled chromatography to be applied in practice.