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

Tetracycline(Tc) was conjμgated with bovine serum albumin(BSA), human serum albumin(HSA) separatedly with the response of Mannich to form artificial antigens. Mice inoculated with different immunogens above produced antibodies against tetracycline. The conjμgants were identified by UV spectrum method. The rate of conjugation were 23:1(Tc-BSA),22:1(Tc-HSA).The results of the indirect Enzyme-linked Immunosorbent Assay(ELISA) showed the titers of the two conjμgatants (Tc-BSA and Tc-HSA) were above 1:3200.Indirect Enzyme-linked Immunosorbent Assay(ELISA) method was developed to detect antibodies against tetracycline. The optimum condition of the ELISA are set as follows: 4μg·mL^-1 of Tc-HSA(Tetracycline-Human Serum Albumin) conjugation used as a coated antigen dissolved in carbonate buffer solution(CBS,pH 9.6,0.05mol·L^-1), 0.5% glutin in PBS(pH 7.4,0.01mol·L^-1) 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^-1) 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 tetracycline was obtained after fuse and named cell strain C4. The ELISA titers of the McAbs in cell supernatant and ascite were 1:12800 and 1:409600, respectively. The McAb's rate of cross reactivity with other antibiotics were below 0.01%. The cell strain C4 also can stably secrete the McAbs after twenty setial passages and freezing ang recicfing three times in three months.Indirect competitive enzyme-linked immunosorbent assay(ciELISA) was developed. 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 tetracycline were 4μg·ml^-1 and 1:5×10⁴. Quantitation of the tetracycline was linear...