Study On Preparation Of Bispecific Monoclonal Antibody Against Furaltadone Metabolite(AMOZ) Derivative And LMG

Furaltadone(FA) and malachite green(MG) are veterinary drugs which are banned in aquaculture in China, and they are rapidly metabolized to 5-morpholinomethyl-3-amino-2-oxazolidone(AMOZ) and leucomalachite green(LMG), respectively. At present, there were some reports about the preparation of polyclonal antibody, monoclonal antibody, and genetically engineered antibody against AMOZ, LMG or their derivatives. FA and MG in aquatic products are usually detected, and bispecific monoclonal antibody(Bs MAb) against AMOZ and LMG is prepared to establish multi-analyte immunoassay, which could significantly improve the convenient and rapidness of single-residue methods. The preparation of bispecific monoclonal antibody(Bs MAb) against AMOZ and LMG was not reported until now. Therefore, in this thesis, we adopted drug-induced methods to constructed a hybrid system for preparation of tetradoma, prepared Bs MAb against AMOZ derivative and LMG by hybrid-hybridoma technology, and established multi-analyte ic-ELISA for AMOZ, MG and LMG analysis and ic-ELISA of determination of total residues of AMOZ, MG and LMG. The main contents and results were shown as below:(1) Mutagenesis and screening of enzyme deficient hybridoma cell lines.Anti-3-nitrobenzaldehyde-derivatized AMOZ(3-NPAMOZ) hybridoma cell line of hypoxanthine-guanine-phosphoribosyltransferase(HGRPT) deficient and anti-LMG hybridoma cell line of thymidine kinase(TK) deficient were prepared based on the mutagenesis of gradient concentration of 8-AG(8-azaguanine) and 5-Brd U(5-bromo-2'-deoxyuridine), respectively. The cell culture supernatants before and after drug treatment were analyzed by ic-ELISA, and there were no obvious differences.(2) Selection and preparation of bispecific monoclonal antibodyA novel tetradoma cell line 4B10-5F1 which could stably secrete Bs MAb both against AMOZ derivative and LMG was selected by fusion of HGRPT-deficient 3-NPAMOZ hybridoma cell line and TK-deficient LMG hybridoma cell line using hybrid-hybridoma technology. Ascites fluids containing Bs MAb were acquired by inoculating into pristine-treated female BALB/c mice and were successively purified by ammonium sulfate precipitation and ion exchange column chromatography. The purified Bs MAb reached to electrophoretic homogeneity. SDS-PAGE, mouse monoclonal antibody isotyping reagents and non-competitive ELISA were adopted respectively to identify the molecular weight of antibody, isotype and affinity constant. The results indicated that Bs MAb was about 150 k Da with Ig G1 isotype, and the affinity constant(Ka) of Bs MAb to AMOZ derivative and LMG were 4.67×108 L/mol and 3.60×108 L/mol, respectively.(3) Establishment of multi-analyte ic-ELISA for AMOZ, MG and LMG analysisMulti-analyte ic-ELISA for AMOZ, MG and LMG analysis was established, and the analytical conditions of the ic-ELISA were determined to be the following: concentrations of coating antigen, Bs MAb dilution, Bs MAb dilution buffer, competition reaction time, HRP-Ig G antibody dilution, HRP-Ig G incubation time, standard dilution buffer and p H of standard dilution buffer. The detection limit of ic-ELISA for AMOZ analysis was 0.2 ng/m L(by the AMOZ derivative 3-NPAMOZ, the same below) with an IC50 value of 1.7 ng/m L, and linearity range was calculated as 0.4-8.3 ng/m L. And the IC50 and LOD(IC10) of ic-ELISA for LMG were 45.3 ng/m L and 4.81 ng/m L. The linear response range of the assay was extended from 7.0 to 291.7 ng/m L. Bs MAb was highly specific with no obvious cross reactivity(CR) with the analogues, except a CR of 32.6% of FA. The recovery of AMOZ in grass carp and tilapia ranged from 72.4% to 101.0% with coefficient of variation less than 15%. Correlation coefficient between the results of ic-ELISA and HPLC-MS/MS exceed 0.97. And also recovery of MG, LMG and MG-LMG ranged from 75.0% to 108.0% with coefficient of variation less than 15%. The correlation coefficient between the results of ic-ELISA and HPLC-FLD was over 0.95. The developed multi-analyte ic-ELISA is suitable for simultaneous determination of AMOZ, MG and LMG in aquatic products.(4) Establishment of ic-ELISA of determination of total residues of AMOZIc-ELISA of determination of total residues of AMOZ, MG and LMG was established, and the analytical conditions of the ic-ELISA were determined to be the following: concentrations of co-coating antigens, Bs MAb dilution, concentrations of standard mixtures based on different proportions of 3-NPAMOZ and LMG, Bs MAb dilution buffer, competition reaction time, HRP-Ig G antibody dilution, HRP-Ig G incubation time, standard dilution buffer and p H of standard dilution buffer. For establishment of ic-ELSIA in our research, AMOZ was derivatized to form 3-NPAMOZ with LMG reduced from MG. The detection limit of ic-ELISA for total 3-NPAMOZ and LMG analysis was 0.07 ng/m L with an IC50 value of 1.8 ng/m L, and linearity range was 0.2-14.5 ng/m L. Bs MAb was highly specific with no obvious CR with the analogues, except cross reactivities of 32.8%, 146.3% respectively for FA and 3-NPAMOZ. The recovery ranged from 72.2% to 107.0% in grass carp with spiked mixture of the two analyte(AMOZ-MG, AMOZ-LMG, AMOZ-MG/LMG), with coefficient of variation less than 15%. The correlation coefficient between the results of ic-ELISA and the results confirmed by HPLC-MS/MS and HPLC-FLD was over 0.93. The developed ic-ELISA of determination of total residues is suitable for aggregate analysis of AMOZ, MG and LMG in aquatic products.