Preparation Of Monoclonal Antibodies Against Cadium Ion And Study On The Immunological Characteristics

Artificial antigen Cd-IEDTA-BSA (HSA) was successfully synthesized using bifunctional chelating agent IEDTA (isothiocyanobenzylethyl enediamine tetraacetic acid) coupling with cadmium and protein carrier. The coupling chelate was analyzed by fluorescence spectrophotometry. Meanwhile, ultraviolet spectrum (UV) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the chelate. The coupling rates were determined by atomic absorption spectroscopy (AAS). The substitution process ofε-amino residue was elucidated using TNBS. The consistent corroboration by these methods showed that the artificial antigen was synthesized successfully. The coupling rates of Cd-IEDTA-BSA and Cd-IEDTA-HAS were 11-12:1 and 14-15:1; the ratios of substitution of free amino groups were 22.75% and 29.71% respectively. The fluorescence intensity of artificial antigen decreased regularly compared to carrier proteins.The fluorescence phase diagram of artificial antigen provided a linear relationship to demonstrate that the folding states of carrier proteins have no basic change. The polarization fluorescence spectra showed some change in the microenvironment of tryptophan residues in carrier proteins that caused a change in protein conformation.Antiserum was obtained from mice immunized with Cd-IEDTA-BSA; the monoclonal antibody was obtained by Hybridoma technique and the class, subclass, antibody cross-sensitivity and response rate of the monoclonal antibody were also determined by this technique. The ascites type of Mcab was induced in vivo and then purified. An indirect competitive ELISA mode was established based on the monoclonal antibody and the optimum conditions have been clarified with standard curves. Results showed: the titer of antiserum was above l: 12800; indirect enzyme linked immunosorbent assay(ELISA) was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones; four cell lines, 1C4, 3E6, 4E2, 6D7 were obtained by the technology of hybridoma all of which were IgG1 subtypes; the light chain wasλ-type; the IC50 of Mcab was 446.68 ng·mL^-1; the detection limit was 16.21 ng·mL^-1;the antibody had no cross reaction with other metal ions but mercury ion; the titer of ascites was 1: 160000; the US Corning 96 enzyme label plate showed superior performances, the optimum condition of coating is 37℃reacting for 2 h with 10% bovine serum or 1% glutin isinglass as closure reagent. The optimum concentration of coating antigen was 2μg·mL^-1 and the optimum diluting concentration of McAb was 1: 3000. The formula is y=27.78x-23.81(R²=0.9950,n=8,SD=2.1235).