Preparation Of The Monoclonal Antibody Against Advanced Glycation End Products

Aims: In order to establish an ELISA method capable of measuring the very low concentration of AGEs in serum, the monoclonal antibody against advanced glycation end products was prepared.Methods: AGE-HSA was prepared by incubating D-glucose and HSA, then was purified by gel chromatography and identified by fluorescence and SDS-PAGE. HSA was incubated as control without D-glucose. Balb/c mice were immunized subcutaneously three times with 100μg of high molecular weight AGE-HSA in 50% Freund's adjuvant, and titers in immunized mice sera were determined by indirect ELISA. The splenic lymphocytes from the immunized mouse were fused to myeloma Sp2/0 cells and cultured selectivly with HAT and HT. Each supernatant was screened by its reactivity to AGE-HSA and HSA. One of antibody-producing wells were selected for further use through two successive subclonings by limiting dilution method. These cell clones were injected into Balb/c mice for production of ascites. Each ascites was purified by mannan binding protein (MBP), then monoclonal antibodies were identified by SDS-PAGE, western blotting and chromosome measurement.Results: AGE-HSA showed the absorption and fluorescent spectra. Five novel murine monoclonal antibodies only against AGE-HSA have been harvested. The subclass of these monoclonal antibodies were determined as IgM.Conclusion: Five anti-AGE-HSA cell lines were obtained, which might be of value for AGE-HSA measurement.