Production of Monoclonal Specific Antibody against Brazzein Fusion Protein

Abstract

Background: The use of a natural sweet protein is important as an attractive alternative to sucrose in the human diet. Rigorous research has identified the use of the artificial sweetener that leads to various diseases, cancer and heart disease. Many functional studies are necessary for examining the structure and biological activity of natural sweeteners such as Brazzein and its receptor targeting. In this report, we produced a panel of monoclonal antibodies against the recombinant brazzein which can be used for functional studies. Methods: Hybridomas cell fusion technology was used for the production of monoclonal antibody. BALB/c mice were immunized twice with purified fusion proteins with Freund adjuvant. Next, 14 days after the last injection, blood sample was collected from the eye and serum was separated and the antibody titer was detected by enzyme-linked immunosorbent assay (ELISA). The mouse with the highest antibody titers was selected. Seven days before fusion, the selected mouse was boosted with 50 µg antigen into the peritoneum to develop a robust immune response and then spleen cells were isolated from the same animal for fusion to myeloma SP2/0 to generate hybridoma cells. Results: Hybridoma clones were screened by ELISA and their monoclonal antibodies were purified. Specificity and reactivity of the monoclonal antibodies were determined by western blot analysis. We produced a panel of monoclonal antibodies that were highly specific and reacted with Brazzein protein that was detected by western blot analysis. Conclusion: These monoclonal antibodies bind to the MBP (Maltose binding protein)-containing recombinant protein in western blotting, so this antibody can be a useful probe for tracing proteins in basic research and pharmaceutical applications.