Introduction: CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. The aim of this study was to produce and characterize a monoclonal antibody against CD45 for use in detection and diagnosis of leukocytes.
Materials and Methods: To produce mAb against CD45 protein, mice were immunized with two KLH-conjugated CD45 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by the limiting dilution method. The desired clones were selected, and supernatants of hybridoma cells were screened by the ELISA method for the antibody. The monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE.Finally, immunoblotting and flow cytometry were recruited to explore the specificity of the mAb.
Results: Our results showed successful production and characterization of anti- CD45 monoclonal antibody. The mAb was IgG2a with Kappa light chain. Immunoblotting and flow cytometry results demonstrated the specific reactivity of this mAb with CD45.
Conclusions: The results show that the produced anti-CD45 mAb is highly specific and functional in biomedical applications (such as flow cytometry and western blotting), and could be utilized for the identification of leukocytes.