Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

Abstract

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock.The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes.To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 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 limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb.Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14.The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.