Preparation and identification of monoclonal antibody against human Atg5

Objective To prepare and identify the monoclonal antibody(MAb) against human Atg5.Methods Splenocytes isolated from Balb/c mice which were immunized with recombinant protein Atg5 were fused with mouse myeloma cells SP2/0.After several rounds of screening and cloning,one stable strains of hybridoma was established.The specificity was evaluated with ELISA,immunohistochemistry and Western blot,and the titer,immunoglobulin subtype and affinity constant of the mAbs were identified.Results One cell line of hybridoma named 1E7B3H9 was obtained,which was identified that the heavy and light chains of anti-human Atg5 MAb were IgG2b and κ,respectively.The mAb titer in ascetic fluid was 1:4.096×105 and the affinity constant reached 7.309×108mol/L,respectively.Western blot demonstrated that the antibody could specifically bind the natural protein of Atg5 in a cervical cancer cell line Hela lysates.The cell immunohistochemistry proved that the antibody could recognize the Atg5 protein expressed in the cytoplasm of Hela cells.Conclusion We have generated a monoclonal antibody,anti-human Atg5 protein with high specificity and affinity,which provides the basis for further study of the role and clinical application of Atg5 in disease with cervical cancer.