Preparation and identification of a monoclonal antibody against delta-like-1

OBJECTIVE To prepare and identify the monoclonal antibody(mAb) against delta-like-1.METHODS Splenocytes isolated from Balb/c mice which were immunized with a peptide of delta-like-1 coupled to bovine serum albumin(BSA) were fused with mouse myeloma cells SP2/0.After several rounds of screening and cloning,two stable strains of hybridoma were established.The specificity was evaluated with ELISA and Western blot,and the titer,immunoglobulin subtype and affinity constant of the mAbs were identified.RESULTS Two stable strains of hybridoma(3H11C11A9E3 and 2D5D11F8F1),that can secrete high specific mAbs against delta-like-1,were obtained.The isotypes of these mAbs were tested to be IgG1 and the subtype of light chains were κ chain.The mAb titers of two strains in ascetic fluid were 1:4.096×105 and 1:1.024×105,respectively.The affinity constants reached 3.98×107 L·mol-1 and 3.28×107 L·mol-1,respectively.Western blot demonstrated that the antibodies could specifically bind the natural protein of delta-like-1 in a glioma cell line U251 lysates.The cell immunohistochemistry proved that the antibodies could recognize the delta-like-1 protein expressed on the nucleus of U251 cells.CONCLUSION Monoclonal antibodies against delta-like-1 were successfully prepared by hybridoma technique.This will provide the basis for further study on glioma and other diseases of nervous system.