Preparation and identification of monoclonal antibody against annexinI

Objective:To prepare monoclonal antibody (mAb) against annexinⅠand identify its properties to establish the basis for future application in malignant tumor diagnosis and therapy.Methods:Prokaryotic expression vector pQE-30 of AnnexinⅠgene (1-480 bp) was successfully constructed.The transformed bacteria bearing pQE30-annexinⅠplasmids were induced by IPTG to express fusion proteins.The expression products was purified by affinity chromatography using Ni-NTA resin.Purified annexinⅠprotein was used as the antigen to immunize BALB/c mice.Splenoeytes from the immunized mice were fused with Sp2/ 0 cells.Monoclonal hybridoma cell lines were obtained by selective culture with the HAT/HT culture systems and isolated by limiting dilution.The mAbs were characterized by ELISA,Western blot and immunohistochemical staining.Results:Five hybri- doma ceil lines named 1A8,1D2,1E2,4E7 and 5G3 were obtained.All of the mAbs belonged to IgG1 where light chains.The affinity constants of mAbs are in the range of 2.3 to 3.03×10~8L/mol.Western blot analysis confirmed that the five mAbs could recognize AnnexinⅠantigen,but had no cross-reaction with other antigens.ImmunohistochemicaI results showed that the mAbs could recognize AnnexinⅠantigen in tumor tissues.Conclusion:Five hybridoma cell lines secreting mAbs against AnnexinⅠpro- tein were obtained successfully,which laid the groundwork for its future application in tumor diagnosis and therapy.