Preparation Of A Monoclonal Antibody Against Chromogranin A For Immunohistochemical Application

Chromogranin A(CgA)is a soluble neuroendocrine acidic protein with a molecular weight of about 49 kDa,which is widely existed in neuroendocrine system and secreted by neuroendocrine cells and other endocrine cells.The value of CgA study gradually increased in the neuroendocrine tumor as tumor marker.Generation and characterization of CgA antibody have significant meaning for clinical diagnosis and science studies.In this study,the synthesized gene of CgA coding area was cloned into pET28a and expressed in procaryotic system.Then the CgA-His protein was purified through affinity chromatography.After that the purified CgA was injected into mice as an immunogen with QuickAntibody-Mouse5W adjuvant.When antiserum titer of mice reached 8×103,the hybridoma cells were generated by fusing mice splenocytes and the myeloma cells with reagent PEG1450.A stable cell line named 4E5,which is secreting antibody against CgA,was secreened.The isotyple of 4E5 hybridoma was determined to be IgG2b,and the chromosome number of 4E5 hybridoma was 102±4.Anti-CgA mAb was purified from ascites fluid with affinity chromatography.The specificity of 4E5 mAb was determined by iELISA and Western Blot.The results of immunohistochemistry showed that the 4E5 antibody is better than LK2H10+PHE5 or SP12 antibody when detecting CgA in SCLC,islet cell carcinoma and ectopic adrenal tissue.The titer of 4E5 antibody is 5.12×105,and the affinity constant reached 9.23×109 L/mol.After 50 d storage at 4? and 37?,the 4E5 antibody still has stronger specificity against CgA in IHC.To determined the epitope for 4E5 antibody,gene fragments from CgA were cloned and expressed in BL21(DE3),and the CgA111-220 fragment was recognized by 4E5 antibody with Werstern blot.This study is to design and produce the CgA antibody with independent intellectual property rights for IHC application.The obtained CgA antibody with high affinity and correspondfing experimental data will provide valuable choices and references for clinical diagnosis.