Preparation and identification of monoclonal antibody against human Cu/Zn SOD

Objective To prepare and identify the monoclonal antibody(MAb) against human Cu/Zn SOD.Methods BALB/C mice were immunized with immunogen human Cu/Zn SOD.The spleen cells of the immunized mice were isolated and fused with SP2/0 cells.After several rounds of detecting and cloning,a hybridoma cell line secreting anti-human Cu/Zn SOD MAb was obtained.Its specificity was evaluated with indirect-ELISA,spot-ELISA,Western blot and immunohistochemistry.The titer,immunoglobulin subtype and affinity of the MAb were measured.Results One cell line of hybridoma named 4H10H4B12 was obtained,which was identified that the heavy and light chains of anti-human Cu/Zn SOD MAb were IgG2a and κ,respectively,with high affinity of 1.101×109 L/mol and high titer of 1∶256,000.The spot-ELISA,Western blot revealed that the MAb was specifically against the immunogen and native proteins expressed in HL-7702 cells,human hematids and didn't react with rat Cu/Zn SOD or human Mn SOD.The cell immunohistochemistry proved that the MAb could recognize the human Cu/Zn SOD located in kytoplasm of HL-7702 cells.Conclusion The success in mouse anti-human Cu/Zn SOD MAb preparation provides the basis for further study of AAA.