title: [Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].
abstract: Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na+-K+-ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na+-K+-ATPase activity was detected by SensoLyteRFDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na+-K+-ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na+-K+-ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit is prepared.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Litao
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: DU
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Fujun
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Shemin
authors.initials: S
authors.lastname: Lyu
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China. *Corresponding author, E-mail: lidongm@mail.xjtu.edu.cn.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000254:Sodium-Potassium-Exchanging ATPase