title: Preparation and characterization of a monoclonal antibody against the refolded and functional extracellular domain of rat P2X4 receptor.
abstract: The P2X4 purinergic receptor is a key molecule in neuropathic pain, particularly in the allodynia after peripheral nerve injury. We therefore sought to establish an anti-P2X4 receptor monoclonal antibody that would be useful for detection and characterization of the P2X4 receptor. We first prepared the refolded extracellular domain of the rat P2X4 receptor expressed in Escherichia coli. Then, we established a B-cell hybridoma producing the monoclonal antibody for the head domain of the rat P2X4 receptor with strict recognition, including S-S bond formation. In addition, we succeeded in the detection and immune precipitation of rat P2X4 receptor molecules on cultured cells. As the antibody specifically binds to the rat P2X4 receptor molecule, it is expected that the established monoclonal antibody will be applicable as a tool for detecting increasing expression levels of the P2X4 receptor molecule accompanied with increasing intensity of neuropathic pain.
authors.affiliation: Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Igawa
authors.forename: Sadayuki
authors.initials: S
authors.lastname: Higashi
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Abe
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Ohkuri
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsuda
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Inoue
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D008969:Molecular Sequence Data; D009154:Mutation; D058849:Protein Refolding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins