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Updated: Jan 30, 2026

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the
Tatsuhiro Igawa1, Shuhei Kishikawa1, Yoshito Abe1
1Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan.
Abstract:
P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.
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