Abstract
This paper focuses on the production of a high-affinity monoclonal antibody (mAb) that can efficiently detect and block purinergic ligand-gated ion channel 7 receptor (P2X7R). To achieve this goal, the extracellular domain of human P2X7R, P2X7R-ECD, was used as an immunogen for BALB/c mice, inducing them to produce spleen lymphocytes that were subsequently fused with myeloma cells. Screening of the resultant hybridoma clones resulted in the selection of one stable positive clone that produced a qualified mAb, named 4B3A4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purity of the purified 4B3A4 mAb was above 85%, with prominent bands corresponding to molecular weights of 55 kDa (heavy chain) and 25 kDa (light chain), and the BCA assay showed that the concentration of the purified 4B3A4 mAb was 0.3 mg/mL. Western blot analysis revealed that the 4B3A4 mAb could specifically recognize and bind both P2X7R-ECD and the full-length P2X7R protein. Laser scanning confocal microscopy (LSCM) revealed that the 4B3A4 mAb specifically bound to P2X7R on the membrane of human peripheral blood mononuclear cells (PBMCs). P2X7R expression was significantly different between healthy individuals and people with certain cancers as determined by flow cytometry (FCM). In addition, the 4B3A4 mAb significantly reduced ATP-stimulated Ca2+ entry and YO-PRO-1 uptake, which indicated that the 4B3A4 mAb effectively blocked P2X7R activity. These data indicate that the 4B3A4 mAb can be further used as not only an antibody to detect cell surface P2X7R but also as a therapeutic antibody to target P2X7R-related signaling pathways.
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This work was supported by the National Natural Science Foundation of China (no. 81770915 and 81301737) and the Major Program of Shandong Province Natural Science Foundation (no. ZR2018ZC1054).
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Design of the study: RLZ and XXP; performed the experiments: RLZ, XXP, MXL, SPL, YFZ, LNJ and CYY; analyzed the results: RLZ, XXP, and MXL; drafted the manuscript: XXP, RLZ, MXL SPL, and YFZ; revised the manuscript: XXP, RLZ, MXL, SPL, and YFZ. All authors approved the manuscript submitted.
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All experiments involving mice were performed in accordance with the Chinese National Laboratory Animal-Guideline for Ethical Review of Animal Welfare and approved by the Institutional Animal Care and Use Committee of Weifang Medical University. All applicable international, national, and institutional guidelines for the care and use of animals were strictly followed. All patients and healthy volunteers enrolled in this study were from the Department of Laboratory at the affiliated hospital of Weifang Medical University. Informed consent was obtained from all subjects. The study was approved by the Ethics Committee of Weifang Medical University, and all research methods were in compliance with the relevant regulations and guidelines.
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Li, M., Luo, S., Zhang, Y. et al. Production, characterization, and application of a monoclonal antibody specific for the extracellular domain of human P2X7R. Appl Microbiol Biotechnol 104, 2017–2028 (2020). https://doi.org/10.1007/s00253-019-10340-0
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DOI: https://doi.org/10.1007/s00253-019-10340-0