Production of anti-human cannabinoid receptor 2 (CB2) monoclonal antibody using non-viral vector induced human CB2 expressing myeloma as an immunogen.

IF 2.3 4区 医学 Q3 ALLERGY Asian Pacific journal of allergy and immunology Pub Date : 2024-01-06 DOI:10.12932/AP-020723-1646
Jindaphun Kanyaruck, Takheaw Nuchjira, Putpim Chaochetdhapada, Laopajon Witida, Kotemul Kamonporn, Chaiwut Ratthakorn, Pata Supansa, Kasinrerk Watchara
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Abstract

Background: Cannabinoid receptor 2 (CB2) of the cannabinoid system is predominantly expressed on immune cells and involved in a diverse range of immune functions. However, the role of CB2 in immunoregulation is still controversial. One of the challenges in the detailed characterization and functional study of CB2 is the lack of CB2-specific monoclonal antibodies (mAbs).

Objective: We aimed to produce mAbs against a native form of human CB2 using human CB2 expressing mouse myeloma cells as immunogens.

Methods: Non-viral vector expression system was used to generate stable human CB2-expressing mouse myeloma cells and were utilized as an immunogen for mouse immunization. Hybridoma technique was employed in the production of mAbs. The produced mAbs were verified by flow cytometry and western blotting.

Results: Using a non-viral vector expression system, myeloma clones, which stable expressed human CB2, were generated and used as immunogen for antibody production. Following mouse immunization process, the anti-CB2 polyclonal antibodies were induced. By hybridoma technique, a mAb against CB2 could be generated. This mAb reacted to CB2-expressing THP-1 cells, but not to non-CB2-expressing SH-SY5Y cells. By western blotting, the generated anti-CB2 mAb reacted with a 42 kDa protein presented in lysates of CB2-expressing THP-1 cells, but not with non-CB2-expressing SH-SY5Y cell lysates.

Conclusion: A new approach using human CB2 expressing myeloma cells as immunogen for production of anti-CB2 mAb was developed. The generated anti-human CB2 mAb is regarded as a valuable tool for CB2 characterization. Moreover, the developed technique can be applied to produce other antibodies of interest.

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以非病毒载体诱导的人 CB2 表达骨髓瘤为免疫原,生产抗人大麻素受体 2(CB2)单克隆抗体。
背景:大麻素系统的大麻素受体 2(CB2)主要在免疫细胞上表达,并参与多种免疫功能。然而,CB2 在免疫调节中的作用仍存在争议。对 CB2 进行详细表征和功能研究的挑战之一是缺乏 CB2 特异性单克隆抗体(mAbs):我们的目的是利用表达人 CB2 的小鼠骨髓瘤细胞作为免疫原,生产针对原生形式的人 CB2 的 mAbs:方法:利用非病毒载体表达系统生成稳定的人 CB2 表达小鼠骨髓瘤细胞,并将其作为免疫原用于小鼠免疫。杂交瘤技术用于生产 mAbs。结果:结果:利用非病毒载体表达系统,产生了能稳定表达人 CB2 的骨髓瘤克隆,并将其作为抗体生产的免疫原。经过小鼠免疫过程,诱导出了抗 CB2 的多克隆抗体。通过杂交瘤技术,可以产生抗 CB2 的 mAb。这种 mAb 能与表达 CB2 的 THP-1 细胞发生反应,但不能与不表达 CB2 的 SH-SY5Y 细胞发生反应。通过 Western 印迹法,生成的抗 CB2 mAb 与表达 CB2 的 THP-1 细胞裂解液中的 42 kDa 蛋白发生反应,但与不表达 CB2 的 SH-SY5Y 细胞裂解液没有反应:结论:利用表达人 CB2 的骨髓瘤细胞作为免疫原生产抗 CB2 mAb 的新方法已经开发成功。生成的抗人 CB2 mAb 被认为是表征 CB2 的重要工具。此外,所开发的技术还可用于生产其他感兴趣的抗体。
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来源期刊
CiteScore
12.80
自引率
0.00%
发文量
74
审稿时长
>12 weeks
期刊介绍: The Asian Pacific Journal of Allergy and Immunology (APJAI) is an online open access journal with the recent impact factor (2018) 1.747 APJAI published 4 times per annum (March, June, September, December). Four issues constitute one volume. APJAI publishes original research articles of basic science, clinical science and reviews on various aspects of allergy and immunology. This journal is an official journal of and published by the Allergy, Asthma and Immunology Association, Thailand. The scopes include mechanism, pathogenesis, host-pathogen interaction, host-environment interaction, allergic diseases, immune-mediated diseases, epidemiology, diagnosis, treatment and prevention, immunotherapy, and vaccine. All papers are published in English and are refereed to international standards.
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