Intracellular Antibody Levels Predict Secretion Efficiency in HEK293T Cells Expressing a Chimeric Anti-CD99 Antibody from a Single-Expression Plasmid

IF 3.1 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Manatchanok Chinakarapong, Kamonporn Kotemul, Sukrueta Hanprathet, Myint Myat Thu, Supansa Pata, Witida Laopajon, Watchara Kasinrerk, Nuchjira Takheaw
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引用次数: 0

Abstract

Recombinant antibody production in mammalian cells is essential for therapeutic, diagnostic, and research applications. Single-expression plasmid systems that co-express both heavy and light chains under balanced promoters offer advantages in improving expression consistency. However, the relationship between intracellular antibody expression levels and secretion efficiency following clonal selection remains unclear. In this study, a single-expression plasmid encoding a chimeric anti-CD99 antibody (ChAbMT99/1) was constructed and used to establish stably expressing HEK293T mini-pool populations through zeocin selection. Intracellular ChAbMT99/1 expression levels in five selected mini-pools were assessed using intracellular immunofluorescence staining and flow cytometry. Antibody secretion into the culture supernatants was quantified by indirect enzyme-linked immunosorbent assay (ELISA) and further validated using indirect immunofluorescence assay and flow cytometry with native CD99-expressing cells. The results revealed a strong positive correlation between intracellular antibody expression and secretion levels. Mini-pools with higher intracellular fluorescence intensities produced greater antibody yields, indicating that intracellular antibody expression levels, within the context of single-plasmid expression systems, can reliably predict secretion efficiency. This finding facilitates the efficient selection of high-producing mini-pools for large-scale recombinant antibody production.

Abstract Image

细胞内抗体水平预测单表达质粒表达嵌合抗cd99抗体的HEK293T细胞分泌效率
在哺乳动物细胞中生产重组抗体对治疗、诊断和研究应用至关重要。在平衡启动子下同时表达重链和轻链的单表达质粒系统在提高表达一致性方面具有优势。然而,克隆选择后细胞内抗体表达水平与分泌效率之间的关系尚不清楚。本研究构建了一种编码嵌合抗cd99抗体(ChAbMT99/1)的单表达质粒,并通过zeocin选择建立了稳定表达HEK293T的迷你池群体。采用细胞内免疫荧光染色和流式细胞术评估5个选定的小池细胞内ChAbMT99/1的表达水平。通过间接酶联免疫吸附法(ELISA)定量抗体分泌到培养上清液中,并通过间接免疫荧光法和流式细胞术进一步验证天然cd99表达细胞。结果显示细胞内抗体表达与分泌水平呈正相关。具有较高细胞内荧光强度的迷你池产生更高的抗体产量,这表明在单质粒表达系统的背景下,细胞内抗体表达水平可以可靠地预测分泌效率。这一发现有助于高效选择用于大规模重组抗体生产的高产小池。
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来源期刊
Biotechnology Journal
Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍: Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.
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