{"title":"Intracellular Antibody Levels Predict Secretion Efficiency in HEK293T Cells Expressing a Chimeric Anti-CD99 Antibody from a Single-Expression Plasmid","authors":"Manatchanok Chinakarapong, Kamonporn Kotemul, Sukrueta Hanprathet, Myint Myat Thu, Supansa Pata, Witida Laopajon, Watchara Kasinrerk, Nuchjira Takheaw","doi":"10.1002/biot.70129","DOIUrl":null,"url":null,"abstract":"<div>\n \n <p>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.</p>\n </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 10","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Journal","FirstCategoryId":"5","ListUrlMain":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/biot.70129","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.
Biotechnology JournalBiochemistry, 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.