Romchat Kraivong1, Prasit Luangaram1, Narodom Phaenthaisong1, Prida Malasit1,2, Watchara Kasinrerk3,4, Chunya Puttikhunt1,2. 1. Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Bangkok, Thailand. 2. Division of Dengue Hemorrhagic Fever Research, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 3. Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Chiang Mai, Thailand. 4. Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Abstract
BACKGROUND: Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. OBJECTIVE: To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. METHODS: VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. RESULTS: Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. CONCLUSIONS: A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.
BACKGROUND: Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. OBJECTIVE: To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. METHODS: VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. RESULTS: Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. CONCLUSIONS: A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.