A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion

Romchat Kraivong,¹ Prasit Luangaram,¹ Narodom Phaenthaisong,¹ Prida Malasit,^1,2 Watchara Kasinrerk,^3,4 Chunya Puttikhunt^1,2

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 (V_H) and kappa light (V_κ) chains from mouse hybridomas.
Methods: V_H and V_κ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. V_κ amplicons were additionally digested with BciVI to eliminate aberrant V_κ transcripts. The productive V_H and V_κ 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 V_κ amplicons were obtained using eight different V_κ primers. Amplicons from productive V_κ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant V_κ transcripts. 500-bp productive V_H amplicons could be obtained from all clones using a set of five V_H primers. The productive V_H/V_κ 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 V_H and V_κ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.
Key words: Aberrant variable transcripts, chimeric antibody, dengue virus, functional variable transcripts, mouse hybridoma cells,