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

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-kappa) chains from mouse hybridomas. Methods: V-H and V-kappa genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. V-kappa amplicons were additionally digested with BciVI to eliminate aberrant V-kappa transcripts. The productive V-H and V-kappa 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-kappa amplicons were obtained using eight different V-kappa primers. Amplicons from productive V-kappa transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant V-kappa 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-kappa 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-kappa genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.