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 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.

An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies

“…An anti-E antibody 4G2 was a gift from Armed Forces Research Institute of Medical Sciences (AFRIMS). , The 2H12 (IgG2b) antibody was generously provided by Dr. Juthathip Mongkolsapaya (Nuffield Department of Medicine, Oxford University) . The coding sequences for V H and V L genes of antibody 513, 4G2, and h38C2_Arg were synthesized or amplified from a hybridoma cell line and subsequently cloned into pVITRO1 expression vector and were expressed in Expi293F cells. ,− The recombinant antibodies were purified with protein A-agarose beads (Sigma-Aldrich) . scFv-derived antibody expression vectors were constructed from the variable regions of the selected phage clones.…”

Blockade-of-Binding Activities toward Envelope-Associated, Type-Specific Epitopes as a Correlative Marker for Dengue Virus-Neutralizing Antibody

“…An engineered and humanized antibody, 513, and a murine antibody, 2H12, recognize distinct common epitopes in the EDIII domain of all four dengue virus serotypes, whereas EDE-1 C10, a human antibody, recognizes the E dimer of all four dengue virus serotypes as well as Zika virus ( 47 , 59 – 63 ). All humanized antibodies were generated from published VH and VL sequences by cloning the synthesized coding sequences into the human IgG expression vector pVitro1-IgG1 as previously described ( 64 , 65 ). The resulting recombinant vectors were transiently transfected into Expi293 cells (Thermo Fisher Scientific).…”

Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques

“…Neutralizing monoclonal antibodies specific for each dengue serotype—DENV-1, clone 1F4 ( 52 ); DENV-2, clone 3H5 ( 53 ); DENV-3, clone 8A1 ( 54 , 55 ), and DENV-4, clone 5H2 ( 56 )—and all serotypes—clones 513 and EDE-1 C10 ( 57 , 58 )—were purified from the cell culture media using protein A/G affinity chromatography. Clones 1F4, 5H2, 513, and EDE-1 C10 were generated based on published VH and VL sequences by cloning synthesized sequences in the human IgG expression vector pVitro1-dV-IgG1/λ and subsequently transfecting the recombinant plasmids into Expi293 cells (Thermo Fisher Scientific) as described previously ( 59 ). Purified antibodies were conjugated with HRP employing a conjugation kit (KPL SureLink HRP conjugation kit, SeraCare) according to the manufacturer’s protocol.…”