Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

Abstract

BackgroundMolecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.ResultsWe developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.ConclusionThe time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

“…Single cells were sorted into each well of U-bottom 96-well plates containing 10 µl cell lysis/binding solution (100 mM Tris HCl (pH 7.5), 500 mM LiCl, 1% lithium dodecyl sulfate, 5 mM dithiothreitol and 10 µg oligo-(dT) 25 magnetic beads). Preparation of the 3'-end homopolymer-tailed cDNA from single ASPCs is performed automatically by a non-contact magnetic power transmission instrument (MAGrahd) [13]. The V H and V L genes were amplified by 5' RACE PCR using the 3'-end homopolymer-tailed cDNA fragments as the templates.…”

“…At 3 days after transfection, the cell culture supernatants were analyzed for the secretion of recombinant antibodies. The antibody concentrations and reactivities were determined by enzymelinked immunosorbent assay (ELISA), as described previously [13]. Large-scale recombinant mAbs were prepared using the FreeStyle™ 293 Expression System (Life Technologies) according to the manufacturer's protocol.…”

“…The manual V H and V L gene amplification from single cells followed by the construction of IgH and immunoglobulin light chain (IgL) gene expression plasmids are also limiting steps of this method [4,[9][10][11][12]. To achieve rapid and scalable automation for the generation of mAbs from a large numbers of single cells, we previously proposed a high-throughput singlecell-based immunoglobulin gene cloning method by developing a non-contact magnetic power transmission system (MAGrahd) for single-cell-based cDNA synthesis and a target-selective joint PCR (TS-jPCR) for IgH and IgL gene expression [13,14].…”

Acquisition and bioactivity analysis of single-chain fragment variable antibody against CSFV

“…Currently, as an emerging technology, single B cell antibody preparation is a technique that clones and expresses single antigenspecific B cell antibody genes in vitro. This technique reserves the native mating of heavy and light chains, and has the advantages of good genetic diversity, high efficiency and low cell requirement [17]. Therefore, in this study, we used a B cell antibody was used for focus to acquire a single-chain antibody to CSFV.…”

Advances in Monoclonal Antibodies Production and Cancer Therapy

“…Currently, for the isolation of high affinity mAbs by somatic hypermutation and affinity maturation technique, the direct molecular cloning of identical pairs of antibody light chain lambda variable (VLλ), heavy chain (IgH) variable (VH) and light chain kappa variable (VLκ) genes from single antigen-specific plasma/plasmablast cells (ASPCs) with the help of polymerase chain reaction (PCR) is an alternative technique being used for the development of mAb from immunized animals [37][38][39][40][41][42] [43][44][45][46][47][48][49][50][51][52][53].…”