Target-selective joint polymerase chain reaction: A robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells

Abstract

BackgroundDuring the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies.ResultsWe have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days.ConclusionOur system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

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

“…DNA fragments encoding the human, rabbit, rat and guinea pig immunoglobulin gamma (IgG), kappa (IgK) and lambda (Igλ) constant regions were amplified using lymphocyte cDNAs as the templates with primers for IgG (IgGC S and IgGC AS), IgK (IgKC S and IgKC AS) and Igλ (IgλC S and IgλC AS), respectively. The amplified DNA fragments digested with XhoI and NotI were inserted into the corresponding sites of pJON-IgG [14]. The DNA fragments encoding the human insulin A and B chains were subcloned into the SpeI/XhoI site of the pET42b vector.…”

“…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].…”

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

“…Traditionally, approaches such as hybridomas 8 and phage display have been implemented to address this issue. 9 More recently, techniques such as single B cell expression cloning, B cell immortalization [10][11][12][13] and deep sequencing of the antibody repertoire are improving the analysis of the human antibody response. The analysis of the antibody repertoire through high-throughput sequencing has been used to study the diversity of the immune response, and for the identification of antibodies of interest.…”

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].…”