![]() Variable region sequence determination by PCR-based approaches is challenging due to difficulties in designing universal primers that amplify all possible variable region sequences. Other methods to sequence antibody variable regions use PCR and Sanger sequencing. Furthermore, the cost of high-throughput library preparation and sequencing can be substantial, and turn-around time at sequencing cores can be weeks to months. However, most labs are not familiar with high-throughput sequencing technologies, which require expertise for the preparation of RNA-seq libraries and for computational analysis. These methods prove highly accurate and allow for the analysis of antibody repertoires to great depths. Some involve the use of high-throughput RNA-sequencing technologies. There are several existing methods to sequence antibody variable regions from hybridoma cells or lymphocytes. In addition, knowledge of the variable region sequences and subsequent recombinant antibody expression reduces the impact of hybridoma cell loss and hybridoma instability caused by mutations, chromosome deletions, or environmental factors. It is therefore critical to obtain the correct sequence of the variable regions to maintain antibody affinity and specificity. These variable regions determine antigen binding. Before the design of recombinant antibody expression plasmids, sequencing of the antibody light and heavy chain variable regions is required. In contrast to monoclonal antibodies generated using traditional hybridoma-based methods and isolated from ascites fluid, recombinant monoclonal antibodies are produced by cloning antibody cDNA or synthetic sequences into expression plasmids and expressing in mammalian cell culture. Recombinant monoclonal antibodies (mAbs) are a multibillion-dollar industry. Recombinant capsid spike domain from human astrovirus serotype 8 Switching mechanism at 5’ end of RNA transcript Spike 8, Reverse transcription polymerase chain reaction SMART, Rapid amplification of 5’ cDNA end RT-PCR, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.Ĭomplementarity-determining region ELISA,Įnzyme-linked immunosorbent assay HEK 293F, received support from the National Institutes of Health under grant #1RO1AI130073-01A1. The custom Python program written to analyze Sanger sequencing data is available at. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the manuscript. Received: ApAccepted: JPublished: June 24, 2019Ĭopyright: © 2019 Meyer et al. Gill, University of Lincoln, UNITED KINGDOM ![]() Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.Ĭitation: Meyer L, López T, Espinosa R, Arias CF, Vollmers C, DuBois RM (2019) A simplified workflow for monoclonal antibody sequencing. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5’ end of the antibody cDNA. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. ![]()
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