To study the diversity of immune receptors and pathogens, multiplex PCR has become a central approach in research and diagnostics. However, insufficient primer design against highly diverse templates often prevents amplification and therefore limits the correct understanding of biological processes. Here, we present openPrimeR, an R-based tool for evaluating and designing multiplex PCR primers. openPrimeR provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage. As proof of concept, we used openPrimeR to find optimal primer sets for the amplification of highly mutated immunoglobulins. Comprehensive analyses on specifically generated immunoglobulin variable gene segment libraries resulted in the composition of highly effective primer sets (oPR-IGHV, oPR-IGKV and oPR-IGLV) that demonstrated to be particularly suitable for the isolation of novel human antibodies.

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As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor-and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12-14 d.