![]() In summary, by coupling Ag-specific single B cell sorting with gene-specific single cell RT-PCR, our method exhibits high efficiency and accuracy, which will facilitate future efforts in isolating mAbs and analyzing B cell responses to infections or immunizations in the guinea pig model.Īntibodies are Y-shaped globular proteins, namely Ig, that are produced by the immune system in most vertebrates ( Boehm, 2012). Furthermore, 85% of the expressed recombinant mAbs bind positively to the antigen probe by enzyme-linked immunosorbent and/or BioLayer Interferometry assays, while five mAbs from four clonal lineages neutralize the HIV-1 tier 1 virus ZM109. Of 88 antigen-specific B cells isolated, we recovered 24 (27%) cells with native-paired heavy and light chains. B cell V(D)J sequences were verified by sequencing and annotated by IgBLAST, followed by cloning into Ig heavy- and light-chain expression vectors containing human IgG1 constant regions and co-transfection into 293F cells to reconstitute full-length antibodies in a guinea pig-human chimeric IgG1 format. We then designed a set of guinea pig immunoglobulin (Ig) gene-specific primers to amplify cDNAs encoding B cell receptor variable regions from the sorted Ag-specific B cells. We stained the peripheral blood mononuclear cells (PBMCs) from a guinea pig immunized with HIV-1 envelope glycoprotein trimer mimic (BG505 SOSIP), using anti-guinea pig IgG and IgM fluorochrome conjugates, along with fluorochrome-conjugated BG505 SOSIP trimer as antigen (Ag) probe to sort for Ag-specific IgG hi IgM lo B cells at single cell density. Here, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for analyzing immunization- or viral infection-elicited antibody response at the clonal level in guinea pigs. 5Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, United States.4Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States.3Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States. ![]() 2IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, United States.1Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.Steinhardt 1, Yongli Xiao 3, Chi-I Chiang 1, Richard T. Lin Lei 1, Karen Tran 2, Yimeng Wang 1, James J.
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