High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

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All clinically approved antibodies are of the IgG isotype and mediate the clearance of target cells via binding to Fcγ receptors and complement (C1q). Even though IgA can elicit powerful cytotoxic action via FcαRI receptor binding, IgA antibodies have not been amenable to therapeutic development. Here, we report the engineering of a "cross-isotype" antibody, IgGA, which combines the effector functions of both IgG and IgA. IgGA binds to FcαRI with an affinity comparable to that of IgA, and to the activating Fcγ receptors, FcγRI and FcγRIIa, with high affinity, and displays increased binding to C1q compared to IgG. Unlike trastuzumab-IgG, trastuzumab-IgGA potently activates both neutrophils and macrophages to kill Her2(+) cancer cells. Furthermore, IgGA mediates greater complement-dependent cytotoxicity than IgG1 or IgA antibodies. The multitude of IgGA effector functions could be important for therapeutic purposes and highlights the concept of engineering antibodies that combine effector functions from multiple antibody isotypes.

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.

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