Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries.

In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette.

A bioinformatic framework for immune repertoire diversity profiling enables detection of immunological status.

Background: Lymphocyte receptor repertoires are continually shaped throughout the lifetime of an individual in response to environmental and pathogenic exposure. Thus, they may serve as a fingerprint of an individual's ongoing immunological status (e.g.

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

Background: Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.

Results: We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations.

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences.