Single cell screening approaches for antibody discovery

Fitzgerald, Valerie; Leonard, Paul

doi:10.1016/j.ymeth.2016.11.006

Cited by 40 publications

(29 citation statements)

References 56 publications

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“…Yeast display has been shown to maintain cognate V H -V L pairing to provide antibodies with higher sensitivity and specificity than random pairing 13 ; it involves multiple steps for library construction, antibody fragment display and multiple rounds of screening, and is restricted to soluble antigens 12,13 . There is, therefore, a need for a system that couples high-throughput single-cell phenotypic screening with paired V H -V L sequencing of IgG-secreting primary cells for both soluble and membrane-bound antigens in a flexible format that enables direct screening for functional activities 19 .…”

Section:  mentioning

confidence: 99%

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

et al. 2020

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Section:  mentioning

confidence: 99%

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

et al. 2020

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“…Although the NanOBlast workflow described here shares some similarities with previously described single-cell ASC discovery methods, [13][14][15][16][17][18][19][20][21][22] it introduces an entirely novel nanofluidic culture and screening paradigm using the Beacon platform. To our knowledge, Beacon is the only tool that enables massively parallel, precise, digitally driven control over the import, culture, screening, analysis, and export of nonimmortalized primary antigen-specific ASCs.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, several approaches that effectively used "micro tools" for direct B cell antibody discovery have been described. [16][17][18] Custom microfluidic chambers, 19 microencapsulation, 20,21 custom microwell devices, [22][23][24] and microcapillary tubes 25 have all been used to discover mAbs, although with variable success. These methods all allow the direct isolation and screening of ASCs as single cells, thereby allowing some phenotypic characterization of the encoded antibody without B cell immortalization or library generation.…”

Section: Introductionmentioning

confidence: 99%

Rapid single B cell antibody discovery using nanopens and structured light

Winters

McFadden

Bergen

et al. 2019

mAbs

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Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of antiidiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

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“…In addition, the antigenic reactivity of single cells can also be determined directly using a variety of methods including microengraving and diverse microfluidics devices such as microdroplets, microfluidic chambers, and microcapillaries. 70,71 New perfusion-based nanofluidic systems combined with light-activated phototransistor arrays enable direct detection of antigen-specific single PC that can then be isolated for additional analysis and mAb generation. 72 As previously discussed however, despite the tremendous pace and promise of these technological developments, the testing of B-cell autoreactivity using these methods lags behind other applications owing at least in part to limitations in the availability of suitable self-antigens of disease relevance and limited commercial applications.…”

Section: Autoreactivity Measurementsmentioning

confidence: 99%

Understanding and measuring human B‐cell tolerance and its breakdown in autoimmune disease

Cashman

Jenks

Woodruff

et al. 2019

Immunological Reviews

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It is well established that normal immune systems are endowed with a significant degree of autoreactivity. Accordingly, effective tolerogenic mechanisms are required to censor autoreactive cells and to ultimately prevent their differentiation into pathogenic effector cells. In this review, we will discuss how immunological tolerance is enforced in the human B-cell compartment. We shall also discuss the breakdown of B-cell tolerance in human autoimmune diseases.Finally, we will review different experimental approaches to measure B-cell tolerance in human disease. | B-CELL TOLER AN CE . CHECK P OINTS AND MECHANIS MS . LE SSONS LE ARNED THROUG H MOUS E S TUD IE SMouse models have been instrumental in defining multiple checkpoints and distinct mechanisms underpinning the enforcement of B-cell tolerance. Tolerance checkpoints are classified into central and peripheral mechanisms based on anatomical location and stage AbstractThe maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B-cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess.Functional autoreactive, flow cytometric, and single-cell cloning assays have proven to be critical in deciphering breaks in B-cell tolerance within autoimmunity; however, newer approaches to assess human B-cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions.

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Single cell screening approaches for antibody discovery

Cited by 40 publications

References 56 publications

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Rapid single B cell antibody discovery using nanopens and structured light

Understanding and measuring human B‐cell tolerance and its breakdown in autoimmune disease

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