Monoclonal antibodies (mAbs) are one of the most successful classes of biologic drugs on the global pharmaceutical market. Since the approval of Orthoclone OKT3 in 1986, over 100 mAbs have been approved by the U.S. FDA for indications including cancer, infectious diseases, autoimmune disorders, and neurological conditions. Their high specificity and predictable developability profiles make them valuable not only in therapeutics but also in diagnostics and research.
Traditionally, monoclonal antibody discovery has relied on hybridoma technology and phage display. While both systems have enabled major therapeutic advances, their limitations, such as low throughput, restricted species compatibility, artificial VH/VL pairing, and long timelines, have driven the development of alternative methods. Among these, single B cell screening offers a more direct and rapid approach to mAb discovery.
B Cell Biology: B cells are integral to the adaptive immune system. They recognize specific antigens through surface-bound B cell receptors (BCRs) and, upon activation, differentiate into antibody-secreting cells. Antibodies generated by B cells are the primary effectors of humoral immunity and are central to both natural and vaccine-induced protection.
Antibody Diversity: Antibodies achieve their diversity through somatic recombination of variable (V), diversity (D), and joining (J) gene segments, followed by somatic hypermutation. While the theoretical diversity of the human B cell repertoire ranges from 10¹² to 10¹⁸ clonotypes, the actual circulating repertoire in an individual is on the order of 10⁷–10⁸ unique B cell clones. Single B cell screening enables direct interrogation of this diversity to identify rare, high-affinity clones.
Single B cell screening involves isolating antigen-specific B cells and recovering their paired VH and VL gene sequences to express as recombinant monoclonal antibodies. Unlike display technologies, this method retains the native pairing of immunoglobulin chains, critical for maintaining physiological binding properties and reducing immunogenicity.
Because these antibodies arise through in vivo development and affinity maturation, they generally demonstrate reduced off-target binding to the human proteome, favorable biophysical properties, and lower immunogenicity, which are key considerations for therapeutic development.
FACS is a widely used method for isolating antigen-specific B cells based on fluorescence intensity. Cells are stained with fluorescently labeled antigens—often using two different fluorophores to minimize false positives—and sorted by gating on both antigen binding and B cell-specific surface markers. Antigen bait can be in the form of labeled proteins, tetramers, antigen-coated beads, or even virus-like particles (VLPs). False positives from nonspecific fluorophore or tag binding are controlled by dual-labeling strategies. The approach is high-throughput, cost-effective, and compatible with standard laboratory instrumentation. However, it depends on the availability of soluble, native-like antigen forms and may not capture plasma cells effectively.
Microfluidics has transformed single B cell screening by enabling high-throughput analysis in miniaturized, compartmentalized systems such as droplets or nanoliter chambers. These platforms include open systems (e.g., Berkeley Lights Beacon, microcapillary arrays) and closed systems (e.g., water-in-oil droplets). Biointron’s AbDrop™ platform utilizes advanced droplet-based microfluidic technology to accelerate antibody discovery from initial screening to validated candidates in just one month.
AbDrop™ integrates single-cell encapsulation, secretion-based screening, and monoclonal cell isolation into a fully automated, sterile, animal component-free workflow. Each B cell is isolated in a picoliter droplet, allowing for ultra-sensitive detection of secreted antibodies at concentrations far higher than in traditional assays. The system rapidly screens hundreds of thousands of individual cells and selectively isolates high-producing clones directly into microtiter plates with high viability and clonality assurance. This approach dramatically shortens timelines, reduces reagent use, and improves throughput, thus making AbDrop™ an ideal solution for next-generation antibody discovery and cell line development.
Following B cell isolation, RT-PCR is used to recover VH and VL genes from single cells. Successful amplification relies on the use of degenerate or nested primer sets. For human B cells, primers from Friedensohn et al. are commonly used; for murine B cells, the von Boehmer et al. set has shown consistent results. The goal is to ensure high coverage across all immunoglobulin gene families. Because plasma cells and plasmablasts have limited ex vivo viability, rapid processing is critical to prevent RNA degradation.
Barcoded beads and sequencing adapters are used to prepare libraries for next-generation sequencing (NGS). Platforms like 10x Chromium and Drop-seq enable single-cell resolution analysis and native VH/VL pairing. Sequencing identifies expanded B cell clones and highly mutated lineages—hallmarks of antigen-experienced cells. This allows prioritization of candidate antibodies based on clonal expansion and somatic hypermutation frequency. However, this approach may miss rare clones, and timelines can extend to several weeks due to the need for bioinformatic processing, gene synthesis, and expression validation.
Once candidate sequences are selected, VH and VL genes are cloned into expression vectors containing human or species-specific constant regions. These constructs are expressed in mammalian cells, often using proprietary high-yield cell lines. Expressed antibodies are screened for specificity and function using ELISA, FACS-based binding assays, or virus/toxin neutralization. Functional screening remains a bottleneck in some high-throughput systems, as most focus on antigen binding rather than activity. Expression variability and developability concerns (e.g., aggregation, poor folding) also require evaluation.
Related: Single B Cell Screening
Sample Collection and Antigen-Specific B Cell Isolation: Samples are typically derived from immunized animals or seropositive human donors. Peripheral blood mononuclear cells (PBMCs) provide access to memory B cells and plasmablasts, the primary sources for antigen-specific mAbs.
Gene Recovery and Cloning: Single B cells are lysed, and their immunoglobulin genes are amplified using nested PCR with optimized primer sets. The sequences are cloned into expression vectors containing constant regions for human IgG subclasses.
Antibody Expression and Functional Assays: Transfected mammalian cells produce recombinant antibodies, which are screened using ELISA, SPR, or virus/toxin neutralization assays. Functional screening is critical for validating the specificity and biological relevance of the antibodies.
Sequence Analysis and Optimization: Bioinformatics pipelines annotate gene usage, mutation frequency, and clonality. Candidates with desirable properties can be further engineered, for example, as scFv, bispecifics, or ADCs.
Therapeutic mAb Development: Single B cell screening is especially suited to identifying therapeutic antibodies directly from human B cells. These antibodies often require no humanization and display better developability profiles due to native in vivo selection.
Infectious Diseases: The COVID-19 pandemic underscored the need for rapid mAb development. Single B cell screening enabled the fast-tracked identification of SARS-CoV-2-neutralizing antibodies, demonstrating its utility in pandemic response.
Oncology and Immuno-Oncology: Antibodies derived from tumor-infiltrating B cells are used to identify cancer-specific epitopes. Variants like scFvs and bispecifics are being developed for T cell engagement and tumor cell targeting.
Diagnostics: High-specificity antibodies from single B cells improve diagnostic assay performance, reduce cross-reactivity, and enable early disease detection.
Research and Biomarker Discovery: Custom single B cell services support projects investigating antigen-specific immune responses, vaccine efficacy, and novel antigen discovery.
Related: Revolutionizing Single B Cell Screening with High-Throughput Antibody Discovery
Single B cell systems deliver high-affinity antibodies with superior developability due to their preservation of native sequences and post-translational modifications. In contrast, phage display systems, while powerful, may produce antibodies with reduced stability or specificity due to their reliance on in vitro selection and artificial pairing.
Ex Vivo Viability: Primary antigen-specific B cells, particularly plasmablasts and plasma cells, are short-lived ex vivo. Protocols must minimize culture time and preserve mRNA integrity for accurate VH–VL recovery.
Antigen Format and Conformation: Effective screening often requires soluble, native-like antigens. This can be problematic for membrane proteins, GPCRs, and other conformationally sensitive targets.
Cost and Complexity: High-throughput systems using microfluidics or droplet encapsulation require significant capital investment and specialized expertise.
Data Analysis: The scale of single-cell sequencing demands robust computational tools for sequence alignment, clonotyping, and mutational profiling.
Diverse Protocols Without a Gold Standard: The field currently lacks standardized protocols. Each single B cell screening platform offers trade-offs in terms of throughput, ease of use, cost, and suitability for specific antigens.
Contract Research Organizations (CROs) have incorporated single B cell technologies to streamline the antibody discovery process for biotech clients.
Services Offered Include:
Donor or immunized animal sourcing and sample collection
FACS or microfluidic-based B cell sorting
Single-cell sequencing and gene recovery
Recombinant antibody expression and screening
Sequence validation and developability assessment
Regulatory documentation and traceability
These CRO services are tailored to support therapeutic, diagnostic, or research-focused antibody programs with defined timelines and deliverables. In particular, single B cell services provide a path for rapid and biologically relevant discovery of high-performance antibodies suited for challenging targets.
At Biointron, we are dedicated to accelerating antibody discovery, optimization, and production. Our team of experts can provide customized solutions that meet your specific research needs, including HTP Recombinant Antibody Production, Bispecific Antibody Production, Large Scale Antibody Production, and Afucosylated Antibody Expression. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.
References:
Pedrioli, A., & Oxenius, A. (2021). Single B cell technologies for monoclonal antibody discovery. Trends in Immunology, 42(12), 1143–1158. https://doi.org/10.1016/j.it.2021.10.008
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