About Us

Nebulabio is a pioneer in next-generation antibody discovery, specializing in advanced Single B Cell Sorting technology. We partner with therapeutic, diagnostic, and research organizations to unlock the full potential of the native immune repertoire for generating superior monoclonal antibodies. Our mission is to bypass the limitations of traditional methods by directly isolating antigen-specific B cells from immunized or convalescent hosts, preserving the natural heavy- and light-chain pairing that is crucial for affinity and specificity. Combining state-of-the-art flow cytometry, automated cell manipulation, and high-throughput molecular biology, we offer an end-to-end service designed to deliver functionally validated, sequence-ready antibody leads with exceptional speed and efficiency. We empower our clients to target complex epitopes, discover rare antibodies, and accelerate the path to clinical candidates.

☞Core Advantages

Nebulabio’s Single B Cell Sorting platform offers distinct, compelling benefits over hybridoma and display technologies:

• Native Pairing Preservation: The number one advantage. We isolate individual B cells, ensuring the natural, in vivo paired heavy-chain and light-chain sequences are recovered together. This preserves the authentic antibody structure and often results in superior affinity and stability compared to randomly recombined libraries.

• Discovery of Rare & High-Quality Antibodies: The technology accesses the complete, naturally matured B cell repertoire, enabling the discovery of rare clones with unique epitope coverage, cross-reactivity, or neutralizing activity that might be lost in bulk fusion or library construction.

• Broad Species Compatibility: We can work with a wide range of immunized or infected hosts, including mice, rats, rabbits, camels (for nanobodies), and humans (from convalescent or vaccinated donors), providing unparalleled flexibility.

• High Efficiency & Speed: By pre-selecting only antigen-reactive B cells, we eliminate the screening of non-productive clones upfront. This focused approach significantly accelerates the timeline from cell sorting to sequence, often yielding leads in weeks.

• Direct Access to Sequence Information: The process yields the complete antibody gene sequences immediately, facilitating rapid downstream steps like humanization, reformatting, and recombinant expression without the need for subcloning from hybridomas.

☞What is Single B Cell Antibody Technology?

Single B Cell Antibody Technology is a cutting-edge methodology for monoclonal antibody discovery that involves the direct identification, isolation, and analysis of individual B lymphocytes from an immune source. Unlike hybridoma technology, which relies on random fusion and bulk culture, or display technologies, which involve artificial library construction and panning, this technique starts with the precise physical separation of B cells that are actively producing antibodies against a target antigen of interest.

The core principle involves labeling B cells with fluorescently tagged target antigens (and other markers for cell viability and type), then using a high-speed Fluorescence-Activated Cell Sorter (FACS) to deposit single, antigen-binding B cells into individual wells of a microtiter plate. Each isolated cell contains the genetic blueprint (VH and VL genes) for a single, unique antibody. These genes are then recovered via reverse transcription and PCR amplification, cloned into expression vectors, and produced recombinantly for characterization. This method captures the antibody in its naturally evolved, paired state.

☞Single B Cell Antibody Discovery Workflow

Our workflow is a streamlined, integrated pipeline from immune response to characterized lead:

• Immune Source Preparation: Preparation of PBMCs or splenocytes from immunized, infected, or vaccinated donors/hosts.

• Antigen-Specific B Cell Staining: Cells are stained with a panel of fluorescent markers, including labeled target antigen(s) (as bait), anti-species B cell markers (e.g., CD19, CD20), and viability dyes. Probes for memory B cells or plasma cells can also be included.

• Fluorescence-Activated Cell Sorting (FACS): Using a high-end sorter, single cells that are positive for both the B cell markers and the target antigen fluorescence are sorted with high purity into 96- or 384-well PCR plates containing lysis buffer.

• Single-Cell RT-PCR & Amplification: Within each well, mRNA from the single B cell is reverse transcribed. Nested or multiplex PCR is performed to specifically amplify the paired immunoglobulin heavy-chain and light-chain variable region genes (VH and VL).

• Sequence Analysis & Cloning: PCR products are sequenced. The paired VH and VL sequences are analyzed for uniqueness, germline origin, and potential issues (e.g., stop codons). Selected sequences are then cloned into IgG expression vectors.

• Recombinant Expression & Screening: Antibodies are expressed transiently in small-scale mammalian cultures (e.g., HEK293). The supernatants are screened in high-throughput for binding (ELISA, FACS), affinity (BLI), and function.

• Lead Characterization: Top hits are expressed and purified for detailed characterization, including kinetics, specificity profiling, and advanced functional assays.

Figure1.Single B cell screening technologies using miniaturized equipment.

☞Single B Cell Antibody Platform

Nebulabio’s platform integrates specialized hardware, reagents, and bioinformatics:

• High-Parameter Cell Sorting: We utilize next-generation FACS instruments capable of multi-color analysis and ultra-pure single-cell deposition into plates, ensuring high efficiency and viability.

• Specialized Staining Reagents: We design and optimize antigen probes (e.g., recombinant proteins with distinct fluorescent tags) and sophisticated staining panels to precisely identify the desired B cell populations (e.g., antigen-specific memory B cells).

• Automated Single-Cell Molecular Biology: We employ automated liquid handling systems for consistent plate setup and PCR assembly, coupled with highly optimized, species-specific primer sets for reliable amplification of full-length, paired VH:VL sequences.

• Bioinformatics Pipeline: A custom software pipeline processes hundreds of sequencing reads, performs germline alignment, identifies clonal families, and annotates sequences, rapidly distilling data into actionable intelligence on lead candidates.

☞Applications of Single B Cell Antibody Screening

This technology is transformative for programs requiring high-fidelity, functional antibodies:

• Therapeutic Antibody Discovery: Ideal for infectious disease (discovery of potent neutralizing antibodies against viruses like SARS-CoV-2, HIV), oncology (finding antibodies against complex membrane targets), and autoimmune disorders.

• Antibody Repertoire Analysis: Profiling the immune response at a clonal level to understand immunodominance, affinity maturation, and epitope spreading in vaccine studies or natural infection.

• Difficult Targets: Highly effective for targeting complex membrane proteins (GPCRs, ion channels) in their native conformation, as B cells from immunized hosts have encountered the antigen in its physiological context.

• Rare Epitope Discovery: Isolating antibodies against subdominant or conserved epitopes that are critical for broad neutralization but may be rare in the overall response.

• Human Antibody Discovery Directly from Donors: Directly cloning antibodies from human convalescent donors or vaccine recipients, rapidly generating clinical leads with fully human sequences.

☞Nebulabio’s Service Process

We guide you through a collaborative, milestone-driven process:

• Consultation & Feasibility: We assess your target, discuss immune source options, and design the antigen staining strategy.

• Sample Reception & Cell Preparation: You provide immune cells (or we coordinate animal immunization). We prepare single-cell suspensions and perform quality control.

• Staining & Single B Cell Sorting: Our experts execute the custom staining panel and perform FACS, delivering a plate of single, antigen-positive B cells.

• Gene Amplification & Sequencing: We perform single-cell RT-PCR, sequence analysis, and provide a report listing unique, paired antibody sequences.

• Expression & Primary Screening: We clone and express selected antibodies, performing initial binding screens and delivering data on positive clones.

• Lead Characterization & Delivery: For selected leads, we provide purified antibody and an extended characterization data package (affinity, specificity, etc.). We deliver the final antibody sequences, expression clones, and materials to seamlessly transition into your development pipeline.

From start to finish, Nebulabio’s service is characterized by transparent communication, rigorous data delivery, and a partnership approach focused on achieving your discovery goals with the most advanced technology available.