I. Single B Cell Antibody Technology

As a next-generation antibody development technology, single B cell antibody technology enables efficient and rapid antibody isolation from individual B cells, representing a breakthrough following hybridoma and phage display technologies.This technology is based on the principle that each B cell contains only one functional heavy-chain variable region (VH) DNA sequence and one light-chain variable region (VL) DNA sequence, and each B cell produces only one specific antibody. Antigen-specific B cells are isolated from immunized animal tissues or peripheral blood, and single-cell PCR is used to amplify the IgG heavy- and light-chain variable region genes from individual antibody-secreting B cells. These genes are then expressed in mammalian cells to produce biologically active monoclonal antibodies.

II. Single B Cell Sorting Platform

This platform integrates microfluidics with various sorting technologies to directly isolate, analyze, and screen B cells at the single-cell level, enabling precise and efficient identification of B cells secreting target antibodies. By combining single-cell sequencing, the target antibody sequences can be obtained.In addition to screening immunized animals, this technology can utilize fully humanized transgenic mice for immunization, allowing direct acquisition of fully human antibody sequences post-screening. Commonly used sorting methods include the following:

1.Isolation of B Cells by MACS (Magnetic-Activated Cell Sorting)

Principle: This method is based on the specificity of antigen-antibody binding.  B cell surface molecules bind to magnetic beads coated with specific antibodies, forming a surface molecule-antibody-magnetic bead complex.  Under an external magnetic field, cells attached to the antibody-coated beads are retained in the magnetic field, while cells that do not bind to the specific antibodies (and thus lack magnetic beads) pass through without retention, achieving cell separation.

Advantages: Simple operation, high stability, and good reproducibility.

Disadvantages: Requires multiple types of antibodies, demands high-quality magnetic beads and columns, and is relatively costly.

This method can be used in combination with other separation techniques.

2.Isolation of B Cells by FACS

The principle of isolating B cells by FACS (Fluorescence-Activated Cell Sorting) is based on the specific antigen-antibody interaction. By using fluorescently labeled antibodies targeting B cell surface molecules and labeled specific antigens, antigen-specific B cells are screened through multicolor flow cytometry analysis and sorting. FACS enables the sorting of individual antigen-specific B cells into single wells of a cell culture plate, allowing direct amplification and characterization of antibody genes. This technique is currently one of the most widely used methods.

Advantages: Rapid, precise, high-throughput isolation of B cells with simultaneous multiparameter analysis.

Disadvantages: High cost, and the electromagnetic field may cause some damage to cells, potentially affecting their viability.

3.Isolation of B Cells by Microfluidic Technology

The integration of microfluidic devices and chip technology is currently the mainstream commercial platform for single B cell isolation. The Beacon Optofluidic System from BLI (Berkeley Lights, Inc.) is favored by many major pharmaceutical companies. The Beacon platform uses a microfluidic system to transport cells secreting antigen-specific antibodies to designated locations on the chip, where individual cells are isolated into separate nanoscale chambers via optoelectronic positioning (OEP) technology. Subsequently, a bead-based dual-color fluorescence binding assay is employed to detect secreted antibodies from each cell. Cells producing antigen-specific antibodies generate fluorescent signals, which are identified by the instrument. Single positive cells are then exported using OEP technology and transferred into a 96-well plate for further sequencing and expression.This system integrates single-cell sorting, detection, and analysis, offering high sensitivity and accuracy.

III. Amplification and Cloning of Antibody Genes

After isolating antigen-specific B cells via FACS, they are sorted into a 96-well plate containing lysis buffer to lyse the cells and release intracellular RNA. Subsequently, RT-PCR and nested PCR are performed to obtain the variable region genes of antigen-specific antibodies from single B cells.

IV. Expression, Screening, and Characterization of Antigen-Specific Antibodies

Following FACS-based single B cell sorting, the essential steps include antibody gene amplification, vector construction, antibody expression, and functional validation.  Common expression systems include prokaryotic systems (e.g., E. coli) and eukaryotic systems.  In E. coli, the antigen-binding fragment (Fab) of the antibody is typically expressed, whereas full-length IgG molecules can be produced in mammalian cells. The biological activity of antibodies is primarily validated using conventional methods such as ELISA, indirect immunofluorescence, and neutralization assays.  Additionally, other techniques may be employed, including flow cytometry analysis, co-immunoprecipitation, plaque assays, and plaque reduction neutralization tests (PRNT) for further characterization.

V. Applications of Single B Cell Antibody Production Technology

Antiviral Therapeutics:Single B cell antibody technology enables the isolation of antibody-secreting B cells from convalescent patients' blood, allowing rapid identification and expression of antibody genes. This approach is particularly valuable for the treatment and prevention of emerging viruses. Numerous single B cell-derived antibodies against viral infections are currently in clinical trials. For instance, HIV—the causative agent of AIDS, which has infected 63.9 million people globally—has been targeted using this technology. Five anti-HIV envelope protein antibodies obtained through single B cell methods are now under evaluation in Phase I/II clinical trials.

Neurological Disease Treatment:The migraine drug Vyepti, a rabbit-derived antibody developed using single B cell technology, targets and blocks the calcitonin gene-related peptide (CGRP) ligand from binding to its receptor. Phase II clinical results demonstrated a 75% reduction in migraine days for many patients. Additionally, another migraine drug from the same company, ALD1910, is currently in preclinical development.

Immunological Disease Therapy:Single B cell antibody technology facilitates the isolation of B cells from any stage of the human immune response, enabling in-depth study of immune system functionality and mechanisms across different phases. This holds significant promise for research into therapeutic antibodies for autoimmune diseases and other immune-related disorders.