Single B cell screening service

With the continuous development of monoclonal antibodies, Alpha Lifetech has introduced a single-cell sorting platform to quickly provide our global customers with highly specific and affinity antibodies. This platform is based on single-cell antibody preparation technology, including a comprehensive solution from antigen selection to antibody application. It can not only enrich a large number of B cells from humans and immune animals, but also perform high-throughput screening, flow cytometry sorting, single-cell sequencing, positive cloning, and in vitro expression research on enriched B cells.

Introduction to single-cell antibodies

In 1996, Babcook JS et al. first reported a method for preparing single-cell antibodies, which involved using single rabbit or mouse lymphocytes to produce immunoglobulin variable region complementary DNA molecular clones and produce specific antibodies. The preparation technology of single-cell antibodies, as an innovative technological achievement in recent years, has significantly accelerated the preparation process of monoclonal antibodies. The core of this technology is to recognize that each B cell contains only one functional heavy chain variable region DNA sequence and one light chain variable region deoxyribonucleic acid sequence, and produces only one antibody with clear specificity. To achieve this goal, researchers first collect B cells from lymphoid tissue or peripheral blood in humans or animals, and then use flow cytometry and efficient screening capabilities, combined with advanced sequencing technology, to accurately amplify the heavy chain and light chain variable region gene sequences of B cells with antibody production ability from individuals. Finally, select an appropriate expression system, such as mammalian cells, to express these genes and obtain monoclonal antibodies with biological activity.

single B cell Screening Service Process

Alpha Lifetech' single B cell sorting platform can screen tens of thousands of plasma cells, greatly reducing the B cell screening time of traditional hybridoma technology and ensuring high cell survival rates during this process. The single-cell sorting platform can provide a detailed process for preparing monoclonal antibodies, including mouse monoclonal antibodies and rabbit monoclonal antibodies, as shown in Figure 1. This process not only preserves the high specificity of antibodies, but also significantly improves preparation efficiency, opening up a new path for antibody research and development in the biomedical field.

Antigen selection and Animal immunzation

Antigen selection: Firstly, it is necessary to determine the target antigen, usually a protein, peptide, or other biomolecule.

Animal immunity: Choose appropriate animal models (such as rabbits, cows, sheep, camels, etc.). The target antigen is injected multiple times into the animal's body, usually several weeks apart, to stimulate the immune system to produce antibodies against the antigen. To enhance immune response, antigens are usually administered together with adjuvants. Differentiated sample collection strategies are adopted for animals of different body types, and for small animals, spleen separation is usually the preferred source of samples. However, for larger animals, analysis mainly focuses on peripheral blood collection. One of the key steps in peripheral blood processing is to isolate peripheral blood mononuclear cells (PBMCs) from peripheral blood.

Fig 1: Process flow chart of single B cell preparation

Enrichment and in vitro culture of B cells

Collection and isolation of B cells

Sample: taken from peripheral blood or lymphoid tissue B cells (such as spleen) of immunized animals (such as rabbits, cows, sheep, camels, etc.).

Cell separation: Effective labeling of separated B cells is achieved through the use of flow cytometry (both FACS) or magnetic bead separation techniques. These techniques can ensure precise separation of target B cells from complex cell mixtures.

Single B cell culture and selection

Single cell culture: Isolated single B cells are cultured in a medium containing appropriate nutrients and growth factors. During the cultivation process, B cells will continuously divide to form a colony of cloned cells.

Antibody screening: Use techniques such as ELISA (enzyme-linked immunosorbent assay) to screen the culture to determine which cell clones produce specific antibodies against the target antigen. Sorting cells through high-throughput screening or FACS.

Single B cell sorting service - FACS

FACS (Flow Cytometry Analysis and Sorting) is a technique for isolating and screening cells based on the expression levels of cell surface markers. The basic principle is to label surface markers on target cells using specific antibodies or fluorescent probes, and then perform high-speed cell analysis using flow cytometry to classify and separate cells based on their fluorescence intensity, size, shape, and other characteristics. This method can simultaneously analyze multiple parameters of a large number of cells, and has the advantages of being fast, accurate, and high-throughput. The FACS process includes:

Cell preparation

Sample collection: Separate cells from living organisms (such as blood, spleen, etc.). Cell processing: Perform steps such as cell lysis, centrifugation, and washing to obtain single-cell suspension.

Labeling cells

Fluorescent labeled antigens or specific antibodies are used to bind to receptors on the surface of B cells to label antigen-specific B cells.

Flow cytometry analysis

The labeled cells pass through the flow chamber of the flow cytometer and form a single-cell laminar flow under the action of sheath fluid. Flow cytometry collects fluorescence signals by irradiating each cell with a laser beam. Classify cells through computer analysis based on fluorescence intensity, cell size, scattered light, and other parameters.

Cell sorting

Set up a gate in a flow cytometer to select cells with specific expression levels. The target cells are separated from the population by static electricity, magnetic field, or mechanical force and collected in designated collection tubes.

Fig 2: Fluorescence-Activated Cell Sorting (FACS) workflow. Reference source: The use of fluorescence-activated cell sorting in studying plant development and environmental responses

Antibody gene amplification and vector construction

Gene amplification: Extract the variable region genes of the heavy and light chains of cloned antibodies from screened positive cells, and amplify them using RT-PCR technology to obtain the variable region sequences.

Vector construction: Clone the amplified antibody gene into an expression vector for in vitro or in vivo antibody expression.

Antibody expression and purification

Antibody expression: Transfect the constructed expression vector into suitable host cells (such as mammalian cells) to express recombinant antibodies. The expression process is usually carried out in cell culture systems.

Antibody purification: Purification and quality control of expressed antibodies through chromatography and other methods to achieve high-purity antibodies.

Antibody validation and application

Antibody validation: The purified antibody undergoes a series of in vitro experiments, including ELISA, Western Blot, and immunofluorescence, to ensure that it has sufficient affinity and specificity.

Antibody applications: Verified antibodies can be widely used in biomedical research, clinical diagnosis, treatment, and biopharmaceutical fields. For example, as a new drug candidate for cancer treatment, improving the efficacy and specificity of vaccines, supporting personalized immunotherapy, etc.

single B cell Sorting Service Workflow

Steps	Service Content	Timeline
Step 1: Animal immunization and ELISA detection	(1)Animals were immunized 4-5 times, starting from the 4th dose, serum was collected for ELISA detection of antibody titer (protein antigen ELISA serum titer >10^5; Peptide antigen ELISA serum titer >10^4). At the same time, 0.1ml of pre-immunization and post-immunization serum are sent to customers for testing; If the titer is not qualified, the immunization will continue or the client requests the termination of the project, and only the immunization part will be charged; (2)If the titer was qualified, PBMC cells were isolated from whole blood.	10-12 weeks
Step 2: Single B cell sorting	(1)Spleen was collected, single B cell suspension was prepared, peripheral blood was collected, and PBMC cells were isolated. (2) Immunoantigen markers FITC and AF648. (3) Double fluorescence flow sorting+ single b cell clone culture. (4)ELISA identification + sequencing of positive clones.	2-3 weeks
Step 3: Antibody expression and validation	(1) 6-10 sequence gene synthesis + mammalian expression test validation. (2) ELISA identification.	3-4 weeks
Step 4: Delivery	(1) Antibody sequences: 4-6 antibody sequences (unexpressed sequences are delivered together). (2) Each expressed sequence delivered 0.2mg of antibody. (3) Experimental report.	1 week