1.Principle of Bovine Single B Cell Technology

Single B cell antibody development technology is a recently developed approach for the rapid preparation of monoclonal antibodies. Based on fluorescence-activated cell sorting (FACS), this technique isolates individual B cells for monoclonal antibody production. Since each B cell contains only one functional heavy-chain variable region DNA sequence and one light-chain variable region DNA sequence—and thus produces only one specific antibody—B cells are collected from the lymphoid tissues of animals. Through monoclonal expansion, the variable region genes of the heavy and light chains are amplified from individual antibody-secreting B cells, allowing the isolated B cells to proliferate into clonal populations of single origin. Subsequently, suitable antibody production hosts are selected to generate the desired antibodies.

Bovine single B cell technology offers advantages such as high specificity and minimal batch-to-batch variation. Monoclonal antibodies produced by this method are widely used as standard monoclonal antibodies. Currently, antibodies prepared via bovine single B cell technology have broad applications in biomedical research, diagnostic tools, and therapeutic drug development.

Figure 1: Preparation of Bovine Recombinant Monoclonal Antibodies

2. Preparation Process of Bovine Single B-Cell Monoclonal Antibodies

①. Animal Immunization and PBMC Isolation. Cattle were selected as the experimental animals and immunized by injecting the target antigen (typically a target protein or peptide) to stimulate an immune response. The cattle were immunized 4-5 times to activate their immune system, prompting B cells to produce antibodies. Subsequently, ELISA titer determination, peripheral blood collection from immunized cattle, and PBMC isolation were performed.

②. Flow Cytometry Sorting. Antigen-specific single B cells were sorted using the PBMC flow cytometry sorting method.

③. Single B Cell Culture and Screening. The sorted single B cells were first cultured in vitro using 5–20 96-well plates for single B cell culture. The supernatant from the cell cultures was then screened via ELISA or preliminary intervention assays such as FACS.

④. Positive Clone Sequencing. At least 20 positive cell clones from the screening were selected for RT-PCR to amplify their antibody variable region genes, followed by sequencing.

⑤. Vector Construction and Antibody Expression/Purification. Finally, mammalian expression vectors for the antibodies were constructed, followed by cell transfection and ELISA validation.

Figure 2: Flowchart of Antibody Preparation Using Bovine Single B Cell Technology

3. Advantages of Monoclonal Antibody Preparation via Single B Cell Technology

The single B cell technology enables rapid monoclonal antibody production based on B cell characteristics, allowing direct acquisition of complete single-source antibodies. This method is species-independent, highly efficient, and offers superior specificity, high antibody affinity, excellent developability, robust genetic diversity, and fully natural origin. Additionally, it provides direct access to antibody gene sequences.

4. Applications of Bovine Single B Cell Technology

Antibodies produced by bovine single B cell technology are widely applied in the following four areas:

① Drug Development – Antibodies generated through bovine single B cell technology can be used in the development of new drugs, particularly showing great potential in the field of cancer treatment.

② Vaccine Development – These antibodies can enhance the efficacy and specificity of vaccines.

③ Immunotherapy – Antibodies prepared using bovine single B cell technology can be utilized in immunotherapy, offering more personalized treatment options for patients.

④ Basic Medical Research – Due to their specificity and diversity, these antibodies are valuable for elucidating biological processes and disease mechanisms.