Development of Single B Cell Screening Technology

In 1975 Milstein[1] first described a method to produce monoclonal antibody by hybridoma technology, which greatly promoted the development of immunology. Monoclonal antibody has become an important scientific research tool and therapeutic molecule, and this technology is still the main technology for the preparation of monoclonal antibody. It plays an important role in cell biology technology. Monoclonal antibody is an important tool in the field of medicine and immunology, and plays a great role in the pathogenesis, diagnosis and treatment of pathogens. At present, the most commonly used methods of monoclonal antibody preparation are hybridoma technology and phage display technology. Hybridoma technology is the fusion of spleen cells and bone marrow cells of immune mice to form hybridoma cells that survive in vitro for a long time and secrete immune proteins, and then produce monoclonal antibodies targeting the same antigen binding site, which is the most classic monoclonal antibody preparation technology. This method is mature and transparent, and can produce strong specific monoclonal antibodies, but the preparation time is long, the repetitive production performance is poor, the difference between antibody batches is large, and hybridoma cells are very easy to appear in the culture process chromosome loss, clonal competition, hybridoma decreased ability to secrete antibodies, etc., increase the risk of antibody development. Phage display technology is to insert variable region genes of antibodies into phage genes, display the expressed antibodies to the surface of phage, construct phage display antibody library, simulate the production of antibodies in vitro, and screen out antibodies against different antigens. The phage display method does not need to immunize animals, shortens the antibody preparation cycle, and has a large screening capacity, which can screen hundreds of millions of clones in a short time. However, the random combination of antibody variable region genes will lead to the loss of natural pairing of antibody heavy chain and light chain, and the modification of antibody is different from that of mammals, so the application of this method in production is limited.

In recent years, the single B cell antibody screening technology has been gradually developed, and then it has been applied to the preparation of monoclonal antibodies. This technology uses flow cell sorting to fluorescentially label specific antigens, and uses the principle that antigens can specifically bind to the BCR on the surface of B cells to screen out single target B cells. Then, the heavy chain and light chain genes of antibodies are obtained by PCR amplification, and the antibody genes are constructed on the expression vector. Transfected into eukaryotic cells to express monoclonal antibodies(Figure 1). Different from traditional hybridoma cell sorting technology, single cell screening technology can directly isolate, culture, analyze and screen single B cells, so as to accurately and efficiently screen out B cells secreting target antibody molecules, and then obtain the target antibody sequence by combining single cell sequencing technology. This technique can quickly produce monoclonal antibodies against humans, rats, rabbits, alpacas, pigs, chickens, dogs and other animals. The method is time-saving and efficient, and retains the natural pairing of antibodies, and has been widely used in the research of infectious diseases such as HIV, COVID-19 , and influenza.

Figure 1: Schematic diagram of preparation of McAbs based on single B cell antibody technology[2]

Significance of Single B Cell Screening Technology inbiotechnology research

Monoclonal antibody plays an important role in the prevention, diagnosis and treatment of diseases, especially in the study of immune mechanism. With the development of monoclonal antibody preparation technology, single B cell antibody preparation technology has become a new generation of rapid monoclonal antibody preparation method. Compared with traditional antibody preparation technology, this technology has the advantages of fast, efficient and high yield, and the antibody expressed has a natural conformation, which can not only be used for the development of antibodies related to pathogenic microorganisms and the study of the mechanism of virus cross-species transmission, but also play an important role in anti-tumor therapy and anti-autoimmune diseases.

Categories Single B Cell Technique Hybridoma Technology Phage Display Technology
Antibody Natural Natural Unnatural
Antibody Affinity High High Medium
Druggability High High Low
Time 16 to 20 Weeks, Optional Rapid Immunization 20 to 26 Weeks, Optional Rapid Immunization 16 to 20 Weeks, Optional Rapid Immunization
Antibody Gene Sequence Direct Acquisition Further Sequencing Required Direct Acquisition
Antibody Diversity High Medium Medium
The species that can be screened Unrestricted Species Mouse and Rabbit Unrestricted Species

References

[1] Köhler, Milstein C .Pillars Article: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256 (5517): 495–497.[J].Journal of Immunology Official Journal of the American Association of Immunologists, 2005, 174(5):2453-2455.

[2] CHEN Yang, LIU Tong, ZHANG Jia-qi, LIAO Hua-xin, LIN Yue-zhi, WANG Xiao-jun, WANG Ya-yu. Screening of Monoclonal Antibodies Targeting the Equine IgG1 Based on Single B Cell Antibodies Gene Amplification Technology. China Biotechnology, 2022, 42(4): 17-23.

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