Monoclonal antibody preparation technology

Monoclonal antibodies are antibodies produced by cloning a single B cell and recognize a single antigenic epitope, which is characterized by high specificity, high uniformity, and mass production.

Monoclonal antibody preparation technology is a key technology in the field of biology and medicine, pioneered in 1975 by German biologist Georges Jean Franz Köhler and Argentine/British biologist César Milstein, for which he was awarded the Nobel Prize in Physiology or Medicine. By fusing B lymphocytes with myeloma cells to form hybridoma cells, cell lines with antibody secretion ability and unlimited proliferation characteristics are screened using HAT selection medium to achieve large-scale production of single antibodies. This is the traditional hybridoma technique that is well known to the public and has been used to this day. Monoclonal antibodies have gone through four stages: murine antibodies, human-mouse chimeric antibodies, humanized antibodies, and fully human antibodies. The corresponding monoclonal antibody production technology has experienced the continuous development and improvement from the pioneering of hybridoma technology to phage display technology, transgenic animal technology and single B cell technology.

The original monoclonal antibodies were generated using mouse-derived proteins, but were not tolerated in humans with long-term use. To overcome the immunogenicity of mouse monoclonal antibodies and optimize their biological effects in humans, scientists subsequently developed mouse-human chimeric antibodies. Through genetic engineering technology, the variable region of mouse antibodies is fused with the constant region of human antibodies, and only the mouse-derived antigen binding site is retained in the structure, while the rest are replaced with human antibody sequences, which greatly improves its tolerance and efficacy in humans. With the continuous advancement of technology, scientists have successfully developed fully humanized antibodies through phage display technology and transgenic mouse technology, further improving the production efficiency and scale of human antibodies, and this innovation has driven the clinical application and market growth of monoclonal antibodies.

1、Hybridoma technology

The basic principle is to take advantage of the fact that B lymphocytes can produce a single specific antibody but cannot proliferate infinitely in vitro, while myeloma cells can proliferate infinitely in vitro but do not produce antibodies. The cells have the advantages of both, and a large number of hybridoma cell lines that produce monospecific antibodies can be obtained through screening and culture, and then monoclonal antibodies can be prepared. As the cornerstone, hybridoma technology has achieved large-scale production of monoclonal antibodies for the first time, and although there are limitations such as murine origin, production efficiency and cell line stability, its mature technology, relatively simple operation, and low cost are still commonly used in laboratories and industries.

2、Phage display technology

Phage display technology The principle is to insert the coding gene fragments of exogenous proteins and peptides into the appropriate position of the gene sequence encoding the bacteriophage shell protein through genetic engineering technology. The foreign protein or peptide fuses with the phage shell protein to form a fusion protein, which is expressed on the surface of the phage with the reassembly of the progeny phage. Fusion proteins maintain independent spatial structure and biological activity, and can recognize and bind to target molecules. The basic steps for preparing monoclonal antibodies using phage display techniques include: (1) Create a phage library containing antibody DNA sequences and evaluate the clonal diversity of the library. (2) The purified target antigen was used to screen out the non-specifically bound phages by screening and screening. (3) Pick out monoclonal phages, detect specificity by ELISA and identify positive clones by sequencing. (4) Subclone the positive antibody gene into the expression vector, transfect the host cell to induce expression, and then purify the antibody by affinity chromatography and other methods, and verify its activity by experiments. The two key parts are the construction of antibody phage libraries and the screening of antibodies.

 3、GMO animal technology

Antibodies prepared from ordinary mice are "mouse-derived". When it is injected into the body, the body's immune system recognizes it as a foreign substance and attacks, causing the antibody drug to fail and potentially cause serious side effects. Although this rejection can be mitigated through "humanized modification", the process is complex and may still retain some of the characteristics of the rat origin. The scientists then developed a mouse-human chimeric antibody. Through genetic engineering, the endogenous genes responsible for encoding the heavy and light chains of antibodies in animals are inactivated or directly knocked out. Import unrearranged antibody gene clusters from humans into the mouse genome. In this way, animal B cells complete a series of complex immune processes such as V(D)J rearrangement of human Ig genes in the body, resulting in the production of "fully human" or highly humanized antibodies. Once such transgenic mice are available, fully human monoclonal antibodies can be prepared using traditional hybridoma technology or single B cell technology.

 4、Single B cell technology

Single B cell technology bypasses the fusion of cells in traditional hybridoma techniques and directly targets a single antigen-specific B cell. Using flow cytometry, microfluidic chips and other technologies, individual B cells that can specifically bind to target antigens are accurately screened and isolated from peripheral blood, spleen and other tissues that are immunized to animals or immune bodies. Either by directly amplifying the heavy chain (VH) and light chain (VL) variable region genes of their antibodies by RT-PCR, or by cultured individual B cells in vitro culture systems to secrete antibodies and then clone the antibody genes. The amplified antibody genes are inserted into suitable expression vectors, transfected into host cells for expression and purification, and finally monoclonal antibodies with specific binding ability are obtained.

These new technologies have their own advantages and play an important role in different application scenarios, jointly promoting the progress and development of monoclonal antibody technology and bringing revolutionary changes to the field of biomedicine. Today, human monoclonal antibodies are the fastest-growing category of monoclonal antibody therapeutics entering clinical trials.

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Comparison of the Four Technologies

Felicia  Felicia is a technical support specialist at EnkiLife, with extensive professional experience in antibody development, optimization, and ELISA assay design and application. She is committed to assisting our clients in selecting suitable antibody products, optimizing ELISA experimental protocols, and resolving technical challenges encountered in the process, thereby supporting the smooth progress of their life science research projects.