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Monoclonal antibody technology (Part One)

The discovery of antibodies originated in 1890. German physiologist Emil von Behring and Japanese microbiologist Shibasaburo Kitasato discovered a substance that can neutralize toxins in the blood of animals exposed to diphtheria toxin and tetanus toxin, and named it antibody (antibody). German scientist Paul Ehrlich, who collaborated with them, was inspired by the “lock-key theory” about the binding of enzymes and substrates at the time, and proposed that the combination of antibodies and toxin antigens is based on similar chemical structural principles. In the 1920s, Michael Heidelberger, the founder of immunochemistry and American microbiologist Oswald Avery discovered that antigens can be precipitated by antibodies and revealed that the chemical nature of antibodies is proteins. In 1948, Swedish immunologist Astrid Fagreaus discovered that plasma cells differentiated from B cells can produce antibodies effector.

Antibodies play an irreplaceable core role in adaptive immunity, and antibodies have both high affinity and high specific binding ability to various antigen molecules. However, we want to obtain antibodies that recognize a specific single antigen Very difficult. For example, to obtain antibodies that can specifically recognize influenza viruses, it is necessary to immunize model animals (such as mice or rabbits) with inactivated influenza viruses, and after a period of time, the antibodies are separated from the sera of the immunized animals. With the existence of a large number of different antibodies, only 0.5% to 5% of the antibody components obtained in this way can specifically bind to influenza virus. Not only is the specificity low, but the reproducibility is also difficult to guarantee. Even the use of affinity chromatography technology cannot purify influenza virus-specific antibodies, because affinity chromatography cannot separate antibodies against different epitopes of the same antigen. In this way, the purified antibodies will be thousands of A mixture of antibodies with different epitopes, also known as polyclonal antibodies, is also limited in scientific research and clinical applications.

How can we produce a large number of high-purity specific antibodies against a single epitope? In the 1960s, multiple myeloma (multiple myeloma) was discovered. As a plasma cell-derived tumor, it does not exist in the human body. Controlled immortal proliferation. Because myeloma cells maintain the high-efficiency secretion of antibodies by plasma cells, a large number of antibodies and antibody fragments called paraproteins (mostly IgA) can be detected in the serum and urine of patients with multiple myeloma. IgG, lambda light chain and kappa light chain). Tumor immunologist Michael Potter found that injection of mineral oil in a specific strain of mice can induce myeloma in mice, which provides animals and cells for myeloma and antibody research. Model. In 1974, when cell fusion technology was popular, Milstein’s team tried to perform cell fusion between some mouse myeloma cell lines and various other cells (such as fibroblasts). However, the antibodies secreted by hybrid cells after cell fusion Poor specificity. It happens that Niels Jerne developed a method that can screen plasma cells that secrete a single antibody, that is, through agar containing sheep red blood cells (SRBC)

Plaques on the plate are used to screen plasma cells that secrete antibodies against a single SRBC antigen. The red blood cells surrounding the plasma cell clusters that secrete and recognize SRBC antibodies will undergo cell lysis due to the action of the antibody binding, thereby forming a block visible to the naked eye. Areas of red plaques. However, due to the lack of immortality, these plasma cells that can secrete a single antibody cannot be cultured, and mass production of antibodies cannot be achieved. Fusion of plasma cells that secret a single antibody with immortal myeloma cells; it can solve the problem of stable and continuous mass production of single antibodies in one fell swoop.

As shown in Figure 1, the spleen cells of mice injected with SRBC were taken out, fused with the P3-X67Ag8 myeloma cell line, and cultured on an agar plate containing SRBC, a large amount of spleen was separated in the area without red patches Cells secrete anti-SRBC antibodies and have immortal fusion cells like myeloma cells. In HAT medium, since aminopterin blocks the de novo synthesis pathway of DNA nucleotides, cells must use their own DNA nucleotide salvage synthesis pathway and the hypoxanthine and thymidine in the medium can survive. But the myeloma cell line used here lacks the necessary enzyme in the DNA salvage synthesis pathway, namely hypoxanthine- Hypoxanthine-guanine phosphoribosyl transferase (HGPRT), so it cannot proliferate normally, and spleen cells themselves are not immortal. In this way, cells that can proliferate normally under the set culture conditions must have both HGPRT and can be normal synthetic DNA, a fusion cell with infinite division characteristics.

Figure 1. Generation of monoclonal antibody in Köhler-Milstein experiment.

The fusion cell cultured by Köhler and Milstein is called the first generation hybridoma (hybridoma) cell, which can be cultured, proliferated and antibody secreted indefinitely. But at the technical level, because the P3-X67Ag8 myeloma cell line they used at the time will Intrinsically secrete antibodies encoded by its own genome, so the plasma cell-derived specific antibodies secreted by the first-generation hybridoma cells will be mixed with non-specific antibodies derived from myeloma. But soon, a class will not be secreted intrinsically The X63-Ag8.653 myeloma cell line with non-specific antibodies derived from myeloma was successfully isolated. Using X63-Ag8.653 myeloma cells to fuse with plasma cells from the spleen was a good solution to the first generation of hybridoma cell culture the problem of mixing two kinds of antibodies in the supernatant. Since then, monoclonal antibodies that can highly specifically bind to various types of antigens that people need can finally be manufactured in vitro, and have been greatly used in laboratory research and medical applications.

The discovery of Köhler and Milstein created an epoch-making monoclonal antibody technology, which has a huge impact on biomedicine. Monoclonal antibodies are mainly used in two aspects in research: First, it is used to study the properties of the antibody itself and it is the mechanism of production; second, it is used to produce reagents that bind to specific proteins or other specific molecules.

Compared with the previous preparation of antibodies that cannot guarantee homogeneity and specificity, the high-purity and high-concentration monoclonal antibodies produced by the new technology will naturally be of greater help to the study of antibody specificity. The variable region of antibodies (V) The constant region (C) and the constant region (C) are encoded by different gene fragments. B cells connect these gene fragments together to form antibodies that recognize different antigens. Among them, the variable region of the antibody that has the ability to bind to the antigen, its gene fragments will perform as many as 1012 possible recombination, the diversity of the antibody library results from this. In addition, due to the somatic hypermutation mediated by antigen stimulation, the affinity of the antibody in the antibody response process continues to increase, that is, the affinity maturation of the antibody (affinity maturation). Monoclonal antibody technology promotes the molecular mechanism of antibody production at the molecular level.

Monoclonal antibody technology provides accurate qualitative, quantitative, and localized means for biological macromolecules (mainly proteins), and has now penetrated into all aspects of biomedical research, such as fluorescence for cell classification and screening analysis through cell surface protein markers Cell sorting technology, high-resolution fluorescence microscopy to display the localization of specific proteins in cells, Western blot to detect specific protein expression in protein mixtures, and enzyme-linked immunosorbent assay commonly used to determine cytokine expression in serum (enzyme-linked immunosorbnent assay, ELISA), etc., the specificity and resolution of these technologies are inseparable from the help of high-quality monoclonal antibody reagents. Thanks to the development of fluorescently-labeled monoclonal antibodies, it is now able to be at the single-cell level High-throughput mapping and analysis of protein marker expression profiles on the cell surface.

To be continued in Part Two…

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