The production of monoclonal antibodies using the hybridoma technology Sbonelo Hadebe 211554629 October 16, 2015 Contents 1 Introduction 1 2 Theory 2 2.1 Monoclonal antibodies...................... 2 2.2 Method of hybridoma technology................ 3 2.3 Purification of monoclonal antibodies.............. 3 2.4 Application of monoclonal antibodies.............. 5 2.5 Monoclonal Antibody in medicine................ 6 3 Conclusion 6 Abstract This article describes the production of monoclonal antibodies using hybridoma technology. The hybridoma technology is a method is described for obtaining antibody-producing hybridomas that are preferentially retained in cultures offused spleen and myeloma cells. Hybridomas are produced by fusing myeloma cells that are deficient in adenosine phosphoribosyltransferase (APRT) with spleen cells carrying Robertsonian 8.12 translocation chromosomes. The cellfusion mixtures are exposed to a culture medium that can be utilized only by APRTpositive cells, which results in the removal of both unfused APRTdeficient myeloma cells and non-antibodyproducing APRT-deficient hybridomas that arise by separatism of the 8.12 translocationchromosomes comprising the APRT genes and the active heavy chain immunoglobulin gene. 1 Introduction Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing an antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its quality to grow in tissue culture and 1
for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single particularity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by César Milstein and Georges J. F. Köhler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with Niels Kaj Jerne, who made other contributions to immunology. The term hybridoma was coined by Leonard Herzenberg during his sabbatical in César Milstein s laboratory in 1976-1977. The hybridoma technology guarantees the unlimited production of monoclona anitbonies of the same isotype with invariable properties. Although it can be foreseen that monoclonal antibodies may eventually be substituted by ret antibodies, hybridoma cells are still required, at least for the time being, as sources for mrna coding for specific. Production of monoclonal antibodies is based on the proliferation of a single antibody-producing cell giving up a uniform population of ab of the same type with identical affinities and specificities. Raising monoclonal antibodies takes advantage of the vertebrate immune system which is able to make antibodies against nearly any chemical structure. However, the hapten property of pesticides such as s-triazines requires coupling of derivatives with reactive groups to an immunogenic carrier protein, e.g., keyhole limpet haemocyanin (KLH). After immunizing mice with the appropriate immunogen, the absecreting B-lymphocytes are fused with suitable myeloma cells.the resulting hybridomas are first selected in a HAT medium containing hypoxanthine,aminopterin and thymidine. Aminopterin poisons the parent myeloma cells which lack an enzyme in the nucleic acid pathway. The hybridomas can survive the HAT selection by using a saivage pathway, due to the complementation of the genetic defect by the B-lymphocyte genome. 2 Theory 2.1 Monoclonal antibodies Monoclonal antibodies (mab or moab) are monospecific antibodies that are made by identical immune cells that are all clones of a incomparable parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that component; they can then serve to detect or purify that substance. This has become an essential tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (Nomenclature of monoclonal antibodies), and many immunotherapy specialists use the word mab anacronymically. 2
2.2 Method of hybridoma technology Laboratory animals (mammals, e.g. chicken or mouse) are first exposed to the antigen that an antibody is to be bring forth against. Normally this is done by a series of injections of the antigen in question, over the course of several weeks. These injections are typically followed by the use of in vivo electroporation, which importantly enhances the immune response. Once splenocytes are isolated from the mammal s spleen, the B cells are fused with immortalised myeloma cells. The fusion of the B cells with myeloma cells can be done using electrofusion. Electrofusion causes the B cells and Myeloma cells to align and fuse with the application of an electric field. The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves and that they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, making them sensitive to the HAT medium.fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for roughly 10 to 14 days. Aminopterin blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the de novo or salvage pathways because they lack HGPRT. Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells, especially weakly established hybridomas. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are thus monoclonal antibodies. 2.3 Purification of monoclonal antibodies After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis. Most of the charged impurities are usually anions such as nucleic acids and endo- 3
Figure 1: The hybridoma technologu from immunisation to monoclonal antibody production and purification.. Figure 2: The types of cells used in hybridoma technology where those which dies or live are observed. 4
Figure 3: The steps in the purification of monoclonal antibodies. toxins. These are often separated by ion exchange chromatography. Either cation exchange chromatography is used at a low enough ph that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough ph that the desired antibody flows through the column while anions bind to it. 2.4 Application of monoclonal antibodies The use of monoclonal antibodies is numerous and includes the prevention, diagnosis, and treatment of disease. For example, monoclonal antibodies can distinguish subsets of B cells and T cells, which is helpful in identifying different types of leukaemias. In addition, specific monoclonal antibodies have been used to define cell surface markers on white blood cells and other cell types. This led to the Cluster of differentiation series of markers. These are often referred to as CD markers and define several hundred different cell surface components of cells, each specified by binding of a particular 5
monoclonal antibody. Such antibodies are extremely useful for fluorescenceactivated cell sorting, the specific isolation of particular types of cells. 2.5 Monoclonal Antibody in medicine Monoclonal antibodies can be used as drugs to fight cancer. When a monoclonal antibody attaches to a cancer cell it can Make the cancer cell more visible to the immune system. The immune system attacks foreign invaders in your body, but it doesn t always recognize cancer cells as enemies. A monoclonal antibody can be directed to attach to certain parts of a cancer cell. In this way, the antibody marks the cancer cell and makes it easier for the immune system to find.the monoclonal antibody drug rituximab (Rituxan) attaches to a specific protein (CD20) found only on B cells, one type of white blood cell. Certain types of lymphomas arise from these same B cells. When rituximab attaches to this protein on the B cells, it makes the cells more visible to the immune system, which can then attack. They can Deliver radiation to cancer cells. By combining a radioactive particle with a monoclonal antibody, doctors can deliver radiation directly to the cancer cells. This way, most of the surrounding healthy cells aren t damaged. Radiation-linked monoclonal antibodies deliver a low level of radiation over a longer period of time, which researchers believe is as effective as the more conventional high-dose external beam radiation. Ibritumomab (Zevalin), approved for non-hodgkin s lymphoma, combines a monoclonal antibody with radioactive particles. The ibritumomab monoclonal antibody attaches to receptors on cancerous blood cells and delivers the radiation. 3 Conclusion In Diagnosis, pregnancy can be discovered by assaying of hormones with monoclonal antibodies. Likewise, pathogens can be detected in a small number of hours sparing few days of culturing of cells earlier needed. Immunopurification includes split-up of one substance from a mixture of very similar molecules. Antibodies are proteins synthesized in blood against specific antigens just to fight and give immunity in blood. They can be collected from the blood serum of an animal. Such antibodies are heterogeneous and contain a mixture of antibody ies (i.e., monoclonal antibodies). Therefore, they do not have characteristics of specificity. If a specific lymphocyte, after the isolation and culture in vitro, the becomes capable of producing a single type of antibody which bears specificity against specific antigen. It is known as monoclonal antibodies. Due to the presence of desired immunity, monoclonal antibodies are used in the diagnosis of diseases. 6
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