Introduction of Hybridoma Technology

Introduction of Hybridoma Technology:
Hybridoma technique is a method of creating pure and uniform antibodies. The hybrid cell or hybridoma resulting from the fusion between myeloma cell and spleen cell of immunized cell of the donor. The term antibodies can be produced in specialized cells through a technique now popularly known as the hybridoma technology. Monoclonal antibodies is valuable for the analysis of parasites antigen and appropriate that WHO should have organized a symposium which brought together those who have establish and refined the technology and those who are using it, or intending to use it for the study of organism responsible for some of the major diseases affecting mankind. Such monoclonal antibodies, as they are known, have opened remarkable new approaches to preventing, diagnosing, and treating disease. Monoclonal antibodies are used to distinguish subsets of B cells and T cells. This knowledge is helpful not only for basic research but also for identifying different types of leukemia and lymphomas and allowing physicians to tailor treatment accordingly. Quantitating the number of B cells and helper T cells is all-important in immune disorders such as AIDS. Monoclonal antibodies are being used to track cancer antigens and, alone or linked to anticancer agents, to attack cancer metastases. The monoclonal antibody saving organ transplants threatened with rejection, and preventing bone marrow transplants from setting off graft-versus-host disease. (Global-research-online.net, 2018) (Anon, 2018)

Principles:
The Hybridoma technology is based on the immortalization of B lymphocyte cells with antibody producing capacity but limited growth characteristics in vitro. The lymphocytes are fused with cells from a non-antibody producing and continuously growing tumour cell line (myeloma), so that the hybrids continue to secrete antibodies while gaining the immortality of the parent tumour cells. Thus, MoAbs are products of ‘hybrid’ cells created by the fusion of two cells, one of which is a myeloma cell with genetic potential of multiplying perpetually, and the other has a gene that codes for the desired antibody. (Anon, 2018)

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(Anon, 2018)

Discovery of Hybridoma Technology:
• 1964- Littlefield developed a way to isolate hybrid cells from two parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media.
• 1975- Hybridoma technology was first discovered by Georges Kohler and Cesar Milstein They were also awarded Nobel Prize along with Niels K Jerne in Physiology & Medicine field during 1984.
• 1990- Milstein produced the first Monoclonal antibodies. (Jana, 2018)

Methodology:
The following steps involved in Hybridoma Technology:
• Immunization and Isolation of B cells from the spleen:Laboratory animals (e.g. mice) are first immunized by exposed to an antigen to which we areinterested in isolating an antibody against and then spleen cells of mouse are removed out.

(Jana, 2018)
• Screening of mice for antibody production: After several weeks of immunization Blood sample of mice for measurement of serum antibodies titre Determined (Technique: – ELISA / Flow cytometry)
Titre is too low mice can be boosted until an adequate response is achieved.
Titre is high mice are commonly boosted by injecting antigen without adjuvant
• Cultivation of myeloma cells: Myeloma cells are immortalized cells that are cultured with 8 Azaguanine to their sensitivity to HAT medium used after cell fusion.
• Generation of B cell hybridomas by fusing prime B cells and myeloma cells: Once splenocytes are isolated from the mammal, the B cells are fused with immortalized myeloma cells using polyethylene glycol. Polyethylene glycol (PEG) is used to increase the somatic cell division.
• Culture in HAT medium: Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin in the myeloma cells die, this is because myeloma cells are not able to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) enzyme. As they cannot produce nucleotides by the de novo or salvage medium blocks the pathway that allows for nucleotide synthesis.
Hence, unfused B cells die as they have a short life span. 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.
• Separation of cell lines and Multiplication: The incubated medium is then “Limiting Dilution” into multiwall plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Once the desired hybridoma clone-it is multiplied either in vivo or in vitro.
• Cloning by propagating the desire hybridomas: Once a hybridoma colony is established, it will continually grow in culture medium and produce antibody. The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. (Global-research-online.net, 2018) (Jana, 2018)

Applications of Hybridoma Technology:
The purpose of hybridoma technique is the production of homogeneous antibody against a specific epitope of an antigen in vitro. These monoclonal antibodies have wide applications such as:
• Immunodiagnostic reagents, either for detection of the causative agents directly in tissues or body fluids or as a reagent used in indirect diagnosis such as serological detection of antibodies to the causative agent.
• For experimental purposes ranging from molecular dissection of antigenic epitopes to monoclonal anti-idiotype antibody utilized as a vaccine to induce protective immunity.
• Immunoprophylaxis or immunotherapeutics applied to infectious, Parasitic, Bacterial and Viral infectious disease diagnosis.
• For Biotechnological application the MoAbs is used for purification of protein, antigens, Drugs treating and Gene cloning & expression.
• MoAbs is applied to medical purpose for pregnancy test, prevention & treatment of diseases, cancer therapy and Bone marrow & organ transplantation.
• Research tools for the recognition of specific amino acid sequences of polypeptides.
• Also applied for diagnostics in vitro and in vivo for identification and localization of disorders. (Anon, 2018)

Advantages of monoclonal antibodies:
• Single MoAbs are chemically defined, single specificity molecules, and can be used easily for standardization of specific assays.
• MoAbs provide a perpetual source of well-defined homogeneous reagents.
• MoAbs could be advantageously used as immunosorbents for antigen purification using immunosorbent column chromatography.
• All the antibodies produced (100%) are active antibodies. Therefore, high specific activity of labelling is possible in radioimmunoassay, enzyme linked immunosorbent assay and fluorescent immunoassay.
• MoAbs specific for a particular target antigen can be obtained even without prior purification of antigens.
• MoAbs are easily manipulatable. For example, bispecific antibodies can be prepared from two MoAbs and used in novel assays.
• Distinct antigenic cross-reactivities can be easily defined and hence most useful in diagnosis. (Anon, 2018)
Problems with Monoclonal Antibody (MoAbs):
Despite the great promise of MoAbs, there are several persistent technical problems to be considered;
• Obtaining MoAbs against certain weak antigens (antigens that do not produce a large immune response) remains difficult.
• Diagnostic assays employing specific MoAbs become ineffective if any alteration in the specific epitope of the target antigen against which the MoAbs are made.
• Low frequency of fusion is a continuing problem in the preparation of hybridomas, as in the stability of the hybridomas and antibodies.
• Some MoAbs are sensitive to small changes in pH, temperature, freezing and thawing and can be inactivated during purification.
• Another problem being addressed is the development of stable hybridomas for specific species which do not have the preferential loss of the spleen B-lymphocyte chromosomes.
• MoAbs are too specific. Limited MAbs might miss important cross-reactive determinants. This could relate to the animal’s immune response to the antigenic stimulus. Polyclonal sera may offer advantage of broad reactivity.
• It is time-consuming to produce MoAbs. The entire process of producing MoAbs takes 3-4 months for each fusion experiment. (Anon, 2018)
Conclusion
Although hybridoma technology has proved useful for the generation of reagent and diagnostic antibodies, the immunogencity of monoclonal antibodies has limited their therapeutic development. This limitation has been largely overcome by the chimerization and humanization of murine mono-clonal antibodies. The clinical use of monoclonal antibodies, however, had to wait for advances in genetic engineering. Currently, many new approaches are being explored to improve the hybridoma technology for making better monoclonal antibodies and for making rodent monoclonal antibodies less immunogenic in humans. These new reagents may be safer and more useful and may fulfil the promise that the hybridoma technology offers for the diagnosis and treatment of diseases. (Global-research-online.net, 2018) (Anon, 2018)