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Producing Antibody Through Hybridoma Technology

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A hybridoma is a hybrid cell that is produced by injecting a specific antigen into a mouse, collecting an antibody-producing cell from the mouse’s spleen, and fusing it with a long-lived cancerous immune cell called a myeloma cell. Individual Hybridoma cells are cloned and tested to find the ones that produce the antibody they desire. Their many identical daughter clones will secrete, over a long period, millions of exact copies of made-to-order “monoclonal” antibodies.

Because of hybridoma technology, scientists are now able to make large quantities of specific antibodies.

Producing Antibody Through Hybridoma Technology

Approximately a hundred years ago, Paul Ehrlich proposed that antibodies be used as “magic bullets” to target and destroy human diseases. This proposal is still being followed today as antibodies combine specificity (the ability to discriminate against different harmful molecules exquisitely) and affinity (the ability to lock on certain targets strongly) with the ability to recruit immune systems effector functions such as antibody- and complement-mediated cytolysis and cell-mediated cytotoxicity (ADCC) based on an antibody.

In the alternative, it is possible to deliver accurately to the target a “toxic payload” (such as a radioactive element or plant toxin) attached to the antibody. This makes them suitable for homing in on and killing cancer cells, infectious diseases as well as modulating the immune system by binding and inhibiting or enhancing its regulatory molecules, thereby curing autoimmune and inflammatory diseases.

White blood cells (B-lymphocytes) of the immune system in higher organisms produce antibodies, which are large, glycoprotein molecules. Antibodies have the function of identifying and adding harmful matter to the organism, thus marking it out for the destruction of certain components of the immune system. The organism generates millions of different types of antibodies, each engineered to bind the foreign body (the antigen) to a surface characteristic (the epitope or antigenic determinant). The most common human antibody, IgG, is shaped like the capital letter “Y,” IgE, IgD, IgA, IgM are other types of antibodies

Overtime, antibodies were developed from animal serum. The serum contains an antibodies (polyclonal) mixture, some of which may be bound to the antigen. Since when, in 1890, Emil Behring published a paper showing that diphtheria antitoxin serum was able to protect against a lethal dose of diphtheria toxin, Antisera has been used for both acute and prophylactic neutralization of pathogens. Antisera is also commonly used as a diagnostic method for identifying and tracking illness in vitro. The concern with using antisera for diagnosis is that it contributes to “serum disease”-essentially, the immune system of the patient responds to the harmful proteins that cause fevers, rashes, joint pains, and sometimes life- Anaphylactic shock threatens. The serum is often an inexperienced extract that includes not only the antibodies to the disease-causing pathogen (often at low concentrations) but also unknown antibodies (plus other non-antibody proteins).

In 1975, César Milstein and Georges Köhler at the Medical Research Council’sCouncil’s (MRC) Laboratory of Molecular Biology (LMB) in Cambridge (UK) developed a way to produce “custom-built” antibodies “in vitro” with relative ease. They injected rodent antibody-producing cells with immortal tumor cells (myelomas) from the bone marrow of mice to produce a hybridoma. A hybridoma has cancer’s ability to reproduce almost indefinitely, plus the immune cell’s ability to make antibodies. If screened, a hybridoma can develop and divide nearly forever, mass-producing, single-type (monoclonal) antibodies to isolate the hybridomas that generate antibodies of a specified antigen specificity and necessary affinity-and given the right nutrients. This resembled a production-line with batch consistency for Ehrlich’sEhrlich’s “magic bullets.” For this breakthrough, these scientists (César Milstein and Georges Köhler) won the Nobel Prize in Medicine in 1984.

Immuno-cytochemical staining has been an essential tool in diagnosis in human pathology since the ’70s ’70s. However, its application was not so common in veterinary diagnostic pathology, due in particular to the lack of specific antibodies. Antibodies which present cross-reactivity with human and animal antigens were applied to overcome this drawback. These antibodies have been created using hybridoma technology. It has been confirmed that many of the antibodies produced for use in human Immunocytochemical staining might be applied in veterinary pathology. However, further studies are proceeding to increase the list of applicability of these antibodies to various animal species.

The hybridoma technology is mainly used in immuno-cytochemical staining in staining. Pretty Poly protocol is a highly flexible, simple, and yet effective technique of staining that essentially solves the co-staining problem with multiple polyclonal rabbit anticorps.

Monoclonal antibodies from this hybridoma technology have been immensely useful in scientific research and diagnostics, especially in the production of antibodies for immune-cytochemical staining. This is possible as the antibodies are cultured and tested to determine what their response to staining looks like. This helps in the diagnostics of diseases. It has helped in the identification and treatment of diseases such as polio, and infections such as E. coli.

 Although monoclonal antibodies (mAbs) from hybridoma technology have proved to be immensely useful scientific research and diagnostic tools, possibilities inherent in Erhlich’sErhlich’s vision have not been realized. The problems included finding better antigenic targets of therapeutic interest to which to lift mAbs; making useful fragments of mAbs (for example, whole antibodies are far too large to reach solid tumors); and adding toxic payloads to mAbs, as rodent antibodies are not as effective as humans in recruiting the other cells of the immune system to complete their function. The big barrier, however, has proved similar to the serum therapy one. This is that when the rodent mAbs are administered in several doses, the patient inevitably elevates an immune reaction to mAbs with similar symptoms to serum sickness and becomes aggressive enough to place life in danger. This is called Human Anti-Mouse Antibody (HAMA), and response can occur within two weeks of the initiation of treatment and does not include long-term therapy. The best thing to do would be to raise human mAbs to the therapeutic targets, but this is difficult both practically and ethically using the route of immortalization of human antibody-producing cells. Human hybridomas are unstable and secrete low levels of IgM class mAbs with poor affinity, besides being difficult to prepare.

Human monoclonal antibody technology has generally been hampered by difficulties related to a lack of suitable immune B cell sources, poor direct B lymphocyte immortalization techniques, a lack of suitable fusion partners, and instability of the rare hybridoma cell lines producing human monoclonal cell lines. Nowadays, several of these matters can be approached by molecular biology approaches involving (semi)synthetic or natural antibody V region libraries, phage display technology, and eucaryotic antibody expression. However, for certain purposes (e.g., when investigating human antibody repertoires found in vivo), direct immortalization of individual human B cells is still the preferred approach.

As stated, the major problem in the development of human monoclonal antibodies is frequently the lack of a suitable source of immune B lymphocytes. This has recently been addressed by the utilization of (semi)synthetic antibody gene libraries, chain shuffling, and selection using phage display technology. An alternative method involves the immunization and expansion of B lymphocytes with suitable specificities from pools of “naive” B cells obtained from non-immunized individuals. Studies have been devoted to the development of such technologies. These investigations have focused on selecting suitable lymphocyte populations to be incorporated into the “in vitro” culture systems, on the immunization process itself and on the “downstream” handling of the “in vitro” immunized lymphocytes for immortalization of the cells by EBV or hybridoma technology or their genes by molecular biology-oriented approaches. Using these approaches, antibodies against HIV-1 glycoproteins have been developed, several of which have biologically important properties (virus neutralization or inhibition of virus spread in cultures) as defined by “in vitro” assays. It has been observed that these techniques may obtain specificities that are not frequently observed in vivo.

In an attempt to realize Erhlich’sErhlich’s vision of a “magic bullet” with high binding affinity, decreased immunogenicity (HAMA response), improved half-life in the body. Good recruitment of effector functions ( i.e., the ability to receive assistance from the body’s body’s natural defenses), scientists have used molecular biology techniques to design, develop, and express mAbs from hybridoma technology to hybridoma technology. The first step was to generate a chimeric antibody in which the xenogeneic variable (V) and human constant (C) domains were formed by linking the encoding genes together and expressing the engineered, recombinant antibodies in myeloma cells. Nevertheless, when these antibodies were used therapeutically in humans, some still produced HAMA response that was directed against the V regions, although the level of immunogenicity differed depending on the chimeric antibody. Greg Winter, also at the MRC Cambridge, noticed that only the human antibody’ antibody’s antigen binding site had to be replaced by the rodent’s rodent’s antigen binding site. As this consisted of the six CDR areas, the human structures were grafted into only those. Antibodies produced in this way are called humanized, reshaped or grafted with a CDRPure CDR-grafting can in some cases produce a humanized antibody with roughly the same antigen specificity and affinity as the original rodent antibody. This wasn’t always true, and it soon became evident that a more thorough design of the engineered antibody would be required before it is designed.

Thus we have looked at the hybridoma technology from the vision of Erhlich to what goes on in the world of molecular biology today. We have also looked at how this technology has helped in the diagnosis and treatment of diseases in both humans and veterinary animals.

No doubt, without advancement in hybridoma technology, we might not have an accurate or even decent diagnosis of diseases. Without proper diagnostic tools, as provided by the hybridoma technology, it could lead to wrong treatment. It should not be forgotten that Erhlich’sErhlich’s dream was of a kind of magic bullet that will cure/prevent diseases. Thus, the use of hybridoma technology in the discovery and manufacture of vaccines to prevent infections is also a significant application of this technology.


  1. Morris TJ, Stanley EF, Journal of Neuroscience Methods. ; Toronto, 2003 Aug 15;127(2):149-55
  2. Ohlin, M., Broliden, P.-A., Danielsson, L., Wahren, B., Rosen, J., Jondal, M. and Borrebaeck, C. A. K. (1989) Human monoclonal antibodies against a recombinant HIV envelope-antigen produced by primary in vitro immunization. Characterization and epitope mapping. Immunology 68, 325-331
  3. Sandusky, G. E.; Wightman, K. A.; Carlton, W. W. Immunocytochemical study of tissues from clinically normal dogs and of neoplasms, using keratin monoclonal antibodies. Am J Vet Res, v. 52, n. 4, p. 613-8, 1991

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