Analysis of Hybridoma Technology - Coursework Example

Hybridoma Technology Hybridoma is a hybrid cell that is produced by injecting a specific antigen into a mouse, collecting an antibody-producing cellfrom 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 those that produce the desired antibody. Their many identical daughter clones will secrete, over a long period of time, millions of identical copies of made-to-order "monoclonal" antibodies. Because of hybridoma technology, scientists are now able to make large quantities of specific antibodies. About a hundred years ago, it was proposed by Paul Ehrlich that antibodies could be used as "magic bullets" to target and destroy human diseases. This proposal is still being pursued today since antibodies combine specificity (the ability to exquisitely discriminate diverse harmful molecules) and affinity (the ability to tightly lock onto those targets) with the ability to recruit effector functions of the immune system such as antibody- and complement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity (ADCC). In the alternative, a "toxic payload" (such as a radioactive element or a plant toxin) attached to the antibody can be accurately delivered to the target. 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. The function of the antibodies is to recognise and attach matter harmful to the organism, thereby marking it out for other components of the immune system to destroy. The organism makes millions of different types of antibodies; each designed to bind a surface feature (the epitope or antigenic determinant) on the foreign body (the antigen). The most common human antibody, IgG, is shaped like the capital letter "Y", IgE, IgD, IgA, IgM are other types of antibodies Overtime, antibodies have been produced from the serum of animals. Serum contains a cocktail of antibodies (polyclonals), some of which will attach to the antigen. Since when Emil Behring, in1890, published a paper demonstrating that diphtheria antitoxin serum could protect against a lethal dose of diphtheria toxin; antisera has been used to neutralise pathogens in acute disease as well as prophylactically. Antisera is also widely used in vitro as a diagnostic tool to establish and monitor disease. The problem with using antisera for treatment is that it leads to "serum sickness" - basically the patient's immune system reacts against the harmful proteins causing fevers, rashes, joint pains and sometimes life-threatening anaphylactic shock. Also, the serum is a crude extract containing not only the antibodies against the disease-causing pathogen (often at low concentration), but also unrelated antibodies (plus other non-antibody proteins). In 1975, Csar Milstein and Georges Khler at the Medical Research Council'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 tumour cells (myelomas) from the bone marrow of mice to produce a hybridoma. A hybridoma has the cancer's ability to reproduce almost indefinitely, plus the immune cell's ability to make antibodies. Once screened, to isolate the hybridomas producing antibodies of a determined antigen specificity and required affinity - and given the right nutrients - a hybridoma will grow and divide almost indefinitely, mass-producing antibodies of a single type (monoclonals). This resembled a production-line with batch consistency for Ehrlich's "magic bullets". For this breakthrough these scientists (Csar Milstein and Georges Khler) won the Nobel Prize in Medicine in 1984. Immuno-cytochemical staining has been an important tool in diagnosis in human pathology since the 70's. However, its application in veterinary diagnostic pathology has not been so common, especially due to the lack of specific antibodies. To overcome this drawback, antibodies which present cross reactivity with human and animal antigens have been applied. These antibodies have been created using the hybridoma technology. It has been confirmed that many of the antibodies produced for use in human Immuno-cytochemical staining might be applied in veterinary pathology. Further studies are however going on to increase the list of applicability of these antibodies to different animal species. The hybridoma technology is used in staining especially in immuno-cytochemical staining. Pretty Poly protocol is a highly flexible, simple and yet effective staining technique that essentially solves the problem of co-staining with multiple polyclonal rabbit antibodies. Monoclonal antibodies from this hybridoma technology have been immensely useful in scientific research and diagnostics, especially in production of antibodies for imuno-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 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's vision have not been realised. The problems included identifying better antigenic targets of therapeutic value with which to raise mAbs against; making useful fragments of mAbs (whole antibodies are rather too large to penetrate solid tumours for instance); and attaching toxic payloads to the mAbs, since rodent antibodies are not as effective as human in recruiting the other cells of the immune system to complete their destructive function. However, the major hurdle has proven to be similar to that of serum therapy. This is that when the rodent mAbs are administered in multiple doses, the patient invariably raises an immune response to the mAbs with similar symptoms to serum sickness and violent enough to endanger life. 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 immortalisation of human antibody-producing cells. Human hybridomas beside being difficult to prepare are unstable and secrete low levels of mAbs of the IgM class with low affinity. 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 an 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 has 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" essays. It has been observed that specificities that are not frequently observed in vivo may be obtained by these techniques. In an effort to realise Erhlich's dream of a "magic bullet" with high binding affinity, reduced immunogenicity (HAMA response), increased half-life in the body and adequate recruitment of effector functions (ie the ability to summon help from the body's own natural defences), scientists have used techniques from molecular biology to design, engineer and express mAbs from hybridoma technology to produce humanised mAbs. The first step was to produce a chimaeric antibody where the xenogeneic variable (V) and human constant (C) domains were constructed by linking together the genes encoding them and expressing the engineered, recombinant antibodies in myeloma cells. However, when these antibodies were used therapeutically in humans, some still generated HAMA response directed against the V regions, although the level of immunogenicity varied depending on the chimaeric antibody. Going one stage further, Greg Winter, also at the MRC Cambridge, realised that only the antigen binding site from the human antibody needed to be replaced by the antigen binding site from the rodent. Since this consisted of the six CDR regions, only these were grafted into the human frameworks. Antibodies made this way are called humanised, reshaped or CDR-grafted. In some cases, pure CDR-grafting could produce a humanised antibody with roughly the same antigen specificity and affinity as the original rodent antibody. This was not always true and it was soon evident that a more detailed design of the engineered antibody will be needed before it is constructed. 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 hasve 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 mightb not have 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's dream was of a kind of magic bullet that will cure/prevent diseases thus the use of the hybridoma technology in the discovery and manufacture of vaccines to prevent diseases is also a major application of this technology. References 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. Read More