The Monoclonal Antibodies

By definition, monoclonal antibodies are mono specific antibodies (antibodies with an affinity for a similar antigen.) which are alike, specifically because their production is by  identical immune cells which  all happen to be clones of a singled-out parent cell-hybridoma, hence a pure single homogeneous antibody (McMichael, Andrew & Fabre 1992). The antibodies possess the monovalent affinity, whereby they bind to the same antigenic determinant, (epitope). Very large numbers of monoclonal antibodies can be produced in laboratories. This gives a high hand to immunology; monoclonal antibodies may be abbreviated as mAb or moAb.

The monoclonal antibodies can be produced from any substance. The monoclonal antibodies are produced solely attach to that substance, hence can be used to purify, or in other cases for detections in that substance (James W 1996). The whole process is currently quite handy in molecular biology and medicine, among other fields.

History of Monoclonal Antibodies

In the early 20th century, the whole monoclonal antibody idea was surfaced by Paul Ehrlich where he talked of a "magic bullet". His postulation was that, a toxin for an organism could certainly be delivered, if one would come up with a compound which would be targeted against a disease-causing organism. He thought that it should be possible to come up with a compound that would identify and kill specific disease cells accordingly. For this work, Paul and Elie Metchnikoff won the 1908 Nobel Prize for Physiology.

 The maze that science was trying to solve for a better part of the 20^th century was settled in 1975 by Cesar Milstein and Georges Kohler, who were then employees at the University of Cambridge. They came up with a laboratory method of monoclonal antibodies creation. Their target was a long-lived cell chain that would produce similar antibodies. The cells that produce the antibodies can be harvested from mice.

They exposed the spleen of the mice to an antigenic protein. However, the cells only grew for a short time. On experiencing this, Milstein and Kohler combined the mice spleen cells with mouse myeloma, tumor cells that in their tests would grow indefinitely. Myeloma produced immunoglobulin, an antibody substance, but wouldn’t produce a pure antibody.  At the end of their lab tests, the combination attained them the specificity they needed in the antibody. This discovery won Milstein and Kohler the 1984 Nobel Prize in Medicine (Alberto & Peter 1995).

Procedure

Monoclonal antibody technology has become very common in the daily operations in various fields, comprising mainly the medical arena, and also quite a contribution in the academic and commercial sectors among others. Hereby, we mention some uses still holding inexhaustibility of the merits of monoclonal antibodies;

• In cancer treatment whereby, the antibodies are used in radio immune detections. Radio immunotherapy also occurs; a new development has seen new methods that only act on the cell membranes of the cancerous cells. Ritoxin is a product of this monoclonal antibody technology development, which is a drug approved by the FDA in November 1997.
• Monoclonal antibodies are used in the detection of toxins, hormones or also small amounts of drugs, for instance, they are used to human chorionic gonadotropin (HCG) in the pregnancy test kits.
• The diagnosis of AIDS by ELISA test also makes use of antibodies.
• In patients with complications after organ transplants, such as organ rejection, an antibody to the T3 antigen of T cells, OKT3 comes in quite handy to make the effects less severe.
• The strains of a pathogen can be classified by the use of monoclonal antibodies.
• In laboratories, during research, experts use monoclonal antibody application to organisms to find specific cells or molecules e.g. the use monoclonal antibodies to determine which proteins are responsible for the differentiation of cells in the respiratory system.
• Monoclonal antibodies are also used in the treatment of viral diseases that have for a very long time been termed and considered as "untreatable". In relation, antibodies actually hold some evidence leading to an AIDS cure.

Monoclonal Antibodies and Cancer Treatment

After the discovery of immunology, efforts of seeking vaccine treatment of cancer have been present. Autologous and allogeneic malignant cells are used to inject cancer patients, and are usually irradiated to prevent the further growth. However, since identification of some tumor antigens and the immune response they provoke has been done, a step towards cancer vaccines has been made, as initially there was a problem in measuring immune responses.

Treatment of cancer may entail binding of monoclonal antibodies to cancer cell. They induce a response against the target cancer cell that is immunological. The immune system in our bodies site foreign invaders and fights accordingly against them, but for our case, cancer cells do not appear as foreigners. This is where the monoclonal antibodies come in handy by attaching themselves onto parts of the cancer cell, hence making it visible to the immune system. Specifically rituximab (the monoclonal antibody drug) gets attached to CD20 (a specific protein in B cells). Rituximab makes visible to the immune system the cell, which can then attack normally, as required (Suresh 1998).  Here, the  target may be antigen and to a conjugate or maybe an effector cell by designing specific antibodies that can then bind with their Fab regions for the results, or whereby such antibodies could in addition be modified to give a radioisotope, cytokine or other conjugate that is active.

Passive immunotherapy thereby entails the antibody-based therapies, whereby an introduction of the molecules or substances is done into the body countering, therefore for the need of the body to create its own immune response. On the other hand, vaccines are taken to be active immunotherapy, as they finally generate an immune response that is intrinsic. In a way they may also be classified under the specific immunotherapy, as they try to arouse an immune response which targets the tumor antigens. It differs from the non-specific approaches, for example, cytokines, whereby the immune system is highly stimulated.

Organ Transplant Rejection

Transplant rejection comes about when the immune system of a recipient rejects a transplanted tissue from the donor. The immune system then destroys the transplanted tissue like all other foreigners. After transplantation, the rejection, therefore, can be reduced through determination of the molecular similitude between recipient and the donor through immunosuppressant drugs.

The main reason for the graft failure is rejection. For instance, in the case of kidneys, if glomeruli and tubule injury is acute, it may not recover. It is necessary, therefore, to diagnose acute rejection early enough, in order to administer prompt antirejection therapy. The success of a transplant is determined by the ability to reverse a rejection by immunosuppressive agents.

During rejection, T cells identify antigens that are foreign, only when the immunogenic peptide or the antigen itself is in association with a HLA molecule, called the antigen-presenting cell. The monoclonal antibody, before administration is evaluated and treated, in any case if there is a need, a physical examination is done, and after a laboratory test its completed administration can now begin. The monoclonal antibody is directed against the molecule of the T lymphocyte.

The antibody is also initially used in transplantation to treat a severe rejection episode or even to prevent a rejection occurrence. Re-use of these antibodies may lead to a development of a host anti- antibody in previously treated patients. To counter this, a development of humanized monoclonal antibodies is done, hence increasing the utility of anti-T-cell antibodies in a recurrent treatment of rejection. The humanized monoclonal antibodies are human immunoglobulin G. The antibodies have a long half-life. They can, therefore, be administered intravenously periodically after a while.

The ELISA Test Kit on HIV

A common serological test, the Enzyme-Linked Immuno-Sorbent Assay (ELISA) is a test on antibody or antigen presence. It is divided into the indirect ELISA and the direct ELISA forms. The indirect form is used in determination of a particular antibody presence, while the direct makes use of monoclonal antibodies to find out the presence of an antigen in a sample.

In the direct ELISA method, antibodies are placed in a well separated by a PVC micro titer plate. A fluid suspension e.g. serum is added to test for a complementary antigen presence. If there is presence of the antigen, the suspension will bind to the antibodies at the well wall, where then the test fluid is removed together with the unbound antigens. The bonds are strong to remain through a change in test fluid upon which another monoclonal antibody aliquot is added to the well.

 The antibodies in the new aliquot are such that attached are reporter enzymes which undergo a color change when a substrate reacts with them. Further rinsing of the sample is done, so as to get rid of any unbound antibodies. A complex is formed, that includes the antibody bound to the well that is, if the antigen is present. If not, then the bound antibody will have been rinsed off. Finally, the enzyme’s substrate is added giving a color change for presence of an enzyme labeled antibody and its bound antigen, whereas no color change indicates that there was no antigen in the test fluid, hence a handy method in the testing of HIV.

Scientific Scholarly Articles Using Monoclonal Antibodies

1. Monoclonal antibodies localization of oestrogen receptor in the nuclei of target cells by monoclonal antibodies done by W. J. King and G. L. Greene^, University of Chicago.

Specific intracellular receptor proteins are very extensively involved in the gene expression and growth in reproductive tissue oestrogenic regulation. The specific composure of the oestrogen regulation is poorly known. The dynamics and distribution of oestrophilin,(an oestrogen receptor protein)  in target tissues in the absence and presence of oestrogen and antioestrogen are among the unresolved issues.

Complication of the use of radio-labeled and unlabeled receptor ligands in measurement and detection of oestrogen receptors in tissues has been brought about by the presence of other intracellular binding proteins and also by the low concentration of receptors in the responsive tissues. When detection of receptor in oestrogen-sensitive tissues was done by monoclonal antibodies to oestrophilin, staining that was specific, was confined to the nucleus of all the stained cells. This showed that both nuclear forms and cytosol of the receptor protein may be found in the nuclear compartment. This is where we report development of an immunocytochemical process that is suitable for the direct localization of oestrophilin in frozen tissue sections or cells from human and non-human origins.

1.  The Monoclonal antibodies against recombinant parts of the Ki-67 antigen.

 A monoclonal antibody undergoes a reaction with human’s antigen which is nuclear cell proliferation-associated. This antigen is in all active cell cycle parts. We raised such antibodies, MIB 1–3, which are against recombinant parts of Ki-67 (Alberto & Peter  1995) . Through immune staining of the biochemistry, fresh specimens, and biological molecular techniques, it was shown that antibodies are the true Ki-67 equivalents. The paraffin-embedded formalin-fixed sections, which processed for immunohistochemistry, have not stained for MIB 2 and Ki-67. The two antibodies MIB 3 and MIB 1 came out as the mitotic figures, hence the proliferating non-mitotic cells were negative under such similar conditions, but as soon as de waxed oven- processed microwave paraffin sections of formalin-fixed tissues were used, MIB 3 and MIB 1gave relatively strong nuclear staining of the cells presumed to proliferate under a variety conditions of normality and neoplastism.

In addition, the routine depigmentation techniques did not change the immune reactivity of MIB I and MIB 3. Moreover such method is highly reproducible, easy to perform, requires low cost, and almost no additional technical skills are needed, as after the microwave treatment, histochemical just routine immune methods are used. Again with success of application of this method, the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on the archival material.

3.Immune enzymatic labeling of monoclonal antibodies using the immune complexes of monoclonal anti-alkaline phosphatase and alkaline phosphatase by B Falini, J L Cordell, K A Pulford, W N Erber,  H Stein, A K Ghosh, D Y Mason, Z Abdulaziz, S MacDonald.

Preparation of a monoclonal murine antibody specific for the alkaline calf intestinal phosphatase was done, and used in an antibody bridge method for monoclonal antibody labeling. The alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) procedure gives perfect immune cyto-chemical labeling of cell smears and tissue sections. This technique is closely comparable in intensity and clarity to the achieved with immunoperoxidase labeling technique.

Suppose, the enzyme label is created with a naphthol salt, a clear red reaction product is produced which is easily visible. Hence the APAAP technique was suitable for labeling the cell smears both for surface-membrane and cytoplasmic antigens, and also for the detection of low number of the antigen-bearing cells in the specimen, such as carcinoma cells. It was also found real to improve the APAAP labeling reaction’s intensity of the by significantly repeating of two incubation steps, namely; the unlabeled "bridge" antibody and the APAAP complexes. It was also found that the APAAP technique was better than the immunoperoxidase labeling, because of the staining of the tissues rich in the endogenous peroxidase Also, the APAAP technique could be used together with methods of immunoperoxidase for immunoenzymatic double staining. The detection of the antigenic molecules, as an outcome of their electrophoretic transferring from the SDS-polyacrylamide gels to the nitrocellulose sheets could also sufficiently utilize the technique. (John G. R 1994).

Conclusion

Monoclonal antibodies prove to be very important in various field applications, including biochemistry, molecular biology and medicine among others and are a recognizable step in the development of science. They most importantly help in solving the big medical question of cancer treatment and other important issues, such as organ transplant complications, and also can serve in detection or purification of a substance. It is a finding, we can say, which was really worth the effort.