monoclonal antibodies

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The procedure of how to make mca and its uses in the field of medicine.

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Saher Abbas : 

Saher Abbas BS(HONS) 4th Semester

Monoclonal Antibodies : 

Monoclonal Antibodies

Contents: : 

Contents: Antibodies Structure Monoclonal vs Polyclonal Discovery How to produce it Applications Conclusion

Antibodies: : 

Antibodies: Antibodies are proteins produced by the B lymphocytes of the immune system in response to foreign proteins, called antigens. Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized and destroyed by phagocytes. The part of the antigen that the antibody binds to is called the epitope. The epitope is thus a short amino acid sequence that the antibody is able to recognize. Antibodies are useful tools for the immune system to combat pathogens.

Antigen : 

Antigen An antigen is a molecule recognized by the immune system. Antigen is any foreign agent that is recognized by the immune system and antibodies produced against to destroy it.

Structure : 

Structure

Monoclonal Abs : 

Monoclonal Abs Monoclonal antibodies are Monospecific Antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. antibody molecules of singular epitope specificity originally produced by one B-cell and sharing identical sequence. a monoclonal antibody is the result of a single B-cell - cancer-cell fusion, that is grown up into a clonal population (thus mono-clonal). have identical idiotype/specificity (it is lots of one antibody).

Polyclonal Abs : 

Polyclonal Abs Antibody molecules which differ in their epitope binding and complementarity region amino acid sequence, however share overall target specificity. Antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.

Discovery : 

Discovery

Beginning of Monoclonal Era : 

Beginning of Monoclonal Era Georg Kohler and Cesar Milstein fuse mouse lymphocytes with neoplastic mouse plasma cells to yield hybridomas that produce specific antibodies. This offers a limitless supply of monoclonal antibodies. Monoclonal antibodies permit diagnostic tests that are specific, and function as probes.

Discovery of Mcab : 

Discovery of Mcab Monoclonal antibodies were produced in mice using a technique described by Köhler and Milstein et al.. They were awarded the Nobel Prize in 1984 (along with Jerne) for their work.

Nobel prize awarded to Köhler, Milstein and Jerne in 1984 : 

Nobel prize awarded to Köhler, Milstein and Jerne in 1984

Study of Myeloma leads to Discovery of Monoclonal antibodies : 

Study of Myeloma leads to Discovery of Monoclonal antibodies

Fusion of Mice spleen cells with Myeloma cells produced Monoclonal antibodies : 

Fusion of Mice spleen cells with Myeloma cells produced Monoclonal antibodies

Hybridoma creates Monoclonal antibodies : 

Hybridoma creates Monoclonal antibodies

Producing Monoclonal Antibodies : 

Producing Monoclonal Antibodies

Method : 

Method Inject the protein into a mouse. Remove the spleen. Identify which spleen cells are producing antibodies. Separate these cells and grow in tissue culture tubes. Screen each Ab for cross reactivity. Select the Ab which doesn't cross react with any other protein.

Method : 

Method First, a mouse is inoculated with the antigen to which the MA are to be produced. After this, the spleen is removed and fused with myeloma cells in order to produce the hybridomas that will be selected according to the antibody produced.

Hybridoma leads to Proliferation : 

Hybridoma leads to Proliferation

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Grow these hybrid cells (called hybridomas) and sequester groups of them in the wells of microtiter dishes. The hybridomas will secrete antibodies into the culture medium, and these "supernatant" antibodies can be collected and tested. Purification: Once a "well" in the plate has been identified as being positive, that is that it has a hybridoma secreting antibody against an antigen of interest, it is important to isolate the hybridoma in pure form. The hybridoma can be grown in vitro, and the culture supernatant collected and purified. This will yield about 10-100 micrograms of antibody per ml of culture.

Slide 22: 

Alternatively, the antibody producing cell can be injected into the mouse (or rat). The ascites fluid can be collected and will contain approximately 1 to 10 mg protein per ml. The in vivo methods are difficult because they cause pain to animals and there is some risk that the ascites will contain endogenous polyclonal antibodies against other specificities, viral pathogens, etc. Hybridoma cells can be cryopreserved for future use.

Applications and uses of Mcab : 

Applications and uses of Mcab

Applications : 

Applications Monoclonal antibodies are widely used as diagnostic and research reagents. an essential tool of much biomedical research and are of great commercial and medical value. Some monoclonal antibodies that have been introduced into human medicine to suppress the immune system. e.gMuromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind to the CD3 molecule on the surface of T cells. Used to prevent acute rejection of organ, e.g., kidney, transplants. (Transweb, 1996).

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Diagnostic tests Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test detect the protein on a membrane. Monoclonal antibodies can also be used to purify a substance with techniques called affinity chromatography. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes and streptococcal infections.

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Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. A monoclonal antibody can be used to detect pregnancy only 14 days after conception. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells Monoclonal Antibodies also used in ELISA( enzyme linked immunosorbant Assay) . A test for detection of viruses.

Monoclonal Abs and Cancer : 

Monoclonal Abs and Cancer By means of genetic engineering, so-called chimeric antibodies are prepared that contain both human and mouse sections. The mouse section is located in the variable region of the antibody that contains the antigen- binding site, while the human section is in the constant region. After the variable region binds to the cancer antigen, the human section activates complement and cytotoxic T cells to destroy the cancer cell.

Slide 30: 

When a monoclonal antibody attaches to a cancer cell, it can: Make the cancer cell more visible to the immune system Block growth signals Deliver radiation to cancer cells. Slip powerful drugs into cancer cells

FDA-approved monoclonal antibodies for cancer treatment : 

FDA-approved monoclonal antibodies for cancer treatment Name of drug Alemtuzumab (Campath) Bevacizumab (Avastin) Cetuximab (Erbitux) Gemtuzumab (Mylotarg) Type of cancer used to treat Chronic lymphocytic leukemia Breast cancerColon cancerLung cancer Colon cancerHead and neck cancers Acute myelogenous leukemia

FDA-approved monoclonal antibodies for cancer treatment : 

FDA-approved monoclonal antibodies for cancer treatment Ibritumomab (Zevalin) Panitumumab (Vectibix) Rituximab (Rituxan) Tositumomab (Bexxar) Trastuzumab (Herceptin) Non-Hodgkin's lymphoma Colon cancer Non-Hodgkin's lymphoma Non-Hodgkin's lymphoma Breast cancer

ELISA : 

ELISA The Enzyme Linked ImmunoSorbent Assay (ELISA) was first developed by Engvall and Perlman in 1971

Monoclonal Abs and ELISA : 

Monoclonal Abs and ELISA The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. ELISA makes it possible to create a simple color test to detect and quantify the presence of an antigen, antibody, or hormone. This system is widely used because it can process large numbers of samples in a short time and can handle complex antigens, such as bacteria.

Steps involve in ELISA : 

Steps involve in ELISA Sandwich ELISA Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface.(Polystyrene) Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.

Slide 36: 

Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal. Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

VideoPregnancy Test : 

VideoPregnancy Test

Disadvantages of Monoclonal Antibodies : 

Disadvantages of Monoclonal Antibodies MAbs are too specific. Limited MAbs might miss important cross-reactive determinants. Single MAb is a single chemical, and physical or chemical treatment that affects one molecule will affect all the MAbs in that population. It is time-consuming to produce MAbs. The entire process of producing MAbs takes 3-4 months for each fusion experiment.

Future and Mcab : 

Future and Mcab Is base upon the role of Biotechnologist’s. Biotechnology field is committed to MAbs and continues to find new ways both to press onward with current processes and to some extent it reduces the economic pressures to develop radically new concepts and technologies that could make monoclonals even more widespread.

References: : 

References: macampbell@davidson.edu Avastin (prescribing information). South San Francisco, Calif.: Genentech, Inc.; 2008. http://www.fda.gov/cder/foi/label/2008/125085s145lbl.pdf. Accessed Dec. 9, 2008. Herceptin (prescribing information). South San Francisco, Calif.: Genentech, Inc.; 2008. http://www.fda.gov/cder/foi/label/2008/103792s5175lbl.pdf. Accessed Dec. 9, 2008. Erbitux (prescribing information). Branchburg, N.J.: ImClone Systems Inc.; 2007. http://www.fda.gov/cder/foi/label/2007/125084s103lbl.pdf. Accessed Dec. 9, 2008. Mylotarg (prescribing information). Philadelphia, Pa.: Wyeth Pharmaceuticals Inc.; 2005. http://www.fda.gov/cder/foi/label/2006/021174s020lbl.pdf. Accessed Dec. 9, 2008.

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www.voet.com www.roit.com www.vadlo.com(biology cartoon) ^ Litman GW, Rast JP, Shamblott MJ (1993). "Phylogenetic diversification of immunoglobulin genes and the antibody repertoire". Mol. Biol. Evol. 10 (1): 60–72. PMID 8450761.  ^ a b c d Eleonora Market, F. Nina Papavasiliou (2003) V(D)J Recombination and the Evolution of the Adaptive Immune System PLoS Biology1(1): e16. ^ a b c d e f g h i Janeway CA, Jr et al. (2001). Immunobiology. (5th ed.). Garland Publishing. ISBN 0-8153-3642-X. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=imm.TOC&depth=10.

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