monoclonal antibodies


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1 : Monoclonal Antibodies AJAY PATIDAR LECTURER SJMIT
2 : The structure of antibodies http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
3 : What are antibodies? An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequence.
4 : Classes of Igs IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid IgA: IgA1 (90%) and IgA2 (10%), external secretions (stomach, intestines, saliva, tears, etc.)? IgM: 5-10% of total serum Ig [1.5mg/ml serum conc.] IgD: 1% of proteins in the plasma membranes of B-lymphocytes, function unknown [30µg/ml serum conc.] 0.2% of total serum Ig IgE: 0.3µg/ml on the surface of plasma membrane of mast cells, play a role in immediate hypersensitivity and denfense for parasite
5 : History of mAb development 1890 Von Behring and Kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated Natural Selection Theory of Antibody Formation. Frank Macfarlane Burnet expanded it. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline (Fc) from rabbit ?-globulin.
6 : History of mAb development 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells 1990 Milstein produced the first monoclonal antibodies
7 : Paul Ehrlich at the beginning of the 20th century theorized that a cell under threat grew additional side-chains to bind the toxin, and that these additional side chains broke off to become the antibodies that are circulated through the body. It was these antibodies that Ehrlich first described as "magic bullets" in search of toxins.
8 : Georges Köhler César Milstein, and Niels Kaj Jerne in 1975 who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery hybridoma technology
9 : MYELOMA CELLS HAVE LOST the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the synthesis of nucleic acids through PURINE SALVAGE PATHWAY.
10 : The selective culture medium is called HAT medium because it contains HYPOXANTHINE, AMINOPTERIN and THYMIDINE. Unfused myeloma cells cannot grow because they lack HGPRT. Unfused normal spleen cells cannot grow indefinitely because of their limited life span.
11 : PRODUCTION OF MONOCLONAL Abs
12 : Types of mAbs designed Murin source mAbs:: rodent mAbs with excellent affinities and specificities, generated using conventional hybridoma technology. Clinical efficacy compromised by HAMA (human anti murine antibody) response, which lead to allergic or immune complex hypersensitivities. Chimeric mAbs:: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource)? Humanized mAbs:: contained only the CDRs of the rodent variable region grafted onto human Framework Regions [FR]
13 : Human mAb :: three currently available approaches to the production of human monoclonal antibodies are described. These include :- the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to immortalize antigen-specific human B lymphocytes; the EBV-hybridoma technique, based on a combination of the first two methods. The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity. Types of mAbs designed . .
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15 : Conventional production of mAbs The hybridoma technology: spleen cells from immunized mice are fused with the murine myeloma cells. The several process had been developed at large scale. According to the different cell culture methods, it can calisifed into four fields Robottle cell culture process. Membrane binded cell culture process Microcarrier cell culture process Suspended cell culture process
16 : Nomenclature of mAbs
17 : Applications of monoclonal Abs Although monoclonal antibodies were first produced in 1975 as research tools, scientists quickly recognized their practical uses, especially in diagnostic tests and in therapy. Several diagnostic procedures that use monoclonal antibodies are now available
18 : A breakthrough in Diagnostics A monoclonal antibody can be used to detect pregnancy in only 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections.
19 : Helps in critical diagnostic decisions 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 and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.
20 : Pictured is a technician's hand, filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form hybrid cell (hybridoma).
21 : Purification of protiens Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.
22 : Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment:: mAbs act directely when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. mAbs may be modified for delivery of a toxin, radioisotope [RADIOIMMUNOTHERAPY], cytokine or other active conjugates. it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell.
23 : Conjugated monoclonal Ab therapy Toxins or radioactive isotopes are bound to the constant region of the mAbs. When the mAb binds to the surface cells of a tumour the toxin or radioactivity will kill the cancer cells and all cells within a certain radius (a killing zone). In this way cancer cells within the tumour will be killed.
24 : Radioimmunotherapy Involves the use of radioactively conjugated Murine antibodies against cellular antigens Eg.; Tositumomab ----- non-Hodgkins lymphoma
25 : mAbs Treatment For Cancer Cells ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129
26 : Dale L Ludwig, et. al. Oncogene(2003) 22, 9097-9106 Strategy of a Direct or Indirect Induction of Apoptosis in Targeted Cancer Cells mAbs target growth factor receptors to exert a direct effect on the growth and survival of the cancer cells by antagonizing ligand-receptor signaling. mAbs can target to cell surface antigens and directly elicit apoptotic signaling.
27 : The first approved mAbs was OKT-3 [1986], which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant. Until Feb 28, 2005, 18 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis. Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23, No.9 Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005 FDA Approval
28 : OKT3 Prevents acute rejection of kidney transplants Prevents autoimmune destruction of islet cells in type I Diabetes mellitus
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31 : Other Applications ELISA Cell Sorting (cell isolation), FACS, or Cell depletion Confocal Microscopy and Imaging
32 : mAbs Development Phage display library: construction of VH and VL gene libraries and expression of them on a filamentous bacteriophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs. Transgenic plants: transgenic tobacco plants to produce IgA Transgenic animals: transgenic mouse to make humanized IgG. (Abgenix,CA)?
33 : Latest News North Carolina-based PPD has established a joint venture with Taijitu Biologics to develop and commercialize a technology platform for the discovery of first- and best-in-class monoclonal antibody therapies in collaboration with MAB Discovery GmbH in Munich. The joint venture will provide drug discovery services based on this technology platform to global biopharmaceutical companies, enabling them to discover best-in-class monoclonal antibodies against both novel and validated targets.
34 : References Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129 Dale L Ludwig, et. al. Oncogene(2003) 22, 9097-9106 Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23, No.9Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005 ^ Schwaber, J; Cohen, EP (1973). "Human x mouse somatic cell hybrid clone secreting immunoglobulins of both parental types". Nature 244 (5416): 444–7. doi:10.1038/244444a0. PMID 4200460. edit ^ Köhler, G.; Milstein, C. (1975). "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature 256 (5517): 495. doi:10.1038/256495a0. PMID 1172191. edit
35 : Acknowledgements I am extremely thankful to our director, Mr. Amit Chakravarty and our entire faculty for giving us such an exuberant chance to present this before everyone . .
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