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Seminar on Monoclonal antibody Prepared By; Kaushik Patel

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Agenda Introduction Antibodies (1) structure (2)class Differences between monoclonal and polyclonal antibody Preparation of monoclonal antibody The types of monoclonal antibody designed

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Monoclonal antibody for cancer treatment Monoclonal antibody development Conventional production of monoclonal antibody Steps of hybridoma technology Application


MONOCLON AL ANTIBODY 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.

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Polyclonal antibodies are antibodies that are derived from different cell lines Ex. Muromonab CD3 , Inactivates T lymphocytes that reject tissue grafts,

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Muromonab CD3 ( Orthoclone OKT3): Muromonab CD3 is a murine Mab that reacts with a T3 (CD3) molecule linked to an antigen receptor on the surface membrane of human T-lymphocytes. It blocks both generation and function of the T- cell in response to antigenic challenge and is indicated for treatment of organ transplant rejection. Uusually it is combined withAzathioprine , Cyclosporine and for Corticosteroids to prevent acute rejection of immunosuppressive drugs

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ANTIBODIES MONOCLONAL. POLYCLONAL. Derived from different B Lymphocytes cell lines Batch to Batch variation affecting Ab reactivity & titre NOT Powerful tools for clinical diagnostic tests Derived from a single B cell clone mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with specificity Enable the development of secure immunoassay systems.

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Preparation of monoclonal antibody: Mabs are used in home pregnancy testing products. There use ensures that a woman can perform the test easily in a short period with high reproducibility and in an inexpensive manner. In this test the monoclonal antibody is highly sensitive to binding on one site on the human chorionic gonadotropic ( HCG) molecule, a specific manner to pregnancy because in healthy woman .

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HCG is synthesized exclusively by the placenta, In medicine monoclonal antibody are being used too stage and to localize malignant cell of cancer and It is anticipated that they will be used in the future to combat disease such as lupus erythematosys , juvenile-on-set disease and Myasthenia gravis.

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The types of monoclonal antibody designed : Murine 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)

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Humanized mAbs: contained only the CDRs of the rodent variable region grafted into human variable region framework

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Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. mAbs act directly 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 was modified for delivery of a toxin , radioisotope , cytokine or other active conjugates.

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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

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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.

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Humanized antibody in clinical trials or introduse into clinical practice:

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M Abs development Phage display library : construction of V H and V L gene libaries and expression of them on a filamentous bacterophage . 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 .

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Conventional production of mAbs The hybridoma technology : S pleen 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 classified into four fields Robottle cell culture process. Membrane binded cell culture process Microcarrier cell culture process Suspended cell culture process

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PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT ( emulsification ) Ab titre reached in Serum Spleen removed ( source of cells)

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PRODUCTION OF MONOCLONAL ANTIBODY Step 2: - Screening Of Mice For Antibody Production HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery ) Titre too low BOOST (Pure antigen) Titre High BOOST (Pure antigen) 2 weeks Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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PRODUCTION OF MONOCLONAL ANTIBODY Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY + 8-Azaguanine High Viability & Rapid Growth Myeloma CellHGPRT - Myeloma Cells Immortal Tumor of Lymphocytes Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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PRODUCTION OF MONOCLONAL ANTIBODY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells HYBRIDOMATECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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PRODUCTION OF MONOCLONAL ANTIBODY Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion HYBRIDOMA TECHNOLOGY A.Clone Each + ve Culture B. Test Each Supernatant for Antibodies C.Expand + ve Clones Mouse Ascites Method Tissue Culture Method

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PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Department of Pharmaceutics,NIMS Institute of Pharmacy, NIMS University, Jaipur

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Applications of Monoclonal Antibodies Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection- Muronomab-CD3 Cardiovascular disease- Abciximab Cancer - Rituximab Infectious Diseases - Palivizumab Inflammatory disease -Infliximab Clinical Applications Pu rification of drugs, Imaging the target Future Applications Fight against Bioterrorism

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