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  2. The Nobel Prize in Physiology or Medicine 1984 For theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies Niels K. Jerne Basel Institute for Immunology Basel, Switzerland 1911-1994 César Milstein MRC Laboratory of Molecular Biology Cambridge, United Kingdom 1927-2002 Georges J.F. Köhler Basel Institute for Immunology Basel, Switzerland 1946-1955

  3. “I looked at the first two plates. I saw these halos. That was fantastic I shouted ,I kissed my wife. It was the best result I could think of.”“I looked at the hybrids growing in the bottles and felt happy with myself for growing them. I was reluctant to test them for their specificity yet. So I weeks before testing.” says kohler

  4. Principle • Monoclonal antibody technology to the basic principles of monoclonal antibody preparation will be required for access to the synthetic-specific monoclonal antibody B lymphocytes, but not in B lymphocytes in vitro growth. The experiment found that myeloma cells in vitro growth and reproduction, application of hybrid technology to myeloma cells and the immune lymphocytes two merged by the myeloma cell hybrids. Two such hybrid cells inherit the characteristics of the pro-cells, B lymphocytes It has the characteristics of specific antibodies, but also in myeloma cells can be cultured in vitro proliferation of perpetuating the use of such cells from a single converged proliferation of cultured cells can be prepared antigenic determinant of a single gram of specificity monoclonal antibody

  5. From the Structure of Antibodies Fig. 1. Antibodies are made of two or more pairs of heavy and light chains joined by disulphide bonds. Each chain has two regions. The variable region differs in structure from one antibody to another and contains the combining site. The antibody combining site is located at the tips of a Yshaped three-dimensional structure. The constant region is invariant within a given class or subclass, and is responsible for effector functions (complement binding, attachment to and transport across membranes etc). The number and position of the interchain disulphide bonds is characteristic for the different classes and subclasses. In this figure, the structure depicted is the mouse myeloma protein MOPC 21 which was the subject of much research in our laboratory.

  6. Antibodies,shows in the illustration at the top of the page as stylized shapes,belong to the family of protein called immunoglobulin G, a hetreogeneous population of molecules sharing a Y-shaped composed of two kinds of molecular chain ,heavy and light ,linked by disulfide bonds .The number and precise position of the disulfide bonds differ and are characteristic of the IgG subclass .Each chain has two regions ,in the variable region amino acid sequence that differ from antibody to antibody provide differently shaped combining sites that specifically to different antigens .constant region of the chains .with the same amino acid sequence in all antibodies of a given subclass ,is responsible for other function.

  7. Preparation Process • Immune response is initiated (a) when an antigen molecule carry several different antigenic determinants enters the body of an animal ,The immune system responds :line of B lymphocytes proliferate ,each secreting an immunoglobulin molecule that fits a single antigenic determinant. A conventional antiserum contains a mixture of these antibodies .Monoclonal antibodies are derived by fusing lymphocytes from the spleen with malignant myeloma cells (b). Individual hybrid cells are cloned , and each of the clones secretes a monoclonal antibody that specifically fits a single antigenic determinant on the antibody molecule.

  8. First hybrid- myeloma cells were prepared by immunizing mice with sheep red blood cell. The cells involved in the experiment and their immunoglobulins are depicted here schematically. Cells from the spleen of immunized mice (which die in any tissue culture)were fused with mouse myeloma cells deficient in HPGRT (which die in the selective medium HAT). Hybrid cells survived in HAT and secreted immunoglobulins derived from both parents ;some secreted active anti-SRBC. Clones of hybrid cells were isolated that secreted only a single species of antibody to SRBC :IgG1,IgG2b and IgM. In other words ,each clone secreted an antibody to the sheep red cells.

  9. Monoclonal antibody preparation process

  10. Total process to Preparae monoclonal antibody • Standard procedure for deriving monoclonal begins with the fusion , mediated by polyethylene glycol , of spleen cells from an immunized mouse with mouse myeloma cells. Hybrids are selected in HAT. The medium is assayed for antibody secretion ,and a portion of each positive culture is frozen as a precaution , positive cultures are cloned and the clones are assayed. The positive ones are are then frozen, recloned and assayed for the presence of immunoglobulin variants. The clones finally selected can be stored frozen. When the samples are thawed ,they can be either grown in culture to produce the antibody or injected into animals to induce myelomas that secrete the antibody.

  11. 正常细胞 HGPRT-细胞 无 药 物 选 择 无 药 物 选 择 主要途径 主要途径 补救途径 补救途径 能存活 能存活 HGPRT-细胞 正常细胞 药 物 选 择 药 物 选 择 主要途径 主要途径 补救途径 补救途径 不能存活 能存活

  12. 骨髓瘤(HGPRT-) B细胞 无 药 物 选 择 无 药 物 选 择 主要途径 细胞融合 主要途径 补救途径 补救途径 在培养中不能存活 能存活 杂交瘤细胞 B细胞 骨髓瘤(HGPRT-) 药 物 选 择 药 物 选 择 主要途径 主要途径 主要途径 补救途径 补救途径 补救途径 不能存活 能存活 在培养中不能存活

  13. Examination the antibody Secondary antibody and enzyme coupling E hybrid E Collection culture medium E E Contain target antigen Colored substrate Antigen and antibody union Put in secondary antibody antibody existence

  14. Application

  15. Application • Crude interferon was purified by immunoadsorption. Spleen cells of mice immunized with a somewhat enriched preparation of interferon were fused with myeloma cells. A hybrid-myeloma clone selected for anti-interferon activity and immunoglobulin secreting was injected to induce myelomas :purified antibody from the serum of tumor-bearing mice was attached to carbohydrate heads to prepare immunoadsorbent columns. When crude interferon was passed through such a column, It bound to the antibody and was retained when other components of the crude mixture were washed out; then the interferon was eluted.

  16. One passage through a column in creased the preparation’s interferon activity about 5000-fold. Electrophoresis of the proteins made the results visible . The partially enriched preparation showed a single band (A), apparently ablumin; over exposure of the same gel (B) showed minor bands, one of them at a position corresponding to the molecular weight of interferon. After the passage of crude interferon through a column (c) there was less contaminant and a strong band at the interferon position . A second passage (D) produced material with a single strong interferon band.

  17. Thank you