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Monoclonal Antibodies

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Monoclonal Antibodies MONOCLONAL ANTIBODIES Most antigens posses multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones , each derived from a B cell that recognizes a particular epitope. The resulting serum antibodies are heterogenous , comprising a mixture of antibodies , each specific for one epitope .Such a polyclonal antibody response facilitates the localization , phagocytosis , and complement- mediated lysis of antigen ; it thus has clear advantages for the organism in vivo . Unfortunately , the antibody heterogeneity that increases immune protection in vivo often reduces the efficacy of an antiserum for various in vitro uses . For most research , diagnostic, and therapeutic purposes , monoclonal antibodies , derived from a single clone and thus specific for a single epitope , are preferable . Direct biochemical purification of a monoclonal antibody from a polyclonal antibody preparation is not feasible . In 1975, Georges Kohler and cesar Milstein devised a method for preparing monoclonal antibody , which quickly became one of immunology’s key technologies . By fusing a normal activated , antibody- producing B cell with a myeloma cell ( a cancerous plasma cell) , they were able to generate a hybrid cell,called a hybridoma, that possessed the immortal – growth properties of the myeloma cell and secreted the antibody produced by the B cell. The resulting clones of hybridoma cells , which secrete large quantities of monoclonal antibody , can be cultured indefinitely . The development of techniques for producing monoclonal antibody gave immunologists ( and molecular biologists in general ) a powerful and versatile research tool. The significance of the work by Kohler and Milstein was acknowledged when each was awarded a Nobel Prize in 1984. Page 1 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal Fig. no. : 1. Formation and selection of hybrid cells : B-cell hybridomas are produced by using polyethylene glycol to fuse myeloma cells with B cells from animals that have been immunized with antigen against which one want to make antibodies . The myeloma cells contribute the immortal growth properties to the fused cells , and the B cells which are only capable of limited growth in vitro , contribute the genetic information for synthesis of the specific Page 2 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal antibody of interest . In practice , not all of the cells fuse , and the procedure gives a complex mixture of unfused myeloma and spleen cells ( “parental cells”) as well as numbers of fused cells.Among the fused cells , there are three different combinations, unwanted fusion of a B-cell with a B-cell , or of a myeloma cell with a myeloma cell ,and the desired fusions of a B cell with a myeloma cell . Thus, the conditions of the procedure must selectively allow the survival and growth of the B-cell × myeloma hybridomas only.One common method requires the use of myeloma cells that are deficient (because of previously selected mutation ) for one of the nucleotide salvage pathways , making them unable to grow in HAT medium ( named for its three components - hypoxanthine , aminopterin , and thymidine ) . If the mixture of hybridomas and unfused parental cells is placed in this medium , the parental myeloma cells cannot survive . The B-cell × myeloma cells can survive because the B cell contributes the missing enzyme for the salvage pathway . Although unfused B cells are able to survive in HAT medium , these cells do not live for extended periods in vitro and thus die out. HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways : the de novo and salvage pathways . The de novo pathway , in which a methyl or formyl group is transferred from an activated form of tetrahydrofolate , is blocked by aminopterin , a folic acid analog . When the denovo pathway is blocked , cells utilize the salvage pathway , which bypasses the aminopterin block by converting purines and pyrimidines directly into nucleotides for synthesis of DNA and RNA . The enzymes catalyzing the salvage pathway include hypoxanthine –guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK). A mutation in either of these two enzyme blocks the ability of the cell to use the salvage pathway . HAT medium contains aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway . Therefore, cells that lack either HGPRT or TK will die in HAT medium , because they lack the ability to use the salvage pathway to acquire essential intermediates for the synthesis of nucleic acids. In hybridoma technology , the myeloma cells used are actually double mutants . They lack the enzyme HGPRTase and therefore are deselected in HAT .They have also lost the ability to produce immunoglobulin (Ig – mutants ). By using Ig-mutants one assures that the antibodies produced by the hybridoma are encoded solely by the spleen cell partner and the myeloma cells only contribute immortal growth properties to the fused cells . The other fusion partner is usually a population of spleen cells containing antigen activated HGPRT + B-cells . These cells contribute the capacity to utilize the salvage pathway for hypoxanthine to the hybridomas, thereby enabling their survival in HAT medium . Page 3 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal Fig. no. : 2. Production of Monoclonal Antibodies : Once antibody –secreting hybridomas are obtained , they must be screened for the desired antigenic specificity. Although some hybridomas will produce antibody specific for the antigen used for immunization ,others will be specific for unwanted antigens .Two of the most common screening techniques are ELISA and RIA. Following identification , hybridomas of the desired specificity are recloned to ensure that the culture is truly monoclonal and are then propagated in one of several ways to produce the desired monoclonal antibody. When a hybridoma is grown in tissue-culture flasks, the antibody is secreted into the medium , usually at fairly low concentrations (1-20 µg/ml).A hybridoma can also be propagated in the peritoneal cavity of histocompatible mice ,where it secretes the monoclonal antibody into ascites fluid at much higher concentrations (usually about 1-10mg/ml);the antibody can be purified from the mouse ascites fluid by chromatography . To meet the increased demand for monoclonal antibodies , in vitro techniques for growth of hybridoma cells at very high densities (>107/ml) have been developed. The production of human monoclonal antibodies has been hampered by a number of technical difficulties . The most important of these has been the lack of human myeloma cells that exhibit immortal growth, are susceptible to HAT selection , do not secrete antibody , and support antibody production in the hybridomas made with them . As an alternative to conferring immortality on human B cells by fusion to myeloma cells , normal human B lymphocytes can be transformed with Epstein –Barr Page 4 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal virus (EBV). When B lymphocytes are cultured with antigen in the presence of EBV, some of them acquire the immortal –growth properties of a transformed cells while continuing to secrete antibody. Cloning of such primed , transformed cells has permitted production of human monoclonal antibody , but the amounts secreted are generally small. Another problem is the difficulty of readily obtaining antigen –activated B cells from the human lymphoid tissues equivalent to the mouse spleen. Generally , human hybridomas are prepared from human peripheral blood, which contains few activated B cells engaged in an immune response . It is possible to obtain B cells that have been activated by antigens present in vaccines that are normally given to people , but human volunteers cannot be immunized with the range of antigens that can be given to mice or other animals . To overcome this difficulty,cultured human cells are sometimes primed with antigen in vitro. However, the in vitro system cannot mimic the normal microenvironment of lymphoid tissue ; as a result , the B cells usually produce only low –affinity IgM antibody. Page 5 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal Fig. no. : 3. Page 6 of 7 Unit#4 : Monoclonal Antibody : Pritha Mondal Clinical Uses For Monoclonal Antibodies : Monoclonal antibodies are proving to be very useful as diagnostic , imaging , and therapeutic reagents in clinical medicine . Initially ,monoclonal antibodies were used primarily as in vitro diagnostic reagents . Among the many monoclonal antibody diagnostic reagents now available are products for detecting pregnancy , diagnosing numerous pathogenic microorganisms, measuring the blood levels of various drugs , matching histocompatibility antigens , and detecting antigens shed by certain tumours . Radiolabeled monoclonal antibodies can also be used in vivo for detecting or locating tumour antigens, permitting earlier diagnosis of some primary or metastatic tumours in patients . For example, monoclonal antibody to breast-cancer cells is labeled with iodine-131 and introduced into the blood to detect the spread of a tumor to a regional lymph nodes .This monoclonal imaging technique can detect breast cancer metastases that would be undetected by other, less sensitive scanning techniques . Immunotoxins composed of tumor –specific monoclonal antibodies coupled to lethal toxins are potentially valuable therapeutic reagents . the toxins used in preparing immunotoxins include ricin,
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