Myeloma antibody

Procec/Mre. - - Determinations on a micro scale are performed in 3-ml conical Pyrex centrifuge tubes. A volume of antiserum (containing antibody or myeloma antibody or crude seed extract or purified lectin) previously centrifuged until it no longer deposits sediment and containing about 6-8 fig of antibody N (AbN) or lectin N in a volume of about 50-100 fil is added to tubes containing a suitable range of accurately mea-... 

Myeloma antibodies to fructosans could be divided into two groups those specific for )8-(2- l)-linked fructosans as evidenced by the ability of inulin, a linear/3-(2- l) linked polymer, to precipitate, and those specific for /3-(2- 6) linkages as seen from the reaction with ryegrass levan, a linear polymer of /3-(2- 6) linkages. The j8-(2- l) specific myeloma proteins did not react with ryegrass levan and the /8-(2- 6) specific myeloma proteins did not react with inulin. 

Although myeloma antibodies provide a substantial advantage for structural studies of antigenic determinants in that they contain homogeneous combining sites, nevertheless the specificities available are limited. Only one myeloma with antiprotein specificity has been found d it is... 

Anti-IL-2-receptor (Anti-CD25) antibodies Daclizumab (Zenapax), a hnmanized murine complementarity-determining region (CDR)/human IgG chimeric monoclonal antibody, and basiliximab (Simulect), a murine-human chimeric monoclonal antibody, have been produced by recombinant DNA technology. The composite daclizumab antibody consists of human (90%) constant domains of IgG and variable framework regions of the En myeloma antibody and murine (10%) CDR of the anti-Tac antibody. 

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

Monoclonal antibodies are derived from a single, monospecific B cell clone. Monoclonal antibodies can be obtained from hybridoma cells that result from the fusion of antibody-producing B cells with immortal cells of a myeloma cell line. 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

Monoclonal antibody technology entails isolation of such B-lymphocytes, with subsequent fusion of these cells with transformed (myeloma) cells. Many of the resultant hybrid cells retain immortal characteristics, while producing large quantities of the monospecific antibody. These hybridoma cells can be cultured long term to effectively produce an inexhaustible supply of the monoclonal antibody of choice. 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

Fusion of human lymphocytes with human lymphoblastoid cell lines is a very inefficient process. Fusion of human lymphocytes with murine myeloma cells lead to very unstable hybrids. Upon fusion, preferential loss of human genetic elements is often observed. Unfortunately, particularly common is the loss of chromosomes 2,14 and 22, which encode antibody light and heavy chain loci. The production yields of human monoclonals upon immortalization of the human B-lymphocyte (by whatever means) are also low. 

In 1975, Kohler and Milstein observed that if an antibody-producing cell was fused with a myeloma tumor cell, a rapidly dividing hybrid was produced that synthesized a monospecific antibody. Each hybridoma formed then became a factory, producing antibodies monospecific to a particular sensitizing antigenic epitope. Cell cloning allows selection of hybrids producing antibodies with the desired characteristics. 

A myeloma is a cancer of the antibody-producing plasma cell and as such is immortal. 

Hybridoma Cell produced by the fusion of antibody-producing plasma cells with myeloma/carcinoma cells. The resultant hybrids have then the capacity to produce antibody (as determined by the properties of the plasma cells), and can be grown in continuous culture indefinitely owing to the immortality of the myeloma fusion partner. This technique enabled the first continuous supply of monoclonal antibodies to be produced. 

Boc-E4 Boc-EAR lR-MCA BJP-B6 + MCA Autoantibodies Bence Jones proteins (BJPs) (monoclonal antibody light chains) isolated from the urine of multiple myeloma patients, were found to hydrolyse peptide methylcoumarin amide peptide-MCA substrates 1.5 x 101 3.3 X 1()-2 nr 5.8... 

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