Hybridoma technique, monoclonal

The next development was the production of monoclonal antibodies (MAbs) in the mid-1970s. This uses hybridoma technology, which involves the fusion of antibody-producing B cells to immortal myeloma cells. Figure 4.4 shows the preparation of MAbs using hybridoma techniques. A more detailed discussion of biopharmaceuticals production is presented in Section 10.5. 

A rat anti-mouse VEGFR2 (Flkl) monoclonal antibody (DClOl) was developed by ImClone Systems (New York, NY, www.imclone.com) using conventional hybridoma technique [194] to conduct proof-of-concept studies. 

Catalytic antibodies, like enzymes, must be isolated and purified to homogeneity before they can be studied. Initially this was done by using the hybridoma technique for isolation of monoclonal antibodies (Box 31-A). After induction of antibody formation by injecting a selected hapten into a mouse, large numbers of monoclonal antibodies had to be tested for catalytic activity. Even if several thousand different monoclonal antibodies were tested, only a few with catalytic properties could be found.1 Newer methods have incorporated recombinant DNA techniques (Box 31-A) and use of combinatorial libraries and phage display.) Incorporation of acidic or basic groups into the haptens used to induce antibody formation may yield antibodies capable of mimicking the acid-base catalysis employed by natural enzymes. 0... 

Fig. 3.2 Hybridoma technique for the production of monoclonal antibodies. Spleen (milt) cells, which have been taken from mice (being immunized with an antigen X) contain anti-X-antibody-producing B cells. These cells are fused with myeloma cells in the presence of polyethylene glycol (PEG) and then taken to the HAT (hypoxanthine-aminopterin-thymi-dine) medium. HAT will induce death to myeloma cells because of the absence of the enzyme hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT). Hybridoma cells, how-...

The development of the hybridoma technique facilitated the development of IHC and the manufacture of abundant, highly specific monoclonal antibodies, many of which found early application in staining of tissues. Initial success in cryostat sections was eventually extended to routinely processed paraffin, celloidin, or other plastic-embedded tissue sections. Only when the IHC technique became applicable to routine EEPE tissue sections did it usher in the brown revolution. The critical significance of rendering the IHC technique suitable for routine paraffin sections was illustrated in 1974 by Taylor and... 

In the hybridoma technique (see Fig. 2), mice are inoculated with a specific protein, the desired antigenic target, over several months. The mouse is then sacrificed, and splenic lymphocytes are harvested. The murine splenic cells are coincubated and fused with immortaUzed human myeloma cells in vitro [32]. The fused cells grow into colonies that effectively serve as antibody-producing biologic factories. Each colony produces molecularly identical antibodies arising from the same original mouse lymphocyte—hence the term monoclonal. Antibodies are then tested for specificity or desired immune effect. 

Although the hybridoma technique does not require pure antigen for the production of specific monoclonal antibodies, it still re-... 

Monoclonal antibodies are most commonly produced by employing the hybridoma technique. Figure 2 illustrates this technique. In this method, splenocytes (normal B-cells) from an immunized animal are fused with myeloma cells (tumor B-cells) using a fusion medium such as polyethylene glycol (PEG). Stock myeloma cell lines selected for the fusion process are specifically chosen because they are derived from a common ancestor MOPC-21 known to have lost the ability to produce IgG, so that the only antibody produced is derived from the antigen-sensitized splenocyte. The resultant hybrid cell is known as a hybridoma and inherits antigen specificity from the splenocytes and immortality from the myeloma cells, thus creating a... 

Immtmoassay systems using monoclonal antibody (MAh) against natural products have become an important tool for studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in animals or plants [75]. Immunogenic forskolin-bovine serum albumin (BSA) conjugate was synthesized via 7-deacetyl-7-hemisuccinyl forskolin. BALB/c male mice were injected intraperitoneally with forskolin-BSA conjugate emulsified with Fretmd s complete adjuvant. Splenocytes were isolated from the hyperimmunized BALB/c mice and fused with the P3-X63-Ag8-Ul myeloma cells. Six hybridomas producing monoclonal antibodies (MAbs) reactive to forskolin were obtained. 

Monoclonal antibodies can be produced not only in a cell culture but also in live animals. When injected into mice (in the peritoneal cavity, the gut), the hybridoma cells produce tumors containing an antibody-rich fluid called ascites fluid. Production in cell culture is usually preferred, as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical. The process of producing monoclonal antibodies described above was invented by Georges Kohler. Cesar Milstein, and Niels Kaj Jeme in 1975 they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery (http //en.wikipedia.org/ wiki/Antibody). 

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. 

Depending on the starting material, preantibody incubation steps may vary and are outlined in Chapters 9-12. The following assay begins with the removal of the slides from the overnight 10% normal serum incubation step. Since the ABC technique is universal in application, and a biotinylated secondary antibody exists for virtually any primary antibody, this protocol will assume a monoclonal assay and a primary antibody from a mouse hybridoma. A good secondary antibody to use in this situation is a biotinylated antimouse antibody made in horse. Therefore, the overnight serum incubation in this case would have been with 10% normal horse serum in PBS (see Note 4). 

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