Hybridoma technique

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. 

Figure 4.4 Production of MAbs using the hybridoma technique.

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... 

A number of non-hybridoma techniques have been developed for in vitro antibody production a review of recombinant antibody production is covered in Chapter 6. 

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-...

In principal, such problems may be overcome by the hybridoma technique of Kohler and Milstein (20), Immunization of a mouse or other animal will stimulate the production of a series of B cells producing antibodies directed at various portions of the immunogen. Such cells do... 

A central engine to Abbott s growth was the diagnostic division within the hospital and medical equipment group headed by James Vincent, another Texas Instruments veteran, who took command of the division in 1974. The following year the division disclosed the Hybridoma technique to... 

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... 

Microbial and cell cultures, including those resulting from recombinant DNA or hybridoma techniques ... 

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... 

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