Hybridoma technology monoclonal antibody production

Since immunoassays utilize antibodies as analytical reagents, the challenge is to obtain antibodies specific for an individual compound that may be present in a milieu of structurally related and unrelated compounds. Antibodies can be produced by in vivo immunization (polyclonal antibodies), hybridoma technology (monoclonal antibodies) and by genetically engineered bacteria (antibody fragments with affinities for specific molecules). Some caveats pertaining to the production and use of such antibodies in immunoassay procedures will be presented. 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

The basis of monoclonal antibody production by hybridoma technology... 

A further advance in antibody technology is the development of transgenic mouse human strains. XenoMouse animals have been engineered in such a way that they now produce exclusively human antibodies rather than murine antibodies when immunized. The use of XenoMouse animals to produce MAbs avoids the need for any engineering of the antibody genes, since the products are already 100% human protein. XenoMouse animals are fully compatible with standard hybridoma technology and can be readily adopted by laboratories experienced in monoclonal antibody production [56]. 

The aim of most technologies is to generate a population of cells each of which secretes a desired product into the medium. For this to occur the cells do not need to be actively dividing and, in fact, this is sometimes counterproductive. Cells can remain viable at high cell densities using perfusion systems (Chapter 3) and the product purified from the spent medium (Spier, 1988). Furthermore, the simpler the growth medium (i.e. the fewer protein factors required) then the easier is the downstream processing and this is one reason for the development of serum-free media especially for the culture of hybridoma cells used for monoclonal antibody production ( 5.8). 

Hayter PM, Kirkby NF Spier RE (1992b) Relationship between hybridoma growth and monoclonal antibody production. Enzyme and Microbial Technology 14 454-461. 

Biotech products based on recombinant DNA, gene expression or hybridoma/monoclonal antibody technologies. 

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. 

The production of polyclonal antibodies by the immunization of animals is a method that has been used for more than a century (1). Hybridoma technology was the next development, allowing the production of monoclonal antibodies (2). However, hybridoma technology has some limitations, some resulting from the instability of the aneuploid cell lines, but most of all, difficulties in producing human antibodies especially to toxic or highly conserved antigens (3). 

In vivo production of hybridoma cells is accomplished by injecting them into the intraperiton. They grow and an ascite is formed which contains large amounts of monoclonal antibodies where the concentration can reach 10 mg/mL and more. This production technology has been banned in several European countries for animal welfare reasons. Moreover, ascites fluid also contains serum proteins and fats that make the separation of antibodies more complex. Protein impurities are less numerous than in serum, but the lipids are removed for better longevity in chromatographic columns. 

EBV), or by fusing it with another immortalized cell. The latter approach is called hybridoma technology because it involves fusion of two cells to produce a hybrid cell. This is the approach used most widely for the production of mouse monoclonal antibodies. 

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