Hybridoma technique, monoclonal antibody production

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-...

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). 

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 mammalian cell culture technique can be employed to produce clinically important biochemicals such as human growth hormones, interferon, plasminogen activator, viral vaccines, and monoclonal antibodies. Traditionally, these biochemicals had been produced using living animals or extracted from human cadavers. As examples, monoclonal antibodies can be produced by cultivating hybridoma cells in the peritoneal cavity of mice, and the human growth hormone to cure dwarfism can be extracted from human cadavers. However, the quantity obtained from these methods is quite limited for the wide clinical usages of the products. 

The most important technique for perfusion culture methods is to separate the concentrated cells and conditioned medium from the suspended culture broth. As noted above, the separation methods chiefly used are filtration with tubular and flat membranes as well as ceramic macroporous filters. These membrane reactors can be employed for both anchorage-dependent and suspension growing cells. Static maintenance type systems are commercially available for disposable reactors, and small size unit reactors from 80 ml to 1 liter are used for continuous production of monoclonal antibodies with hybridoma cells. The maintainable cell densities are about 10 -10 cells/ ml which is essentially mouse ascites level. However, in these systems, the cell numbers cannot be counted directly because the cells adhere to membranes or hollow fibers. Therefore, the measurement of cell density must use indirect methods. Such indirect methods include the assaying of the quantities of glucose consumption and the accumulation of lactate. The parameters of scale-up have not yet been established for these static methods. 

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

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