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4 - Engineering antibodies page 3
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2. Understanding specific immunity Link to the Medical Research Council web site

Figure 5.
a) An antibody fragment against a human tumour (made by phage display technology) has been radioactively labelled and used to detect cancer in the liver of a patient.
b) A CT scan of the same patient with a lobe of liver on the left.
Monoclonal antibodies?
Monoclonal (belonging to a single clone) antibodies (mAbs for short) are antibodies of just one sort which recognise and can seek out one particular antigen. They are pure and highly specific and lend themselves to many diagnostic, therapeutic and research uses (see Figure 5). In the twenty five years since their development at the MRC Laboratory of Molecular Biology in Cambridge, mAbs have transformed biological research and created an international multi-million pound business.
During an immune response, a mixture of small amounts of many different antibodies is produced. Purifying a single antibody from this polyclonal mixture is difficult, if not impossible. In 1975, Kb hler and Milstein devised a method of making large amounts of mAbs - for this they won a Nobel Prize in 1984. Their method is shown in Figure 8. It uses B cells from a mouse which has been injected with a specific antigen.

Figure 6. Production of monoclonal antibodies
How are mAbs produced?
Stage 1. A mouse is injected with some of the antigen for which the mAb is required. The antigen stimulates immune production of the mAb.

B cells produce antibodies but are unable to divide and they survive only a few days in a culture medium. However, myeloma (cancer) cells, divide indefinitely and grow well in cell culture.

Stage 2. B cells are fused with myeloma cells to form hybridomas (hybrid cells). These can be cultured and used to produce monoclonal antibodies - but only 1 hybridoma is produced for every 10 000 cells in the mixture.

Stage 3. The hybridomas continue to grow successfully on a selection medium. The many unfused cells die after a few days. Each hybridoma cell is screened to find out which antibody it produces.
Stage 4. The hybridoma cell producing the desired antibody is propagated in a fermenter (Figure 7). A large population of clones (genetically identical cells) is generated, each of which produces the required antibody.

Figure 7. Monoclonal antibodies on a huge scale: a 2000-litre stainless steel fermenter at Celltech Ltd., the world's largest manufacturer of monoclonal antibodies. Hybridoma cells are grown in a sterile, controlled environment and produce vast quantities of a desired mAb.

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Question 3

Figure 7 refers to the 'controlled environment' of the fermenter in which mAbs are produced. List three key factors to be monitored and, for each factor, explain how it may be controlled.

Factor Method of control