Research updates
4 - Engineering antibodies page 6
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5. Abs without immunisation Link to the Medical Research Council web site
 
An innovative development from Dr Greg Winter's Cambridge team involves the production of human antibody fragments. These are the antigen-binding fragments (Fab) shown in Figure 2. These fragments can be made outside the body, without the need for immunisation. Genetic engineering has been used to build an elegant and revolutionary new system for producing antibody fragments of high affinity and specificity in a test-tube.

Figure 10. A genetically engineered bacteriophage displaying antibodies.
 How is this done?
A blood sample is taken from an unimmunised human donor and the genes coding for the variable regions of antibody molecules (V genes) are isolated from the B cells. The arrangement of genes within a B cell is unique and codes for the antigen binding site of one specific antibody. Once isolated, the V genes from each B cell are genetically engineered into the DNA of a bacteriophage (a virus which infects bacteria) (Figure 10).
The engineered bacteriophages are allowed to infect their bacterial host. Using the protein synthesis machinery of the bacterium, each phage particle then codes for the synthesis of a specific antibody fragment. The antibody fragment is fused to a phage coat protein and so is displayed on the surface of the phage. (Compare this with a B cell which displays its particular antibody molecule on its surface).
 
The system results in the production of a test tube full of a vast array of phage particles, each displaying a unique antibody fragment. This, the scientists refer to as their 'phage display library'.

Human antibody fragments have been isolated for a variety of human antigens. These include fragments against antigenic molecules which are specific to the surface of the tumour cells. The antibody fragments show high binding affinity for their antigen and could prove invaluable for locating cancerous cells (see Figure 5 on page 3).


Figure 11. Separating a particular antibody fragment from the mixture of engineered phage particles using an antibody affinity column.

Figure 2 (copy). Basic structure of an antibody (IgG) shown here binding to an antigen through the antigen binding regions. [Original figure 2 is on page 2]
Selecting an antibody fragment
Any particular antibody fragment can be isolated from the 'library' using the technique of affinity purification, shown in Figure 11. This technique is based on the highly specific binding of an antibody with its antigen. The phage particles can be recovered from the column and cloned to produce many copies of the antibody fragment. 

• A mixture of phage particles is passed into the top of the antigen affinity column. The column's surface holds antigen molecules specific to the required antibody fragments.

• Phages displaying the required antibody fragment bind to the antigen molecules and are held in the column.

• Having been separated from the mixture, the desired phage molecules are washed free from the column. They can then be cloned.

Question

7. efer back to Figure 2 and explain the following.

a) The phage/bacterium system can be used to produce fragments but not the complete antibody molecule.

b) The antibody fragments can bind antigen but not activate the complement system.

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