Affinity screening


production of gene



Recovery of desired gene

Phage elution

Phage elution

Immobilized target molecule

Immobilized target molecule

Figure 13.2 Phage display technology and the screening of a library for a protein capable of binding to a desired target molecule. In this simplified example an initial library of three genes is inserted into three phages. One (gene 2) codes for the desired protein. In reality, libraries typically consist of hundreds of thousands/millions of different genes. Refer to text for further details

ligand with the highest specificity/affinity. Once this is achieved, the gene coding for the protein of interest can be excised from the phage genome by standard techniques of molecular biology. It can then be incorporated into an appropriate microbial/animal cell/transgenic expression system (Chapter 3), facilitating large-scale production of the gene product. Variations of the phage display approach have been developed, some of which utilize engineered phage (phagemids), whereas others achieve library expression not on the surface of phage but on the surface of bacteria.

Amongst the first and still most prominent application of phage display technology is the production and screening of antibody libraries in order to isolate/identify an antibody capable of binding to a desired target molecule. As such, this technology has now come to the fore in identifying antibodies suited to clinical application.

Genes/cDNA libraries coding for antibodies/antigen-binding antibody fragments have been obtained from human and animal (e.g. mice, rabbit and chicken) sources. Two types of library can be generated. 'Immune libraries' are obtained by cloning antibody/antibody fragment coding sequences derived from B-lymphocytes (usually from spleens) of donors previously immunized with the target antigen. A high number of hits (positive clones) should be obtained from such libraries.

Non-immune libraries are produced in a similar fashion, but using B-lymphocytes from non-immunized donors as a source of antibody genes. This approach becomes necessary if initial immunization with the antigen of interest is not possible (e.g. due to ethical considerations). Although such libraries will generate a lower number of positive clones, they can be screened against multiple antigens. Such native non-immune libraries can also be the starting point for the generation of so-called synthetic immune libraries. Their generation entails initial in vitro engineering of the non-immune library of antibody genes in order to increase still further the level of antibody diversity generated.

Supplements For Diabetics

Supplements For Diabetics

All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.

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