B

Figure 3 Schematic outline of the basic selection procedure. The antibody phage display repertoire is grown in E. coli bacteria (A) and purified from the medium (B). The phage antibodies are allowed to bind to the antigen, which may be either coated to a solid surface or expressed on a cell surface (C). Nonbinding phages are removed by washing (D) after which the remaining phage antibodies are eluted (E), propagated in bacteria and used for a next round of selection.

Figure 3 Schematic outline of the basic selection procedure. The antibody phage display repertoire is grown in E. coli bacteria (A) and purified from the medium (B). The phage antibodies are allowed to bind to the antigen, which may be either coated to a solid surface or expressed on a cell surface (C). Nonbinding phages are removed by washing (D) after which the remaining phage antibodies are eluted (E), propagated in bacteria and used for a next round of selection.

tion of bound phages and 4) infection and propagation of eluted phages in E. coli bacteria. Phage selections have been extensively performed on purified antigens coated to plastic surfaces. Although the antibodies obtained through this procedure perform well in ELISA, their effectiveness in other assays with antigens in their native conformation is usually limited. Presumably the coating procedure leads to the partial destruction of the conformational integrity of the antigen and the subsequent selection of phages against neo-epitopes.

Phage antibodies may also be selected using intact eukaryotic cells in suspension or cells growing in monolayers as targets. Selection procedures on eukaryotic target cells may be extended by an absorption step in which the library is incubated with absorber cells to remove undesired specificities. For example, selection of phages for binding to malignant melanoma cells followed by absorption of eluted phages on normal melananocytes yielded antibodies that exclusively recognized the melanoma cells. Along this line, the combination of flow cytometry and phage display library technology has proven to be a successful approach for isolating antibodies directed against surface markers present on subpopulations of cells in a heterogeneous mixture. In this procedure, the phage library is incubated with the heterogeneous population of cells, the subpopulation of interest is subsequently stained with fluorochrome-labeled monoclonal antibodies, and sorted from the heterogeneous mixture by fluorescence activated cell sorting. The nonselected cells in the mixture act as an absorber population.

Preparation of cells for phage selections may require treatment of tissues with proteolytic enzymes and/or cell culture, potentially resulting in the loss of phenotypic characteristics of the target cells. To circumvent this problem, intact fragments of tissues can be used as targets for phage selection. In this manner, scFv antibodies have been obtained binding to either membrane-bound or intracellular proteins.

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