Antigen presentation for the generation of binding molecules.

In the last few decades, several new methods have been established to isolate full antibodies and fragments thereof, some even using alternative scaffolds from in vivo and in vitro sources. These methods encompass robust techniques including immunization and hybridoma technology or phage display and also more laborious and novel approaches including ribosome display or B-cell immortalization. All methodologies are dependent upon proper antigen presentation for isolation, screening, and further characterization of the selected binding molecules. Here, antigens are classes of molecules including soluble or membrane proteins, part or domains thereof (extracellular domains of GPCRs), peptides, carbohydrates, and small-molecular-weight moieties. Presentation of the antigen in a functional state or perhaps even mimicking the intended application is crucial for successful isolation of useful binding molecules. Moreover, it is also necessary to consider the expression host and any posttranslational modifications of target proteins. The increasing demand to target more complex antigens, for instance, receptors and ion channels, is leading to the development of alternative procedures to present these proteins appropriately, for example by the use of virus-like particles and DNA immunization. This chapter describes in general approaches for the preparation of different forms of immunogens including synthetic peptides, proteins, cell-based antigens for immunization and in vitro display systems and in detail the preparation of a soluble protein as antigen