Selection of Antibodies Against Biotinylated Antigens

Phage antibody (Ab) library selections on peptides or proteins are usually carried out using antigens (Ags) directly coated onto a plastic surface (e.g., Petri dishes, microtiter plate wells, and immunotubes). This straightforward method is easy to perform and has been shown to be successful for a diverse set of Ags (for review, see ref. 1 ). However, phage Ab selections on some proteins and especially on peptides are not always successful, which is often caused by immobilization-associated features. The main problem observed for selection on peptides is the poor coating efficiency of some peptides and the altered availability of epitopes on plastic-coated peptides. The direct coating of proteins on plastic is usually more efficient, but may also be problematic because the passive adsorption on plastic at pH 9.6 is a mechanism of protein denaturation. Under these conditions, 95% of adsorbed proteins are nonfunctional (2 ,3 ) This problem is not important for a classical enzyme-linked immunosorbant assay (ELISA) mostly because a small fraction of proteins having a native conformation are still detectable. However, this phenomenon can be troublesome for phage Ab library selections because phage Abs binding to epitopes, only present in denatured molecules may be selected.