FIGURE 3.

Schematic comparison of the classic approach with the in vitro approaches to obtain phosphospecific antibodies. For both approaches the antigen (Ag) is produced in full length and then phosphorylated (top portion, shaded gray). In the classic in vivo method (left, shaded blue), phospho-epitope mapping through mass spectrometry (mass spec), selection of the phospho-epitope to be targeted, and synthesis of a short phosphopeptide to be injected are all performed before immunization of animals. Additionally, downstream affinity purification or, alternatively, screening of a large number of hybridoma clones must be performed. Overall, this process takes months and yields one serum or one monoclonal antibody (monocl. Ab) directed against one predefined epitope. In the in vitro approach (right, shaded red), up-front phosphoamino acid identification is omitted, and the full-length antigen is used directly for antibody selection. Random clones are analyzed, and eventually Fc portion-containing antibodies are produced. In total, this approach takes only weeks, allows direct access to the DNA of the antibody, and has the potential of leading to several distinct monoclonal antibodies in endless supply directed against multiple epitopes.