to be tested in each case the first step is to identify the helical elements or the corresponding loops if no helices are present in the immunoglobulin domain that has been selected as the target for optimisation in the case of constant antibody domains the helices are always located for example between the u03b2 pleated sheet strand a and b and e and f fig4 after identification of these regions optimisation is carried out according to the following plan 1 all the proline and or glycine groups are replaced by another amino acid preferably alanine if there is no conflict with point 2 that is to be prioritised the substitution is only carried out if the group that is to be replaced is not the first group after the preceding u03b2 pleated sheet strand or the last group before the succeeding u03b2 pleated sheet strand 2 then the helices are stabilised by the insertion of additional salt bridges this is done by replacing previously existing amino acids with amino acids having charged side chains of a different charge any combination of arginine lysine histidine aspartate or glutamate may be used the groups that are to be replaced must be separated by two three or four amino acids to ensure the formation of the salt bridge in the helix so that the charged groups inserted will for example have the numbering i and i 3 i and i 4 or i and i 5 all permutations of the above mentioned groups are possible preferably however arginine lysine or histidine is inserted closer to the c terminus than aspartate or glutamate double salt bridges are also theoretically.