Alanine scan of potential linear epitopes of Klebsiella pneumoniae MGH 78578

Sequences of 11 amino acids belonging to the KPN_00363 protein and KPN_00459 protein from Klebsiella pneumoniae MGH 78578 which was previously identified as potentially immunogenic was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. 26 peptides, two representing the original sequences, 22 peptides with each residue replaced by alanine (or glycin, if the original sequence contained alanine) as well as two related peptides were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was seperated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interaction rabbit polyclonal IgG to K. pneumoniae (Abcam ab20947) was used, while non-specific binding to the epitope sequences was checked using rabbit polyclonal IgG to E. coli (Abcam ab137967) and S. enterica (Abcam ab35156).