Substitutional Analysis on Influenza PeB Binding

In search for peptides with higher or special binding affinity and for further understanding of the mode of action, a full substitutional analysis of peptide PeB using microarrays was performed. Thus, 152 PeB mutant variants were generated. In each of them, the full-length sequence was preserved except for only one amino acid from the eight loop-forming amino acids of the original PeB peptide (ARDFYDYDVFYYAMD) which was substituted with the 19 remaining natural amino acids. To assess binding, influenza material was labeled with a protein reacting fluorophore. Microarray-based substitutional analysis of peptide PeB was performed using a PepStar® peptide library spotted on glass slides by JPT Peptide Technologies. The slides were used without additional treatment. For the labeling of proteins with a fluorescent dye, Dyomics DY-634 (λex = 635 nm, λem = 654 nm, Fluoro-spin 634 Kit (emp Biotech) was used, according to the manufacturer´s instructions. The following materials were labeled: NewYork H3N2, Aichi H3N2, Victoria H3N2 and California H1N1. Labeled analytes were incubated several hours or overnight at indicated concentrations using Femtotip buffer (FTP)30 (20 mM Tris, 30% glycerol, 3% polyvinylpyrrolidon 90, 0.1% Tween 20, pH 8.4) for dilution. The slides were washed twice in FTP and twice in ultrapure water and subsequently dried under a stream of nitrogen. Experiments were performed in triplicates using glycans (2,3'-/2,6'-sialyllactose) and proteins (Anti-H1/Anti-H3 antibodies, fetuin) as positive and negative controls.