Figure 1.
A small section of the peptide array used for identification and fine specificity mapping of HSA epitopes: The section shows approximately 300 of the total 220.428 peptide fields in this array. The peptides were synthesized in predefined, addressable fields generated by 2×2 mirrors on the DMD each measuring 10×10 µm resulting in peptide fields with the size 20×20 of µm. The peptide fields were spaced by 10 µm wide empty zones. Binding of polyclonal rabbit anti-HSA antibody to the fields was recorded by fluorescence microscopy after incubation with Cy3-conjugated goat anti-rabbit IgG.
Figure 2.
Bar chart displaying the fluorescence signal obtained from binding of polyclonal anti-HSA to 15-mer peptides with 14 residue overlap (average signal from 5 copies of each peptide). A: peptides from HSA, B: peptides from BSA and C: peptides from RSA. The peptides are numbered on the x-axis according to the position of their n-terminal residue in the protein sequence. The y-axis denotes the average intensity of the signal (AU) after background subtraction.
Figure 3.
The figure shows two examples example of epitope sequences identified using ANOVA followed by Tukey’s HSD post-hoc analysis. The leftmost column shows overlapping 15-mer peptides from HSA from position 510–527 in figure 3A and from position 309 to 333 in figure 3b. The rightmost column highlights the amino acids identified as being important for antibody binding (dashes indicate non-important positions). The important amino acids were determined on the p<0.01 level in the HSD post-hoc analysis.
Figure 4.
The figure shows two examples using the Rq-values. Figure 4a shows residue 510–527 in the HSA sequence (top row) a sequence containing the epitope important residues LEVDETYV identified by Tukey’s HSD. Figure 4b shows residue 317 to residue 338 containing the epitope important residues DEMPADLP-LAADFVESKD. The column below each HSA residue lists the Rq-values calculated in each of the 15 overlapping peptides in which the residue is represented. Rq values from peptides with no positions identified by Tukey’s HSD test are set as non significant (NS) in the figure. The color indicates the size of the Rq value; darker color indicates higher Rq values.