Mapping of IgE-binding Epitopes on Parvalbumin
Mapping of linear IgE-binding epitopes on parvalbumin was performed by
PEPperPRINT. Epitope mapping was based on peptides of the sequences of
parvalbumin from salmon (β-1, Accession# CAA66403.1 and β-2,
Accession# Q91483.3), cod (Gad m 1, Accession# AAK63089.1), grouper
(Accession# XP_033500493.1) and grass carp (Accession# QCY53440.1).
The peptides ranged from 12 to 15 amino acids in length, offset by three
amino acid residuals and printed in duplicate and framed by additional
HA (YPYDVPDYAG, 60 spots) control peptides on microarray. Peptide
microarray was pre-stained with the secondary antibodies in incubation
buffer to determine background interactions that could interfere with
the main assays. Incubation of further peptide microarrays with 29 serum
samples (one pool of non-atopic control sample and 28 subjects who
completed food challenge to salmon, grouper or cod, and grass carp) at
successive dilutions of 1:20 was followed by staining with the
anti-human IgE secondary antibody and read-out with an Innopsys InnoScan
710-IR Microarray Scanner. The additional HA peptides framing the
peptide microarrays were subsequently stained with the control antibody
as internal quality control to confirm assay performance and peptide
microarray integrity.
Quantification of spot intensities and peptide annotation were based on
16-bit gray scale tiff files that exhibit a higher dynamic range than
the 24-bit colorized tiff files shown in or provided with this report.
Microarray image analysis was done with Mapix. A software algorithm
breaks down fluorescence intensities of each spot into raw, foreground
and background signal, and takes into account any flagging of artifacts
by assigning the values -100 (artifact), 0 (standard) or 100 (very
clear) to individual peptide spots. Based on artifact-corrected
foreground median intensities, intensity maps were generated and
interactions in the peptide maps highlighted by an intensity color code
with green for high and white for low spot intensities. We tolerated a
maximum spot-to-spot deviation of 40%, otherwise the corresponding
intensity value was zeroed. This can be bypassed by manual flagging of
peptides as “Artifact” or “Valid“.