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“.