Journal: bioRxiv

Article Title: An integrative approach for profiling antibody responses in bats to human pathogens

doi: 10.1101/2024.11.29.626104

Figure Lengend Snippet: High throughput analyses of reactivity of bat antibodies. a . Plots depicting the mean fluorescence intensity of the raw signal from microarray chips. Each spot indicates the binding intensity of bat antibodies to a single peptide. b . Heat-map of MFI values obtained after subtraction of the background. Each line in the graph represents the reactivity to a single peptide in the array. Statistical analyses were performed by a Wilcoxon two-tailed test to compare the mean antibody binding reactivity for the indicated conditions. c . Plots depicting the values of Z scores. The dashed gray lines correspond to Z score of 2, which is considered as a threshold of significance. Each dot in the plots corresponds to the Z score of antibody reactivity to a single peptide. d . Heat map and hierarchical clustering of Z scores depicting the reactivity of bat antibodies to peptides in microarrays. Each column in the heat maps displays the binding intensity of a single peptide. The average of each column in the map is always equal to 0. The map distinguished six clusters of peptides.

Article Snippet: To this end, we used a high-density peptide array, namely the PEPperCHIP ® Infectious Disease Epitope Microarray (PEPperPRINT), which was originally developed for the purpose of serological profiling of infections in humans.

Techniques: High Throughput Screening Assay, Fluorescence, Microarray, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping and sequence analysis

doi: 10.1016/j.isci.2023.107394

Figure Lengend Snippet: High-density peptide arrays (HDPA) provide high-resolution antibody epitope maps across the SARS-CoV-2 proteome (A) Overview of analytical pipeline. The proteome of SARS-CoV-2 was translated into 15-mer overlapping peptides with a peptide-to-peptide overlap of 13 amino acids. The resulting individual peptides were printed in duplicates on the microarray. Sera from confirmed SARS-CoV-2-positive and -negative individuals were incubated on PEPperCHIP the HDPA. Serum antibody binding was visualized using respective fluorescently labeled secondary antibodies (anti-human IgG and anti-human IgA). Image acquisition and data quantification resulted in epitope-specific antibody profiles for SARS-CoV-2. (B) Average relative fluorescent units (RFU) profiles and peptide coverages are plotted across the SARS-CoV-2 proteome (ORF1A, ORF1B, Spike (S) protein, Envelope (E) protein, Membrane (M) glycoprotein, Nucleocapsid (N) phosphoprotein). Antibody responses to each linear 15-mer peptide were mapped across the SARS-CoV-2 proteome and average RFU calculated for each amino acid residue. The normalized positional ‘epitope coverage’ at each protein residue location is defined as the ratio of total peptides mapped to each position by the total expected peptides (see section). ‘Hotspots’ can be seen as spiked in the RFU or coverage distributions. (C) Comparison of mean RFU (log-scale) between SARS-CoV-2-positive and -negative sample groups for each viral protein. (unpaired t-test, ns: p > 0.05; ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001).

Article Snippet: To analyze the antibody responses to SARS-CoV-2 at the epitope level we used a recently developed high-density peptide array (HDPA), the PEPperCHIP Microarray (PEPperPRINT GmbH, Germany), covering the proteome of the SARS-CoV-2 isolate Wuhan-Hu-1 as well as the four seasonal hCoVs OC43, HKU1, NL63 and 229E (see for accession numbers used).

Techniques: Microarray, Incubation, Binding Assay, Labeling, Membrane, Residue, Comparison