PEPperMAP ® Type 1 Epitope Mapping of Mouse Monoclonal Antibody anti-HA (12CA5) PEPperPRINT GmbH Heidelberg, 10/2013

Experimental Conditions Microarray Content: The sequence of hemagglutinin (Influenza A virus, Uniprot ID P03438) was translated into 13 aa peptides with a peptide-peptide overlap of 12 aa. The resulting peptide microarray contained 1,031 different peptides as duplicates (2,062 peptide spots) and was framed by Flag (DYKDDDDKGG) and HA (YPYDVPDYAG) control peptides (82 spots each control). Sample: Mouse monoclonal antibody anti-HA (12CA5) Washing Buffer:PBS, pH 7.4 with 0.05% Tween 20 (2x1 min after each assay) Blocking Buffer:Rockland blocking buffer MB-070 (60 min before the first assay) Incubation Buffer: PBS, pH 7.4 with 0.05% Tween 20 and 10% Rockland blocking buffer Assay Conditions: Antibody concentration of 1 µg/ml in incubation buffer; incubation for 16 h at 4°C and shaking at 500 rpm Secondary Antibody: Goat anti-mouse IgG (H+L) DyLight680; 30 min staining at RT and a dilution of 1:5000 Control Antibodies: Monoclonal anti-HA (12CA5)-LL-DyLight680 (1:1000), monoclonal anti-FLAG(M2)-LL- DyLight800; staining in incubation buffer for 1 h at RT and a dilution of 1:1000 Scanner: LI-COR Odyssey Imaging System; scanning offset 1 mm, resolution 21 µm, scanning intensities of 7/5 (green/red) Microarray Data: MicroarrayData_mAb_anti-HA.xlsx Microarray Identifier: _04 (three array copies per slide for one-by-one assays)

Data Analysis Pre-staining of the peptide array was done with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody at a dilution of 1:5000 to investigate background interactions that could interfere with the anti-HA (12CA5) assays. Subsequent incubation of the peptide microarray with mouse monoclonal antibody anti-HA (12CA5) at a concentration of 1 µg/ml in incubation buffer was followed by staining with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody and read-out at scanning intensities of 5 (red). HA and Flag control peptides framing the peptide arrays were subsequently stained with high and homogeneous spot intensities (scanning intensities red/green: 5/7). Quantification of spot intensities and peptide annotation were done with PepSlide ® Analyzer and listed in the Excel file MicroarrayData_mAb_anti-HA.xlsx. A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal (see “Mapping Raw Data” tab), and calculates the standard deviation of foreground median intensities (see “Mapping Summary” tab). Based on averaged foreground median intensities, a intensity map was generated and binders in the peptide map highlighted by an intensity color code with red for high and white for low spot intensities. We further plotted averaged spot intensities of the anti-HA (12CA5) assay against the antigen sequence from the N- to the C-terminus of hemagglutinin to visualize overall spot intensities and signal to noise ratios (see “Intensity Plot” tab). The intensity plots were finally correlated with peptide and intensity maps as well as with visual inspection of the microarray scans to identify the peptides and consensus motif that interacted with the antibody sample. In case it was not clear if a certain amino acid contributed to antibody binding, the corresponding letters were written in grey.

anti-HA (12CA5) Microarray Scans After 10 min pre-swelling in standard buffer and 60 min in blocking buffer, the peptide array was initially incubated with the secondary goat anti-mouse IgG(H+L) conj. DyLight680 antibody at a dilution of 1:5000 for 60 min at room temperature to analyze background interactions with the antigen-derived peptides. At a scanning intensity of 7, we did not observed any background due to non-specific binding of the secondary antibody (left). Data quantification with PepSlide ® Analyzer was neither possible nor required, since the absence of any spot pattern hampered alignment of the microarray grid. Subsequently, the peptide microarray was incubated with mouse monoclonal antibody anti-HA (12CA5) at a concentration of 1 µg/ml in incubation buffer. Staining with the secondary goat anti-mouse IgG(H+L) conj. DyLight680 antibody was followed read-out at a scanning intensity of 5 (red; middle). We observed a clear epitope-like spot pattern formed by a row of neighbored peptides with a consensus motif. Furthermore, antibody anti-HA (12CA5) strongly reacted with the HA control peptides framing the hemagglutinin array content. After the main assays, the peptide microarray was pre-swelled in standard buffer followed by incubation with the labeled HA and Flag control antibodies for 1 h at room temperature at a dilution of 1:1000. Read-out at a scanning intensity of 7/5 (green/red) resulted in the well-defined expected spot pattern of HA and Flag control peptides framing the peptide microarray (right). The green spot in the array resulted from a cross-reaction of the anti-Flag control antibody with IKGVELKSGYKDW Pre-StainingControl Staining anti-HA (12CA5), 1 µg/ml

anti-HA (12CA5) Intensity Plot Data quantification was followed by generation of peptide and intensity maps as well as of an intensity plot. In accordance with the microarray scans, we observed a clear and strong epitope-like spot pattern after incubation with anti-HA (12CA5) at a concentration of 1 µg/ml in incubation buffer. The row of neighbored spots was correlated with the consensus motif DVPDYA that formed the epitope of mouse monoclonal antibody anti-HA (12CA5). Since peptide AFSNCYPYDVPDY was significantly less reactive than neighbored peptide FSNCYPYDVPDYA, the C-terminal alanine was regarded as part of the anti-HA (12CA5) epitope. DVPDYA

Conclusion The Type 1 Epitope Mapping of mouse monoclonal antibody anti-HA (12CA5) was done against hemagglutinin (Influenza A virus, Uniprot ID P03438) translated into 13 aa peptides with a peptide-peptide overlap of 12 aa. The peptide microarray with the antigen-derived peptides was incubated with the mouse monoclonal antibody anti-HA (12CA5) at a concentration of 1 µg/ml in incubation buffer followed by staining with a secondary goat anti-mouse IgG (H+L) DyLight680 antibody and read-out with a LI-COR Odyssey Imaging System. Quantification of spot intensities and peptide annotation were done with PepSlide ® Analyzer. We did not detect any background interaction of the secondary goat anti-mouse IgG (H+L) DyLight680 antibody with the antigen-derived peptides. Mouse monoclonal antibody anti-HA (12CA5), however, showed a very strong response against peptides with the consensus motif DVPDYA at excellent signal to noise ratios. The pronounced increase of spot intensities from peptide AFSNCYPYDVPDY to peptide FSNCYPYDVPDYA underlined the contribution of C-terminal alanine to binding of antibody anti-HA (12CA5). For further epitope validation, the investigation of sequence conservation and the determination of the actual epitope length we recommend an in-depth analysis of the suggested epitope DVPDYA by a PEPperMAP ® Full Substitution Scan of e.g. peptide FSNCYPYDVPDYA with all amino acids at all amino acid positions. PEPperMAP ® Type 1 Epitope Mappings are available as full service offer for less than 1,000 Euro per sample. Even more details can be obtained by PEPperMAP ® Type 2 Epitope Mappings with an antigen translated into peptides of different lengths (e.g. 10, 12 and 15 amino acids with peptide-peptide overlaps of 9, 11 and 14 amino acids) to investigate the dependency of antibody binding on peptide length, and to clearly differentiate between neighbored epitopes in polyclonal samples.

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