Antibodies are essential tools for therapeutic, diagnostic and research purposes. However, antibodies are often poorly characterized in terms of specificity and cross-reactivity (see Nature Feature Reproducibility crisis: Blame it on the antibodies). For instance, researchers at Mount Sinai Hospital in Toronto, Canada, had been chasing a protein called CUZD1, a supposed diagnostic marker protein for pancreatic cancer. They bought a protein-detection kit comprising a CUZD1 specific antibody and wasted two years, $500,000 and thousands of patient samples before they realized that the antibody actually recognized a different cancer protein, CA125, but did not bind to CUZD1 at all.
This single example of the Nature Feature strongly underlines the urgent need for antibody validation and cross-reactivity testing. To address this topic, we developed a new three-step approach for the cross-reactivity analysis of your antibody samples based on the new PEPperCHIP® Human Epitome Microarray in a single assay.
The PEPperCHIP® Human Epitome Microarray with 29,128 different peptides covers all linear B-cell epitopes of the Immune Epitope Database with the host “human”. Therefore, this microarray is an ideal tool to screen for antibody responses against tens of thousands of different antigenic peptides that were described in literature and linked with antibody responses in human serum or plasma. The three-step approach further includes bioinformatics analysis of the top cross-reactions of an antibody using the MEME (Multiple Em for Motif Elicitation) and FIMO (Find Individual Motif Occurrences) tools of the MEME Suite for motif discovery as well as a correlation of top hits and common motifs with protein databases.
- Cross-reactivity profiling of mono- and polyclonal antibodies
- Bioinformatic analysis of antibody response profiles
- Identification of essential core amino acids
- Epitope and mimotope discovery
- Differentiation between specific cross-reactions and non-specific interactions
- Risk assessment of antibody cross-reactions by database blasting
The cross-reactivity analysis of a mono- or polyclonal antibody with the PEPperCHIP® Human Epitome Microarray is based on the three-step approach sketched above:
(1) The antibody sample is reacted with the PEPperCHIP® Human Epitome Microarray followed by staining with a suited secondary antibody and data read-out.
(2) Data quantification is followed by generation of a peptide hit list. The top peptide hits are uploaded to the MEME tool (Multiple Em for Motif Elicitation) of the MEME Suite to discover common motifs, essential and conserved amino acid positions in these top hits. The search for common motifs also enables the unambiguous differentiation between specific cross-reactions and less specific interactions e.g. with highly basic or acidic peptides.
(3) The MEME consensus motif is uploaded into the FIMO tool (Find Individual Motif Occurrences), and processed in a protein database blast search to identify protein candidates for cross-reactions sorted by decreasing cross-reaction probabilities.
Incubation of a PEPperCHIP® Human Epitome Microarray with an antibody sample typically results in few but clear interactions with the 29,128 different peptides covering all linear B-cell epitopes of the Immune Epitope Database (left); the resulting peptide hit list is directly linked with the corresponding database entries and is used for MEME analysis of the top peptides to identify common motifs, essential and conserved amino acid positions (right).
The antibody responses found with the PEPperCHIP® Human Epitome Microarray as well as the MEME/FIMO-based bioinformatic analysis enable a detailed cross-reactivity evaluation of a given antibody in a single assay in a uniquely fast and efficient manner with minimal sample consumption. Compared to protein- or cell-based assays, the three-step peptide microarray-based approach enables the unambiguous differentiation between specific cross-reactions and non-specific or less-specific interactions.
Sample Need and Timelines
- Sample need: ca. 100 - 200 µg each antibody
- Timeline: Reporting ca. 2 weeks after sample delivery
- Deliverable: Scientific report on antibody cross-reactions including all raw data and a specificity evaluation of a given antibody