Antibodies are applied in many research areas such as antibody-based proteomics or cellular biology. However, the specificity of antibodies and hence their ability to generate reliable data is under scrutiny in the scientific community. Highly specific antibodies are a prerequisite to generate reliable data, going along with the urgent need for appropriate validation methods to determine specificity and cross-reactivity.

In this webinar, we present a straightforward approach for the unambiguous validation of antibodies by combining protein and peptide microarrays: An initial screening of antibodies against HuProt™ Human Proteome Microarrays with more than 20,000 human proteins enabled the determination of the target specificity and off-target binding on the protein level. For further validation of the antibody responses, we applied a multiplexed high resolution epitope mapping of the top cross-reactions and the target protein by PEPperCHIP® Peptide Microarrays. This resulted not only in the identification of the actual epitopes of the antibodies, but also provided more insight on the observed cross-reactions.

We compared two different commercial research antibodies against human transglutaminase 2, and observed a high cross-reactivity particularly with a highly validated one. This outcome raises questions about the reliability of some methods for antibody validation. The combined protein and peptide microarray screening is not only an ideal tool for antibody validation, but also for the identification and validation of unknown antigens.