Multiplexed Antigen Bead Arrays for the Assessment of Antibody Selectivity and Epitope Mapping
With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.
Key wordsAffinity proteomics Antigen arrays Peptide arrays Protein arrays Suspension bead arrays Antibody selectivity Epitope mapping
We thank the entire staff of the Human Protein Atlas for producing all the protein fragments and antibodies utilized in this study. This work was supported by the ProNova VINN Excellence Centre for Protein Technology (VINNOVA, Swedish Governmental Agency for Innovation Systems) and by grants from the Knut and Alice Wallenberg Foundation, SciLifeLab Stockholm and the KTH Center for Applied Proteomics funded by the Erling-Persson Family Foundation. The authors declare no conflict of interest.
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