Fast Protocols for Characterizing Antibody-Peptide Binding.

Microarray assay formats gained popularity in the 1990s, first implemented in DNA-based arrays but later adopted for use with proteins, namely antibodies, peptides, low molecular weight (LMW) molecules, such as lipids, and even tissues. In nucleic acid-based affinity assays and arrays, but not in protein or peptide arrays, the specificity and affinity of complementary strand interactions can be deduced from or adjusted through modifications to the nucleotide sequence. Arrays of LMW molecules are characterized by largely uniform but low binding affinities. Multiplexed protein-based affinity assays, such as microarrays, might present an additional challenge due to heterogeneity of antigen properties and of their binding affinities. The use of peptides instead of proteins reduces physical heterogeneity of these reagents through either the widened peptide selection options or rational sequence engineering. However, rational engineering of binding affinities remains an unmet need, and peptide-binding affinities to the respective antipeptide antibodies could vary by orders of magnitude. Hence, multiplexing of such assays by using a microarray format and data analysis and interpretation requires some knowledge of their binding affinities. Low-throughput binding assays to characterize such peptide-antipeptide antibodies interactions are widely available, but scaling-up of traditional protein- and peptide-binding assays might present practical challenges. Here, we describe fast label-free practical approach especially suitable for estimating peptide-binding affinities. The method in question relies on commercially available biolayer interferometry-based equipment with a protocol which can be easily scaled-up, subject to user needs and equipment availability.

Peptides and Anti-peptide Antibodies for Small- and Medium-Scale Peptide and Anti-peptide Affinity Microarrays.

This chapter describes the principles for selection of antigenic peptides for the development of anti-peptide antibodies suitable for microarray-based multiplex affinity assays and optional mass spectrometry detection. The methods described here are mostly applicable to small- and medium-scale multiplex affinity assay and microarrays. Although the same principles of peptide selection may also be applied to larger-scale arrays (with 100+ features), informatics software and printing methods may well differ. Due to the sheer number of proteins/peptides to be processed and analyzed, dedicated software with high processing capacity and enterprise-level array robotics may be required for larger-scale efforts. This report aims to provide practical advice to those seeking to develop or use arrays with up to ~100 different peptide or protein features.