ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

Abstract

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity.B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

MAbTope: A Method for Improved Epitope Mapping

“…Aside from three-dimensional (3D) structures, most experimental methods available for epitope determination are based either on the following: 1) site-directed mutagenesis; 2) peptide arrays (3)(4)(5); or 3) mass spectrometry (6). Most peptide-based methods use 15-30 aa overlapping peptides of the target arrayed on solid support, which are then exposed to the Ab (4,5). This identification of interacting peptides can then be completed by alanine scanning to define the epitope more precisely (3).…”

Polymorphisms in Cha o 1 and Cha o 2, major allergens of Japanese cypress (Chamaecyparis obtusa) pollen from a restricted region in Japan

“…We conclude that isoallergen Cha o 1.0101 is a major variant of Cha o 1 from artificial forests in Chiba Prefecture. The presence of the L262I substitution in Cha o 1 should have a limited effect on recognition by antibodies because Leu and Ile share the same molecular weight and hydrophobic properties [ 25 , 26 ], and the average frequency of the allele among the 47 plus trees is only 0.11. Because no substitutions other than L262I were found in Cha o 1 from plus trees and pollen, Cha o 1 derived from pollen collected in Chiba Prefecture may behave uniformly when interacting with its antibodies that target Cha o 1.…”

Reductionist Approach in Peptide-Based Nanotechnology

“…Another very important technological advancement facilitating the exploitation of functional peptide sequences is related to the production of thousands of peptides in the format of custom-made peptide arrays. An array of peptides can now be produced at a cost of less than 0.1 cent per peptide (e.g., PEPperCHIP® peptide microarray technology), resulting in the cost-effective production of thousands of peptides on a standard solid support chip (54–59). Peptide arrays allow the mapping of molecular recognition between peptide elements and the identification of interaction modules.…”