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Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies
Author(s) -
Vernet Thierry,
Choulier Laurence,
Nominé Yves,
Bellard Laure,
Baltzinger Mireille,
Travé Gilles,
Altschuh Danièle
Publication year - 2015
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2477
Subject(s) - surface plasmon resonance , peptide , chemistry , combinatorial chemistry , epitope , computational biology , selectivity , peptide library , antibody , antigen , high throughput screening , dna microarray , binding selectivity , protein array analysis , epitope mapping , peptide sequence , biochemistry , biology , gene , nanotechnology , genetics , nanoparticle , materials science , gene expression , catalysis
Antibody selectivity represents a major issue in the development of efficient immuno‐therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high‐throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross‐reactive capacity of an antibody fragment using the PEPperCHIP ® technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore ® technology; GE‐Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N‐terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence 6 TAMFQDPQER 15 ) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross‐reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity. Copyright © 2015 John Wiley & Sons, Ltd.