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J Mol Recognit. 2015 Oct;28(10):635-44. doi: 10.1002/jmr.2477. Epub 2015 May 11.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies.

Author information

1
IBS, Université Grenoble Alpes, F-38044, Grenoble, France.
2
IBS, CNRS, F-38044, Grenoble, France.
3
IBS, CEA, F-38044, Grenoble, France.
4
Biotechnologie et signalisation cellulaire, Université de Strasbourg, CNRS, ESBS, Boulevard Sébastien Brant BP10413, 67412, Illkirch, France.
5
Faculté de Pharmacie, CNRS UMR 7213, Université de Strasbourg, 74 route du Rhin, 67401, Illkirch, France.
6
IBMC, CNRS UPR 9002 - ARN, Université de Strasbourg, 15 rue René Descartes, 67084, Strasbourg Cedex, France.

Abstract

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.

KEYWORDS:

antibody selectivity; antigen-antibody recognition; cross-reactivity; peptide array; spot peptide; surface plasmon resonance

PMID:
25960426
DOI:
10.1002/jmr.2477
[Indexed for MEDLINE]

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