title: Validation of an ELISA for determination of antibodies induced in monkeys against Epi-hNE4, a recombinant protein inhibitor of human neutrophil elastase.
abstract: The engineered protein inhibitor of human neutrophil elastase, Epi-hNE4, is being developed for the treatment of cystic fibrosis. Like many recombinant proteins, Epi-hNE4 may induce antibodies in pre-clinical species and in humans. The aim of this report was to validate an ELISA to assess its immunogenicity in monkeys. We have designed and optimized a classical ELISA in which Epi-hNE4 was coated directly on microtitre plates and the antibodies were detected using a secondary antibody labelled with peroxidase. We report implementation of the recent recommendations proposed for the validation of immunogenicity assessment. The cut-off point was determined by means of statistical analysis of negative samples. Linearity, reproducibility, stability and specificity were estimated using quality control samples obtained from a pool of positive samples. The method was applied to monkeys given Epi-hNE4 by inhalation. A confirmation test and a neutralization assay were developed in order to further assess positive samples. In conclusion, we present here one of the first examples of validation in application of recent recommendations [A.R. Mire-Sluis, Y.C. Barrett, V. Devanarayan, E. Koren, H. Liu, M. Maia, T. Parish, G. Scott, G. Shankar, E. Shores, S.J. Swanson, G. Taniguchi, D. Wierda, L.A. Zuckerman, J. Immunol. Methods 289 (2004) 1-16].
authors.affiliation: CEA, Service de Pharmacologie et d'Immunologie, CE Saclay, 91191 Gif-sur Yvette, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Dubois
authors.forename: Valérie
authors.initials: V
authors.lastname: Delaunay
authors.forename: Laurent
authors.initials: L
authors.lastname: Delestre
authors.forename: Eric
authors.initials: E
authors.lastname: Ezan
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D000882:Haplorhini; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D008297:Male; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D010455:Peptides; D011994:Recombinant Proteins; C512995:depelestat; D019272:Leukocyte Elastase