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Validation of an ELISA for determination of antibodies induced in monkeys against Epi-hNE4, a recombinant protein inhibitor of human neutrophil elastase.
- Source :
-
Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2007 Mar 12; Vol. 43 (4), pp. 1423-9. Date of Electronic Publication: 2006 Nov 21. - Publication Year :
- 2007
-
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].
Details
- Language :
- English
- ISSN :
- 0731-7085
- Volume :
- 43
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Journal of pharmaceutical and biomedical analysis
- Publication Type :
- Academic Journal
- Accession number :
- 17118611
- Full Text :
- https://doi.org/10.1016/j.jpba.2006.10.022