期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 362, 期 1-2, 页码 161-167出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2010.09.027
关键词
EGF; E/K coiled-coil interactions; ELISA test
资金
- Canada Research Chairs
- Natural Sciences and Engineering Research Council of Canada
- Groupe de Recherche en Sciences et Technologies Biomedicales (GRSTB)
The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 52 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as polyhistidine for the development of ELISA tests aiming at quantitating coil-tagged proteins. (C) 2010 Elsevier B.V. All rights reserved.
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