New ELISA approach based on coiled-coil interactions

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• 作者：Benoî ; t Liberelle ; Laurence Bartholin ; Cyril Boucher ; Fré ; dé ; ric Murschel ; Mario Jolicoeur ; Yves Durocher ; Abderrazzak Merzouki ; Gregory De Crescenzo
• 关键词：EGF ; E/K coiled-coil interactions ; ELISA test
• 刊名：Journal of Immunological Methods
• 出版年：2010
• 出版时间：31 October 2010
• 年：2010
• 卷：362
• 期：1-2
• 页码：161-167
• 全文大小：442 K

文摘

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 5.2 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 poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins.