New ELISA approach based on coiled-coil interactions.
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.
Liberelle
Benoît
B
Department of Chemical Engineering, Bio-P2 Research Unit, Institute of Biomedical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales, Ecole Polytechnique de Montréal, PO BOX 6079, Station Centre-ville, Montréal (QC) Canada H3C 3A7.
Bartholin
Laurence
L
Boucher
Cyril
C
Murschel
Frédéric
F
Jolicoeur
Mario
M
Durocher
Yves
Y
Merzouki
Abderrazzak
A
De Crescenzo
Gregory
G
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004815:Epidermal Growth Factor / Q000032:analysis* / Q000276:immunology; D006801:Humans; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity
D004815:Epidermal Growth Factor