C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection / S. Oloketuyi, R. Bernedo, A. Christmann, J. Borkowska, G. Cazzaniga, H.W. Schuchmann, J. Niedziółka-Jönsson, K. Szot-Karpińska, H. Kolmar, A. de Marco. - In: BIOSENSORS. - ISSN 2079-6374. - 11:12(2021 Dec 03), pp. 496.1-496.14. [10.3390/bios11120496]

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

G. Cazzaniga;
2021

Abstract

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.
phage display; yeast display; nanobodies; biopanning; CRP
Settore BIO/19 - Microbiologia Generale
Settore BIO/10 - Biochimica
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/888793
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