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

Sandra Oloketuyi,Harald Kolmar,Robert Bernedo,Ario De Marco,Horst Wilhelm Schuchmann,Joanna Niedziółka-Jönsson,Giulia Cazzaniga,Justyna Borkowska,Andreas Christmann,Katarzyna Szot-Karpińska

doi:10.3390/bios11120496

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.

Highlights

C-reactive protein (CRP) is a plasma protein that participates in the systemic response to inflammation and the synthesis of which rapidly increases within hours of an infection or tissue injuries and apoptosis [1,2]
We used a llama pre-immune phage display library [14] for isolating nanobodies that are able to bind to CRP
This work had the following several objectives: (i) isolating nanobodies as recombinant immunocapture reagents for affordable CRP diagnostics; (ii) comparing the efficiency of a new selection method that integrates the phage and yeast display with the standard panning protocol based on phage display only; (iii) showing the proof-of-principle that engineered recombinant nanobodies may be used for the effective immunocapture of soluble antigens on biosensor surfaces

Summary

Introduction

C-reactive protein (CRP) is a plasma protein that participates in the systemic response to inflammation and the synthesis of which rapidly increases within hours of an infection or tissue injuries and apoptosis [1,2]. Recent reports indicate that CRP content variation could represent a complementary biomarker to evaluate microvascular risk in patients with type 2 diabetes, the prognosis of pancreatic neoplasm and to follow the progression of a COVID-19 infection [3,4,5]. The involvement of such protein in several physiological disorders has urged the development of diagnostic tools for its quantification. Due to their expansive production process and heterogeneity after functionalization, alternative immunocapture reagents have been proposed as peptides, aptamers and bacteriophages [8,9,10]

Methods

Results

Conclusion