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

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.

Investigation of Peptides for Molecular Recognition of C-Reactive Protein–Theoretical and Experimental Studies

“…One reason can be the higher surface density of the peptide as compared to the antibodies, which can lead to increased steric hindrance . The obtained value of K d for P3 peptide is comparable with the other platforms modified with aptamers or nanobodies, and lower for anti-CRP antibodies-based systems. , In the case of mAb, the K d value is relatively uncertain because of the large error bars. But the shape of the curve clearly indicates a n larger than 1.…”

“…Antibodies are currently the most frequently used CRP receptors. ,, In the literature, there are also examples of CRP determination using bioreceptors such as aptamers, – bacteriophages, affimers, peptides, , or nanobodies …”

“…A solution might be characterization of the molecular interactions of CRP with receptors using various physicochemical techniques. Some examples are 2D DNA origami nanostructures for quantitative single-molecule biosensing, SERS, fluorescence microscopy, atomic force microscopy, biolayer interferometry, or SPR ,, and electrochemical methods. ,,– Not all of these are easy to perform, but they can give deeper analysis and insights into understanding protein–peptide interactions.…”

“…24,29,30 In the literature, there are also examples of CRP determination using bioreceptors such as aptamers, 31−35 bacteriophages, 36 affimers, 37 peptides, 38,39 or nanobodies. 40 CRP assays are often performed using biological methods such as ELISA, 41 test-based lateral flow immunoassays, and other immunoassays. 42 These methods are costly and laborious and require expensive equipment and specialized staff.…”

Comparing nanobody and aptamer-based capacitive sensing for detection of interleukin-6 (IL-6) at physiologically relevant levels

“…They have some advantages such as simple engineering, lower price production, better stability, high solubility, and the possibility to obtain higher density onto the surfaces and greater analytical signal gain. Their sequence can be transferred to bacterial expression systems for their production [ 29 , 30 ]. Aptamers are synthetic single-stranded oligonucleotides which are currently gaining in popularity in bioanalysis because of advantages such as easy chemical manufacturing processes, thermal and chemical stability, and simple modification with different labels or marker molecules.…”