Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 6:13:33.
doi: 10.1186/s12951-015-0091-7.

Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library

Junrong Yan  1 , Pingyan Wang  2 , Min Zhu  3 , Guanghui Li  4 , Ema Romão  5 , Sheng Xiong  6 , Yakun Wan  7   8
Affiliations

Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library

Junrong Yan et al. J Nanobiotechnology. .

Abstract

Background: Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems.

Results: We constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 10(11) clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT.

Conclusion: We have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Scheme of strategy to construct the naïve library.
Figure 2
Figure 2
Construction of the naïve library. (A)The VHH genes were obtained by two steps PCR. (B) The library size was measured by counting the colonies number after serial dilution. (C) 24 colonies were randomly picked to estimate the correct insertion rate of VHH genes by PCR amplification.
Figure 3
Figure 3
Non-immune library derived PCT Nbs. (A) Amino acid sequence alignment of three PCT-specific Nb families as classified by CDR3. Amino acids positions of the framework region (FR) and of the three antigen-binding loops (CDR1, CDR2 and CDR3) are numbered according to the IMGT Scientific chart for the V-Domain and are indicated at bottom. (B) SDS-PAGE analysis of purified Nbs.
Figure 4
Figure 4
Binding thermostability analysis. The PCT Nbs from the naïve and the immune libraries were incubated at (A) 37°C, (B) 60°C or (C) 90°C for different times to analyze the binding thermostability by ELISA. The activity of the Nbs never been treated was regarded as 100% and three independent experiments were performed.
Figure 5
Figure 5
SPRi binding assay. Affinity between PCT and the three Nbs was determined by SPRi binding assay. (A) Nb1, (B) Nb2 and (C) Nb3 were immobilized on the chip surface and PCT in PBST were injected at the concentrations of 9.7, 29.2, 87.6 and 263 nM.
Figure 6
Figure 6
Detection of PCT by the sandwich ELISA based on BiNb2. (A) The purified BiNb2 was analyzed by SDS-PAGE. (B) Schematic drawing of the proposed sandwich ELISA. (C) Calibration curve toward different concentrations of PCT. The linear relationship was in the range from 10 to 1000 ng/mL.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fernandez LA, Muyldermans S. Recent developments in engineering and delivery of protein and antibody therapeutics. Curr Opin Biotechnol. 2011;22:839–42. doi: 10.1016/j.copbio.2011.08.001. - DOI - PubMed
    1. Saerens D, Frederix F, Reekmans G, Conrath K, Jans K, Brys L, et al. Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal Chem. 2005;77:7547–55. doi: 10.1021/ac051092j. - DOI - PubMed
    1. Market E, Papavasiliou FN. V(D)J recombination and the evolution of the adaptive immune system. PLoS Biol. 2003;1 doi: 10.1371/journal.pbio.0000016. - DOI - PMC - PubMed
    1. Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P, Vincke C, Muyldermans S. Nanobodies and their potential applications. Nanomedicine (Lond) 2013;8:1013–26. doi: 10.2217/nnm.13.86. - DOI - PubMed
    1. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM. Domain antibodies: proteins for therapy. Trends Biotechnol. 2003;21:484–90. doi: 10.1016/j.tibtech.2003.08.007. - DOI - PubMed

Publication types