AUTHOR=Murad Hossam , Assaad Jana Mir , Al-Shemali Rasha , Abbady Abdul Qader 

TITLE=Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

JOURNAL=Frontiers in Endocrinology

VOLUME=Volume 8 - 2017

YEAR=2017

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2017.00115

DOI=10.3389/fendo.2017.00115

ISSN=1664-2392

ABSTRACT=Background: Monitoring blood-levels of human growth hormone (hGH) in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin®). Besides, misusing of rhGH by sportsmen is banned by the world anti-doping agency (WADA) and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies (HCAbs). They were successfully generated against numerous antigens including hormones.
Methods: A fully nanobody-based sandwich-ELISA method was developed in this work for direct measurement of GH in biological samples. 
Results: Two major characteristics of nanobody were exploited for this goal; the robust and stable structure of the nanobody (NbGH04) used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07) on the tip of M13-phage. Such huge, stable and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide “immune” cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~ 0.5 ng/ml) and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants.
Conclusions: In regard to all advantages of nanobodies; stability, solubility, production affordability in E. coli and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.