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

Abstract

BackgroundMonitoring 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 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. They were successfully generated against numerous antigens including hormones.MethodsA fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples.ResultsTwo 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.ConclusionIn regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

Application of Single-Domain Antibodies (“Nanobodies”) to Laboratory Diagnosis

“…This contrasts with the higher detection limits of various automated platforms using conventional antibodies [44] (Continued to the next page) [47]. Similar advantages in sensitivity were seen with a growth hormone assay utilizing a nanobody, wherein a detection range of 0.5-110 ng/mL was achieved [48]. Our efforts to design better performing assays culminated in a recent highly reproducible sensitive and specific assay for Toxocara excretory secretory components [49,50].…”

Production and engineering of nanobody‐based quenchbody sensors for detecting recombinant human growth hormone and its isoforms

“…NbRec (NbGH04) and NbPit (NbGH06) derived from the heavy chain-only antibody (HCAb) of camelids (Figure S1A) against GH were used in this study. 22 The sequences of NbRec and NbPit gene were used in this study developed from, 23 and synthesized from Macrogen.…”

Engineered Yeast Displaying Specific Norovirus-Binding Nanobodies for the Concentration and Detection of Human Norovirus in Food Matrix

“…Taking the most commonly used filamentous M13 bacteriophage as an example, each engineered M13 phage can only display 3−5 copies of nanobody fused to the N-terminus of capsid pIII. 23 As a comparison, the larger surface area available on yeast cells and the eukaryotic machinery-mediated expression allow for greater expression levels and higher yields of nanobodies for downstream applications. As reported, the display level of nanobodies on the yeast surface is variable but has been estimated at approximately 10 4 −10 5 copies per cell.…”

“…Though the phage display systems have traditionally been used for the selection and screening of nanobodies and are well-established for this purpose, nanobody expression on phage-displayed systems is less efficient than that based on the yeast-displayed systems. Taking the most commonly used filamentous M13 bacteriophage as an example, each engineered M13 phage can only display 3–5 copies of nanobody fused to the N-terminus of capsid pIII . As a comparison, the larger surface area available on yeast cells and the eukaryotic machinery-mediated expression allow for greater expression levels and higher yields of nanobodies for downstream applications.…”