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

Abstract

Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti‐Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence‐incorporated antibody sensor “Quenchbody (Q‐body)” that activates upon antigen binding. Camelid‐derived nanobodies were used to produce stable Q‐bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q‐bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis‐tag, and Q‐bodies were obtained using a maleimide‐thiol reaction between the N‐terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58‐fold to 3.04‐fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA‐labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520‐labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q‐bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti‐doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.

Nanobody Engineering: Computational Modelling and Design for Biomedical and Therapeutic Applications

“…Detecting antigens through fluorescence intensity changes is simple, easy to operate, and highly sensitive. The choice of the dye structure [148] the composition of the linker that attaches the dye to the antibody fragment (length and flexibility) [148][149][150], and the position of key quenching tryptophans [151,152] has been studied for maximising the fluorescence quenching and antigen-dependent de-quenching. To date, various formats of Q-bodies have been developed targeting antigens of interest.…”

“…Finally, nanobody-based mini Q-bodies that detect the small hapten methotrexate [148], lysozyme [157] and recombinant human growth hormone and its isoforms [151] have been developed. Fab-based Q-bodies generally exhibit a more substantial increase in fluorescence upon binding to the antigen compared to the scFv-based ones [158].…”