A1 Refereed original research article in a scientific journal
Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid
Authors: Luque-Uría Álvaro, Peltomaa Riikka, Nevanen Tarja K, Arola Henri O, Iljin Kristiina, Benito-Peña Elena, Moreno-Bondi María C
Publisher: AMER CHEMICAL SOC
Publication year: 2021
Journal: Analytical Chemistry
Journal name in source: ANALYTICAL CHEMISTRY
Journal acronym: ANAL CHEM
Volume: 93
Issue: 29
First page : 10358
Last page: 10364
Number of pages: 7
ISSN: 0003-2700
DOI: https://doi.org/10.1021/acs.analchem.1c02109
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/66911601
Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL(-1) and an IC50 of 2.9 +/- 0.5 ng mL(-1). The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.
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