posted on 2021-07-14, 15:40authored byÁlvaro Luque-Uría, Riikka Peltomaa, Tarja K. Nevanen, Henri O. Arola, Kristiina Iljin, Elena Benito-Peña, María C. Moreno-Bondi
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.