Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid

Abstract

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 IC 50 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.

Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies

“…Furthermore, the growing market of immunotherapies has provided a new purpose to antibody detection: therapeutic drug monitoring (TDM). This type of monitoring is usually seen alongside any drug treatments that have severe adverse reactions (SARs) and a narrow therapeutic window ( Luque-Uría et al, 2021 ). Anticoagulant, immunosuppressive, and cytotoxic drugs are among those that need vigilant monitoring ( Jossen and Dubinsky, 2016 ; Konkle, 2016 ; Paci et al, 2014 ).…”

Ultrathin Cu–TCPP Nanosheet-Based Electrochemical Microsensor for Detecting the Immunosuppressive Drug Mycophenolic Acid

“…These methods generally require large-scale instruments, multiple treatment stages, and professionally trained staff and cannot guarantee the avoidance of cross-reactivity due to interfering substances. Electrochemical sensing technology can be used for target analysis because it has the advantages of good repeatability and accuracy, high sensitivity, convenience, and real-time detection. − Additionally, the integration, intelligence, and miniaturization of electrochemical sensors can be achieved by combining them with the micro-electromechanical system (MEMS) or printed electronics technology . Electrochemical microsensors can not only be used to detect biological macromolecules but also directly detect molecules with electrochemical activity based on the electrochemical catalytic principle. , With the advantages of miniaturization, rapid response, convenience, and portability, electrochemical microsensors would also have great application prospects for continuously monitoring immunosuppressive drugs …”

Front Cover: Comparative Study of the Performance of Two Different Luciferases for the Analysis of Fumonisin B1 in Wheat Samples (Anal. Sens. 4/2022)

“…They are able to catalyze reactions that emit light, and are currently applied in highly relevant fields, such as food testing, diagnostics or drug screening. [4,5] Two of the smallest luciferases currently known are Gaussia luciferase (GLuc) and NanoLuc luciferase (NLuc), with a size of only 19.9 kDa and 19 kDa, respectively. These two luciferases offer an exceptional performance in protein À protein interactions or bioluminescence resonance energy transfer (BRET) immunoassays, as they are considered two of the brightest luciferases yet discovered.…”