Abstract
This study describes, for the first time, the development of two platforms of competitive fluorescent immunoassays for bioanalysis of crizotinib (CZT), a potent drug used for the treatment of non-small cell lung cancer (NSCLC). These platforms were microwell-based heterogeneous fluoroimmunoassay (FIA) and a kinetic exclusion assay (KinExA) with KinExA™ 3200 immunosensor. Both FIA and KinExA were developed using same reagents; mouse anti-CZT antibody and a capturing reagent of CZT conjugated with bovine serum albumin (CZT-BSA). In the FIA, the CZT-BSA coated onto the microwells of the assay plate was present simultaneously in the assay mixture (CZT and its antibody). In the KinExA, the antibody was allowed to pre-equilibrate with CZT, and then the incubation mixture was rapidly passed through a microcolumn containing CZT-BSA coated onto polymethyl methacrylate (PMMA) beads. The analytical performances of both assays were comparatively evaluated in terms of assay working range, limit of detection, precision profile, and accuracy. The results revealed that KinExA yielded higher sensitivity and better precision than FIA; whereas, both assays had comparable accuracies. Both FIA and KinExA were superior to all the existing chromatographic methods for CZT in terms of the assay sensitivity, convenience, analysis throughputs. The proposed FIA and KinExA are anticipated to effectively contribute to the therapeutic drug monitoring (TDM) of CZT in clinical settings.
Operational steps of microwell-based fluoroimmunoassay (FIA) on plate reader (A) and kinetic exclusion assay (KinExA) on KinExA™ 3200 immunosensor (B) for bioanalysis of crizotinib. [Display omitted]
•Two platforms of fluorescent immunoassays were developed for crizotinib (CZT).•These platforms were fluoroimmunoassay (FIA) and kinetic exclusion assay (KinExA).•FIA and KinExA adapted microwells and KinExA™ 3200 immunosensor, respectively.•KinExA yielded higher sensitivity and better precision than FIA.•Both FIA and KinExA were superior to the existing chromatographic methods for CZT.