Development of two different formats of heterogeneous fluorescence immunoassay for bioanalysis of afatinib by employing fluorescence plate reader and KinExA 3200 immunosensor

Abstract

Afatinib (AFT) is a potent and highly selective drug used to treat various solid tumors including non-small cell lung cancer (NSCLC). To ensure safe and effective treatment of cancer patients with AFT, its plasma concentrations should be monitored. Thus, sensitive immunoassays are required for measuring AFT concentrations in plasma samples. In this study, two different formats of heterogeneous fluorescent immunoassays were developed and validated for AFT bioanalysis. These assays were microwell-based fluorescence immunoassay (FIA) using fluorescence plate reader and kinetic exclusion assay (KinExA) using KinExA 3200 immunosensor. Both FIA and KinExA were developed using the same reagents: mouse anti-AFT antibody, solid-phase immobilized AFT conjugated with bovine serum albumin protein (AFT-BSA), and goat anti-mouse IgG labelled with fluorescein isothiocyanate (FITC-IgG) for signal generation. The analytical performances of both assays were comparatively evaluated, and the results revealed that although both assays had comparable accuracies, KinExA was superior to FIA in terms of sensitivity and precisions. Moreover, both FIA and KinExA were better alternatives to the existing chromatographic methods for bioanalysis of AFT. The proposed FIA and KinExA are anticipated to effectively contribute in ensuring safe and effective treatment with AFT in clinical settings.

Parallel detection of multiple biomarkers in a point-of-care-competent device for the prediction of exacerbations in chronic inflammatory lung disease

“…Thus, even lower spiking concentrations such as 1 ng/ml should be feasible. This would translate into limits of detection (LODs) of about 40–100 pM analyte, which is in accordance with previous reports where LODs in the picomolar to low nanomolar range were observed for fluoroimmunoassays (FIA) conducted on plain surfaces 24 , 25 .…”

Novel ultrasensitive automated kinetic exclusion assay for measurement of plasma levels of soluble PD-L1, the predictive and prognostic biomarker in cancer patients treated with immune checkpoint inhibitors

“…The KinExA-based assays have proven to be highly sensitive and versatile in detecting and quantifying various molecules of interest across a wide range of applications [ [40] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] ]. These molecules encompass bacteria [ 44 ], chemotherapeutic drugs targeting tyrosine kinase inhibitors [ 45 ], therapeutic monoclonal antibodies [ 46 ], hormones [ 47 ], biomarkers associated with different diseases and conditions [ 40 ], opiates [ 48 ], environmental contaminants [ 49 , 50 ], food toxins [ 51 ], and heavy metals [ 52 ].…”

“…KinExA enables the real-time determination of true-solution-based binding affinities and kinetics of molecular interactions [ 34 ]. In previous studies [ [35] , [36] , [37] , [38] ], our laboratory have developed successful KinExA-based assays for quantitative analysis of diverse analytes, showcasing advantages over conventional immunoassays like ELISA. These advantages include the elimination of immobilization effects, overcoming mass transport limitations, and providing improved performance.…”

New Immunosensing-Fluorescence Detection of Tumor Marker Cytokeratin-19 Fragment (CYFRA 21-1) Via Carbon Quantum Dots/Zinc Oxide Nanocomposite

“…The heterogeneous competitive assay is conducted by immobilizing capture molecules on the surface and then incubated with fluorophore-conjugated biomarkers. The competition between the free and conjugated biomarkers for binding to the capture molecules decreases the fluorescence intensity with biomarker concentration [16]. The heterogeneous sandwich assay is based on the incubation of capture molecules and solution of interest forming a complex with biomarkers.…”