Development and validation of generic heterogeneous fluoroimmunoassay for bioanalysis of bevacizumab and cetuximab monoclonal antibodies used for cancer immunotherapy

Hamidaddin, Mohammed A.; AlRabiah, Haitham; Darwish, Ibrahim A.

doi:10.1016/j.talanta.2018.05.091

Cited by 4 publications

(6 citation statements)

References 36 publications

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“…Working solutions of goat anti-mouse FITC-IgG were prepared at concentrations of 0.4 and 0.25 µg mL −1 for use in FIA and KinExA, respectively. These solutions were prepared as described in our previous studies 19,20 . The solutions were prepared fresh at the start of each experiment.…”

Section: Methodsmentioning

confidence: 99%

“…The coated and blocked plates could be stored for at least 6 weeks at 4 or −20 °C without any noticeable deterioration of the coated AFT-BSA conjugate. The details of coating the FIA plates were described in our previous study 19 .…”

Section: Methodsmentioning

confidence: 99%

“…Standard AFT was spiked in AFT-free plasma samples (50 μL) and the AFT-spiked samples were diluted to 2 mL with PBS or PBS containing BSA (1%, w/v) for analysis by FIA or KinExA, respectively. AFT was spiked to final concentration in the range of 0.1−200 and 0.01−100 ng mL −1 for FIA and KinExA, respectively 19,20 .…”

Section: Methodsmentioning

confidence: 99%

“…The analysis by FIA was conducted according to the detailed procedures described in our previous study 19 . Briefly, aliquots (50 µL) of samples (standard solutions or spiked plasma samples) containing AFT (0.1 − 200 ng mL −1 ) were mixed with equal volumes of anti-AFT antibody (diluted 160-fold in PBS).…”

Section: Methodsmentioning

confidence: 99%

“…A previous study 16 in our laboratory has described the production of a polyclonal antibody recognizing AFT with high selectivity and its use in the developed an enzyme-linked immunosorbent assay (ELISA) for AFT. However, heterogeneous microwell-based fluorescence immunoassay (FIA) with fluorescence plate reader is more powerful than ELISA because it is characterized with of higher sensitivity, time-saving in samples processing, wide working assay range, and high precision 17–19 . Furthermore, kinetic exclusion assay (KinExA) developed using KinExA immunosensor offers the same advantages as those of FIA in addition to convenient automation, and devoid from the common problems and limitations of ELISA 20–22 .…”

Section: Introductionmentioning

confidence: 99%

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

Darwish

AlRabiah

Hamidaddin

2019

Sci Rep

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%