Abstract
Crizotinib (CZT) is a potent drug used for treatment of non-small cell lung cancer (NSCLC); however, its circulating concentration variability has been associated with acquired resistance and toxicity, restricting the success of cancer treatment. As such, the development of an assay that monitors CZT plasma concentrations in patients is a valuable tool in cancer treatment. In this study, a hapten of CZT was synthesized by introducing the acetohydrazide moiety as a spacer into the chemical structure of CZT. The chemical structure of the CZT acetohydrazide (hapten) was confirmed by mass, 1H-, and 13C-NMR spectrometric techniques. The hapten was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. CZT-KLH conjugate was used for immunization and generation of a polyclonal antibody recognizing CZT with high affinity (IC50 = 0.5 ng/mL). The polyclonal antibody was used in the development of an ELISA for determination of CZT. The ELISA involved a competitive binding reaction between CZT, in its samples, and immobilized CZT-BSA conjugate for the binding sites on a limited amount of the anti-CZT antibody. The assay limit of detection was 0.03 ng/mL and the working range was 0.05 − 24 ng/mL. Analytical recovery of CZT from spiked plasma was 101.98 ± 2.99%. The precisions of the assay were satisfactory; RSD was 3.2 − 6.5% and 4.8 − 8.2%, for the intra- and inter-assay precision, respectively. The assay is superior to all the existing chromatographic methods for CZT in terms of its procedure simplicity, convenience, and does not require treatment of plasma samples prior to the analysis. The proposed ELISA is anticipated to effectively contribute to the therapeutic monitoring of CZT in clinical settings.
Highlights
Lung cancer is the most common cancer in terms of both incidence and mortality in men and women [1]
Solutions of CZT hapten, CZT-bovine serum albumin (BSA), BSA, CZT-keyhole limpet hemocyanin (KLH) and KLH were prepared in phosphate buffer saline (PBS) solution (50 mM, pH 7.2)
In order to convert CZT to ELISA for therapeutic monitoring of crizotinib immunogenic molecule, it should be linked to some immunogenic macromolecules such as carrier proteins [21]
Summary
Lung cancer is the most common cancer in terms of both incidence and mortality in men and women [1]. CZT has demonstrated high response rates in non-small cell lung cancer (NSCLC) patients carrying anaplastic lymphoma kinase This gene results in constitutive kinase activity that contributes to carcinogenesis and drive the malignant phenotype [6,7]. Immunoassays (e.g. ELISA) are more preferable alternative techniques in the field of clinical analysis [19] This was attributed to the facts that they are specific for the analyte, they usually do not require pretreatment for the specimens of complex matrix (e.g. plasma, urine, etc.), they have high analytical throughputs are suited for clinical setting processing large number of samples, and the analysis by these assays is not expensive. All other chemicals and solvents used throughout the work were of analytical grade
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