Synthesis of hapten, generation of specific polyclonal antibody and development of ELISA with high sensitivity for therapeutic monitoring of crizotinib

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.