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.

克唑替尼（Crizotinib, CZT）是一款用于治疗非小细胞肺癌（non-small cell lung cancer, NSCLC）的强效抗肿瘤药物，但其循环血药浓度的变异度与获得性耐药及毒性反应密切相关，限制了癌症治疗的临床成效。鉴于此，开发一种可精准监测患者血浆中CZT浓度的检测方法，成为癌症治疗领域极具价值的技术手段。本研究通过在CZT的化学结构中引入乙酰肼基团作为间隔臂，成功合成了CZT的半抗原（hapten）。采用质谱法、氢核磁共振谱（¹H-NMR）及碳核磁共振谱（¹³C-NMR）光谱技术，对该CZT乙酰肼半抗原的化学结构进行了确证。以1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（ethyl-3-(3-dimethylaminopropyl) carbodiimide）作为偶联试剂，将该半抗原分别与牛血清白蛋白（bovine serum albumin, BSA）和钥孔戚血蓝蛋白（keyhole limpet hemocyanin, KLH）进行共价偶联。以CZT-KLH偶联物作为免疫原开展动物免疫，成功获得了对CZT具有高亲和力的多克隆抗体（polyclonal antibody），其半抑制浓度（IC50）为0.5 ng/mL。利用该多克隆抗体，开发了用于定量检测CZT的酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）方法。该ELISA方法基于竞争性结合反应原理：样品中的CZT与固定化的CZT-BSA偶联物竞争有限量的抗CZT抗体结合位点。本检测方法的检出限为0.03 ng/mL，有效工作范围为0.05 ~ 24 ng/mL。对加标血浆样品中CZT的分析回收率为101.98 ± 2.99%。方法精密度表现良好：批内精密度的相对标准偏差（relative standard deviation, RSD）为3.2% ~ 6.5%，批间精密度的RSD为4.8% ~ 8.2%。相较于现有的所有CZT色谱检测方法，本方法操作简便快捷，且无需对血浆样品进行前处理，优势显著。本研究所提出的ELISA方法有望为临床场景下CZT的治疗药物监测提供有力支撑。