College of Chemistry, Chemical Engineering & Materials Science, Soochow University, Renai Road 199, Suzhou, 215123, China.
Changzhou Cancer Hospital Affiliated to Soochow University, Honghe Road 68, Changzhou, 213032, China.
Talanta. 2022 Nov 1;249:123655. doi: 10.1016/j.talanta.2022.123655. Epub 2022 Jun 7.
5-Fluorouracil (5-FU) is an effective anticancer drug widely used in cancer treatment. In this study, two 5-FU derivatives containing a spacer arm with the carboxylic group at the end were synthesized, which were linked to the carrier proteins to form 5-FU-protein conjugates used as the immunogens for the production of monoclonal antibody (mAb). Based on the produced mAb, the highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for 5-FU detection was established. The IC and LOD values of the assay were found to be 19.5 ng mL and 0.5 ng mL, respectively. There was no cross-reactivity (CR) of the ELISA with cytosine, thymine and uracil, which avoided the interference from inherent pyrimidines. The CR values of the assay with three substitutes of 5-FU (tegafur, 5-fluoro-2'-deoxyuridine, carmofur) were within 9.7%-17.6%. The produced mAb was also applied in sample extraction. The immuno-affinity column capable specific capturing 5-FU was prepared by immobilizing the mAb on Sepharose-4B gel and filling into a SPE column. The recoveries of 5-FU in spiked samples measured by ELISA were 72.4%-90.7% with RSD of 3.6%-8.3%. Five blood samples collected from patients were extracted by immuno-affinity column, then measured by ELISA and confirmed by HPLC-MS/MS. There was a good correlation between HPLC-MS/MS and ELISA. It is demonstrated that the developed ELISA combined with immuno-affinity extraction can be a powerful alternative method for the detection of 5-FU in blood samples.
5-氟尿嘧啶（5-FU）是一种广泛用于癌症治疗的有效抗癌药物。在本研究中，合成了两种含有末端带有羧基间隔臂的 5-FU 衍生物，将其与载体蛋白连接形成 5-FU-蛋白缀合物，用作产生单克隆抗体（mAb）的免疫原。基于产生的 mAb，建立了用于 5-FU 检测的高灵敏度和特异性酶联免疫吸附测定（ELISA）。该测定的 IC 和 LOD 值分别为 19.5ng/mL 和 0.5ng/mL。该 ELISA 与胞嘧啶、胸腺嘧啶和尿嘧啶无交叉反应（CR），避免了内源性嘧啶的干扰。该测定与 5-FU 的三种替代品（替加氟、5-氟-2'-脱氧尿嘧啶、卡莫氟）的 CR 值在 9.7%-17.6%之间。所产生的 mAb 也应用于样品提取。通过将 mAb 固定在 Sepharose-4B 凝胶上并填充到 SPE 柱中，制备了能够特异性捕获 5-FU 的免疫亲和柱。通过 ELISA 测量的加标样品中 5-FU 的回收率为 72.4%-90.7%，RSD 为 3.6%-8.3%。从 5 名患者采集的 5 份血液样本通过免疫亲和柱提取，然后通过 ELISA 测量并通过 HPLC-MS/MS 确认。HPLC-MS/MS 与 ELISA 之间存在良好的相关性。结果表明，开发的 ELISA 结合免疫亲和提取可以成为血液样本中 5-FU 检测的有力替代方法。
