Development of a highly sensitive and specific monoclonal antibody-based ELISA coupled with immuno-affinity extraction for the detection of anticancer drug 5-fluorouracil in blood samples

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 IC₅₀ and LOD values of the assay were found to be 19.5 ng mL^-1 and 0.5 ng mL^-1, 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.