Magnetic particle-based time-resolved fluoroimmunoassay for the simultaneous determination of α-fetoprotein and the free β-subunit of human chorionic gonadotropin

Abstract

In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic particles for the simultaneous determination of α-fetoprotein (AFP) and the free β-subunit of human chorionic gonadotropin (free β-hCG) in human serum is described. The new approach uses magnetic particles as an immobilization matrix and means of separation, while the luminescent europium and samarium chelates are used as probes. The proposed method was evaluated via a single-step, sandwich-type TRFIA immunoassay of AFP and free β-hCG as model analytes in serum. With the advantages of magnetic particles, the TRFIA immunoassay exhibited a wide dynamic range for AFP of 0.1–750 ng mL⁻¹, with a lower detection limit of 0.05 ng mL⁻¹. The dynamic range for free β-hCG was 0.16–450 ng mL⁻¹, with a lower detection limit of 0.08 ng mL⁻¹. Satisfactory specificity, reproducibility, and recovery of the immunoassay were demonstrated. Good correlations were obtained in the analysis of 446 human serum samples between the proposed method and a commercial TRFIA kit. These results demonstrate the feasibility and potential of the new method as a rapid and highly sensitive immunoassay that could be developed into a platform for multi-analyte determinations in clinical practice.