Enzyme-free chemiluminescence immunoassay for the determination of thyroid stimulating hormone

Abstract

Based on the chemiluminescent resonance energy transfer (CRET) from high-energy intermediate formed in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction to fluorescent microsphere-conjugated detection antibody, a cost-effective and highly sensitive enzyme-free sandwich immunoassay with chemiluminescence detection was developed for the early diagnosis of thyroid cancer. Thyroid stimulating hormone (TSH) primary antibody immobilized on paramagnetic bead captures rapidly TSH in human serum within 15min. Also, TSH detection antibody-conjugated yellow-green microsphere, capable of emitting green light, rapidly interacted with TSH-bound TSH primary antibody immobilized on paramagnetic bead during the 30-min incubation at room temperature. The sandwich complexes immobilized on paramagnetic in PBS buffer (pH 7.4) emit bright green light with the addition of ODI-CL reagents (e.g., H₂O₂, ODI). The dynamic range of enzyme-free sandwich immunoassay was 0.037-18μIUml^-1 with 3-5% coefficient of variation. The limit of detection (LOD=background+3σ) of the enzyme free sandwich immunoassay was as low as 0.011μIUml^-1. We expect that the enzyme free sandwich immunoassay with ODI-CL detection can be a new assay method for the early diagnosis of thyroid cancer with statistically acceptable accuracy, precision, and reproducibility.

Keywords: 1,1′-oxalyldiimidazole chemiluminescence; Enzyme-free immunoassay; Fluorescence; Microsphere; Thyroid cancer; Thyroid stimulating hormone (TSH).