Preparation and characterization of a homogeneous immunoassay for point-of-care testing (POCT) of procalcitonin (PCT).

Abstract

Procalcitonin (PCT) has been recognized as a specific and early marker for microbial infection and sepsis. Sensitive measuring interaction-triggered luminescence experiment (SMILE), a homogeneous immunoassay method, was established for point-of-care testing (POCT) of PCT. SMILE is achieved through the principle of double antibody sandwich, where two antibodies immobilized on the surface of polystyrene microspheres (donor and acceptor beads) bind to the PCT antigen. The donor bead contains phthalocyanine dye (luminol chemiluminescent substance) and the acceptor bead contains dimethylthiophene derivatives and Eu chelates. Therefore, singlet oxygen can be transferred when the distance between donor and acceptor beads is within 200 nm, generating detectable luminescent signals. Scanning electron microscopy (SEM) was used to detect the diameter and polymer dispersity index (PDI) of microspheres before and after binding with antibodies to characterize the immobilization of antibodies. The reaction conditions for antibody immobilization including pH, mass ratio and reaction time have also been optimized. The limit of quantitation (LOQ) of the SMILE method (0.01 ng mL-1) was lower than that of the LFI method (0.1 ng mL-1), the working range (0.01-500 ng mL-1) was wider than that of the LFI method (0.1-50 ng mL-1), and the assay time (10 min) was shorter than that of the LFI method (15 min). So, SMILE is more suitable for POCT of PCT compared with lateral flow immunochromatography (LFI), which is the most used measuring method, due to its advantages of simple operation, saving time, convenience, wide detection range, and high sensitivity and accuracy.