Development of a novel immunoassay for the simple and fast quantitation of neutrophil gelatinase-associated lipocalin using europium(III) chelate microparticles and magnetic beads.

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for diagnosing acute kidney injury (AKI). Currently, there are few assays for determining NGAL and they are complex, time-consuming or expensive. We aimed to establish an efficient immunoassay to measure NGAL in human urine simply and rapidly. A novel immunoassay for NGAL determination was established by combining a dissociation-enhanced-free time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a "sandwich"-type immunoassay format, analytes in samples were captured by a pair of monoclonal antibodies (mAb) in which one mAb was coated in magnetic beads and the other mAb was labeled with europium(III) chelate microparticles (CM-EUs) as "fluorescent reporters". NGAL concentrations were determined in a linear range (10-1500 ng mL) with a limit of detection of 0.32 ng mL. The reproducibility, recovery, and specificity of our TRFIA were acceptable. Our method was compared with that of a chemiluminescence immunoassay (CMIA) using 115 urine samples, and the results showed good correlation (R = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.