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

J Immunol Methods. 2019 Jul:470:15-19. doi: 10.1016/j.jim.2019.04.004. Epub 2019 Apr 18.

Abstract

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-1) with a limit of detection of 0.32 ng mL-1. 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 (R2 = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.

Keywords: Europium(III) chelate microparticles; Magnetic beads; Neutrophil gelatinase-associated lipocalin; Time-resolved fluoroimmunoassay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Kidney Injury / diagnosis*
  • Acute Kidney Injury / physiopathology
  • Acute Kidney Injury / urine
  • Antibodies, Monoclonal / chemistry
  • Biomarkers / urine
  • Early Diagnosis
  • Fluoroimmunoassay / methods*
  • Fluoroimmunoassay / standards
  • Humans
  • Immunoconjugates / chemistry*
  • Immunomagnetic Separation / methods*
  • Immunomagnetic Separation / standards
  • Limit of Detection
  • Lipocalin-2 / urine*
  • Magnets / chemistry
  • Organometallic Compounds / chemistry
  • Reproducibility of Results

Substances

  • Antibodies, Monoclonal
  • Biomarkers
  • Immunoconjugates
  • LCN2 protein, human
  • Lipocalin-2
  • Organometallic Compounds
  • europium(III) chelate-2,2',2'',2'''-((4-((4-isothiocyanatophenyl)ethynyl)pyridine-2,6-diyl)bis(methylenenitrilo))tetrakis(acetic acid)