Quantum Dot-Based Fluorescent Lateral Flow Immunoassay Strip for Rapid Detection of Anti-nephrin Autoantibodies in Idiopathic Nephrotic Syndrome.

Abstract

Idiopathic nephrotic syndrome is a common primary glomerular disease in children. Circulating anti-nephrin autoantibodies have emerged as clinically relevant biomarkers, but existing detection methods are complex and time-consuming. We developed a quantum dot microsphere-based lateral flow immunoassay (QD-LFIA) for rapid serum anti-nephrin autoantibody detection. Quantum dot microspheres were conjugated to recombinant human nephrin protein, and the reading time, T-line coating concentration, and nephrin protein labeling amount were optimized. Analytical performance was evaluated by receiver operating characteristic (ROC) analysis based on test line/control line ratios generated by the dry fluorescence immunoanalyzer, together with interference and accelerated stability testing. Clinical performance was assessed in 191 pediatric serum samples, with qualitative QD-LFIA results compared against immunoprecipitation-Western blotting (IP-WB). Discordant samples were evaluated by recombinant nephrin protein inhibition assay. Clinical indices were compared between anti-nephrin autoantibody positive and negative patients. The optimized reaction time, T-line coating concentration, and recombinant nephrin protein labeling amount were 10 min, 0.5 mg/mL, and 92.4 μg, respectively. ROC analysis yielded an AUC of 0.90 (95% CI 0.84-0.97), with 93% sensitivity and 92% specificity at a test line/control line cutoff of 0.003. The QD-LFIA resisted the tested interferents and maintained stable performance for 40 days at 37 °C. QD-LFIA achieved overall, positive, and negative percent agreements of 92%, 89%, and 93%, respectively, relative to IP-WB. Recombinant nephrin protein inhibition abolished the signals in all 12 QD-LFIA-positive/IP-WB-negative samples. Anti-nephrin autoantibody positive patients had higher 24-h urinary protein excretion and lower serum albumin levels than negative patients. These findings suggest that the QD-LFIA enabled rapid detection of anti-nephrin autoantibodies in children with idiopathic nephrotic syndrome. The QD-LFIA showed good agreement with IP-WB and identified additional anti-nephrin-positive samples undetected by IP-WB.