Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin

Background: Microalbuminuria is an established early marker of diabetic nephropathy and an important cardiovascular risk factor in diabetes and hypertension. We aimed to develop a rapid point-of-care assay for the measurement of urine albumin. Methods: The competitive homogeneous assay used an albumin-specific monoclonal antibody labeled with a stable fluorescent europium chelate as donor and an albumin labeled with cyanine 5 (Cy5) as acceptor. The assay was performed at room temperature in single microtitration wells that contained all the required dry-form reagents. The close proximity between the two labels in the immune complex allowed fluorescence resonance energy to be transferred from the pulse-excited europium chelate to the acceptor Cy5. The emission of long-lived energy transfer signal from the sensitized Cy5 was measured at 665 nm with time-resolved fluorometry that eliminated short-lived background. Results: The assay procedure required 12 min for a 10-μL urine sample. The working range was from 10 to ∼320 mg/L, and the lower limit of detection was 5.5 mg/L. The within- and between-run CVs were 6.9–10% and 7.5–13%, respectively. Recovery was 103–122%. The assay correlated well (r2 = 0.98; n = 37) with a laboratory-based immunoassay, although mean (SD) results were 7 (29)% lower. Conclusions: The speed and ease of performance of this assay recommend it for near-patient use. The assay is the first to combine a fluorescence resonance energy transfer-type rapid competitive assay with an all-in-one dry reagent.