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- micro L urine sample. The working range was from 10 to approximately 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 (r(2) = 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.
Qin
Qiu-Ping
QP
Department of Biotechnology, University of Turku, Tykistökatu 6, 20520 Turku, Finland. qiqin@utu.fi
Peltola
Olli
O
Pettersson
Kim
K
D000418:Albumins / Q000032:analysis* / Q000737:chemistry; D000419:Albuminuria / Q000175:diagnosis* / Q000652:urine; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015415:Biomarkers / Q000652:urine; D002232:Carbocyanines / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D004735:Energy Transfer; D005063:Europium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015200:Fluoroimmunoassay; D006801:Humans; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D019095:Point-of-Care Systems
D000418:Albumins; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D002232:Carbocyanines; D002614:Chelating Agents; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D005063:Europium