On-site quantification of human urinary albumin by a fluorescence immunoassay.

Abstract

Microalbuminuria (MAU), defined as a urinary albumin excretion of 30–300 mg/day, indicates a high probability of renal damage and is an accepted predictor for the early diagnosis of nephropathy in diabetic patients (1)(2). In addition, MAU has diagnostic implications in pregnancy as a predictive marker of preeclampsia (3)(4) and may play a role in identifying high risk of developing complications from cardiovascular diseases even in nondiabetic patients (5)(6)(7). Dye-binding assays can measure serum albumin but are too insensitive for MAU testing, making immunochemical assays the most widely used MAU methods (8). These immunoassays include immunoturbidimetry, immunofluorescence, ELISA, RIA, and zone immunoelectrophoresis. Recently, Kessler and coworkers (9)(10) introduced a laser-induced fluorescence system coupled to an automated centrifugal analyzer as a nonimmunologic assay for urinary albumin. Their system was based on the probe Albumin Blue 670/580, which becomes highly fluorescent on binding to albumin. We report a fluorescence immunochromatography assay (ICA) for quantitative determination of albumin in urine. The assay system consists of an ICA test strip in a disposable cartridge, a fluorescently labeled detector, and a laser fluorescence reader. Basically, the assay system adopts the inherent simplicity of a lateral-flow ICA and uses a competitive immunoassay mode with a simple, one-step operation (11). Briefly, fluorescently labeled albumin in the detector buffer competes with albumin in the sample for binding to an anti-albumin antibody immobilized on the test strip matrix. The more albumin is in the sample, the less the fluorescently labeled albumin reacts with the anti-albumin antibody and, thus, the lower the accumulation of fluorescence in the test line of a test strip. We generated a monoclonal antibody against human albumin (Sigma A8763) as an immunogen and conducted immunizations, cell fusion, and screening of hybridoma cells according to a standard …