A combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine

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Abstract

A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings. Polyclonal rabbit anti-human albumin was used as a capture antibody and monoclonal mouse anti-human albumin as a detection antibody in a two-site immunometric assay requiring no additional washing procedures. The polyclonal anti-human albumin was conjugated to silica microparticles (solid phase) and the monoclonal antibody to dextran-coated nanoscaled superparamagnetic particles (tracer). Quantification of human albumin in undiluted urine was performed by adding 2 μL urine to a measuring vial containing solid-phase, superparamagnetic tracer and reaction buffer and then inverting the vial by hand for 20 s. The measuring vial was allowed to stand for 6 min prior to detection, in order for the solid-phase sediment to form at the bottom of the vial. Lastly, the measuring vial was placed into a magnetic permeability detector, which measured the enrichment of superparamagnetic tracer in the sediment due to complex formation with human albumin. Total analysis time was 6.5 min. A linear response was obtained for 0–400 mg/L albumin with a detection limit of 5 mg/L. The total coefficient of variation (CV) was 11% calculated from four consecutive runs on a urine sample containing 11.1 mg/L human albumin during 3 consecutive days. Human urinary albumin analysis was performed on 149 patient samples using the MIA technique and the obtained results showed good correlation with the hospital immunoturbidimetric reference method (y = 1.004x + 4.01, R2 = 0.978, N = 149) and a commercially available point of care albumin analysis provided by HemoCue Inc. (y = 0.98x + 5.8, R2 = 0.833, N = 90).

Introduction

Human serum albumin is a 66.5 kDa protein composed of one long polypeptide chain and is a major constituent of plasma proteins (Dockal et al., 2000). During normal renal processing of the blood, a small concentration of albumin is excreted with the urine. Microalbuminuria, a slightly increased albumin excretion in urine, is a risk indicator for nephropathy in patients suffering from diabetes and hypertension (Mogensen, 1987, Vigstrup and Mogensen, 1985). It is also a predictive factor for microvascular disease (Rowe et al., 1990, Lydakis and Lip, 1998, Bakris, 2001). Microalbuminuria has been defined as a persistent urinary albumin excretion of 30–300 mg/day in timed urine collections or 30–300 mg/L in spot early morning urine (Mogensen et al., 1995, Rowe et al., 1990). A ratio of albumin to creatinine is also used to define microalbuminuria (Mangili, 1998, Bakris, 2001). Early control of microalbuminuria decreases the risk of further complications including clinical nephropathy and renal failure. Thus, routine screening of albumin excretion has been recommended for patients suffering from diabetes and hypertension (Lydakis and Lip, 1998, Bakris, 2001).

Different immunoassay technologies, involving labeled and non-labeled approaches, have been developed for the detection of albumin in urine, including, radioimmunoassay (Stoica et al., 1983, Rowe et al., 1990), immunoturbidimetric assay (Lloyd et al., 1987, Bakker, 1988, Tencer et al., 1996), immunonephelometric assay (Marre et al., 1987), fluorescent immunoassay (Chavers et al., 1984, Silver et al., 1986), ELISA (Aybay and Karakus, 2003), immunoelectrophoresis (Coimbra et al., 1984), chemiluminescence immunoassay (Horton et al., 1989) and dye reaction-based analysis (Blagg et al., 1993). Although the techniques have shown to be extremely sensitive and accurate, many require expensive equipment for detection or laborious and time-consuming steps, limiting their use for laboratory applications run by highly skilled personnel.

For decentralized clinical analysis in near patient settings, rapid and on-the-spot semi-quantitative detection methods for measuring human albumin have been developed involving urinary dipstick (Gilbert et al., 1997, Zheng et al., 1999), reagent tablets (AL-Kassab, 1990) and/or quantitative absorbance techniques (Schenck, 2003).

The work presented herein involves the development of a rapid detection method for human albumin in urine using nanoscaled superparamagnetic tracer in a one-step, two-site immunometric assay. Our group has been pioneers in the development of magnetic permeability-based immunoassays (Kriz et al., 1996, Larsson et al., 1999). The use of superparamagnetic nanoparticles as labels to proteins (Kriz et al., 1998) and DNA (Abrahamsson et al., 2004) in bioassay formats has been previously reported. Recently, the detection of high-sensitive C-reactive protein (hsCRP), a cardiovascular risk indicator, in 103 patient whole blood samples was reported using the magneto immunoassay (MIA) technique (Kriz et al., 2005). In this study, urine samples obtained from 149 unidentified patients were analyzed with a magneto immunoassay developed for human urinary albumin analysis. The obtained results were compared with a commercial immunoturbidimetric analysis (Cobas Mira S System by Roche at Diagnostics, Basel, Switzerland) performed at a hospital central laboratory as well as with a commercial point of care albumin analysis (HemoCue, Ängelholm, Sweden) performed in-house. The accuracy and sensitivity of the MIA assay was also discussed.

Section snippets

Chemicals

Polyclonal rabbit anti-human albumin and human serum protein calibrator were obtained from Dakocytomation (Glostrup, Denmark). Monoclonal mouse anti-human albumin (Clone: 1A9) was purchased from HyTest Ltd. (Turku, Filand). BCA protein assay reagent kit was obtained from Pierce (Rockford, USA). The following chemicals were obtained from Sigma (St. Louis, MO, USA): dextran (500) with an average molecular weight of 464,000 g/mol; bovine serum albumin (BSA); sodium periodate and triton X-100. The

Evaluation of antibody-conjugated silica microparticles and superparamagnetic tracer

It was calculated, after antibody conjugation to the silica microparticles there was approximately 0.46 mg antibody per gram silica. Similarly, after antibody conjugation to the superparamagnetic tracer, there was approximately 118 μg antibody per milliliter tracer. The magnetic permeability of the superparamagnetic tracer before and after conjugation was μr = 1.00154 ± 0.00005 and μr = 1.00162 ± 0.00005, respectively.

Optimization of silica microparticle and superparamagnetic tracer concentrations

The magneto immunoassay for human urinary analysis was optimized with a series of

Conclusions

The one-step magneto immunoassay provides a new method for accurate and rapid analysis of human urinary albumin. The operation is very simple and the detection range for human albumin is rather broad compared with other commercially available techniques designed for near patient testing. One distinct advantage of this detector system is the versatility in the analyses it can perform. The same instrument can be used to measure a wide range of analytes simply by changing the reagents provided in

Acknowledgement

We kindly thank professor Anders Grubb at Lund University Hospital for providing urine samples for urinary albumin analysis.

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