Annals of Clinical & Laboratory Science 35:149-154 (2005)
© 2005 Association of Clinical Scientists
Microplate ELISA for Urine Microalbumin: Reference Values and Results in Patients with Type 2 Diabetes and Cardiovascular Disease
Tsu-Lan Wu1,
Pi-Yueh Chang1,
Chia-Chi Li1,
Kuo-Chien Tsao1,
Chien-Feng Sun1 and
James T. Wu2
1 Department of Pathology, Chang Gung Memorial Hospital, Taipei, Taiwan; 2 ARUP Laboratories, University of Utah Health Sciences Center, Salt Lake City, Utah
Address correspondence to James T. Wu, Ph.D., ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA; tel 801 583 2787; fax 801 584 5207; e-mail wuj{at}aruplab.com.
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Abstract
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An ELISA for urine microalbumin using microtiter plates has been developed. The assay uses polyclonal anti-human albumin antibody for coating the microtiter plates and the same antibody conjugated with horseradish peroxidase for detection. The assay sensitivity is 1.6 µg/ml. Results by this in-house ELISA show good correlation (r = 0.99) with those obtained by a commercial assay using the Behring BNII autoanalyzer. Within-day and between-day CVs are 10%. R eference values for microalbumin in 769 urine specimens from healthy Chinese subjects were higher in women than men and higher in subjects 50 yr than in those <50 yr of age. Elevated mean concentrations of urine microalbumin were observed in patients with type 2 diabetes and CVD. This in-house ELISA is simple, sensitive, precise, and especially suited for laboratories without expensive autoanalyzers.
(received 18 November 2004; accepted 24 December 2004)
Keywords: enzyme-linked immunosorbent assay, type 2 diabetes, cardiovascular disease, microtiter plate assay, urine microalbumin
Abbreviations: BSA-PBS: phosphate buffer, 0.01 mol/L, pH 7.2, containing bovine serum albumin, 10 g/L; BNII, Behring Nephelometric Analyzer II; CRP, C-reactive protein; CVD, cardiovascular disease; ELISA, enzyme-linked immunosorbent assay; HRP, horse radish peroxidase; IL-6, interleukin-6; TNF-
, tumor necrosis factor-alpha; PBS, phosphate buffer, 0.1 mol/L, pH 7.2, containing NaCl, 0.14 mol/L, K-Blue, tetramethylbenzidine.
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Introduction
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Albumin is generally not detectable in the urine by use of the common dipstick. When the excretion rate of urine albumin becomes slightly increased (eg, >15–20 µg/min), the condition is referred to as "microalbuminuria." Microalbuminuria is an important risk factor for diabetic nephropathy in patients with type 2 diabetes [1,2]. A slight increase in urine microalbumin precedes the development of clinical diabetic nephropathy by several years [3]. Studies have also indicated that at an early stage of microalbuminuria, renal complications are reversible with improved glycemic control and reduced blood pressure [4]. Early intervention may be beneficial in preventing diabetic patients from developing renal dysfunction and avoiding the progression to end-stage renal failure. Therefore assays capable of detecting microalbuminuria at an early stage are clinically useful [5].
Recent findings have indicated that the clinical significance of detecting microalbuminuria extends far beyond detecting diabetic nephropathy [4,5]. Hypertensive or diabetic individuals with microalbuminuria have been found at risk for cardiovascular diseases. In addition, patients with microalbuminuria may be at risk for carcinogenesis [6,7]. Many cancer patients have been found to suffer from glomerular leakage of albumin, as measured by urinary microalbumin excretion. Microalbuminuria appears to be associated with inflammation, a known risk factor for type 2 diabetes, cardiovascular disease (CVD), and cancer [8]. For example, strong correlation between microalbuminuria and serum levels of inflammation markers (eg, C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor- (TNF-)) has been reported in patients with non-Hodgkin’s lymphoma [9]. Stuveling et al [10] have noted that CRP and microalbuminuria may be risk markers for vascular disease, with different risk profiles for different vascular beds. They found that CRP is independently associated with all three domains of vascular disease, whereas microalbuminuria is independently associated only with coronary heart disease. Evidently, testing for urine microalbumin is useful for risk assessment of diabetic nephropathy, CVD, and cancer, and is a marker for inflammation.
To measure urine microalbumin, a more sensitive method is required than those used in clinical laboratories for measuring serum albumin. At present, urine microalbumin is commonly measured in clinical laboratories by immnoturbidimetry. In this investigation, we developed a simple, sensitive ELISA on microtiter plates for use in laboratories, especially in underdeveloped countries, that are unequipped with autoanalyzers or nephelometers. Results by our microplate ELISA correlate well with those obtained by a commercial assay for urine microalbumin. Our ELISA can either be performed manually or by use of an automated microtiter plate analyzer.
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Materials and Methods
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Rabbit anti-human albumin polyclonal antibody (pAb) and HRP-conjugated rabbit anti-albumin polyclonal antibody were purchased from DAKO (Carpinteria, CA). The calibrator (purified human albumin) was purchased from Sigma (Saint Louis, MO). Maxisorp F8 removable well strips were from Nalge Nunc International (Rochester, NY). K-blue substrate was from Neogen Corp (Lexington, KY).
Normal random urine specimens.
Urine specimens were collected from 769 apparently healthy normal Chinese subjects who visited Chang Gung Memorial Hospital for an annual health check-up. Individuals with an abnormal serum chemistry profile were excluded from this study. Urine specimens that gave positive results by the urine dip-stick teststick were also excluded. Urine specimens were stored at –80°C and assayed within one yr.
Random urine specimens from patients.
Left-over urine samples from patients with CVD or type 2 diabetes were stored at –75°C for up to 2 yr before assay. The criterion for CVD was a serum troponin I level >2 ng/ml; the criterion for type 2 diabetes was a blood HbA1c level >6.5%.
ELISA procedure.
The 96-microwell plate was coated with polyclonal anti-human albumin antibody for capturing urine microalbumin. At the beginning of the ELISA, the calibrator and the urine sample were both prediluted 100-fold with 1% BSA-PBS with 0.1%Tween 20. Exactly 10 µl of pre-diluted calibrator or sample was added to each antibody-coated well and incubated for 30 min at 30°C with gentle agitation. After washing 3 times with the washing buffer, 100 µl of diluted, HRP-conjugated detecting antibody was added and incubated at 30°C for 1 hr with gentle agitation. Wells were washed 3 times with washing buffer and the enzyme substrate, K-Blue, was added. After incubation for 10 min at room temperature, the reaction was stopped by adding 100 µl of 1 mol/L H2SO4 and the absorbances at 450 nm and 620 nm were read. The ELISA is diagrammed in Fig. 1
and the assay details are outlined in Table 1.
Other measurements.
Urine microalbumin concentrations were also determined by a nephelometric assay using the Behring BNII apparatus, with commercial reagents supplied by Behring Corp (Marburg, Germany). Urine creatinine concentrations were determined with an Olympus HITACHI analyzer.
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Results
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Assay characteristics.
Assay characteristics of the ELISA are listed in Table 2 and a calibration curve is shown in Fig. 2. The calibration curve covers the microalbumin concentration range from 1.6 to 100 µg/ml. The sensitivity of the ELISA was 1.6 µg/ml, which was slightly better than that of the commercial assay with the BNII autoanalyzer (sensitivity, 2.2 µg/ml). Within-day and day-to-day CVs of the ELISA assay were <10 % (Table 2).
Comparison with commercial kit.
Measurements of urine microalbumin by the in-house ELISA were compared with those obtained by a commercial kit using the BNII analyzer. The commercial assay is used in our laboratory for analysis of patients’ specimens. Because the concentration of urine microalbumin is normally in the range 20 µg/ml, the comparison data are plotted in 2 graphs that cover microalbumin concentrations in the lower and elevated levels. In Fig. 3, the upper panel shows data from 0 to 40 µg/ml and the lower panel shows data from 0 to 240 µg/ml. The slopes, correlation coefficients, and standard errors indicate that the in-house ELISA should be acceptable for routine use in clinical laboratories.
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Fig. 3. Comparison of the in-house ELISA and a commercial assay on the BNII analyzer for urine microalbumin. The data are plotted in 2 different scales to show the details at lower and higher ranges of microalbumin concentrations. For the range from 2.3 to 40 µg/ml (n = 48), SE of slope = 0.033, SE of intercept = 0.57, and SE of regression = 2.51. For the range from 2.3 to 231 µg/ml (n = 63), SE of slope = 0.017, SE of intercept = 1.11, and SE of regression = 7.13.
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Reference values.
In Table 3 are listed the reference values for urine microalbumin concentrations (µg/mg creatinine) for 769 healthy men and women at different ages. In Table 4 these reference values are categorized for men and women with ages <50 yr and 50 yr . The results show that, at least in this Chinese population, the reference values are gender-and age-dependent. At all ages, women have higher concentrations of urine microalbumin than men. Higher levels of urine microalbumin were associated with older ages regardless of gender.
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Table 4. Comparisons of reference values for urine micro-albumin in men vs women and those under/over 50 yr of age.
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Association with CVD and diabetes mellitus.
Micro-albumin concentrations were measured by our in-house ELISA in urine specimens from 40 patients with CVD and 100 patients with type 2 diabetes (Fig. 4). The ages of the patients were not available for analysis. Urine microalbumin concentrations averaged 317.4 µg/mg creatinine, and 291.9 µg/mg creatinine for patients with CVD and type 2 diabetes, respectively. These results are consistent with previous reports that urine microalbumin concentrations can serve as a risk marker in patients with type 2 diabetes for the development of nephropathy and cardiovascular disease.
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Fig. 4. Mean concentrations of urine microalbumin (plotted on a log scale) were elevated in specimens from 40 patients with cardiovascular disease (CVD) and 100 patients with type 2 diabetes, compared to 769 normal healthy subjects. The effects of gender and age are not considered in this plot.
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Discussion
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The ELISA for urine microalbumin was developed for the following reasons: (a) in underdeveloped countries, many laboratories have no access to automated instruments; (b) reagents sold in the USA and Europe are often too expensive for purchase by laboratories in underdeveloped countries; (c) testing for urine microalbumin assists in the detection of nephropathy at an early stage, which may be reversible when treated; and (d) such biomarkers for nephropathy, CVD, and cancer are likely to be more widely used in the near future.
The ELISA for urine microalbumin has additional advantages: (a) the test is simple, sensitive, precise, inexpensive, and easy to perform; (b) albumin concentrations are stable in frozen urine specimens; and (c) the test can either be performed manually or adapted to automated microplate analyzers
Techniques generally used for quantifying serum albumin are insufficiently sensitive for analysis of the low concentrations of albumin that are present in urine specimens of healthy subjects. The most popular assays for urine microalbumin use nephelometry, such as the Beckman Array, Behring BNII (immunonephelometric), Dade aca particle-enhanced turbidimetric inhibition immunoassay, and INCSTAR SPQ (immunoturbidimetric method) [11]. There are several other techniques, in addition to nephelometry and ELISA, that can be used to measure urine microalbumin, such as electroimmunodiffusion and radioimmunodiffusion [12]. The microplate sandwich ELISA described here has better precision, covers a wider concentration range, and requires the least labor.
The same anti-human serum albumin antibody can be used to capture the albumin and also for its detection in the sandwich ELISA. Apparently there are multiple antigenic sites on the albumin molecule, so that some sites remain available on the albumin molecule for binding to the detecting antibody after being fixed on the microwell. ELISA on microplates is unsuitable for measuring µg or mg concentrations of analytes because of the limited surface area of the microwell that is available for antibody coating. Even though we use only a 10 µl specimen for the assay, predilution is required. Less dilution may be possible if a smaller aliquot of the urine specimen is used. A similar enzyme immunoassay for urine albumin using microtiter plates was described by Fleiding et al [13] in 1983, using a slightly different assay format. The sensitivity of their ELISA was sufficient for the measurement of microalbumin.
Since urine volume is influenced by the subject’s liquid intake, urine analytes such as microalbumin are customarily normalized on the basis of the urine creatinine concentration. The level of urine microalbumin is therefore highly dependent upon the urine creatinine concentration.
The reference values for urine microalbumin that are reported herein are derived from a healthy Chinese population. These reference values are both gender- and age-dependent. The authors are unaware of prior publications that show the effect of age on reference values for urine microalbumin. Since many risks are associated with elderly men and women, it is obviously important to employ the appropriate age- and gender-specific reference values.
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Acknowledgement
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This study was supported by a research grant from Chang Gung Memorial Hospital (CMRP G32097
[GenBank]
).
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References
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Abstract
Introduction
