Abstract
Background: Better diagnostic methods to detect urinary proteinuria or albuminuria, important indicators of diabetic kidney disease, are needed. Here, we describe the development and performance qualification of a new immunoassay for the quantitation of non-albumin urinary proteins of 20 to 90 kilodaltons. Methods: Urinary proteins, purified from pooled, 24-hour, diabetic urines, were immunoabsorbed to remove albumin, electrophoretically characterized, and identified by mass spectrometry. Sheep anti-urinary protein polyclonal antibodies were immunoabsorbed to remove albumin reactivity. Major immunoreactive specificities of the polyclonal antibody were identified by Western blot. A polyclonal antibody-based competitive immunoassay was developed and performanceevaluated. An unpaired t-test (α = 0.05) and a receiver-operating characteristic curve were used to evaluate the measurement of 24-hour urinary protein excretion rates in distinguishing between normal, proteinuric, and albuminuric samples. Results: Approximately 380 mg of urinary protein was purified from 6.0 g of total urinary proteins. Mass spectrometry identified more than 36 different proteins in the purified preparation. The anti-urinary protein polyclonal antibody possessed significant immunoreactivity towards transferrin, IgG chains, alpha-1-acid glycoprotein, zinc-alpha-2-glycoprotein, prostaglandin-H2-d-isomerase, and alpha-1-microglobulin. The competitive immunoassay exhibited excellent analytical and clinical performance. Measurement of urinary protein excretion rates could distinguish between normoproteinuric and proteinuric samples (p < 0.0001; area under the curve = 0.6900) and between normoalbuminuric and albuminuric samples (p < 0.0001; area under the curve = 0.8782). Conclusion: Measurement of urinary protein excretion rates using the urinary protein immunoassay is clinically equivalent to laboratory methods of quantitating total urinary protein or albumin in identifying proteinuria and albuminuria.
Highlights
Diabetes is the principal cause of end stage renal disease (ESRD) and kidney failure [1, 2]
Numerous studies have shown that early therapeutic intervention in type II diabetic patients can decrease the level of urinary proteinuria and albuminuria and can delay the onset of ESRD [7,8,9]
Measurement of 24 hour UPER by the urinary protein immunoassay was able to distinguish between normoproteinuric (n = 291; m + standard error of the mean (SEM) = 50.8 + 2.3 mg UPER per 24 hours) and proteinuric (n = 52; m + SEM = 74.3 + 6.4 mg UPER per 24 hours ) patient samples with statistical significance (p < 0.0001, α = 0.05), while a receiver-operating characteristic (ROC) curve of 24 hour UPER results in this sample collection had an area under the curves (AUC) of 0.6900
Summary
Diabetes is the principal cause of end stage renal disease (ESRD) and kidney failure [1, 2]. Among patients initiating treatment for ESRD, 14% to 60% are diabetic [3]. Urinary proteinuria is an important clinical indicator of diabetic glomerular kidney disease and disease progression. The excretion of both total urinary proteins and specific proteins, such as albumin, increases and becomes diagnostically significant [6]. Numerous studies have shown that early therapeutic intervention in type II diabetic patients can decrease the level of urinary proteinuria and albuminuria and can delay the onset of ESRD [7,8,9]. Better diagnostic methods to detect urinary proteinuria or albuminuria, important indicators of diabetic kidney disease, are needed. We describe the development and performance qualification of a new immunoassay for the quantitation of non-albumin urinary proteins of 20 to 90 kilodaltons
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