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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.
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.
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).
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.