Development of a Novel Polyclonal Antibody-Based Immunoassay for the Quantitation of Non-Albumin Urinary Proteins
Wallace B. Haigh1, Donna M. Guralski1, Lisa M. Arrigo1, Jack T. Letsinger1, Michele Clarke2, 3, Richard J. MacIsaac2, 4, George Jerums2, 3, Donald L. Very*, 1
Identifiers and Pagination:Year: 2013
First Page: 1
Last Page: 12
Publisher Id: TOCCHEMJ-6-1
Article History:Received Date: 24/8/2013
Revision Received Date: 20/10/2013
Acceptance Date: 25/10/2013
Electronic publication date: 15/11/2013
Collection year: 2013
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.