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
1 The Institute for Bioanalytics, L.L.C., Branford, CT, 06456 USA
2 University of Melbourne, Melbourne, Victoria, Australia
3 Department of Endocrinology & Diabetes, St. Vincent’s Hospital, Melbourne, Victoria, Australia
4 Endocrine Centre, Austin Health, Melbourne, Victoria, Australia


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© 2013 Haigh et al;

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.

* Address correspondence to this author at the PharmLogic, LLC; 85 Tollgate Road; Warwick, RI, 02886 USA; Tel: 908-489-2850; Fax: 401-732-8512; E-mail: donaldvery@hotmail.com


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.

Keywords: Albuminuria, glomerular filtration, immunoassay, proteinuria, tubular reabsorption.