Qualification and application of an ELISA for the determination of Tamm Horsfall Protein (THP) in human urine and its use for screening of Kidney Stone Disease

Abstract

Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r2 > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 μg/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.

Common variants of the UMOD promoter associated with blood pressure in a community-based Chinese cohort

“…15 First, 96-well microtiter plates were coated with polyclonal mouse anti-human UMOD antibodies (Biomedical Technologies Inc., Stoughton, MA, USA; BT-590). Urinary samples and UMOD standards (Biomedical Technologies) were then diluted and added to wells in duplicate.…”

Immunohistochemical Identification of Kidney Nephron Segments in the Dog, Rat, Mouse, and Cynomolgus Monkey

“…Precocious and reliable detection of renal toxicity in preclinical safety assessment has therefore stimulated intense research to develop novel biomakers (Boudonck et al 2009). Among studied molecules, N-acetyl-b-glucosaminidase (NAG), glutathione S-transferases (GST), b2-microglobulin, cystatin-C, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), fatty acid binding protein (FABP) (Ferguson, Vaidya, and Bonventre 2008;Togashi et al forthcoming), Calbindin-D 28K (Hoffmann et al 2010), and Tamm-Horsfall protein (Lau et al 2008) have given promising results; and seven of them (Kim-1, albumin, total protein, b2-microglobulin, cystatin C, clusterin, and trefoil factor-3) have been qualified in 2008 for renal safety by the Food and Drug Administration and the European Medecines Agency (see http://www.c-path.org/pdf/ PSTC_nephro_VXDS_summary_final.pdf).…”

Untitled

“…Uromodulin is a glycoprotein that is encoded by the UMOD gene [13,14]. Uromodulin is the most plentiful protein expelled in familiar urine [15]. This protein acts as a constitutive inhibitor of calcium crystallization in renal fluids.…”