Product Protocol: RABBIT ANTI HUMAN TIMP-1 antibody used to evaluate urinary matrix metalloproteinases reflect renal damage in anti-neutrophil cytoplasm autoantibody-associated vasculitis (OASA11651)

Specificity: TIMP-1
Product Page: RABBIT ANTI HUMAN TIMP-1 antibody (OASA11651)
Antibody Pair Available: OASA11651 Capture OASA11652 Detection
Experiment Type: ELISA

Protocol:
Urinary and plasma levels of MMP-2, MMP-9, and TIMP-1 were determined by ELISA. In brief, for MMP-9 and TIMP-1 ELISA, 96-well plates (M129A, Greiner) were precoated with F(ab)2 fragments of goat anti-mouse IgG-Fc (Jackson, West Grove, PA) in 0.1 M carbonated buffer (pH = 9.6) for at least 48 h. After a washing, plates were coated with 100 ng/ml mouse anti-human MMP-9 (clone 36020.111, R&D Systems, Oxon, UK) or 500 ng/ml mouse anti-human TIMP-1 (clone 63515.111, R&D Systems), respectively. Human urine samples and recombinant MMP-9 and TIMP-1 were diluted in 1% BSA, 0.05% Tween 20 in 50 mM Tris·HCl (pH = 8.0), and 300 mM NaCl, and plasma samples were diluted in HPE buffer (Sanquin, Amsterdam, The Netherlands). For MMP-2 ELISA, 96-well plates (MaxiSorp, Nunc) were coated overnight with 2 μg/ml mouse anti-human MMP-2 (clone 36006.211, R&D Systems), and, after being washed, blocked with 1% BSA in PBS for 60 min. Human urine samples, plasma samples, and recombinant MMP-2 were diluted in 0.1% BSA, 0.05% Tween 20 in PBS. For MMP-2, MMP-9, and TIMP-1, an ELISA of both urine and plasma samples was performed in duplicate, and the samples were incubated at room temperature for 1 h. After washing, bound MMP-2, MMP-9, or TIMP-1 was detected by a 1-h incubation with, respectively, biotinylated rabbit anti-human MMP-2, rabbit anti-human MMP-9, or rabbit anti-human TIMP-1 (all R&D Systems). After being washed, samples were incubated with 0.125 μg/ml peroxidase-conjugated streptavidin (Sanquin) for 30 min, and the color reaction was performed with tetramethylbenzidine (Roth, Karlsruhe, Germany). The colorimetric reaction was stopped by the addition of 100 μl/well 0.5 M 2N H2SO4, and adsorption at 450/575 nm was measured with a microplate reader.

Sensitivity of the ELISA was 3.9 ng/ml for MMP-2, 0.039 ng/ml for MMP-9, and 0.39 ng/ml for TIMP-1. Urine and plasma concentrations were determined by interpolation from a standard curve. Urinary concentrations were adjusted for the creatinine concentration and expressed in picograms per millimole creatinine.

Summary:
1. Urinary MMP-2 and TIMP-1 levels showed a positive correlation with tubulointerstitial damage and a negative correlation with creatinine clearance. 2. TIMP-1 levels in urine samples of patients (median 2.86 pg/mmol creatinine; range 0.00–23.50) were significantly higher than in urine samples of healthy controls (median 1.10 pg/mmol creatinine; range 0.30–5.20) (P = 0.001). Plasma TIMP-1 levels were significantly higher in patients with ANCA-associated vasculitis (median 649 ng/ml, range 86–1253) than in healthy controls (median 380 ng/ml; range 50–620) (P < 0.0001). Plasma levels of MMP-2, -9, and TIMP-1 did not correlate significantly with urinary MMP activity or levels and with TIMP-1 levels, respectively. Plasma levels of MMP-2, -9, and TIMP-1 did not correlate with CRP or BVAS. References:
1: Sanders JS, Huitema MG, Hanemaaijer R, van Goor H, Kallenberg CG, Stegeman CA. Urinary matrix metalloproteinases reflect renal damage in anti-neutrophil cytoplasm autoantibody-associated vasculitis. Am J Physiol Renal Physiol. 2007 Dec;293(6):F1927-34. Epub 2007 Sep 26. PubMed PMID: 17898039.

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