Modular e170 instrument

Total protein concentration in urines was determined by Bradford assay (Coomassie Protein Assay Reagent, Pierce). The quantitative ELISA determination of human LYVE-1 (Cat# SEB049Hu, Uscn Life Science Inc.) and human TFF1 (Cat# ELH-LYVE1-001, RayBiotech Inc.) was performed according to the manufacturer’s instructions; human REG1A levels were initially assessed in our laboratory, and afterwards by BioVendor Analytical Testing Service (BioVendor - Laboratorní medicína a.s). Calibration curves were prepared using purified standards for each protein assessed. Curve fitting was accomplished by a four-parameter logistic regression following the manufacturer’s instructions. The limits of detection and the coefficient of variation (CV) for each of the ELISA assays were as follows: LYVE-1 - 8.19 pg/ml, intra-assay CV - 9%, inter-assay CV - 12%, TFF1 - 0.037 ng/ml, intra-assay CV -9%, inter-assay CV - 12%. REG1A - 0.094 ng/ml, intra-assay CV - 9%, inter-assay CV 20%; REG1B- 3.13 pg/ml, intra-assay CV - 3.9%, inter-assay CV - 2.7%. Urine creatinine was measured by the Jaffé method using the Roche Cobas 8000 system (Roche Diagnostics, Mannheim, Germany) and plasma CA19.9 using a Roche Modular E170 instrument according to the routine protocols at the Clinical Biochemistry Laboratory, RLH (London, UK).