Dcrp00

Blood samples were taken from the antecubital vein into the vacutainer tubes with silica clot activator. After centrifugation at 2000 × g for 10 min at 4°C, the serum was collected and stored in aliquots at -80°C until analysis.
Serum 25(OH)D₃ level was determined using the 25-OH Vitamin D total ELISA kit (DE1971, Demeditec Diagnostics, Germany) according to the manufacturer’s instructions. The intra-assay coefficients of variability (CVs) and inter-assay CVs reported by the manufacturer were 2–8% and 4–9%, respectively. Serum CRP, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) concentration were measured using the ELISA kit (DCRP00, HS600B, HSTA00E, and HS100C, respectively: R&D, United States) according to the manufacturer’s instructions. The intra-assay coefficients of variability (CVs) and inter-assay CVs reported by the manufacturer were 2–8% and 4–9%, respectively.

Cytometric bead arrays (Becton Dickinson Biosciences, San Jose) were used to measure TNF (Flex Set C4 100Tst), IL‐13 (Flex Set E6 100Tst), IL‐8 (Flex Set A9 100Tst), IL‐6 (Flex Set A7 100Tst) and IL‐5 (Flex Set A6 100Tst). Enzyme‐linked immunosorbent assays were used to measure neutrophil elastase (BMS269; Invitrogen, Waltham; measured in sputum only), sIL‐6R (SR600; R&D Systems, Minneapolis) and CRP (R&D Systems, DCRP00; measured in serum only). Cytokine levels in sputum were determined using the DTT‐free supernatant samples.

The CRP concentration in the CKD patients’ plasma and serum samples was measured by a commercial Human CRP ELISA Kit (DCRP00, R&D Systems Inc., Minneapolis, MN, USA) and a multi-plate reader at 450 nm (Synergy 4 HT). All the serum and plasma samples were initially diluted 100 times using the sample diluent buffer for the test according to the manufacturer’s recommendation. For the samples with high CRP concentration exceeding the ELISA kit’s detection range, the test was repeated with a 1000× dilution of the sample.

The levels of inflammatory cytokines and chemokines were established in plasma samples and the levels of cortisol and adiponectin were established in serum samples. Undiluted samples were used for inflammatory cytokines and chemokines, and samples for CRP and adiponectin were diluted 200-fold. All biomarkers were analyzed in duplicate using the following enzyme-linked immunosorbent assay (ELISA) kits: IL-1β (KET6013; Abbkine, Wuhan, China), IL-6 (KET6017; Abbkine, Wuhan, China), TNF-α (ADI-900-099; Enzo, Madison Avenue, New York, NY, USA), IL-17 (KET6022; Abbkine, Wuhan, China), CRP (DCRP00; R&D Systems, Minneapolis, MN, USA), adiponectin (DRP300; R&D Systems, Minneapolis, MN, USA), cortisol (ADI-900-071; Enzo, New York, NY, USA), CCL1 (MBS824930; MyBioSource, San Diego, CA, USA), and CCL2 (LS-F146; LSBio, Seattle, WA, USA), according to the manufacturer’s instructions. The standard solutions were respectively prepared using the reagents provided in each kit. Plates were read at 450 nm using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Protein quantification was performed using the mean value of the duplicate samples.

Plasma IL-6, TNFα, IL-1β, and MIP1α (neat) (R&D Systems, HS600C, DTA00D, DLB50, DMA00), IL-8 (neat) (Abcam, ab46032), and CRP (1:100) (R&D Systems, DCRP00) were quantified following manufacturer’s instructions. LBP (1:100) (R&D Systems, DY870-05) and iFABP (1:10) (R&D Systems, DY3078) ELISA kits were supplemented with the DuoSet Ancillary Reagent Kit (R&D Systems, DY008). Fecal LCN2 was quantified following manufacturer’s instructions (Eagle Bioscience, NGL35-K01).

Cryopreserved plasma was thawed and analysed for C-reactive protein (CRP) using a highly sensitive kit manufactured by R&D systems a biotech brand Catalogue number DCRP00.