Enzyme linked immunosorbent assay kit

Wnt 3a concentration was detected using an Enzyme-linked immunosorbent assay kit (Catalogue No. # MBS1608561, MyBioSource, USA) using a wavelength of 450 nm. Results were expressed as ng/mg protein.

MUC5B was measured with a sandwich immunoassay, the Enzyme-Linked Immunosorbent Assay Kit (MyBioSource, Inc., San Diego, CA, USA), following the instructions of the manufacturer (Number: MBS2024599 96) with SPECTROstar Nano (BMG Labtech GmbH, Ostenberg, Germany). The levels of MUC5B were reported in ng/mL of saliva. The detection ranges of MUC5B were 0.312–20 ng/mL.

IFNg was detected by enzyme-linked immunosorbent assay kit (MyBioSource, San Diego, California, United States) according to the manufacturer's instructions. Briefly, 50 μL of standard reagent was added to each standard well and 50 μL of the sample to each sample well. Adding sample diluent 50 μL to each black/control well, 20% of the samples were duplicated. The Horseradish peroxidase-conjugate reagent was added 100 μL to each well, then covered with a closure plate membrane and incubated for 60 minutes at 37°C. The plate was then washed four times. Chromogen solution was added to each well, then protected from light and incubated for 15 minutes at 37°C. After adding stop solution of 50 μL to each well, the color in the wells changed from blue to yellow. Finally, the optical density was read using Universal Microplate Reader (Sunrise, Tecan, Austria) at 450 nm within 5 to 15 minutes after adding the stop solution. The IFNg level was determined by comparing it with the standard curve. The detection range was from 31.2to 1,000 pg/mL, but the lowest standard diluted twice so the final detection range was from 7.8 to 1,000 pg/mL. The detected samples of more than 1,000 pg/mL or out of range were diluted and re-examined. The final measurement was multiplied by the number of dilutions.

Mononuclear cells from the peripheral blood and spleens of humanized mice and PBMCs from patients were reacted with fluorescent antibodies (BD Biosciences, San Diego, CA, USA) against CD4 (clone RPA-T4), CD8 (clone SK1), CD19 (clone HIB19), CD25 (clone M-A251), CD14 (clone MφP9), IL-17 (clone SCPL1362), phosphorylated STAT3 (Tyr705, pSTAT3, clone 4/P-STAT3), and collagen type I (COL1A1, R&D Systems, Minneapolis, Minn). Prior to intracellular staining, the cells were restimulated for 4 h with phorbol myristate acetate (25 ng/mL, Sigma-Aldrich) and ionomycin (250 ng/mL, Sigma-Aldrich) in the presence of GolgiSTOP (BD Biosciences, San Diego, CA, USA). Intracellular staining was performed using a kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Flow cytometry was performed using a FACSCalibur instrument (BD Biosciences, San Diego, CA, USA). Mouse antibodies targeting human endothelin receptor A (ETAR) were measured using a commercial enzyme-linked immunosorbent assay kit (MyBioSource Inc., San Diego, CA, USA). Detailed measuring protocols followed the ref. ^{19 (link)}.

Serum 25(OH)VD₃ levels were analyzed through enzyme immunoassay using the 25-Hydroxy Vitamin D3 ELISA kit (Cat No. LS-F5643; LifeSpan Biosciences, USA). Serum VDR concentrations were detected using Enzyme-linked Immunosorbent Assay Kit (Cat No. MBS022460; MyBioSource, USA). The levels of CYP24A1 were determined by Cytochrome P450 24A1 (CYP24A1) ELISA kit (Cat No. abx508211; Abbexa, USA). The Defensin 5 (Sandwich ELISA) kit (Cat No. LS-F33773; LifeSpan Biosciences) was applied to measure the levels of DEFA5 in human blood samples. The DEFA5 level of rat intestinal tissues was determined using the reverse transcription polymerase chain reaction (RT-PCR) analysis.

Human urinary SEMA3A levels were measured by an enzyme-linked immunosorbent assay kit (MyBioSource, Inc., San Diego, CA, USA), as previously described [12 (link)]. The levels of SEMA3A were determined by comparing the optical density (O.D) at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA).