ELISA was conducted to verify the biomarker candidate proteins identified in transcriptomic and proteomic analyses. An additional 12 AH samples from each group were subjected to ELISA. AH proteins were measured using the human metallopeptidase inhibitor 1 (TIMP1) (DTM100; R&D Systems), angiopoietin-related protein 7 (ANGPTL7), and Fc fragment of IgG binding protein (FCGBP) ELISA Kit (CSB-EL001715HU, CSB-EL008536HU; Cusabio Technology) according to the manufacturer’s instructions [48 (link),49 (link)]. Briefly, all samples were brought to room temperature before use and were assayed in duplicate. 50 μL of samples or standard was added into the wells pre-coated with an antibody specific to the antigen (human TIMP-1, human ANGPTL7, or human FCGBP) and incubated for 2 h at room temperature. This was followed by incubation for 2 h with the target antibody conjugates. Substrate solution was added to the samples and was incubated for 30 min. Stop solution was added, and the absorbance of color at 450 nm was measured using a microplate reader (Bio-Rad® Microplate Absorbance Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA). The intra-assay and inter-assay coefficients of variation within and between ELISA tests were <8%. All absorbance results are expressed as nanogram per milliliter.
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