Dtm100

Serum levels of brain natriuretic peptide (BNP) were measured using an enzyme immunoassay kit (BNP-32, Phoenix pharmaceuticals, Belmont, USA). The intra- and inter-assay variations were < 5% and < 14%, respectively. Serum levels of type I aminoterminal propeptide of procollagen (PINP) were measured using a rapid equilibrium radioimmunoassay kit (No. 67034, Orion Diagnostica, Espoo, Finland). The intra- and inter-assay variations were both < 7%, and the detection limit was 2 μg/l. Serum PIIINP was determined using a coated-tube radioimmunoassay method (No. 68570, Orion Diagnostica, Espoo, Finland). The intra- and inter-assay variations of serum PIIINP were both < 5%, and the detection limit was 0.3 μg/l. TIMP-1 was measured using an ELISA kit (DTM100, R & D Systems, Minneapolis, USA). The intra- and inter-assay variations of serum TIMP-1 were both < 5%, and the detection limit was 0.08 ng/ml. Serum MMP-2 was measured using an ELISA kit (DMP200, R & D Systems, Minneapolis, USA). The intra- and inter-assay variations of this method were < 6% and < 8%, respectively, with a detection limit of 0.16 ng/ml. MMP-9 was also measured using an ELISA kit (DMP900, R & D Systems, Minneapolis, USA). The detection limit was 0.156 ng/mL, and the intra- and inter-assay variations were < 3% and < 8%, respectively.

Serum biomarker analysis was performed in 17 STEMI patients and 37 control patients. Blood was drawn into tubes containing sodium citrate. After centrifugation, plasma was collected and stored at −80 °C until analysis. Plasma TIMP-1 was measured using an enzyme immunoassay kit (DTM100, R & D Systems, Minneapolis, USA). The intra- and inter-assay variations were <5%, and the detection limit of this method was 0.08 ng/ml. Plasma NT-proBNP was measured using an enzyme immunoassay kit (SEA485Hu, USCN Life Science, Inc., Houston, TX, USA). The intra-assay variation was <10% and inter-assay variation was <12%, and the range of detection was 39.06–2500 pg/mL.

MMP-14, TIMP-2, MMP-9, and TIMP-1 tissue levels were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits. A standard curve was run in each assay; all samples and standards were measured in duplicate and findings were averaged. The TIMP-2, MMP-9, and TIMP-1 assay procedures were done according to the manufacturer's instructions (DTM200, DMP900, and DTM100, R&D Systems). The MMP-14 assay procedure was done according to the manufacturer's instructions with the samples in the antibody cocktail's incubation time increasing to two hours (ab197747, Abcam).

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.

Measurement of active MMP-9, total MMP-9, and tissue inhibitor of metalloproteinase 1 (TIMP-1) were done using established fluorometric assays (F9M00 and DTM100, respectively; R&D Systems). Interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) were analyzed via electrochemiluminescence using the Meso Scale Discovery V-PLEX Cytokine Panel 1 Human Kit (K15049D-1; Meso Scale Diagnostics). We measured RSV loads by reverse-transcription quantitative polymerase chain reaction using known concentrations of RSV to derive a standard curve.^{19 (link)} Standards and negative controls were included and tested with each polymerase chain reaction assay. We reported RSV quantification as log₁₀ copies per mL.