Serum biomarker levels were determined using a double-antibody sandwich ELISA according to the manufacturer's instructions (CHI3L1, R&D systems, USA; MMP13, CUSABIO, China; SPP1, ebioscience, USA). They were analyzed blinded to clinical parameters and study endpoints at the end of the study.
Briefly, 100 μl of the test samples (1:100 diluted for CHI3L1, original for MMP13, 1:5 diluted for SPP1) were added and incubated for 2 h at room temperature. Subsequently, 100 μl/well of the detection antibody was added and incubated for 2 h at room temperature. Next, 100 μl/well of Streptavidin-HRP was added and incubated for 20 min at room temperature. Finally, the substrate (tetramethylbenzidine) solution was added, and the reaction was stopped with 2 N H2SO4 and read at an OD of 450 nm. For Intra-assay Precision: CHI3L1, CV: 5.2% (0.25 ng/ml) and CV: 3.6% (2 ng/ml); MMP13, 4.9% (2 ng/ml) and 2.3% (10 ng/ml); SPP1, 4.1% (2 ng/ml) and 2.9% (10 ng/ml). For Inter-assay Precision: CHI3L1, 10.1% and 7.5%; MMP13, 8.7% and 6.1%; SPP1, 6.6% and 5.6%, respectively.
Briefly, 100 μl of the test samples (1:100 diluted for CHI3L1, original for MMP13, 1:5 diluted for SPP1) were added and incubated for 2 h at room temperature. Subsequently, 100 μl/well of the detection antibody was added and incubated for 2 h at room temperature. Next, 100 μl/well of Streptavidin-HRP was added and incubated for 20 min at room temperature. Finally, the substrate (tetramethylbenzidine) solution was added, and the reaction was stopped with 2 N H2SO4 and read at an OD of 450 nm. For Intra-assay Precision: CHI3L1, CV: 5.2% (0.25 ng/ml) and CV: 3.6% (2 ng/ml); MMP13, 4.9% (2 ng/ml) and 2.3% (10 ng/ml); SPP1, 4.1% (2 ng/ml) and 2.9% (10 ng/ml). For Inter-assay Precision: CHI3L1, 10.1% and 7.5%; MMP13, 8.7% and 6.1%; SPP1, 6.6% and 5.6%, respectively.