Ehigfbp3

A total of 4 mL venous blood was collected from each patient and HC in a blood collection tube with coagulating agent, and the samples were allowed to clot at room temperature for approximately 2 h. After clot formation, the samples were centrifuged at 2000g for 10 min. Then, the sera were collected and stored at −80°C.
Serum IGFBP and CTGF levels were detected using the following human ELISA kits: IGFBP-1 (ab213789, Abcam Cambridge, UK), IGFBP-2 (ab215082, Abcam), IGFBP-3 (EHIGFBP3, Thermo Fisher Scientific, Waltham, MA, USA), IGFBP-4 (ab230936, Abcam), IGFBP-6 (EHIGFBP6, Thermo Fisher Scientific), and CTGF (ab261851, Abcam).

The levels of IGFBP-3 were quantitated using the human IGFBP-3 (EHIGFBP3, ThermoFisher) ELISA kit. In this assay, the amount of IGFBP-3 bound between a matched pair of antibodies is measured. In brief, samples were added to wells precoated with an IGFBP-3-specific antibody. After addition of the second detector antibody conjugated to biotin, Streptavidin-HRP, and a TMB substrate solution, a signal was developed, and the absorbance measured at 450 nm. The signal was directly proportional to the concentration of IGFBP-3 in the sample.