Aia system

Blood samples were obtained on the same day or within 14 days after liver biopsy. Separated serum samples were immediately stored at −20 °C until testing.
Serum ATX antigen concentration was determined using a specific 2-site enzyme immunoassay. For preparation of the immunoassay, R10.23 was digested with pepsin, and the purified F(ab)₂ form was isolated using phenyl-5PW hydrophobic column chromatography (Tosoh Co., Tokyo, Japan) to avoid the nonspecific binding of human antibodies to animal IgG types, such as human anti-mouse antibodies. In-house magnetic beads were coated with R10.23 F(ab)₂ and placed in a reaction cup, and then 35 ng of alkaline phosphatase-labeled R10.21 in assay buffer (5% BSA, 5% sucrose, 10 mmol/l Tris–HCl, 10 mmol/l MgCl₂, pH 7.4) was added. The ATX assay reagent was prepared by immediate freeze-drying of the reaction cup for subsequent use in a commercial automated immunoassay analyzer AIA-system (Tosoh Co.). The AIA-system process included automated specimen dispensation, incubation of the reaction cup, bound/free washing procedure, 4-methylumbelliferyl phosphate substrate dispensation, fluorometric detection, and result report. Antigen-antibody reaction time was 10 min and the first result was reported within 22 min. The throughput of the system was 60 samples/h using the AIA-600 II system26 (link).

AH samples were collected as described previously^{4 (link),10 (link)}. Levels of ATX in the AH were determined via a two-site immunoenzymatic assay with an ATX assay reagent using a Tosoh AIA system (Tosoh, Tokyo, Japan). TGF-β levels in the AH were measured using a Bio-Plex Pro TGF-β assay (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer’s protocol.

We measured ATX levels by a specific two-site enzyme immunoassay using the commercial automated immunoassay analyzer AIA-system (Tosoh Co., Tokyo, Japan) for serum samples obtained before the start of treatment, at 4 weeks after the start of treatment, and at 24 weeks after treatment completion. The AIA-system includes automated 10-microliter specimen dispensation, incubation of the reaction cup, a bound/free washing procedure, 4-methylumbelliferyl phosphate substrate dispensation, fluorometric detection, and a result report. The antigen-antibody reaction time is 10 min and the first result is reported within 20 min. The throughput of the AIA-2000 system is 200 samples/h [27 (link)]. The assay was conducted according to the optimized protocols used in previous studies [22 (link), 23 (link)].