2.2. Reagents and instruments

First, 2 mL of anticoagulated venous blood was collected from the 4 samples taken from each patient, centrifuged at 2000 × g for 15 min and stored at −80 °C until use. The total antibody detection reagent used was InnoDx reagent (magnetic particle chemiluminescence method, batch number: 20210101) with an automatic chemiluminescence immunoanalyzer (Xiamen InnoDx Biotechnology Co., Ltd., Caris 200). The NAb detection reagent used was YHLO reagent (Shenzhen YHLO Biotech Co., Ltd., Reagent, magnetic particle chemiluminescence method, batch number: 20210101) with an automatic chemiluminescence analyzer (iFlash 3000-a). A result for the total antibody signal/cutoff (S/Co) ratio less than 1.00 was considered negative, and a S/Co ratio greater than or equal to 1.00 was considered positive. A concentration of NAbs less than 10.00 AU/mL was regarded as nonreactive, and a concentration greater than or equal to 10.00 AU/mL was considered reactive. Then, 2 mL of anticoagulated venous blood was collected twice to count the absolute number of lymphocyte subsets, which employed a lymphocyte subgroup detection reagent (BD Multitest 6-color TBNK reagent) using a flow cytometer (BD FACSCanto II). Cytokines were analyzed for the serum sample using cytokine assay kits (Weimi Bio-Tech Co., Guangzhou, China; batch number: 20201102) using a BD FACS CantoII flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, US) following the manufacturer’s’ instructions. The kits included 14 types of microbeads with distinct fluorescence intensities and were coated with, respectively, specific antibodies against IL-17F, IL-21, IL-2, IL-4, IL-5, IL-6, IL-8, IL-1β, IL-17A, IL-10, TNF-α, TNF-β, IL-12p70, and IFN-γ. After incubation with the serum sample, the immunocomplex was further combined with phycoerythrin fluorescently labeled detection antibody to form a double-antibody sandwich complex, and the fluorescence intensity of the complex was analyzed using a flow cytometer to quantify the cytokines. Next, 2 mL of EDTA-K2 anticoagulant and 2 mL of nonanticoagulated venous blood were collected for the CMP. A Siemens 2400 automatic biochemical analyzer (Siemens Healthcare, Erlangen, Germany) and supporting reagents were used to measure liver and kidney function, and a Sysmex XE2100 automatic blood cell analyzer (Sysmex Corp., Kobe, Japan) and supporting reagents were used to measure routine blood parameters.