Quantikine human elisa assay

The concentrations of S100A9 protein in CM from mono-culture or co-culture and those in patient sera were measured in triplicate by CircuLex S100A9/MRP14 ELISA kit (Abnova Corp., New Taipei City, Taiwan). The detection sensitivity for this kit was 6.55-3200 pg/mL without the cross-reactivity to the other S100 proteins. The IL-6 level in the CM and patient serum was individually quantified in triplicate by using the Quantikine human ELISA assay (R&D systems, Minneapolis, MN, USA). We calculated the serum concentration of S100A9 or IL-6 based on serial dilutions of a recombinant protein at known concentrations.

Anti-GAD65, anti-IA2, and anti-ZnT8 antibodies were determined through enzyme immunoassay (ELISA) using the Medizym commercial kits (Berlin, Germany). Antibody detection was carried out semiquantitatively through reference to the value of 5 IU/mL for GAD65, 10 IU/mL for IA2, and 15 IU/ml for ZnT8. The analysis of inflammatory markers included human ultrasensitive C Reactive Protein (usCRP, BioVendor, Czech Republic) and the measurements of TNFα, IL-6, vCAM, and C-peptide concentrations determined by ELISA (R&D Quantikine Human ELISA Assay, UK). HbA1c levels were measured using a commercially available automatic system (DCA 2000, Bayer Diagnostics, Tarrytown, NY, USA).