Quanta lite ccp3

Anti-CCP was detected by Quanta Lite CCP3 enzyme-linked immunosorbent assay (ELISA) kits (Inova Diagnostics, San Diego, CA, USA) in accordance to the manufacturer’s recommendations. ELISA plates (BD Biosciences, San Jose, CA, USA) were coated for 2 h at 37 °C with 1 μg/mL GRP78 or CarGRP78 in coating buffer containing 35 mM NaHCO₃ and 15 mM Na₂CO₃, pH 9.6. The plates were washed with phosphate-buffered saline (PBS) for four times and blocked with PBS containing 2% skim milk overnight. Then, Quanta Lite reagents (Inova Diagnostics, San Diego, CA, USA) were used for performing ELISA according to the supplier’s protocol. The plates were read at 405 nm on an Anthos Zenyth 3100 multimode fluorometer (Anthos Labtec Instruments GmbH, Salzburg, Austria). All samples were analyzed at the same time. The absorbance values of control wells were low in all cases. The cutoff values for the anti-GRP78 and anti-CarGRP78 antibody ELISA was defined as the mean plus two standard deviations of the controls.