Lectin Microarray Validation in RA-ILD

To validate the results of the lectin microarray, serum samples from different groups were randomly chosen from the lectin microarray analysis cohort and a new cohort of 50 RA-ILD patients was included. Briefly, to determine the location of IgG in immunoblotting, 1:100 diluted serum proteins mixed with loading buffer (CW biotech, Beijing, China) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking non-specific binding sites with 10 × Carbo-Free Blocking Solution (1:10; Vector Laboratories Inc., US) at room temperature for 2 h, the membranes were incubated with 20 μg/mL Cy3 (1:1,000; GE Healthcare, Chicago, IL, USA)-labeled lectins including SBA, STL, PHA-E, SNA, Jacalin, SNA-I, MNA-M, AAL, ConA, PHA-L, DBA (EY Laboratories, Inc., US and Vector Laboratories Inc. US) at 4 °C overnight in the dark. Excess lectins were removed by washing three times with PBST. The washed and dried membranes were detected by a fluorescence signal system of Typhoon FLA 9500 (GE Healthcare, Chicago, IL, USA). Finally, ImageJ software was used for signal intensity analysis.