Interestingly, the diabetes sera in which anti-GAD65 reactivity was not completely removed were sera that contained both anti-GAD65 and anti-GAD67 [26], (and unpublished observations).
For immunohistochemistry, patient and normal sera (300 µl) were purified using the Melon Gel IgG purification kit according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL) to remove IgM, and purified sera were concentrated by Amicon Ultra 100 (Millipore, MA).
After incubation, cell medium was removed and 10% patient sera, control sera or sera with 5 to 320 μg/ml hydroxychloroquine (Sigma-Aldrich) in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 2 mM CaCl2 were added to NETs and incubated for 60 minutes at 37°C.
After incubation, cell medium was removed and 10%% patient sera, control sera or fractionated sera in DNase buffer was added and incubated for 60 minutes at 37 °C.
A negative biopanning selection was done using 10 pooled sera from healthy controls to remove non-specific IgG, while 2 sarcoidosis sera were used for positive selective enrichment.
Differential biopanning for negative and positive selection was performed using sera from healthy controls to remove the non-specific IgG, and sarcoidosis sera for selective enrichment according to manufacturer's suggestions (T7 Select System, TB178; EMD Biosciences-Novagen). Protein G Plus-agarose beads (Santa Cruz Biotechnology) were used for serum IgG immobilization.
The blood was allowed to clot and then centrifuged at 12 000 r.p.m. for 5 min and the top layer of sera removed.
To determine whether any of the antibodies to EBNA-1generated in rEBNA-1 injected mice also cross-reacted with dsDNA, week 12 sera from all 5 EBNA-1 injected mice were adsorbed over dsDNA-cellulose beads to remove dsDNA reactive antibodies and then sera were tested by ELISA to determine if adsorbed sera showed reduced binding to EBNA-1.
To remove possible background sero-reactivity, sera were also tested against bovine serum albumin (BSA).
miRNA microarray data from cancer patients and normal donor sera were coded to remove any indication of disease status and submitted for analysis.
