Sample diluent

Multiplexed addressable laser bead immunoassays (ALBIA) provided by TheraDiag (CTD-13 profile, Paris, France) and Inova Diagnostics Inc. (San Diego, CA, USA) were used to identify the specific ANA targets which included: U1RNP, dsDNA, histone, Jo-1, ribosomal P protein, Sm, Scl-70, PM-Scl, CENP-B, PCNA, SSA/Ro60, Ro52/TRIM21, SSB/La, centromere, RNA polymerase III, Th/To-Rpp25, and Th/To-Rpp38. Twenty microliter of suspended beads, 25 μl of sample diluent (Inova Diagnostics Inc.) and 5 μl of diluted mouse serum were added into the wells of 96-well plate. The plate was incubated with agitation at 600 rpm for 30 min at RT, followed by incubation in goat anti-mouse IgG phycoerythrin conjugated secondary antibody (0.5 μg/ml, Jackson ImmunoResearch Lab. Inc.) for 30 min and 600 rpm in the dark. The antibodies to survival of motor neuron (SMN), Gemin3 (DEAD box RNA helicase), Mup44/NT5c1A (cytosolic 5-nucleotidase 1A), and RUVB1/2 (AAA+ ATPases) were detected using a laboratory developed test as previously described (11 (link)). Plates were analyzed by using a Luminex-100 plate reader (Luminex Corp., Austin, TX, USA). Cutoff values were established on negative and positive controls in each run and was set at three standard deviations (SD) above the mean for WT mice.

A group of AILD-related autoantibodies (AMA-MIT3 (against PDC-E2, BCOADC-E2, and OGDC-E2), LKM, sp100, gp210, SLA, LC-1) were identified by Euroimmun Line Immunoassay (LIA; Euroimmun, Lübeck, Germany), as previously reported (19 (link)). Antibodies directed to HK and KL were measured by QUANTA Lite enzyme-linked immunosorbent assay (ELISA) (Inova Diagnostics Inc., San Diego, CA, USA) (20 (link)). Autoantibodies to GW182, Ago2, Ge-1, EEA1, VCP, and lamin B1 were detected using a laboratory developed multiplexed addressable laser bead immunoassay (ALBIA), as previously described (10 (link), 19 (link), 20 (link)). Briefly, 20 microliters (µls) of suspended beads bearing the covalently coupled antigen analyte, 25 μl of sample diluent (Inova Diagnostics Inc., San Diego, CA, USA) and 5 μl of diluted mouse serum were added into the wells of 96-well plate. The plate was incubated with agitation at 600 rpm for 30 min at room temperature (RT), followed by incubation in goat anti-mouse IgG phycoerythrin conjugated secondary antibody (0.5 μg/ml, Jackson ImmunoResearch Lab. Inc., West Grove, PA, USA) for 30 min and 600 rpm in the dark. Plates were analyzed by using a Luminex-100 plate reader (Luminex Corp., Austin, TX, USA). Cutoff levels were determined on positive and negative controls in each run and were set at three standard deviations (SD) above the mean for WT mice.