Filter plate

SpAwt beads were prepared as already described above. Commercial Human IgGs (Sigma-Aldrich) were biotinylated and preincubated 30 minutes at room temperature in a 96-well Filter Plates (Millipore)at a final concentration of 8 μg/mL with vigorous shaking, to saturate 20 µl of SpAwt-coated beads. Beads were washed from excessive Hu-IgGs and then incubated with 50 μl of IgG3 mAbs 1-4 or IgG3 isotype control, serially diluted in PBS. After a final washing in PBS, the displacement of human antibodies was revealed with 50 µl of 0.1 mg/mL PE-conjugated streptavidin (Invitrogen), and was acquired with FLEXMAP 3D (Luminex Corporation). Median fluorescence intensity (MFI) was subtracted from the background signal (SpA beads + secondary antibody + streptavidin-PE) and was evaluated by using Graphpad Prism 8.1.2 (GraphPad Software Inc, CA).

Immunoprecipitation of radiolabeled antigens was used to screen sera for autoantibodies against tryptophan hydroxylase-1. Human complementary DNA from the antigen in expression vector was used to perform the assay. In vitro transcription and translation were performed in the presence of ³⁵S-methionine, according to the manufacturer’s protocol (Promega TNT Systems). Immunoprecipitation was performed in 96-well plates overnight at 4°C at 300 rpm with serum samples (2.5 µl) and 30,000 cpm of radiolabeled protein. A positive control (positive patient serum to each antigen) and a negative control, 4% bovine serum albumin, were included in each plate. All samples were analyzed in duplicates. The immune reaction was transferred to filter plates (Millipore) and immune complexes were captured to Protein A sepharose (nProtein A Sepharose 4 fast flow, GE Healthcare) during a 45 min incubation at 4°C at 300 rpm. After ten washing steps with wash buffer (150mM NaCl, 20 mM TrisHCl pH 8, 0.15% Tween 20, and 0.1% BSA), plates were dried and scintillation fluid (Optiphase, HiSafe 3, PerkinElmer) was added. Radioactivity was then measured in a beta counter (1450 Microbeta Trilux, Wallac). Autoantibody index values were calculated according to the following: (sample value – negative control value)/(positive control value – negative control value) x 100.

CC3-Fc concentrations in plasma were analysed by quantitative LC-MC method described by Steven et al. [50 (link)]. Briefly, 50 μl Protein A-Sepharose was added to wells of a Millipore filter plate and conditioned with PBS pH 7.4. Plasma samples were diluted 1 : 4 with PBS and added to the filter plate, incubated at room temperature and agitated at 700 rpm for 1 h. Wells were washed four times with 200 μl PBS using a vacuum manifold. Bound Fc protein was eluted twice with 25 μl of 100 nM glycine pH 2.0 into a low-bind deep-well block and neutralised by adding 7.5 μl 2M Tris pH 8.0. Eluted protein samples were then trypsin-digested by addition of 20 μl of proteomics-grade trypsin (made up at 20 μg/mL in 10 mM CaCl₂, 50 mM Tris-HCl, pH 8.0) followed by incubation at 37°C for 18 h prior to LC-MS/MS analysis. Samples were analysed using LC-MS/MS by monitoring tryptic signature peptides resulting from CC3-Fc present in the samples.