Polystyrene plates

An aCL ELISA was performed according to our previous publication [19 ] and was sensitive to β2GP1-independent and β2GP1-dependent aCLa. Polystyrene plates (polySorp, Nunc, Roskilde, Denmark) were covered with either 20 µL/well of 50 µg/mL cardiolipin (CL) (Sigma-Aldrich, Steinheim, Germany) in ethanol or ethanol alone (as a control well). After that, the wells were blocked by 1.0% bovine serum albumin (BSA) in 0.05 M of Trizma buffer pH-8.5. The serum samples were instilled in the wells for an hour after diluting to 1:50 in RPMI medium with 0.5% BSA and 0.01% Tween-20. The amount of bounded aCLAb was determined by incubation with horseradish peroxidase-conjugated goat anti-human IgG (λ chain specific) and a reaction of peroxidase with TMB. The optical density (OD) was read at λ = 450 nm using a Multiscan device (LabSystems, Finland). Values below 10 GPL for CL were considered as negative (>20 GPL were considered as positive) and were excluded from the investigation.

Hyaluronic acid (Sigma) or α-IgM (Zymed) coated 96 well polystyrene plates (Nalge Nunc International) were incubated for 1 h at 37°C. The plates were then washed twice with PBS before adding 4 × 10⁵ panning-enriched B cells in 200 µl of RPMI 1640 per well. The cells were adhered for 1 h at 37°C. The plates were then washed with 300 µl of PBS, fixed with 4% paraformaldehyde for 10 min, before adding crystal-violet (7.5 g/l crystal-violet, 2.5 g/l NaCl, 1.57% formaldehyde, 50% methanol) for an additional 5 min. The cells were washed extensively eight times with distilled water, solubilized with 10% SDS, and the amount of dye remaining in the plates was recorded at 540 nm (Multiskan Ascent, Thermo Scientific, Waltham, MA, USA). After subtraction of the non-specific dye bound to empty wells, absolute binding was calculated. The absorbance was determined in at least four wells per condition, in three independent experiments.

Fibrinogen, methylglyoxal, oxalic acid, thiobarbituric acid (TBA), trichloroacetic acid (TCA), sodium chloride (NaCl), sodium hydroxide (NaOH), phosphotungstic acid, bovine serum albumin (BSA), ammonium persulphate (APS), bis-acrylamide, and tetramethylethylenediamine (TEMED) were purchased from Hi-Media. Sodium dodecyl sulfate (SDS) and 2,4,-trinitrobenzene-1-sulphonic acid (TNBS) were obtained from G Biosciences, whereas ethyl acetate and dinitrophenylhydrazine (DNPH) were purchased from Rankem. Guanidium hydrochloride was obtained from S. D. Fine-Chem Limited, and hydrochloric acid (HCl) was purchased from Fisher Scientific. A protein A agarose column was purchased from Sigma, and polystyrene plates were obtained from Nunc (Denmark). Analytical grade chemicals were used in this study. All the solutions were filtered under aseptic conditions using a sterilized Puradisc™ 0.2 mM syringe filter (Whatman, GE Healthcare UK Limited, UK).

An in-house ELISA was used to determine anti-Hsp60 and anti-Hsp70 IgM and IgG autoantibodies according to the protocol described previously [21 (link)]. Briefly, ninety-six-well polystyrene plates (Nunc, Roskilde, Denmark) were coated with human Hsp60 (Abcam, Waltham, Boston, MA, USA) and human Hsp70 (Abcam, Waltham, Boston, MA, USA) at a concentration of 1 μg/mL in ELISA coating buffer (Bio-Rad, Hercules, CA, USA) at 4 °C overnight. After blocking the wells with a 2:1 solution of 0.5% polyvinyl alcohol and bovine gelatin, serum samples were incubated in duplicate at 1:200 dilution in washing buffer (PBS, 0.05% Tween 20) for 1 h at 37 °C. After incubation with HRP-conjugated anti-human IgG- and IgM-specific secondary antibodies (Agilent-Dako Santa Clara, CA, USA) for 40 min at 37 °C, the reaction was visualized using TMB solution, and the OD was read at λ = 450/620 nm using the BEP 2000 Advance automated system (Siemens, Marburg, Germany).

Wells in polystyrene plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with rOmp19, rOmp25, or rOmp31 at 5.0 μg/mL in bicarbonate buffer, pH 9.6 and incubated at 4°C overnight. The plate was then washed 3 times sequentially with PBS and PBS supplemented with Tween-20 (PBS-T). Antisera dilutions were prepared in PBS-T (1:100–1:12,800) which were added to 8 wells coated with each of the proteins; the plate was incubated at 37°C for 1 h. After washing, (HRP)-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted with PBS-T was added to each well. The plate was incubated for another 1 h at 37°C, washed 3 times, and then developed with O-phenylenediamine (Sigma-Aldrich, St. Louis, USA). The reactions were stopped after 3-5 min by adding an equal amount of 2 M sulfuric acid to each well. The absorbance was measured at 490 nm using a plate reader (Bio-Rad 680, Redmond, WA, USA). The cutoff value for the assay was calculated as the mean optical density (OD) plus 3 standard deviations for 18 negative control sera obtained from mice before immunization.

Three µg/mL of α-synuclein and TDP-43 diluted in PBS were coated on separate polystyrene plates (ThermoFisher, Waltham, MA, USA), and incubated overnight at 4 °C. The plates were then washed three times with PBST and blocked with 200 µL of 3% bovine serum albumin (BSA; Millipore, Burlington, MA, USA) diluted in PBS for 1 h at room temperature. After washing, 100 µL of serially diluted (50, 10, and 2 ng/mL) of recombinant TDP-43 and α-synuclein in PBST were applied on to α-synuclein and TDP-43 coated wells, respectively. The plates were then left to incubate for 1 h at 37 °C. After washing, 100 µL of 0.1 µg/mL of biotinylated TDP-43 antibodies (10782-2-AP and 12892-1-AP; Abcam, Cambridge, UK) diluted in PBST with 10% BlockAce, and anti-α-synuclein antibodies (FL-140; Santa Cruz Biotechnology, Dallas, TX, USA) were applied on wells treated with TDP-43 and α-synuclein, respectively, and left to incubate for 1 h at 37 °C. After washing, the wells were treated with 100 µL of HRP-conjugated streptavidin diluted 10,000 times in PBST with 10% BlockAce and left to incubate for 30 min at room temperature. After washing, chemiluminescent substrate (SuperSignal ELISA Pico, Thermo Scientific) was added to achieve greater sensitivity. The resulting luminescence was then measured using a Victor 3 multilabel reader.