Nunc immuno maxisorp plates

Total IgG, IgG_2b, IgG_2c, IgG₃, IgE, and IgA concentrations were determined by ELISA in serum samples as previously described (26 (link)). Briefly, Nunc-Immuno Maxisorp plates (Fisher Scientific) were coated overnight with 2 µg/ml MHC class II SLPs in bicarbonate buffer, and after blocking (skim milk powder, Fluka BioChemika) sera from mice (i) chronically infected, (ii) long term vaccinated with MHC class II SLP vaccines and anti-OX40 mAb treated (i.p. during booster vaccination only), or (iii) uninfected were added. Next, plates were incubated with various HRP-conjugated antibodies (SouthernBiotech) to detect different immunoglobulin isotypes. Plates were developed with TMB substrate (Sigma Aldrich), and the color reaction was stopped by the addition of 1 M H₂SO₄. To serve as a positive control, a peptide from the M2 protein (eM2) of influenza A virus with known ability to induce antibodies and corresponding serum was used. Optical density was read at 450 nm (OD₄₅₀) using a Microplate reader (Model 680, Bio-Rad).

Blood was collected from the retro-orbital plexus and after brief centrifugation, sera were obtained and stored at −20°C. Specific immunoglobulin levels in serum were measured by ELISA as described [39 (link)]. Briefly, Nunc-Immuno Maxisorp plates (Fisher Scientific) were coated either with 2 μg/ml SLPs or with MCMV-Smith in bicarbonate buffer, and after blocking with skim milk powder (Fluka BioChemika) diluted sera were added. Next, plates were incubated with HRP-conjugated antibodies (SouthernBiotech) to detect different Ab isotypes. Plates were developed with TMB substrate (Sigma Aldrich) and the colour reaction was stopped by addition of 1M H₂SO₄. To serve as a positive control, a peptide from the M2 protein (eM2) of influenza A virus with identified ability to induce antibodies and corresponding serum was used. Optical density was read at 450 nm (OD₄₅₀) using a Microplate reader (Model 680, Bio-Rad).

Dynamic evaluation of brain and global biomarkers was performed at D1 and D2 post-injury in the PBBI model. Serum concentrations of brain and global biomarkers pNF-H (cat no. RD191138300R; Biovendor), NF-L (item 103345; Quanterix), HSP70 (cat no. ab133060; abcam), and inflammation markers HMGB1 (ABIN416082; Antibodies-online.com) and αII-spectrin (cat no. ABIN1572517; Antibodies-online) were determined with sandwich enzyme-linked immunosorbent assays (ELISA) at the University of Florida. Sandwich ELISAs were conducted using standard 96-well, flat-bottom, Nunc Immuno Maxisorp plates (Fisher, Pittsburgh, PA, USA), according to the manufacturer's protocol. Selected capture and detection antibody pairs were used.
Briefly, ELISA plates were passively coated overnight at 4°C with capture antibody, then washed and blocked. Serum samples were added and incubated at room temperature with shaking. After washing, peroxidase-conjugated detection antibody or HRP-conjugated streptavidin were added, which catalyzed the reaction with a colorimetric substrate (TMB; Pierce). The product was quantified by absorbance at 450 nm in a microplate spectrophotometer. Standard curves were generated using recombinant proteins corresponding to the biomarker measured in each assay. Four parameter-fit non-linear regression analyses were applied to determine biomarker quantities.

An in-house ELISA method was set-up and validated for measuring lubricin concentrations in SF, adapted from others^{54 (link),55 (link)}. Monoclonal antibody 9G3 (1 μg/mL in PBS) was coated on 96-well Nunc-Immuno maxisorp plates (Thermo Fisher Scientific) at 4 °C over-night. After blocking with 3% BSA in PBS + 0.05% Tween, SF samples were added as a dilution series (1/50) in assay buffer (1% BSA in PBS-Tween) and incubated for 1 hour at room temperature (RT). Bound proteins were then incubated with biotinylated PNA (Vector laboratories) (1 μg/mL, 1 hour at RT), followed by HRP-streptavidin (Vector laboratories) at 0.1 μg/mL (1 hour at RT). Between each incubation, the wells were washed three times with PBS-Tween to remove unbound reagents. Proteins were stained with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) until blue colour was fully generated and the reaction stopped by adding 1 M H₂SO₄. Absorbances were read at 450 nm, and compared with a standard curve using recombinant lubricin (dilution series of rhPRG4 (1 mg/mL) in assay buffer). Samples were measured in duplicates and mean values are reported. The lubricin ELISA had an intra plate CV = 7.5% (n = 1 SF sample with 10 repeats) and a inter plate CV% = 7.8% (n = 1 SF sample, tested on 4 plates). Technical performance of the assay is summarized in Supplementary Table 3.