Maxisorp nunc immuno plate

Antigen-specific antibodies were examined by enzyme-linked immunosorbent assay (ELISA). Briefly, recombinant N protein purified from E. coli and recombinant orf8 protein purified from Expi293 cells were coated with 100 ng and 250 ng of protein per well in 50 mM coating buffer (pH 9.6 Na₂CO₃/NaHCO₃) on ELISA plates (Maxisorp nuncimmuno plate, Thermo Fisher Scientific) and incubated overnight at 4°C. Plates were blocked with 0.5% (wt/vol) bovine serum albumin (BSA) and 0.5% gelatin in 0.05% Tween 20 (Sigma-Aldrich) in PBS at room temperature for 1 h and washed one time in 0.05% Tween 20 (Sigma) in PBS. Diluted sera were added and incubated for 1 h at 37°C. Plates were washed three times and incubated with HRP-conjugated goat anti-human IgM (Thermo Fisher Scientific, catalog no. A18841), goat anti-human IgG (Thermo Fisher Scientific, catalog no. A2290), or goat anti-human IgA (Thermo Fisher Scientific, catalog no. A18781) for 1 h at 37°C. The color was developed using trimethyl borane (TMB) solution (Thermo Fisher Scientific), and absorbance was measured at 450 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). We used archived anonymous serum samples as the negative control. The cutoff for seropositivity was set as the mean value of 100 control serum samples plus two times the standard deviation.

MAXISORP Nunc-Immuno plates (Thermo Scientific, Waltham, MA) were coated overnight at 4 °C with CD3 mAb (5 µg/ml) in combination with CD28 (5 µg/ml) mAb. The plates were then washed to remove unbound mAbs and purified T cells (2 × 10⁵/well) were then added to the respective wells. T cell proliferation was assessed via cellular incorporation of [methyl-3H]-thymidine. Cells were labeled with 0.05 mCi/well of [methyl-3H]-thymidine on day 3 and cultured for another 18 h prior to harvesting. Detection was performed on a microplate counter (MicroBeta2; Perkin). All assays were performed in triplicates and readings are displayed as counts per minute.

To determine the Aβ_1–42 levels in the CSF of APP/PS1 mice, we performed Aβ ELISA as we described previously [14 (link)]. In brief, 96-well Maxisorp Nunc Immunoplates (430314; Thermo Scientific) were coated ON at 4 °C on a shaker with 50 µl anti-Aβ_1–42 antibody (1.5 μg/ml; JRF/cAb42/26) in coating buffer (10 mM Tris–HCl, 10 mM NaCl, 10 mM NaN₃ in 500 ml distilled H₂O, pH 8.5) per well. Plates were washed five times with PBST (PBS + 0.05% Tween-20), after which residual protein binding sites were blocked for 4 h at RT with 100 µl blocking buffer (0.1% casein buffer). 30 µl of either Aβ_1–42 standard (A-1163-1; rPeptide) or CSF (1:100 in sterile PBS) was mixed with 30 µl detection antibody (JRF/AbN/25 coupled to HRPO (Janssen Pharmaceutica); 1:2000 in blocking buffer). After blocking, ELISA plates were washed five times with PBST and 50 µl of the standard/sample-detection mixtures was added to the ELISA plates. Plates were incubated ON at 4 °C, while slowly shaking. Absorption at 450 nm was measured after adding 50 µl of TMB substrate solution (555214; BD Biosciences OptEIA™), followed by stopping buffer (50 µl 1 M H₂SO₄). The amount of Aβ_1–42 was determined with GraphPad Prism 8.3 using the nonlinear regression model.

Anti-S IgG was captured through affinity purification with recombinant trimerized S protein-coated Maxisorp NUNC-Immuno plates (Thermo Fisher Scientific, Roskilde, Denmark) (18 (link)), while total IgG was enriched using protein G Sepharose Fast Flow 4 beads (32 (link)). A 100 mM formic acid solution was used for antibody elution, followed by sample drying through vacuum centrifugation and reconstitution in 25 mM ammonium bicarbonate. The purified antibodies were subjected to tryptic digestion to obtain glycopeptides, as described previously (29 (link), 32 (link)). For the samples from Brazil, a minimum of 2 Visucon-F standards, 4 pooled anti-S IgG samples and 2 blanks were included per plate. For the German samples at least 1 Visucon-F standard and 1 blank was included on each plate.

The amount of hyaluronan in conditioned media of cultured cells was quantified by an assay based on the specific interaction of hyaluronan with the G1 global domain of aggrecan, immobilized to 96-well microtiter plate (MaxiSorp Nunc-Immuno plates, Thermo Fischer Scientific, Gothenburg, Sweden).^{36 (link)} The amount of hyaluronan in the samples was normalized to the amount of cellular protein.

Antibodies against bovine-CD63 (1μg/ml) (MCA2042G, Bioconnect, Huissen, The Netherlands) were coated onto Maxisorp Nunc-Immuno plates (Thermo Scientific, Roskilde, Denmark) overnight at 4°C. Coated plates were blocked with 1% OVA 1h prior to addition of milk-derived EVs. Samples were diluted in blocking buffer and incubated overnight at 4°C. After incubation, the plates were washed multiple times with PBS-Tween. IgG1 was coated onto plates as a negative control for aspecific binding. After washing the plates 100μl of 100mM glycine-HCl (pH 2.4) was added to the wells to elute the bound EVs. After 30 minutes, the eluted samples were neutralized by the addition of NaOH. The diameters and concentration of the EVs were measured using a NanoSight LM12 (Nanosight Ltd., Amesbury, UK).

MAXISORP Nunc‐Immuno plates (Thermo Scientific, Waltham, MA) were coated overnight at 4° with either CD3 mAb (OKT3) alone or in combination with CD28 mAb (10F3) or one of the CD43 mAbs (6E5 or 10G7). All mAbs were used at 5 μg/ml. The plates were then washed to remove unbound mAbs and purified T cells (2 × 10⁵/well) were added to the respective wells. T‐cell proliferation was monitored, measuring [methyl‐³H]thymidine (PerkinElmer, Inc. Waltham, MA) incorporation at day 3. Cells were harvested 18 hr after adding [methyl‐³H]thymidine (0·05 mCi/well) and incorporated thymidine was detected on a microplate scintillation counter (Topcount; Packard, Meriden, CT) as counts per minute. Assays were performed in triplicates.