Hydrospeed microplate washer

SIVmac₂₅₁ g140-foldon-type glycoproteins were produced by transient transfection of FreeStyle™ 293-F cells and purified by affinity chromatography using Ni Sepharose® Excel beads (GE Healthcare)^{74 (link)}. High-binding 96-well ELISA plates (Costar, Corning) were coated overnight with 250 ng/well of purified recombinant SIV gp140 protein. After washing with 0.05% Tween 20-PBS (washing buffer), plates were blocked for 2 h with 2% BSA, 1 mM EDTA, and 0.05% Tween 20-PBS (blocking buffer), washed, and incubated with serially diluted NHP sera in duplicate at 1:250 or 1:50 followed by 7 consecutive 1:4 or 1:3 dilutions in PBS for IgG or IgA detection, respectively. After washing, the plates were revealed by incubation for 1 h with goat HRP-conjugated anti-human IgG or IgA antibodies (Jackson ImmunoReseach, 0.8 µg/ml final) and by adding 100 µl of HRP chromogenic substrate (ABTS solution, Euromedex). Optical densities were measured at 405 nm (OD405 nm), and background values based on incubation of PBS alone in coated wells were subtracted. Experiments were performed using a HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Männedorf, Switzerland).

Total IgA and IgG2b antibody titres in blood serum and intestinal tissue explant supernatants were quantified via sandwich ELISA. Briefly, 96-well flat bottom Maxisorp plates (Thermo Fischer Scientific, MA, USA) were coated with 50 μL goat anti-mouse IgA (Southern Biotech, AL, USA) and incubated overnight at 4 °C. Plates were washed using a Tecan Hydrospeed microplate washer and blocked with 200 μL 3% BSA in PBS for 1 h before 50 μL samples and standards were added. Plates were incubated with samples and standards for 2 h at room temperature, after which 50 μL goat anti-mouse AP-coupled IgA detection antibody (Southern Biotech, AL, USA) was added for 1 h. 50 μL phosphatase substrate was then added and plates were incubated for 30 min at 37 °C. To stop the enzymatic reaction, 25 μL 100 mM EDTA was added and the signal was measured at 405 nm minus reference wavelength 630 nm on a Biotek Synergy H1 Hybrid Reader.

The concentrations of serum amyloid A (SAA) and serum calprotectin were determined using commercial sheep ELISA kits (MyBioSource Ltd., San Diego, USA). Undiluted serum samples were analyzed following the manufacturer’s instructions. The SAA and calprotectin concentrations had coefficients of variation < 10 and < 15% (intra- and interassay), respectively. The sensitivities of the Elisa kits were 0.1 ng/mL for calprotectin and 0.1 μg/mL for SAA. The detection range of the SAA kit was 3.12–100 μg/mL. All samples were analyzed in duplicate. The optical density of the samples was determined at 450 nm using an Apollo 11 LB913 Elisa absorbance reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). We analyzed both parameters on D7, D35 and D49. Serum immunoglobulin A (IgA) was measured with a sheep IgA enzyme-linked immunosorbent assay (Sheep Immunoglobulin A ELISA Kit, Cusabio, Wuhan Huamei Biotech Co., LTD, Wuhan, China). The sensitivity of the kit was 1.87 μg/mL. A HydroSpeed microplate washer (Tecan Austria GmbH, Grödig/Salzburg, Austria) was used for improving assay precision. The IgA values were determined on D7, D35, D49 and D70 using a microplate reader, with the same optical density described above.

For GFAP detection in the cell culture medium and in EV lysates, high-binding 96 well polystyrene microplates (Corning) were coated with mouse monoclonal GFAP antibody (clone GA5, Sigma-Aldrich, 5 µg/ml in PBS pH 7.4) as the capture antibody at 4 °C overnight. Blocking of the plate was performed with 1% BSA in PBS pH 7.4 with 0.15% ProClin (all Sigma-Aldrich) for a minimum of 1.5 h at RT. Samples (medium samples 1:10, EV lysate samples 1:24) were diluted in ELISA buffer (0.1% BSA, 0.05% Tween 20, 0.15% ProClin in PBS pH 7.4, all Sigma-Aldrich) and incubated for 2 h at RT. The detection antibody rabbit polyclonal GFAP (DAKO, 1 µg/ml in ELISA buffer) was incubated for 1 h at RT, followed by an incubation with anti-rabbit IgG-HRP (#A0545, Sigma-Aldrich, 1:2000 in ELISA buffer) for 1 h at RT. K-Blue Aqueous TMB substrate (Neogen) was applied as HRP substrate and the reaction stopped with 1 M H₂SO₄ (Sigma-Aldrich). Washes were performed after every incubation (except between coat and block) with 0.1% Tween 20 and 0.15% ProClin in PBS pH 7.5 using a HydroSpeed micro-plate washer (TECAN). An Infinite M200 Pro microplate reader (Tecan) was used to measure the absorbance at 450 nm. ELISAs were repeated three times and the average reported.