Microplate manager 5

Serum and tracheal fluid C5a was measured using a custom sandwich enzyme-linked immunosorbent assay (ELISA). Monoclonal anti-human C5a antibody (Hycult Biotech, Uden, The Netherlands) was coated on 96-well, medium-binding microplates (Greiner Bio-One, St. Gallen, Switzerland). Bound C5a was detected using a biotinylated monoclonal anti-human C5a antibody [28 (link)] and developed using avidin-alkaline phosphatase (MilliporeSigma, Merck KGaA, Darmstadt, Germany). The optical density signal was acquired using the Bio-Rad Model 680 Microplate Reader and quantified using the Microplate Manager 5.2.1 software (Bio-Rad, Hercules, CA, USA). Zymosan-induced activation of normal human serum was used as standard.

One hundred and fifty mg of hair were weighted from each sample and placed into a 50-ml conical tube. After three washes with 3 ml of isopropanol, all samples were left to dry in an extractor hood during 12 h. Dried hair samples were cut into 2–3 mm pieces using scissors, and 50 mg were transferred into 2 ml eppendorf. One ml of methanol was added to each sample and incubated 18 h at 37 °C under moderate shaking (100 rpm). After incubation, extracted samples were centrifuged at 7000g for 2 min and 700 µl of supernatant was transferred to a new 1.5 ml tube. The supernatant was then placed into a speed vac for 2 h to evaporate the methanol. The dried extracts were stored at − 20 °C until analysis. Total concentrations of cortisol (CORT) were measured by ELISA kit (Cusabio Technology LLC., Bionova, Spain) with dried samples reconstituted with 210 µl of sample diluent. Samples were quantified by reference to standard curves constructed with known concentrations of pig cortisol dilutions of the Standard. Absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad) and analysed using the Microplate manager 5.2.1 software (Bio-Rad).

96-well Maxisorp plates (Nunc, Fisher Scientific, Vienna, Austria) were coated with 50 ng/well of a 1086.C gp140 protein [30 (link)] in 0.2 M Na₂CO₃- NaHCO₃, pH 9.5, overnight at 4 °C. Blocking was performed with superblock (PBS with 4% Whey protein, 15% goat serum, 0.5% Tween20) for 1 h at 4 °C. All washing steps were performed with PBS containing 0.1% Tween20 (PBST). Mouse and rabbit plasma samples were heat inactivated for 1 h at 56 °C and pre-diluted in superblock 1:100. Samples were further serially diluted 1:3-fold and transferred in duplicates to the pre-coated 96-well plate and incubated for 1–1.5 h at room temperature. Detection was performed using a horseradish peroxidase conjugated mouse or rabbit IgG-specific antibody from goat (incubation of a 1:10,000 dilution for 1 h at 37 °C) and Sure Blue TMB detection reagent and TMB stop solution (KPL, Gaithersburg, MD, USA). Plates were analyzed at 450 nm (signal) and 650 nm (background) on a model 680 microplate reader (Bio-Rad, Hercules, CA, USA) using Microplate Manager 5.2.1 software (Bio-Rad, Hercules, CA, USA). Endpoint titers were determined as the reciprocal maximum dilution at which the mean optical density (OD) at 450 nm minus the background (OD450–OD650) of duplicates was greater than the mean OD450–OD650 plus 3 standard deviations of naive sera, the double mean of naive sera, and 0.1 for reliable values.

Serum and tracheal fluid TCC was measured using a custom sandwich ELISA. Monoclonal anti-human C9 neoantigen antibody (Hycult Biotech) was coated on 96-well, medium-binding microplates (Greiner Bio-One). Bound TCC was detected using a custom biotinylated polyclonal anti-human C7 antibody [29 (link)] and developed using avidin-alkaline phosphatase (MilliporeSigma, Merck KGaA). The optical density signal was acquired using the Bio-Rad Model 680 Microplate Reader and quantified using the Microplate Manager 5.2.1 software (Bio-Rad). Zymosan-induced activation of normal human serum was used as standard.

100 µL of peripheral blood was collected 1 week before vaccination and 15 days post-vaccination. Blood was left to clot at 4 °C for 5 h. Serum samples were obtained by centrifugation at 8,000 xg for 10 min and stored at -20 °C until use. Serial dilutions (1/3) of sera were prepared with 1% BSA in PBS-0.05% Tween 20. 96-well flat-bottom microplates were coated with 50 μL of 50 μg/mL recombinant S1+S2 ECD-His protein (Sino Biological, Beijing, China) and RBD (RayBiotech Life, Peachtree Corners, GA) in 3.36 mM carbonate–10 mM bicarbonate buffer at 4° C for 16 h. After three washes with 1% BSA in PBS-0.05% Tween 20, blocking was performed in PBS-0.05% Tween 20 containing 3% BSA for 1 h at room temperature. Next, the washes were repeated, and 100 μL of diluted serum samples were added, incubating for 1 h. After repeating the wash step, incubations with 1:8,000-diluted HRP-conjugated protein A (Invitrogen, Waltham, MA), 1:20,000 goat anti-mouse IgG1, or 1:20,000 goat anti-mouse IgG2c (Bethyl Laboratories, Montgomery, TX) were performed for 1 h, followed by three final washes. Color development was executed with TMB Substrate Kit (Thermo Scientific, Waltham, MA) for 10 min in 100 µL. The reactions were stopped by adding 100 μL of 2 N H₂SO₄. A₄₅₀ was registered with Microplate Reader 680 (BioRad, Hercules, CA) and Microplate Manager 5.2.1 software (BioRad, Hercules, CA).

Secreted Gal-3 concentrations in plasma samples were quantified by ELISA (Bender MedSystems, Austria) using provided manufacturer protocol. Briefly, various dilutions and undiluted plasma from each patient or control sample were incubated for 2 hours with biotin-conjugated Gal-3 antibody followed by addition of streptavidin-HRP. Standard curve to extrapolate actual amount in samples was drawn using serial dilutions of provided recombinant Gal-3. All samples were tested in triplicate. The ELISA used incorporated TMB substrate to reach a chromagenic end point detected at 450 nm (reference wavelength = 620 nm) on BioRad microplate reader (Microplate manager 5.2.1).