Maxisorp 96 well flat bottom plates

Titration of HA-specific Immunoglobulin G (IgG) was performed on serum from individual mice collected at 3 weeks after the 1st or 10 days after the second immunization. Maxisorp 96-well flat-bottom plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 0.6 μg/mL HA in phosphate-buffered saline pH 7.4 (PBS). The coated wells were blocked for 1 h at room temperature with 300 μL of PBS pH 7.4, 0.1% BSA, and 0.05% Tween-20 with 1% goat serum. The plates were washed with PBS pH 7.4, 0.1% BSA, and 0.05% Tween-20, tapped. Serum samples were initially diluted 1:300 with the dilution buffer (PBS pH 7.4, 1% BSA, 0.05% Tween-20), then transferred into coated-blocked plates in which the samples were serially diluted three-fold with the same buffer. Antigen specific IgG1, IgG2b, IgG2a, and IgA titers were revealed with HRP-conjugated goat anti-mouse IgG1, IgG2b, IgG2a, and IgA (Southern Biotech Associates, Al, USA). Antibody titers are expressed as the logarithm of the enzyme-linked immunosorbent assay titers that give an optical density (OD) higher than the mean plus five times the standard deviation (SD) of the average OD obtained in the pre-immune or naïve sera.

For detection of Bos d 5-specific IgE in sera of milk allergic patients (10 patients positive and 10 patients negative to oral cows milk allergen challenge; sera were retrospectively collected in accordance with the Helsinki Declaration of 1975 and under approval of the ethical committee of the Bambino Gesù Pediatric Hospital, Rome; individual informed consent from all donors was collected by Dr. Alessandro Fiocchi, Children’s hospital Bambino Gesú, Rome, Italy), duplicate wells of MaxiSorp 96 well flat-bottom plates (Nunc, Rochester, NY, USA) were coated with 100 µl/well of apo-Bos d 5 or holo-Bos d 5 diluted at 5 µg/ml in coating buffer and incubated overnight at 4 °C. After 2 h blocking at room temperature with 200 µl Tris buffered saline (TBS)+ 0.05% Tween 20 (TBST), wells were incubated with 100 µl of human serum diluted 1:5 in TBST overnight at 4 °C. Alkaline-phosphatase (AP) conjugated mouse anti-human IgE antibody (BD Pharmingen) diluted at 1:1000 in TBST was used as secondary antibody. AP substrate (100 µl/well; SIGMAFAST p-Nitrophenyl phosphate Tablets, Sigma) was added to detect bound AP conjugated antibodies. The optical density was measured at 405 nm using an Infinite M200Pro microplate reader (Tecan, Austria).

Maxisorp 96-well flat bottom plates (Nunc) were coated with 30 μL of the relevant antigens at 20 μg/mL (in 2% BSA-PBS) for both vimentin and laminin. The protein was adsorbed to the plates during storage at 4°C overnight. Each well was washed with three times 200 μL PBS using a multi-channel pipette, before the plates were blocked with 300 μL 2% BSA in PBS (BSA-PBS) for 1 hour at room temperature under gentle agitation. The washing step was then repeated, followed by the addition of serial dilutions of scFv-rFcs in 100 μL 2% BSA-PBS. The antibodies were incubated in the antigen-coated wells for 1.5 hour at room temperature, and subsequently the plates were washed three times with PBS, each time for 5 min. Detection of bound scFv-rFcs was performed by incubating the plates with polyclonal swine anti-Rabbit Immunoglobulins (Dako) in a 1:2000 dilution in 2% BSA-PBS for 1 hour. Finally, the plates were washed three times with PBS, each time for 5 minutes. The amount of bound antibody was visualised by the addition of 80 uL TMB single solution (Sigma Aldrich), and the colour reaction was terminated after 7 minutes by the addition of 50 μL 1 M H₂SO₄ to each well. Absorbance was measured at 450 nm and corrected for at 655 nm. The absorbance was conducted in a microplate reader; Model 550 (Bio Rad).