Maxisorp microtiter plate

Hybridoma cell supernatants were screened for PFD1235w DBLβ3_D4-reactive Abs using ELISA. Duplicate wells of MaxiSorp microtiter plates (Nunc) were coated with DBLβ3_D4 (50 μl; 1 μg/ml; 0.1 M glycine/HCl buffer pH 2.75; overnight; 4°C) and blocked with blocking buffer (PBS, 0.5 M NaCl, 1% Triton X-100, 1% BSA, pH 7.2). A total of 100 μl undiluted cell supernatant was added (1 h; room temperature). The plates were washed in PBS + 1% Triton X-100, and bound Ab was detected with an anti-mouse Ig-HRP (Dako; 1:3000 in blocking buffer). After 1 h of incubation, plates were developed using OPD tablets (Dako) according to the manufacturer’s instructions. The OD value was read at 490 nm using a VERSAmax microplate reader (Molecular Devices) and Softmax Pro v4.7.1.

Serum H56-specific IgG were determined by ELISA. Flat bottomed Maxisorp microtiter plates (Nunc, Denmark) were coated with H56 (0.5 µg/ml) for 3 h at 37°C and overnight at 4°C in a volume of 100 µl/well. Plates were washed and blocked with 200 µl/well of PBS containing 1% BSA (Sigma-Aldrich) for 2 h at 37°C. Serum samples were added and titrated in twofold dilution in duplicate in PBS supplemented with 0.05% Tween 20 and 0.1% BSA (diluent buffer) in 100 µl/well. After 2 h at 37°C, samples were incubated with the alkaline phosphatase-conjugate goat anti-mouse IgG (diluted 1:1,000 in diluent buffer, Southern Biotechnology, USA) for 2 h at 37°C in 100 µl/well and developed by adding 1 mg/ml of alkaline phosphatase substrate (Sigma-Aldrich) in 200 µl/well. The optical density was recorded using Multiskan FC Microplate Photometer (Thermo Scientific). Antibody titers were expressed as the reciprocal of the highest serum dilution with an OD value ≥0.2, after subtraction of background values measured with diluent buffer alone.

The synthesized glycans and neo-glycoproteins were analyzed for binding of Gal-3 and Gal-1 in 96-well microtiter plate formats [37 (link),41 (link)]. Glycans 11 and 12, as well as the corresponding LacNAc-LacNAc-linker-NH₂1a and LacdiNAc-LacNAc-linker-NH₂2a (deprotected forms of 1 and 2) were immobilized via the amino group in aminoreactive microtiter plates (Immobilizer Amino, Nunc, Wiesbaden, Germany). The immobilization of 5 nmol glycan in sodium carbonate buffer (100 mM, pH 9.6) was done overnight. For immobilizing neo-glycoproteins, 5 pmol protein were incubated in PBS (pH 7.5) in MaxiSorp microtiter plates (Nunc) overnight. Wells were then washed with PBS-Tween (0.05% (v/v)) and blocked with 2% BSA in PBS followed by incubation for one hour with galectins diluted in EPBS. Incubation with anti-His₆-peroxidase (Roche) was done subsequently. Microtiter plates were read out at 495 nm after conversion of OPD substrate (o-phenylenediamine, Dako, Hamburg, Germany). Measured data were analyzed using Sigma Plot (Systat software GmbH, Erkrath, Germany).

The ELISA was essentially performed as described previously [51 (link)], using either the purified native pE6 protein or the CaSki cells as the plate-bound antigen. Briefly, 96-well maxisorp microtiter plates (Nunc, Naperville, IL, USA) were coated with pE6 (300 ng/well) in PBS⁻ or with CaSki lysates (10⁵ cells/well) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. CaSki cells were disrupted by freeze-thawing three times and MRC-5 cells were used as a negative control. The sera were then added at a 1:50 dilution, and the binding was revealed by a 1:2000 dilution of goat anti-mouse HRP-conjugated sera (Dako) and tetramethylbenzidine substrate (Sigma-Aldrich Italia, Milan, Italy). The absorbance for each well was measured at 450 nm with a 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Gluten proteins were extracted according to the manufacturer’s instructions using Universal Gluten Extraction Solution UGES (Biomedal SL, Seville, Spain). Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with Prolamin Working Group (PWG) gliadin solution and incubated overnight at 4°C. The plates were washed with PBS-Tween 20 buffer and blocked with blocking solution (phosphate-buffered saline (PBS)-5% non-fat dry milk) for 1 h at RT. Different dilutions of each sample as well as standard solution of PWG gliadin were made in PBS-bovine serum albumin 3%, to each of which was added horseradish peroxidase–conjugated G12 mAb solution. The samples were pre-incubated at RT and added to the wells. After 30 min of incubation at RT, the plates were washed, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Sigma, St. Louis, Missouri, USA) was added. After 30 min of incubation at RT in the dark, the reaction was stopped with 1 M sulfuric acid, and the absorbance at 450 nm was measured (microplate reader UVM340, Asys Hitech GmbH, Eugendorf, Austria). Results were expressed in parts per million (ppm) in dry matter for the flour, and in a 35% humidity basis for the bread loaves. In order to estimate the total gluten content the results obtained were multiplied by two. Data were obtained from three independent experiments with samples run in triplicate.