Peroxidase labelled streptavidin

For cytokine assays, colons were weighed and homogenized in PBS containing 0.05% (v/v) Tween-20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KIU AprotininA using a tissue homogenizer (1 mL/0.1 g). Suspensions were centrifuged at 600 g for 10 min at 4°C and the supernatants collected for cytokine assay. Concentrations of IL-10, TNF-α, IL-6, IL-17 were measured by ELISA as described previously [23 (link)]. Briefly, after coating microtitre plates (NUNC, Thermo Scientific, Waltham, MA, USA) with purified monoclonal antibodies reactive to mouse cytokines IL-10, TNF-α, IL-6, IL-17 (BD, New Jersey, USA), standards and samples were added and incubated overnight at 4°C. Biotinylated monoclonal antibodies anti-mouse IL-10, TNF-α, IL-6, IL-17 were added and incubated for 1 h at room temperature, after which peroxidase-labelled streptavidin (Sigma, St. Louis, MO, USA) was added. A colour reaction was developed at room temperature with 100 μL/well of OPD (1 mg/mL) and 0.04% H₂O₂ substrate in sodium citrate buffer. The reaction was stopped by the addition of 20 μL/well of 2 N H₂SO₄. Absorbance was measured at 492 nm using a Bio-Rad Model 450 Microplate Reader. Results were expressed as concentration of each cytokine (pg/mL), according to the respective standard curve.

The generation 2.0 PAMAM dendrimer with a cystamine core (647829, Sigma Aldrich) was conjugated to three different glycans via reductive amination. 20 equivalents of LS-Tetrasaccharide d (LSTd, Elicityl) and D-(+)-Galactose (G0750 Sigma Aldrich) per dendrimer were dissolved in Dimethylsulphoxide (DMSO) and acetic acid (8:2). Per dendrimer, 160 equivalents 2-Methylpyridine borane complex (65421 Sigma Aldrich) was added to a total volume of 200 µL. The reaction was incubated at 65 °C for 2 h with frequent vortexing. The reaction products were purified over disposable PD10 desalting columns (GE17-0851-01 GE Healthcare) in 50 mM Ammonium Formate pH 4.4 (NH₄HCO₃).
The presence of α2-3 sialic acid was validated using an ELISA-type assay using Maackia Amurensis Lectin I (MAL-I) (Vector Laboratories, Peterborough, UK). Briefly, NUNC maxisorb plates (RosKilde) were coated overnight at 4 °C with 5 µM of the products. The wells were subsequently blocked for 2 h at room temperature with carbo-free blocking buffer (Vector, SP5040). Incubation with the biotinylated MAL-I and peroxidase-labelled streptavidin (Sigma-Aldrich) allowed spectrophotometric quantification of the binding with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) at 450 nm on the iMark^TM Microplate Absorbance Reader (Bio-RAD).

Specificity of the anti-cathepsin antibodies produced in mice was verified on PVDF membrane immunoblots of soluble worm extracts previously separated by SDS-PAGE in 12% gels (20 μg/well). The experiment was performed according to a modified protocol [40 (link)]. The membranes were first incubated for 1 h with mouse anti-EnCL3/anti-EnCL1/anti-EnCB /control sera diluted 1:100 in PBS-T and then for 1 h with horseradish peroxidase-labelled goat anti-mouse IgG (Sigma-Aldrich) diluted 1:5000 in PBS-T. Finally, the membranes were developed by the Opti-4CN Substrate Kit (Bio-Rad, Hercules, California).
Purified recombinant proteins (1 μg/well) were resolved by SDS-PAGE in 12% gels and either stained by CBB or trans-blotted onto a PVDF membrane. His-tagged enzymes were detected on blots either with a mouse biotinylated monoclonal Anti-polyhistidine antibody (Sigma-Aldrich), alternatively with 5 nM iBody4, which is a biotinylated copolymer containing nitrilotriacetic acid-bound nickel cations [46 (link)]. In both cases, detection was finalized using peroxidase-labelled streptavidin (Sigma-Aldrich) and the Opti-4CN™ Substrate kit.