Horseradish peroxidase coupled streptavidin

The levels of Dp-specific antibody in plasma were determined by ELISA. To detect IgG, IgG1, IgG2a, IgG2b and IgG3, we coated ELISA plates (Maxisorp, type 96 F; Nunc A/S, Roskilde, Denmark) with Dp in PBS (50 μg mL⁻¹). The coated plates were incubated with 2 % Block Ace (Dainippon Sumitomo Pharmaceuticals, Osaka, Japan). Plasma samples were diluted by 2 % Block Ace and these dilutions were added to the Dp-coated plates. After incubation with plasma, the coated plates were incubated with a horseradish peroxidase–conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b or IgG3 solution (SouthernBiotech, Birmingham, AL, USA) for two hours at room temperature. After the incubation, the color reaction was developed with tetramethyl benzidine (Moss, Inc.; Pasadena, MD, USA), stopped with 2 N H₂SO₄, and measured at OD_450–620 on a microplate reader.
To detect Dp-specific IgE, we coated ELISA plates (Maxisorp, type 96 F) with purified rat anti-mouse IgE (2 μg mL⁻¹; R35-72, BD Biosciences;), incubated them with Block Ace, and added samples of diluted plasma as described earlier. Treated plates were incubated with biotin-conjugated Dp (5 μg mL⁻¹) followed by horseradish peroxidase–coupled streptavidin (Southern Biotechnology Associates). Color detection was performed as described earlier.

For assay optimization phosphate-, tris(hydroxymethyl)aminomethan (TRIS)- and triethanolamine (TEA)-based buffers with different concentrations of dimethyl sulfoxide (DMSO) were compared. Furthermore, different reporter molecules, reagent concentrations, detection systems, and the use of detergents (Tween-20, Triton X-100, Triton X-114, IGEPAL, Brij 35, CHAPSO; Sigma-Aldrich) were evaluated. Following these multiple optimization steps, a new standard protocol was defined: MaxiSorp immunoassay plates were coated with 5 μg/ml of mAb JD5.1 overnight at 4°C. After washing the plate twice with PBST, the plate was blocked with SuperBlock T20 (TBS) blocking buffer (Thermo Scientific) for 1 h at 37°C. After another washing step, serial dilutions of the samples in LW buffer (0.2 M TEA, pH 7.5, with 20% DMSO) were added to the plates and incubated in the dark for 2 h at 37°C. Without washing, 100 μL of an 80 ng/ml solution of the reporter molecule MG-161 (Fig 1) in LW buffer was added to the plate and incubated for an additional 45 min. Subsequently, plates were washed four times, and bound MG-161 was detected after 1 h incubation at 37°C by horseradish peroxidase-coupled streptavidin (SouthernBiotech) diluted 1:5000 in PBST. Signal development was done with 3,3’,5,5’-tetramethylbenzidine (TMB) for 5 min after which the reaction was stopped with 0.5 M sulphuric acid.

Plasma levels of antigen-specific antibodies were determined by ELISA. To detect OVA-specific IgG1, LPS, or NP-specific IgM or IgG3, we coated ELISA plates (Maxisorp; Nunc) with OVA (10 μg/mL), LPS (25 μg/mL), or NP₃₀-BSA (5 μg/mL). The coated plates were incubated with 2 % Block Ace (for OVA-coated plates) (Dainippon Sumitomo Pharmaceuticals, Osaka, Japan) or 1 % BSA (for LPS or NP₃₀-BSA-coated plates) for 2 h at room temperature. Plasma dilutions were added to the antigen-coated plates. After incubation with the plasma for 2 h at room temperature, the coated plates were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG1, IgM, or IgG3 solution (SouthernBiotech, Birmingham, AL, USA) for 2 h at room temperature. After the incubation, the color reaction was developed with tetramethylbenzidine (Moss, Inc.; Pasadena, MD, USA), stopped with 2 N H₂SO₄, and measured at OD_450–620 on a microplate reader. To detect OVA-specific IgE, we coated ELISA plates with purified rat anti-mouse IgE (2 μg/mL) and detected OVA-specific IgE with biotin-conjugated OVA (5 μg/mL) followed by horseradish peroxidase-coupled streptavidin (Southern Biotechnology Associates, Birmingham, AL).