Horseradish peroxidase hrp labeled streptavidin

A non-neutralizing anti-IL-6R Nanobody (20 μg/mL) was immobilized on 96-well microtiter plates, after which the coated plates were blocked with Superblock T20 (ThermoFisher Scientific, Waltham, MA, USA). A dilution series of ALX-0061 in the absence or presence of 1 mg/mL HSA was first pre-incubated for one hour with 100 ng/mL recombinant hIL-6 (Gentaur, Kampenhout, Belgium) and 4 ng/mL recombinant hIL-6R, after which the mixtures were transferred to the coated wells. Bound IL-6R/IL-6 complexes were detected with 100 ng/mL of a biotinylated polyclonal anti-hIL-6 mAb (R&D Systems, Minneapolis, MN, USA), followed by horseradish peroxidase (HRP)-labeled streptavidin (Dako, Glostrup, Denmark, 1/5,000 or ThermoFisher Scientific, Waltham, MA, 1/25,000). Visualization was performed with esTMB (Stereospecific Detection Technologies reagents, Baesweiler, Germany), after which the coloring reaction was stopped with 1 M HCl. The absorbance was determined at 450 nm using 620 nm as reference wavelength.

Serum samples were obtained from blood collected at the indicated time points during experiments using Microtainer tubes (BD, 365967). Corning half area 96 well-plates were coated with 50 μl of 1 μg/ml Qβ VLPs overnight at 4°C. Sera were 1:10 pre-diluted and 1:4 further serial diluted to analyse a total of 7 dilutions per sample. Qβ specific antibodies were detected using mouse anti-mouse IgG for both allotypes. IgHa-specific (biotin ms anti-ms IgG1[a] (10.9), biotin ms anti-ms IgG2a[a] (8.3) from BD) and IgHb-specific (biotin ms anti-ms IgG1[b] (B68-2), biotin ms anti-ms IgG2a[b] (5.7) from BD) antibodies were detected using horseradish peroxidase (HRP) labeled streptavidin (Dako).
Cell supernatants were used undiluted and a 1:2 serial dilution was performed. An anti-Qβ monoclonal antibody (purified from hybridoma cells) was used as a standard to quantify specific antibodies in the supernatants. Qβ specific antibodies were detected using goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-035-071).
The absorbance readings of the tetramethylbenzidine (TMB) color reaction at 450 nm for the serum samples were interpreted as OD50 antibody titers. The OD50 antibody titers are defined as the reciprocal of the dilution that reaches half of the OD max. The anti-Qβ monoclonal antibody standard curve was used to calculate antibody concentrations in the cell supernatants.

Serum samples were obtained from blood collected at the indicated time points during experiments using Microtainer tubes (BD, 365967). Corning half area 96 well-plates were coated with 50 μl of 1 μg/ml Qβ VLPs overnight at 4°C. Sera of the different time points were applied with a 1:20 pre-dilution and 1:4 further serial diluted. After 1 h incubation, the sera were washed off and the plates washed 3 times 5 min either with 7M urea in PBST (PBS containing 0.05% tween 20) or PBST only. Qβ specific antibodies were detected using mouse anti-mouse IgG for both allotypes. IgHa-specific (biotin ms anti-ms IgG1[a] (10.9), biotin ms anti-ms IgG2a[a] (8.3) from BD) and IgHb-specific (biotin ms anti-ms IgG1[b] (B68-2), biotin ms anti-ms IgG2a[b] (5.7) from BD) antibodies were detected using horseradish peroxidase (HRP) labeled streptavidin (Dako). The absorbance readings of the tetramethylbenzidine (TMB) color reaction at 450 nm served as basis for avidity index calculation. The avidity index (AI) was calculated by AI_x = OD (dilution x) + urea / OD (dilution x)–urea.