Horseradish peroxidase hrp labeled streptavidin

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 1 μg/ml Qβ VLPs overnight at 4°C. Sera of the different time points were applied with a 1:10 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 7 M urea in PBST (PBS containing 0.05% Tween20) 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 (Jackson ImmunoResearch, 016-030-084). Total 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 served as basis for avidity index calculation. The avidity index (AI) was calculated by AI_x = OD (dilution x) + urea/OD (dilution x)–urea.

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 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 (Jackson ImmunoResearch, 016-030-084).
Total 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 or endpoint antibody titers. The OD50 antibody titers are defined as the reciprocal of the dilution that reaches half of the maximal optical density (ODmax). The endpoint antibody titers are defined as the reciprocal of the last dilution above the threshold, which is set above the background level.