Biotin rat anti mouse igg2b

Whole blood was collected on day 21. Clotting was allowed overnight at 4°C and serum was recovered after centrifugation for 10 min at 8000 xg. Samples were serially diluted 1:1 in assay diluent and incubated on antigen-medium-binding pre-coated plates (10 μg/mL OVA or 50 μg/mL ClfA in sodium carbonate buffer, pH 9.5). Ag-specific serum Ab titers were measured using a standard ELISA protocol and the following antibodies: goat anti-mouse total IgG-HRP from Southern Biotech (1/5000 dilution), biotin rat anti-mouse IgG1 from BD (1/4000 dilution), biotin rat anti-mouse IgG2b from BD (1/4000 dilution), and goat anti-mouse IgG2c-HRP from AbD Serotec (1/2000 dilution) and developed using streptavidin-HRP or OPD substrate directly as required. OD values were obtained using a VersaMax microplate reader at 492nm. Antibody concentrations were expressed as Log10 endpoint titers calculated by regression of a curve of OD values versus reciprocal serum levels to a cutoff point of 4 SD above the blank.