Alkaline phosphatase conjugated goat anti mouse igg h l secondary detection antibody

Where applicable, immunized mice were bled via saphenous vein 5 weeks post-immunization. Anti-Ova serum IgG titers were measured via indirect ELISA. Costar 3590 96-well EIA/RIA high binding plates (Corning) were coated with 100 µL of Ova (5 µg/mL PBS) or PA (0.5 µg/mL PBS) and incubated overnight at 4°C. Plates were blocked using 2.5% (w/v) powdered skim milk PBS containing 0.05% Tween-20 (PBS-T), that has been heat inactivated at 56°C for hours to inactivate any phosphatase activity, for two hours at room temperature. After three washes using PBS-T, serum samples were titrated across the plate using two-fold serial dilutions, starting at 1:100, in PBS-T and 1% (v/v) normal goat serum. Samples were incubated overnight at 4° C. After three washes in PBS-T, an alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) secondary detection antibody (Cat# 115–005-003, Jackson ImmunoResearch, West Grove, PA) was diluted 1:1000 in PBS-T and added to the wells and allowed to incubate at room temperature for two hours. Plates were washed three times with PBS-T and alkaline phosphatase substrate was added at 1 mg/mL in buffer containing 50 mM sodium carbonate, 2 mM magnesium chloride, and sodium bicarbonate was titrated into the solution in order to achieve a pH of 9.3. Plates were allowed to develop for 30 min and analyzed using the SpectraMAX 190 at a wavelength of 405 nm.