Alkaline phosphatase conjugated anti mouse igg antibody

Enzyme-Linked ImmunoSorbent Assay (ELISA) was performed using both E. coli and CHO rPvs48/45 proteins, as described before [27 (link)]. Briefly, 96-well plates (Nunc-Immuno Plate, Maxisorp, Roskilde, Denmark) were coated with one μg/mL of rPvs48/45 produced in CHO or E. coli in PBS, pH 7.4 at 4°C, overnight. After plates were blocked with 5% skim milk solution [PBS 1X, 0.05% Tween 20, (PBS-T)], sera samples were added at three-fold serial dilutions starting at 1:300 in 2.5% skim milk in PBS-T and were incubated for 1 hour. All sera were pre-adsorbed in E. coli powder to remove any anti-E. coli antibodies potentially present. After the reaction, plates were washed and incubated with alkaline phosphatase-conjugated anti-mouse IgG antibody (Sigma Chemical Co., St Louis, MO) at a 1:1000 dilution for 1 hour. Reactions were revealed with para-nitrophenyl phosphate substrate (p-NPP) (Sigma Aldrich) and read at 405 nm wavelength (Dynex Technologies, Inc., MRX Chantilly, VA). Cut-off points for ELISA were calculated as three SD above the mean absorbance value at 405 nm of sera from naive mice.