Goat anti mouse igg alkaline phosphatase antibody

To analyse serum antibody responses in sera from immunized animals, Immuno MaxiSorp 96-well microplates (Nunc) were coated with soluble gp120AG (2 µg/mL), M.CON.S D11 gp120 (2 µg/mL), C2V3C3 polypeptide (2 µg/mL) or supernatant from VV_WR-infected cells. After overnight incubation at 4 °C, the microplates were blocked with 2% gelatin (Bio-Rad). Mouse sera from days 0, 15, 30, 45 and 60 were heat-inactivated at 56 °C for 1 h, added to the microplates (1:200 final dilution in a total volume of 100 µL) and incubated for 2 h at room temperature. Goat anti-mouse IgG alkaline phosphatase antibody (Sigma) at 1:2000 dilution was added to the microplates as a secondary antibody. The colourimetric reaction was developed as described above. Negative controls were pre-immune serum and serum from mice immunised with VV_WR. Positive controls were sera from individuals with HIV-1 and HIV-2. In this case, the secondary antibody was a goat anti-human IgG alkaline phosphatase antibody (Sigma). Sera with an optical density (OD) greater than that of the preimmune controls were considered positive.

Phenol-water extracts and total cell lysates were analyzed by 12% Bis‐Tris Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and silver stained using the SilverQuest Silver Staining Kit (Invitrogen). Samples were transferred to nitrocellulose membranes and incubated with a 1:1000 dilution of the primary antibody, washed and then incubated with a 1:5000 dilution of the appropriate secondary antibody (Goat anti-Mouse IgG-Alkaline Phosphatase antibody, Sigma-Aldrich, or Goat anti-Rabbit IgG-Alkaline Phosphatase conjugate, Invitrogen). Immunoblots were developed using the SIGMAFAST BCIP/NBT tablet (Sigma-Aldrich) solution. At least two independent experiments were performed. Representative blots are shown.

Vaccine samples were prepared in Laemmli buffer (BioRad) and proteins separated by SDS-PAGE on pre-cast NuPAGE 4-12% Bis-Tris Midi Protein Gels (Invitrogen) using NuPAGE MES SDS Running Buffer (Invitrogen). Total protein was either visualized by silver staining (Pierce Silver Stain Kit, Thermo Scientific) or blotted on nitrocellulose membranes using the BioRad TransBlot Turbo Transfer System (BioRad). Fusion proteins were detected using mouse anti-HBsAg (BioRad, MCA4658) and goat anti-mouse IgG-Alkaline Phosphatase antibody (Sigma, A3562). Western blots were developed with SIGMAFAST BCIP/NBT (Sigma).