The monoclonal antibody (MAb) 3C2 (18) recognizes the viral GAG protein and can be used to assess the extent of infection following transfection of line 0 CEF cells (Subheading 3.3.) and/or to determine the viral titer of concentrated supernatant (Subheading 3.4.).
1. Rinse cells twice with 1X PBS.
2. Fix for 15-30 min at room temperature with 3.5% (v/v) paraformaldehyde in PBS.
3. Rinse cells twice with 1X PBS.
4. Block 3X 30' at room temperature in CEF media containing 0.05% Triton X-100.
5. Incubate in 3C2 1° antibody (diluted 1:4 in CEF plus 0.05% Triton X-100) overnight at 4°C. The 1° antibody can be reused several times, and between uses should be kept at 4°C in the presence of 0.02% (w/v) sodium azide.
6. Rinse 3X 30' with 1X PBS containing 10% serum (e.g., goat serum).
7. Incubate in 2° antibody (peroxidase-conjugated antimouse IgG, IgM diluted 1:200 in 1X PBS plus 10% serum) for at least 1 h at room temperature.
9. Develop with activated DAB. To enhance the signal, 0.5 mg/mL nickel chloride can be added to the DAB solution giving a black precipitate, which is easier to visualize (see Fig. 1).
10. The reaction is stopped by rinsing several times with 1X PBS.
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