2.8. Nef Antibodies and Detection Reagents

Different anti-Nef antibodies and reagents were used: (1) anti-Nef JR6, a mouse monoclonal antibody (Abcam ab42358); (2) anti-Nef 2A3, a mouse monoclonal antibody (Abcam ab77172); (3 and 4) anti-Nef sheep serum, either as a purified biotinylated polyclonal antibody or non-labeled (both from Targeted Affinity Oy, Helsinki); (5) anti-Nef polyclonal serum (provided by Mark Harris, Leed University). All Nef-antibodies were used to demonstrate the presence of Nef in pEV. For immunoblotting JR6 turned out to have the highest sensitivity and specificity as judged by the ratio of Nef vs. background staining. For detection in tissue we used the biotinylated anti-Nef sheep serum and the JR6 antibody.