Hrp coupled polyclonal rabbit anti human immunoglobulins

For Western blot analysis and quantification, VLP samples were prepared as previously described [32 (link)]. SIVmac239 Gag was detected using either anti-SIV mac251 p27 Gag antibody [33 (link)] (MyBioSource) followed by HRP coupled polyclonal rabbit anti-mouse immunoglobulins (Dako) or SIV mac251 antiserum [34 (link)] (NARRP) followed by HRP coupled polyclonal rabbit anti-human immunoglobulins (Dako). HIV-1 M CON-S Env variants were probed with an antibody cocktail consisting of VRC01 (1 µg/ml) [27 (link)] and b12 (1 µg/ml) [35 (link)], and 2G12 [36 (link), 37 (link)] (1 µg/ml) (NARRP), and then detected with HRP coupled polyclonal rabbit anti-human immunoglobulins (Dako). The density of Gag bands were quantified using Image Studio Lite software (LI-COR Biosciences).

For Env quantification, the HIV-1 M CON-S gp160 ELISA was adapted. Purified VLPs were prepared in lysing buffer (Zeptometrix) and added in two-fold dilutions to a ConA coated MaxiSorp plate. HIV-1 M CON-S gp140 CFI avi was used as a standard. After blocking, Env was detected with an anti-gp120 cocktail at 1 µg/ml consisting of VRC01, b12 and 2G12 (NARRP). HRP coupled polyclonal rabbit anti-human immunoglobulins (Dako) were used to detect the mAbs.