Goat anti mouse immunoglobulin antibody

VLPs purified from Ad5 infected vero cells were prepared as previously described [31 (link)] and characterized by western blot. Env was detected with the SIVmac251 gp120 specific monoclonal antibody (mAb) KK46 [34 (link)] [NIH AIDS Research and Reference Reagent Program (NARRRP)] followed by HRP coupled goat anti-mouse immunoglobulin antibody (Dako). The same blot was analysed for the presence of Gag using HIV-1 anti-p24 mAb 183-H12-5C [35 (link)] (NARRRP), and goat anti-mouse immunoglobulin antibody (Dako). SIVmac239 gp130 [36 (link)] and SIVmac251 BK28 pr55 Gag were loaded as positive controls (NARRRP). The blots were developed using ChemiLucent Detection System kit (Pierce). Analysis was performed using Image Studio Lite software (LI-COR Biosciences).

T cell analyses were performed in purified peripheral blood mononuclear cells (PBMCs). A primary stain with unlabelled murine anti-human antibodies directed against PD-1 (clone EH12.2H7) or isotype control followed with secondary phycoerythrin (PE)-labelled goat-anti-mouse immunoglobulin antibody (Dako, Glostrup, Denmark) was followed by primary labelled surface stains as follows: CD4 (Pacific Blue); CD5 (allophycocyanin (APC)-AlexaFluor 700); CD25 (fluorescein isothiocyanate, FITC); CD45RA (energy-coupled dye, ECD or FITC); CD62L (PE or phycoerythrin-cyanin 5, PC5); CD127 (PE) (all from Beckman Coulter, CA, USA); CD8 (Pacific Orange) (Invitrogen, MD, USA); CD183 (APC) and CD194 (phycoerythrin-cyanin 7, PC7) (all from BD Biosciences, CA, USA). Analyses were done on a Gallios flow cytometer and T cell subsets were defined as follows: naïve (CD62L+CD45RA+), central memory (CM, CD62L−CD45RA+), effector memory (EM, CD62L−CD45RA−), TEMRA (CD62L+CD45RA−), TH1 (CD183+CD194−), TH2 (CD183−CD194+) and Treg (CD25+CD127−). Statistical analyses were performed using unpaired t tests for all subgroup results. A simple Bonferroni correction was applied to adjust for multiple testing, leading to a necessary p value of < 0.0025 for the definition of significance.