Hrp conjugated goat anti human igg antibody

The antibodies (and their Env epitopes) used in this study include the bNAbs VRC01, VRC03, 3BNC117 and b12 (CD4-binding site, CD4BS); PGT145 and PG9 (V2 quaternary, V2q); PGT151 and 35O22 (gp120–gp41 interface); 2G12 (gp120 outer domain glycans); and 10E8 (gp41 MPER). The pNAbs used in this study include 19b and 39F (gp120 V3); b6 and F105 (gp120 CD4BS); 902090 (gp120 V2 linear); 17b and E51 (gp120 CD4-induced, CD4i); and F240 (gp41 Cluster I). CD4-Ig is a fusion protein consisting of the N-terminal two domains of human CD4 and the Fc portion of an antibody^{14 (link)}. Antibodies against HIV-1 Env were kindly supplied by Dennis Burton (Scripps), Peter Kwong and John Mascola (Vaccine Research Center, NIH), Barton Haynes (Duke University), Michel Nussenzweig (Rockefeller University), Hermann Katinger (Polymun), James Robinson (Tulane University) and Marshall Posner (Mount Sinai Medical Center). In some cases, anti-Env antibodies were obtained through the NIH AIDS Reagent Program. Antibodies for Western blotting included goat anti-gp120 polyclonal antibody (Thermo Fisher) and the 4E10 anti-gp41 antibody (Polymun). A horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (Thermo Fisher) and an HRP-conjugated goat anti-human IgG antibody (Santa Cruz) were used as secondary antibodies for Western blotting.

The AE2 Env was purified as described above, except that counterselection with the 19b and F240 pNAbs was omitted. The purified AE2 Env was incubated with antibodies or CD4-Ig together with Protein A-Sepharose beads for one hour at 4 °C. The precipitated Envs were analyzed by Western blotting with a 1:5000 dilution of a goat anti-gp120 polyclonal antibody (Thermo Fisher) and 0.5 μg/ml 4E10 anti-gp41 antibody (Polymun). Secondary antibodies at a 1:5000 dilution were an HRP-conjugated rabbit anti-goat antibody (Thermo Fisher) and an HRP-conjugated goat anti-human IgG antibody (Santa Cruz), respectively.

The purified protein was dialyzed to remove urea and subsequently coated on nitrocellulose membrane (50 mm × 5 mm). The control strip was coated with PBS only. After drying, the strips were blocked with 3% BSA in PBS followed by incubation overnight at 4°C. Human sera serially diluted in PBS (1: 500, 1: 1000, 1: 2000, 1: 3000 and 1: 5000) were used as primary antibody for detection of anti-TTV antibodies. A serum without TTV-DNA (1: 500 dilution) was used in negative control. Diluted sera were added to the NC strips and incubated at room temperature for 2 hours with constant shaking. Secondary antibody (goat anti-human IgG-HRP conjugated antibody; Santa Cruz Biotechnology, USA) was added in 1: 4000 dilution in PBS and incubated for 2 hours at room temperature. The strip treated with anti-His (His probe H-3) primary antibody was used as positive control. The blot was developed using HRP-conjugated anti-mouse IgG antibody as detailed earlier. Finally, the substrate (DAB) was used for detection of antigen-antibody complex in all strips. The development of colour in the coated region of strips confirms the binding of anti-TTV antibodies to expressed N22 translational product.