1 Supporting Information SI Materials and Methods Plasma IgG adsorptions on SF162 gp120- and SF162 gp120 D368R -coated beads. Plasma adsorptions were performed as previously described (1, 3), with some modifications, and the flow of the adsorptions is outlined in Figure S1. Briefly, total IgG was isolated by protein A chromatography (Pierce, Rockford Il, USA) from VC10042 plasma drawn 22 years post-infection. The purified total IgG was serially adsorbed onto SF162 gp120-coated beads (MyOne Tosylactivated Dynabeads, Invitrogen, Carlsbad, CA, USA), and the flow through collected. The antibodies bound to the gp120 coated beads were eluted by vortexing in increasingly acidic 0.1M glycine solutions, followed by buffer exchange into PBS. The anti-gp120 Ab fraction was then serially adsorbed onto SF162 gp120 D368R -coupled beads to remove Abs that do not bind the CD4-BS. Each fraction described above was tested for residual neutralizing activity against 4 clade B, 3 clade C, and 2 clade A isolates in the TZM-bl neutralization assay. The depleted total IgG and the gp120 D368R depleted anti-gp120 fractions were tested for the presence of anti-CD4-BS antibodies, and the absence of non-CD4-BS gp120 Abs by Luminex assay (Luminex Corporation, Austin, TX, USA) against both wild type SF162 gp120 and SF162 gp120 D368R .
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Supporting Information SI Materials and Methods Plasma IgG adsorptions on SF162 gp120- and SF162 gp120D368R-coated beads. Plasma adsorptions were performed as previously described (1, 3), with some modifications, and the flow of the adsorptions is outlined in Figure S1. Briefly, total IgG was isolated by protein A chromatography (Pierce, Rockford Il, USA) from VC10042 plasma drawn 22 years post-infection. The purified total IgG was serially adsorbed onto SF162 gp120-coated beads (MyOne Tosylactivated Dynabeads, Invitrogen, Carlsbad, CA, USA), and the flow through collected. The antibodies bound to the gp120 coated beads were eluted by vortexing in increasingly acidic 0.1M glycine solutions, followed by buffer exchange into PBS. The anti-gp120 Ab fraction was then serially adsorbed onto SF162 gp120D368R-coupled beads to remove Abs that do not bind the CD4-BS. Each fraction described above was tested for residual neutralizing activity against 4 clade B, 3 clade C, and 2 clade A isolates in the TZM-bl neutralization assay. The depleted total IgG and the gp120D368R depleted anti-gp120 fractions were tested for the presence of anti-CD4-BS antibodies, and the absence of non-CD4-BS gp120 Abs by Luminex assay (Luminex Corporation, Austin, TX, USA) against both wild type SF162 gp120 and SF162 gp120D368R.
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