Sera were tested for HIV-1 gp140-specific and V2-specific Abs by ELISA on plates coated with gp140 protein or linear V2 peptide, respectively. HIV-1 strain Du172 gp140 protein was produced in baculovirus[19 (link)]. V2 peptide was based on a 19 amino acid immunodominant linear epitope within V2 of HIV-1 strain Du172: TTTEITDKKKKEYALFYKL (Genscript, Piscataway, NJ)[20 (link)]. Gp140-specific IgG was tested in 2-fold serum dilutions from 1:1000–1:16,000. V2-specific IgG was tested using 2-fold serum dilutions from 1:100–1:6400. Briefly, 96-well plates (Nunc-Immuno™) were coated overnight at 4°C with (A)gp140 protein (200ng/well) or (B)V2 peptide (400ng/well). Wells were blocked overnight at 4°C with (A)PBS/3% BSA/0.1% Tween-20 or (B)PBS/8% BSA/0.1% Tween-20. Duplicate samples and a standard were incubated at RT for (A)2h or (B)90 min. Bound IgG was detected with goat anti-mouse IgG alkaline phosphatase conjugate (Sigma-Aldrich) at (A)1:30,000 or (B)1:15,000. Bound enzyme was detected with phosphatase substrate tablets (Sigma-Aldrich) in DEA substrate buffer (KPL) by optical density (OD) at 405 nm. Gp140-specific IgG is reported as area under the curve (AUC) for 1:2000–1:16,000 dilution curves. V2-specific IgG is reported as OD at 1:100 dilution.