Biophys. binding to Galcer liposomes and the subsequent dissociation phases are shown. The PBS-treated Galcer liposome binding curve was normalized to the anti-Galcer binding endpoint. Signal from a blank sensor treated similarly was subtracted from the experimental to obtain the specific binding signal shown here. For the Galcer-blocking assay, Galcer-liposome binding of 1086.C gp140 at 50 g/ml was monitored for 30 min, and dissociation was monitored for 1 h. In parallel, Galcer-liposome binding of 1086.C gp140 (50 g/ml) incubated with a 3 M excess of antibodies was monitored. The Galcer-liposome-binding responses (after subtracting the signal found with blank sensors) at the end of a 1-h dissociation phase were averaged (over a 20-s window). The percent Galcer blocking was calculated using the following equation: [(A ? B)/A] 100%, where A is the Galcer binding response of 1086.C gp140 and B is the Galcer binding response of 1086.C gp140 in the presence of a 3 M excess of the antibody of interest. The binding of 1086.C gp140 to CH38 IgG and IgA2 antibodies pairs was measured by coupling the antibodies to amine-reactive sensors (AR2G sensors) as per the manufacturer’s instruction. The antibody-coupled AR2G sensors were dipped into wells containing 1086.C gp140 at various concentrations and subsequently in PBS buffer for monitoring association and dissociation, respectively. CH65 IgG (a broadly neutralizing influenza virus antibody [48]) antibody-immobilized AR2G sensors were used as blank sensors so that nonspecific binding signal could be subtracted. The experiments were performed in triplicate. Titration curves were fit to a Langmuir 1:1 binding model using ForteBio Data Analysis software. SPR assay. All surface plasmon resonance (SPR) assays were performed using a Biacore 3000 instrument at 25C, and data analyses were done using BIAEvaluation 4.1 software. The 17B MAb upregulation assay was performed using a CM5 chip immobilized with 17B MAb (6,000 to 7,000 resonance units [RU]) by a standard amine-coupling procedure in three flow cells. The fourth flow cell was immobilized with 6,000 (E)-Alprenoxime RU of Synagis (47) and used as a negative control surface to determine responses due to nonspecific interactions to be subtracted as background. The T/F HIV Env 1086.C gp140 (40 g/ml) was set up to flow over the antibody surfaces at a 20-l/minute flow rate for 2 min. Dissociation was followed for 500 s after the injection of Env protein was complete. In order to measure 17B upregulation, 1086.C gp140 (40 g/ml) was mixed with the antibodies (100 g/ml) listed in Table 1, below, and injected over antibody surfaces. The binding data were processed to obtain the specific binding response by subtracting the binding response on the RSV-specific MAb Synagis surface. The specific binding responses from three 17B surfaces were averaged and are presented in Table 1. The percentage of 17B upregulation was calculated using the following equation: [(D ? C)/C] 100%, where C and D are the Rabbit polyclonal to PHF7 17B-binding responses of 1086.C gp140 in the absence and in the presence of antibody, respectively. TABLE 1 1086.C gp140 binding to CD4i epitope-specific MAb 17B in the absence or presence of various C1-specific antibodies (E)-Alprenoxime and control antibodiesand and the derived values. The black lines are the best fit of the time courses. Titrations were carried out in duplicate. Representative data with fits are shown. (C and D) Representative raw data and isotherms of ITC measurements of the interaction of CH38 IgG (C) and CH38 IgA2 (D). Statistical analysis. A Spearman correlation analysis was used to determine the nonparametric statistical dependence between 1086.C gp140-binding and Galcer-blocking abilities. The apparent outliers were not included in this analysis. Binding to Env on the surface of HIV-1 infected CD4+ cells. Primary CD4+ T cells were isolated from an HIV-1-seronegative donor, activated, and infected with an infectious molecular clone that encodes the HIV-1 subtype C Env from isolate 1086.C (GenBank accession number ACS67968) in an isogenic backbone that contains the luciferase reporter gene and all viral open reading frames (49). Cell activation and infection were conducted as previously described (50). Mock-infected and HIV-1 1086.C-infected cells were incubated with the RSV-specific negative control Palivizumab or with MAb CH38 at 1 g/ml for 2 h at 37C. Primary Ab binding was detected by secondary labeling with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (KPL Inc., Gaithersburg, MD). Live and HIV-1-infected cells were identified by staining with a viability dye and analyzed for intracellular expression of p24 by (E)-Alprenoxime using standard methods. ADCC assays. ADCC activity was determined in a luciferase-based assay as previously described (51). Briefly, CEM.NKRCCR5 cells (from Alexandra Trkola; NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) infected with the HIV-1 1086.C infectious.
