In the post-exposure model, protection was complete for up to three hours, which is comparable to what was demonstrated with FP complexes and other polyclonal antibody mixtures (Al-Saleem et al., 2011; Cheng et al., 2009; Sepulveda et al., 2010). mAbs into HPs on toxin neutralization and handling and 2 HPs, rather than an HP + mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2 hours, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages of HP and HP complexes binding to RBCs Blood from Tg-hCR1 mice was collected in heparinized tubes and RBCs were isolated. The RBCs were washed with 200 l PBS/1% BSA (PBSA) and centrifuged at 326 g in a microfuge. HC50A, the 50 kD C-terminal domain name of BoNT serotype A (13), was biotinylated using a FluoReporter Mini-biotin-XX protein labeling kit (Invitrogen, Carlsbad, CA). Biotinylated HC50A (BIOT-A) was incubated with 1:100 diluted PE-Streptavidin (PE-SA; Jackson ImmunoResearch, West Grove, PA), rotating for 30 min at 4 C. BIOT-A with PE-SA was then added to RBCs with 20 ng HP and anti-human IgG APC (Jackson Immunoresearch), incubated at RT for 30 min, washed twice in PBSA, resuspended in a final volume of 1 ml PBSA, and analyzed by flow cytometry for RBCs that were double positive, thus indicating that both HP and biotinylated HC50A were bound to the RBCs. 2.4. BoNT proteins Serotype A1 BoNT (BoNT/A) was obtained from Metabiologics, Inc. (Madison, WI). The recombinant 50 kD C-terminal domain (HC50A) and a recombinant inactive BoNT/A (RI-BoNT) were produced in following published YM-90709 methods (Pier et al., 2008; Ravichandran et al., 2007). 2.5. Analysis of RBC binding by HPs as unmodified mAbs and in studies of immune adherence induced by the FP (Adekar et al., 2011; Adekar et al., 2008b). Both mAbs were converted into HPs by cross-linking with murine mAbs, 7G9 or HB8592 or 7B7. 7G9 and HB8592 are specific for the hCR1, but bind different CR1 epitopes; 7B7 is an isotype control mAb that does not bind CR1. Following cross-linking, the HPs were separated from monomeric IgG by chromatography Thbs1 using a Superose 6 column (M.A. Lindorfer and R. P. Taylor, data not shown). HPs YM-90709 incorporating the 7G9 were named 6A-HP and 4LCA-HP, those with the HB8592 mAb were named 6A-HP-HB and 4LCA-HP-HB, and those with the control mAb 7B7 were named 6A-HP-CTRL and 4LCA-HP-CTRL. To test the binding and activity of the HPs, we used the transgenic mouse Tg-hCR1, which expresses the human CR1 protein (hCR1) on the surface of its RBCs (Repik et al., 2005). Murine RBCs do not express a CR1 receptor that can YM-90709 bind complement-opsonized immune complexes, rather, their platelets perform this function using platelet-associated factor H (Alexander et al., 2001). We tested the ability of the HPs to adhere BoNT to the Tg-hCR1 RBC surface by mixing the HPs and biotinylated RI-BoNT holotoxin with RBCs and detecting the bound complexes with PE:SA and an APC anti-human Fc secondary (Figure 1). A double positive population of RBCs was only seen with the CR1-specific HPs 6A-HP (75.5%), 6A-HP-HB (76.4%), 4LCA-HP (75.4%), 4LCA-HP-HB (73.3%). Substantially less binding was observed with the two non-binding HPs, 6A-HP-CTRL (12.8%) and 4LCA-HP-CTRL (17.6%). Open in a separate window Figure 1 Binding of HP + BoNT complexes to Tg-hCR1 RBCs. Tg-hCR1 RBCs were incubated with biotinylated RI-BoNT/A, PE-SA, anti-human IgG APC, along with the following mAbs or HPs: 6A mAb, 6A-HP, 6A-HP-HB, 6A-HP-CTRL, 4LCA mAb, 4LCA-HP, 4LCA-HP-HB, or 4LCA-HP-CTRL. Percent double positive RBC values are given in the upper right quadrant of each diagram. 3.2. Protection conferred by HPs We first tested whether conversion of the mAbs to HPs improved their ability to neutralize toxin to activated peritoneal macrophages from Tg-hCR1 mice. Nuclei were stained with DAPI. Preparations include: a) Cells only; b) BoNT antiserum; c) 6A + 4LCA-HP; d) 6A-HP + 4LCA; e) BoNT alone (no HPs or mAbs); f) 6A + 4LCA YM-90709 mAb; g) 6A-HP-CTRL + 4LCA-HP-CTRL YM-90709 ; h) 6A-HP + 4LCA-HP. 80X. We quantitated these results by measuring the Alexa-fluor corrected total cell fluorescence (CTCF) for each.