, 2000, 2003) but not for clearance of established deposits Prev

, 2000, 2003) but not for clearance of established deposits. Previous studies demonstrated that the modified Aβp3-42 peptide

accumulates early in the deposition cascade (Iwatsubo et al., 1996; Saido et al., 1995) and probably was specific for plaque (i.e., no soluble peptide found in physiological fluids). We immunized mice with the Aβp3-42 peptide and subsequently screened clones for Aβp3-x binding and counterscreened against Aβ1-42. A low-affinity Aβp3-42 monoclonal antibody was affinity matured to yield the high-affinity (140 pM) anti-Aβp3-x antibody mE8 (koff < 1 × 10−5/s at 25°C). Characterization of the binding properties of mE8 demonstrated that it specifically recognized the modified amino terminus of Aβp3-x in that it does not recognize full-length Aβ or unmodified Aβ3-x

http://www.selleckchem.com/products/3-methyladenine.html (see Figure S1 available online). In order to evaluate the impact of effector function on in vivo plaque clearance, mE8 was made in both mouse IgG1 (minimal effector function) and IgG2a (maximal effector function) isotypes. The affinity-matured mE8 was first used to investigate levels of the Aβp3-42 http://www.selleckchem.com/btk.html peptide in PDAPP and AD brain lysates. ELISA analyses demonstrated that low levels of the Aβp3-42 peptide could be detected in both PDAPP and AD brains (Figure 2A). Interestingly, the prevalence of the Aβp3-42 peptide was quite low (∼0.6%) with respect to the overall amount of Aβ42 deposited in these brains (Figure 2B). The analyses also showed an age-dependent accumulation of Aβp3-42 peptide in PDAPP brains that increased 47-fold between 12 and 23 months

of age (Figure 2A). The similar prevalence of Aβp3-42 in AD patients and PDAPP mouse brains demonstrates that our transgenic model recapitulates the generation of this neuropathological target. Immunohistochemical analyses were performed with anti-Aβp3-x antibodies in order to determine whether the epitope is accessible in aged PDAPP and AD brain sections. Robust Aβ staining was observed in brain sections from a 24-month-old PDAPP mouse with 3D6 (Figure 2C) and more discrete staining was observed for the mE8 antibody (Figure 2D). Histological analyses performed on fresh-frozen AD brain resulted in similar staining between the 3D6 and mE8 antibodies, where again the labeling was more intense and widespread for the 3D6 antibody Unoprostone (Figures 2E and 2F). We next investigated whether the relatively low levels of the Aβp3-42 antigen would be sufficient to enable opsonization and Fc receptor-mediated phagocytosis. Ex vivo phagocytosis studies were performed with exogenously added Aβ antibodies preincubated with AD brain sections that were subsequently treated with primary murine microglial cells (Figure 2G). The following murine Aβ antibodies were investigated: 3D6 (anti-Aβ1-x, IgG2b), mE8 (anti-Aβp3-x, IgG1), mE8 (anti-Aβp3-x, IgG2a), 21F12 (anti-Aβx-42, IgG1), 2G3 (anti-Aβx-40, IgG1), and a murine control antibody (IgG2b, same effector function as 3D6).

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