IgG2a antibodies, which exhibit higher affinity than additional isotypes for phagocytic Fc receptors (in particular FcRI), provided the highest level of plaque clearance and were the only anti-A antibodies to provide neuronal protection under the conditions tested. with reactivity against different A epitopes and monoclonal antibodies with different isotypes were examined for effectiveness both and assay with sections of PDAPP or Alzheimer’s disease (AD) brain, there was a strong correlation between those that produced efficacy and those that were efficacious by mechanisms that are self-employed of Fc relationships (8). Studies possess indicated that an antibody directed against the midportion of A, which cannot identify amyloid plaques, appears to bind to soluble A and reduce plaque deposition (6). In addition, it has been reported recently that short-term treatment with this antibody improved overall performance in an object-recognition task without influencing amyloid burden (9). To understand the parameters of an antibody response that are required for neuronal safety, several questions should be considered. Is neuronal safety associated with plaque clearance, or is it necessary for antibodies to capture soluble aggregates of A to protect neurons against the directly toxic effects of the peptide? Does a clearance response depend on Fc receptor-mediated phagocytosis of A after antibody binding or on match receptor-mediated phagocytosis after antibody binding and match activation? Alternatively, is definitely a clearance response self-employed of antibody Fc receptor function? In the current study we approached these questions by analyzing the influence of different antibody epitopes and isotypes on plaque clearance and neuronal safety. The studies required advantage of the fact that some epitopes of A are preferentially available for antibody binding within plaques, whereas others are only available for antibody capture of the soluble peptide. In addition, the isotype of an antibody is important for either Fc- or complement-mediated phagocytosis of A by microglial cells, because antibody isotype defines its affinity for Fc receptors as well as its ability to activate match. If plaque clearance and/or neuronal safety do not depend on Fc-mediated processes, then the isotype of an antibody against A should have little impact on efficacy. These studies provide insight for the design CALN of antibodies with restorative potential. Materials and Methods A Fragments. Peptides related to A1C5, A3C9, A5C11, and A15C24 and the reverse sequence A5C1 were synthesized contiguous to a 17-aa T cell epitope derived from ovalbumin ML221 (amino acids 323C339, ISQAVHAAHAEINEAGR) on a branched peptide platform (triple-lysine core with four peptide arms) to produce a multiantigen peptide as explained (10). Polyclonal antibodies against A1C42 (pAb 1C42) were raised and the Ig portion was isolated as explained (7). pAb-EL16, pAb-EL17, and pAb-EL20 were from the sera of PDAPP mice immunized with peptides related to A1C7, A15C24, and A3C9, respectively, which had been synthesized on a branched platform as explained above. pAb-EL26 was from the sera of mice immunized having a(7C1)-42. The peptides were synthesized by AnaSpec (San Jose, CA). Monoclonal Antibodies (mAbs). The production of mAbs 10D5 and 6C6, which were raised against synthetic A1C28 coupled to a carrier protein, has been explained (11). mAbs 12B4, 2C1, 12A11, and 3A3 were raised against synthetic A1C42 by using similar strategy except that hybridoma supernatants were screened by an RIA. All antibodies were purified by HPLC and were free of endotoxin (<1 endotoxin unit/mg protein) as determined by the amoebocyte gel-clot assay (Associates of Cape Cod). The mAbs 3D6 and 21F12 were obtained as explained (7), and 22D12 and 266 were raised against synthetic A13C28 (12). Epitope Mapping. Epitope mapping of the mAbs and pAbs was performed by using an ELISA that measured antibody binding to overlapping peptides (10 amino acid peptides offset by 1 residue) covering the entire A1C42 sequence. The 1st 32 peptides were biotinylated in the C terminus, and the last 10 peptides were biotinylated in the N terminus. The biotinylated peptides were synthesized by Mimotopes (Clayton, Victoria, Australia) and captured on streptavidin-coated wells of a 96-well plate (Pierce). Passive and Active Immunization Methods. mAbs in PBS were given via passive administration (i.p. injection) at a dose of 10 mg/kg weekly for 6 months. For active immunization, 100 g of A fragment was given by i.p. injection in total Freund's adjuvant followed by boosts with 100 g of peptide in incomplete Freund's adjuvant at 2 and 4 weeks, and regular monthly thereafter. Antibody Binding to Aggregated and Soluble A1C42. Serum titers (determined by serial dilution) and mAbs binding ML221 to aggregated synthetic A1C42 were performed by ELISA as explained (1). Soluble A1C42 refers to the synthetic A1C42 peptide sonicated ML221 in dimethyl sulfoxide. Serial dilutions of sera or mAb at 20 g/ml were incubated with 50,000 cpm [125]A1C42 (190 Ci/mol; labeling with Iodogen reagent, Pierce) over night at space temp. Fifty microliters of a slurry comprising 75 mg/ml protein A Sepharose (Amersham Pharmacia) and ML221 200 g of rabbit anti-mouse IgG (H+L) (Jackson ImmunoResearch) was incubated with the diluted antibodies for 1 h at space temperature, washed twice, and counted on a Wallac gamma counter (PerkinCElmer)..