specific target epitope of OMAB is located within Ab residues 1– 16, a flexible portion of the Ab sequence that becomes partly structured within mature fibrils [27,28]. The dissociation constant between Ab and OMAB was investigated using monomeric Ab(1– 16), an Ab fragment not prone to aggregate. The dissociation constant was determined to 0.5 mM by fitting to a single site binding isotherm (Fig. 1B). Affinity of OMAB for different Ab assemblies Ab(1–42) peptides rapidly self-assemble into a range of different small and soluble oligomers at 37uC in PBS, a reaction easily monitored using SEC. The predominant species are monomers, dimers, and trimers; higher multimers are also detected, but they are present at considerably lower concentrations. Using an indirect ELISA setup where OMAB was probed against various Ab species, we showed that OMAB exhibited considerable selectivity towards oligomeric species. OMAB identified oligomeric Ab species with a molecular weight between 15–100 kDa, but failed to detect the much more abundant fractions of monomers and dimers (Fig. 2). The second anti-Ab antibody, AS08 328, used in the indirect ELISA, binds Ab within the N-terminus, as does OMAB. The N- terminal localization of these antibodies is a deliberate choice since the N-terminal part of Ab that remains exposed after aggregation. It is important to note that OMAB and AS08 328 competed for Ab to only a minor extent (20%, data not shown). Indirect ELISA illustrated the binding pattern of OMAB to Ab, but failed to elucidate the binding kinetics. Using SPR, the binding kinetics between OMAB and the different Ab species was examined. OMAB immobilized on the surface of a biocore CM5 chip, was probed with different Ab species obtained through SEC. A dramatic difference in dissociation rates was observed between the different species. The monomer-dimer fractions displayed, in accordance with the different monomeric Ab variants, a very rapid dissociation rate that could be fitted to an exponential decay function with a T1/2 corresponding to 75 s, indicating a low binding affinity (Fig. 3a). A considerably stronger Figure 2. OMAB selectively reacts with oligomeric Ab. A sample containing different oligomeric species of Ab(1–42) was separated by SEC (-). Fractions, collected throughout the SEC, were tested in an indirect ELISA for reactivity with OMAB (---). In connection to the SEC of Ab(1–42), a sample containing proteins with known molecular weights was analyzed. The exclusion volume of the peak maxima of these proteins is illustrated in the figure (&). doi:10.1371/journal.pone.0013928.g002 Targeting Ab Oligomers binding was observed to occur between OMAB and the oligomeric fractions containing Ab(1–40) assemblies of approximately 10–20 Ab peptides. The dissociation rate is clearly bi-modal and a double exponential decay model was needed for an accurate fit: the initial rapid phase has a T1/2 of 42s, likely reflecting a partial monovalent binding, while the second phase has a 5000X slower dissociation rate, with a T1/2 of 4.2 days (Fig. 3B). These results clearly illustrate a binding mechanism for OMAB strongly dependent upon avidity, where a low affinity at each antigen-binding site (i.e. between a single Ab monomer and the antibody) is compensated for by an increase in the number of interactions. Regarding the Ab(1–42) this effect was even more striking where no biphasic dissociation pattern was noted. The T1/2 of Ab(1–42) monomers was measured to 57s (Fig. 3C), which is in the same range as monomeric Ab(1–40). However, the binding between OMAB and Ab(1–42) oligomers, constituted by approximately 10–20 Ab peptides, was extremely strong and no dissociation could be noted throughout the duration of the experiment (Fig. 3D). The lack of avidity to dimers, and to some extent, trimers may be explained by the relatively smaller size of these moieties, as the antigenic aggregate must be large enough to bridge the antigen binding sites on the IgM molecule. OMAB binds selectively to Ab fibril ends and inhibits fibril growth To further evaluate the binding specificity of OMAB, we probed its interaction towards mature Ab fibrils. A convenient method for the monitoring of fibril polymerization in real time is to immobilize mature Ab(1–40) fibrils on a Biacore CM5 sensor chip and incubate with an excess of monomeric Ab(1–40) peptide, an event that will give a continuous propagation of the sensor chip signal as a result of fibril growth [29]. Upon the addition of OMAB to the immobilized Ab fibrils, a strong, but highly substoichiometric binding was detected, which corresponded to a molar ratio of about 1:2000 OMAB:Ab. This substoichiometric binding suggested that only a small fraction of the Ab fibrils expose the epitopes required for OMAB to bind, thus OMAB likely binds predominantly to exposed fibrillar ends rather than to the lateral fibril. This hypothesis is supported by the finding that immobilized fibrils, saturated with OMAB, were unable to bind monomeric Ab (data not shown). Thus, OMAB effectively inhibited the Ab polymerization process. OMAB effectively inhibits the cytotoxicity of Ab in a cell- viability assay The cytotoxicity of Ab was studied using a cell-based viability assay. Freshly prepared Ab(1–42) was added to cultured SH-SY5Y cells, resulting in 35% of cells remaining viable after 48 hours of Ab exposure. Co-incubation of Ab and OMAB essentially eliminated the cytotoxic effect at a substoichiometric OMAB:Ab ratio corresponding to 1:50 (Fig. 4). Human IgM auto-antibodies can selectively target the oligomeric fraction of Ab Auto-antibodies against Ab are common in humans, and both IgG and IgM isotypes have been described [16,18,19,20,21, 22,23,24]. In a previous study, an immortalized cell line that produces an anti-Ab human IgM monoclonal antibody was established from a healthy human blood donor [17]. This specific antibody, denoted L11.3, was able to prevent the phenotype of impaired spatial cognition in aged SAMP mice, similar to previous findings using IgG antibodies [6,7,30,31]. To compare the binding properties of the L11.3 antibody with the mouse-derived OMAB, PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13928