anti-Ab antibody of the IgM isotype. Using indirect ELISA in combination with SEC-separated Ab(1–42), we discovered OMAB to have a much higher affinity for Ab oligomers than for Ab monomers and dimers. SPR studies demonstrated that the avidity effect of OMAB induced a very strong binding to, and a high selectivity for, Ab oligomers. The most striking effect was seen when comparing dissociation rates of the different Ab species. The dissociation rate for Ab monomers and dimers regarding Ab(1–40) and Ab(1–42) is rapid where T1/2 equals approximately 1 minute (Fig. 3A and 3C, respectively). On the contrary the corresponding oligomers, having an apparent size of a 10–20 mer, shows a dramatic decrease in dissociation rate where T1/2 for Ab(1–40) equals 4.2 days, while T1/2 regarding Ab(1–42) became to slow to be measured (Fig. 3B and 3D, respectively). This finding suggests that the IgM isotype introduces the potential of multivalent binding, allowing oligomers to bind with a high affinity, while monomers and dimers bind only transiently with fast off-rate kinetics. When using IgM antibodies to trap Ab oligomers, unwanted binding to Ab monomers and APP is, to a great extent, omitted. It is important to emphasize that the high selectivity towards oligomers only can be obtained at a relevant time-scale, if the off-rate at each single binding site is high. A to high single valence affinity would slow down the process of identifying oligomers in a large excess of monomeric Ab since the sites will be blocked by monomers. As a result the ‘‘scanning’’ effect for oligomeric species within the sample would be lost. A potentially complication of generating oligomer-specific antibodies is the possible cross-reactivity to mature Ab fibrils. SPR was used to investigate if OMAB detected amyloid fibrils. We found that only a small fraction of the OMAB antibody, corresponding to a molar ratio of 1:2000 OMAB:Ab, bound to the immobilized amyloid fibrils. We conclude that OMAB is unable to perform a lateral binding along the Ab fibril, but instead binds to the more exposed fibrillar ends. This was verified through competition studies, where OMAB effectively inhibited a continu- ous polymerization of the fibril upon addition of free Ab monomers. Specific assemblies of Ab(1–42) efficiently kill human neuronal cells in culture. Due to the kinetic instability of Ab(1–42) in cell culture media, the initial oligomeric state of Ab is not maintained throughout the duration of the experiment. As a consequence, it is difficult to elucidate the exact size of the cytotoxic Ab assembly, as well as to determine the molar concentration of cytotoxic Ab. We have shown that the addition of OMAB, in a substoichiometric ratio corresponding to 1:50 (OMAB:Ab monomer), completely blocks the neurotoxic effect exerted by the Ab(1–42) peptide. This result also demonstrates the specificity of OMAB in a more complex matrix. To date, a detailed characterization regarding the molecular properties and size of cytotoxic Ab has been lacking. A cytotoxic effect of Ab-derived diffusible ligands (ADDLs) has been described, comprising a rather broad spectrum of Ab assemblies [43]. A cytotoxic effect has also been reported from spheroids, large Ab assemblies with a molecular weight of 500 kDa [44]. An acute cytotoxic response is, however, a dramatic cellular effect and, in vivo, subtler changes may better reflect the pathological events underlying disease. Essentially all soluble forms of Ab have the ability to inhibit long-term potentiation (LTP), a cellular correlate of memory (LTP) [13,45,46,47,48]. LTP is a long-lasting enhancement in signal transmission between two neurons and is one of several phenomena underlying synaptic plasticity, the ability of synapses to change their strength. As the ability to form memories is thought to be encoded by modifications in synaptic strength, LTP is considered to be a major cellular mechanism underlying learning and memory. In AD models, initial observa- Targeting Ab Oligomers tions correlated impairment in spatial memory with the presence of Ab nonamers and dodecamers [39], species that are readily detected by the OMAB antibody. However, impaired LTP, as well as a decreased dendritic spine density in the hippocampal dentate gyrus, has been noted prior to the detection of nonamers and dodecamers [49], suggesting that even smaller Ab assemblies are able to exert a physiological effect in vivo. Engineered disulphide- linked dimers of Ab have also been shown to inhibit LTP both in vitro and in vivo [47]. As OMAB fails to detect assemblies smaller than a trimer, possibly due to steric hindrance as a result of the difference in size, alternative smaller binding structures should be considered to accomplish an oligomer-specific effect targeting e.g. dimeric species. The initial events regarding Ab assembly may moreover be intracellular and design intracellular antibodies, delivered through an adenoviral vector, have resulted in partial clearance of Ab(1– 42) deposits in a mouse model for AD [50]. The IgM isotype has previously been evaluated in vivo using a transgenic AD mouse model [17]. Using brain injections of a human-derived anti-Ab IgM antibody (L11.3), both memory and learning deficits could be restored. In this study, the injection of high intravenous doses of the IgM antibody resulted in a fraction crossing the blood brain barrier and ameliorating the learning deficiency [17]. In the current investigation, we have evaluated the binding properties of L11.3 using indirect ELISA and probed it against a spectrum of different Ab assemblies in parallel with the murine-derived OMAB. Our results show L11.3 to exhibit a selectivity for the oligomeric Ab fraction (Fig. 5A), similar to OMAB, and support the notion that the multiple antigen-binding sites of IgM induce a high specificity towards oligomeric Ab assemblies. Epitope mapping of L11.3 demonstrated that antibody binding occurs within the 16 most N-terminal residues of Ab (data not shown). Endogenous autoantibodies against Ab are common in humans [3,4,5,6,7,8,51,52] and passive immunization using total isolate of human immunoglobulins, given to AD individuals has resulted in a slight cognitive improvement [53,54]. Intriguingly, IgM anti-Ab autoantibodies are found in essentially all individuals, although in different concentrations [17,18,23]. These human IgM autoanti- bodies have previously been studied with the aim of identifying differences between healthy controls and individuals affected by mild cognitive impairment or AD [18,23]. Differences in IgM auto-antibodies have been suggested as a potential diagnostic marker, as the levels of specific IgM auto-antibodies were lower in AD patients compared to controls [18]. Interestingly also a catalytic degradation of Ab has been described from IgM autoantibodies [20,55]. However, regarding OMAB no such effect could be noted, data not shown. All plasma samples from young healthy individuals screened by an indirect ELISA contained anti-Ab IgM auto-antibodies with a strong preferential binding to oligomers as compared to monomers and dimers (Fig. 5B). The fact that these antibodies are ubiquitously present in normal plasma suggests that they have a possible role in the normal homeostasis of Ab. The L11.3 antibody is derived from an Epstein-Barr virus immortalized cell line established from B lymphocytes of a healthy individual. Thus, L11.3 resembles an anti-Ab antibody naturally present in the sera of healthy humans. Our finding that OMAB, L11.3, and, IgM auto-antibodies from eight plasma samples showed a very high selectivity towards Ab oligomers suggests that human IgM auto- antibodies may confer a protective effect in vivo, both normally and when used for AD passive immune therapy. The current work proposes that the IgM molecule provides an efficient architecture for the selective trapping of Ab oligomers. PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13928