preserving Ab monomers and APP. Intriguingly, conformational- dependent anti-Ab antibodies, of the IgG isotype, have been shown to selectively target Ab oligomers, suggesting that an oligomer-specific architecture exists [12,13,14,15]. However, to avoid the difficult task of identifying a specific architecture, only present within the oligomeric form, we have employed a different rationale based on the assumption that all oligomeric variants of Ab must expose several similar epitopes. The binding of a multivalent target to a corresponding multivalent receptor will be influenced by the avidity that, as opposed to affinity, represents the product of all single valence interactions. Avidity and affinity are, however, correlated, as the strength of avidity depends both on the affinity of each interaction, as well as on the number of interactions. We propose that an IgM antibody, containing 10 identical binding sites, fulfils the requirements to be a multivalent receptor for oligomers. We have therefore generated an oligomer- specific IgM anti-Ab antibody (OMAB) that binds Ab oligomers with a high specificity and a high affinity, whereas its binding to Ab monomers and dimers is comparatively weak. OMAB has a high specificity for oligomeric Ab even in a complex medium (containing many different forms of Ab), demonstrated by its ability to completely block, at a highly substoichiometric ratio, the cytotoxic effects exerted by oligomeric Ab(1–42). The specificity of a previously identified human anti-Ab IgM antibody (L11.3) was also evaluated in the current study, and the results were analogous to those obtained with OMAB, demonstrating a high affinity for Ab oligomers as compared to Ab monomers and dimers. Interestingly, anti-Ab auto-antibodies of both IgG and IgM isotypes are commonly found in the sera of the healthy human population [16,18,19,20,21,22,23,24]. To further study the Ab binding pattern of endogenous anti-Ab antibodies having the IgM isotype, we have screened a set of plasma samples from healthy donors. These studies found that the isolated endogenous anti-Ab IgM antibodies all displayed a much higher affinity towards the oligomeric Ab as compared to monomeric Ab. Our findings suggest a simple mechanism whereby avid IgM binding can be used to specifically target oligomeric assemblies of Ab. With such an approach, antibody binding is not restricted to specific epitope conformations: the epitope recognized by the IgM antibody need only be exposed within the oligomeric state. Moreover, our findings suggest a more specific and possibly protective role for the abundant spectra of anti-Ab IgM auto- antibodies present in humans. Materials and Methods Ethical statement The use of all human samples, within this study, was verbally approved by the donors. The study has been fully anonymized and no data regarding specific individuals has been recorded. This implies no possibility to identify and connect specific individuals to the acquired result. All work and procedures involving human samples was approved by the Committee of Research involving Human subjects of the Hebrow University Hadassah medical school, Jerusalem, Israel, according to the declaration of Helsinki (permit number: 211-09.02.07). All immunisations of mice was approved by the Umea˚ Ethical Board of Animal Research, Va¨sterbottens la¨n, hovra¨tten o¨vre norrland, box 384, 901 08, Umea˚, Sweden, according to the declaration of Helsinki (permit number A47-07). Immunization and cell fusion Mice (BALB/c, female, 8–10 weeks old) were immunized with the recombinant Ab(1–40) (Agrisera AB, Va¨nna¨s, Sweden). Six Targeting Ab Oligomers weeks following immunization, sera were tested for antibodies against Ab. Mice testing positive for anti-Ab antibodies were then given booster i.v. injections of 50 mg of Ab(1–40). Fusion of spleenocytes with mouse B-cell myeloma cells was performed 3 days later according to standard procedures and cells were seeded in 96-well plates at a density of 300 000 cells/well. Selection for hybrid cells was performed using Dulbecco’s Modified Eagle Medium/DMEM) with high glucose (4500 mg/L D-Glucose) containing 10% (v/v) fetal calf serum and hypoxanthine/ aminopterin/thymidine (Gibco). Supernatants from wells with growing cells were screened for antibodies reactive to Ab. Isotype determination was performed using the IsoQuickTM strips for Mouse Monoclonal Isotyping (Sigma-Aldrich, Germany). Cell culture SH-SY5Y neuroblastoma cells were obtained from the European Collection of Cell Cultures (Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK). Cells were cultured in MEM with Earle’s salts (Gibco), supplemented with 10% (v/v) foetal bovine serum, 2 mM L-glutamine (Gibco), 100 units/ml penicillin, 100 mg/ml streptomycin (Gibco), and 1% non-essential amino acid solution (Gibco). Cultures were maintained in an incubator at 37uC, with a humidified atmosphere of 5% (v/v) CO2/air. Cytotoxicity measurement Ab(1–42) (AlexoTech AB, Umea˚, Sweden) was dissolved in 10 mM NaOH to 160 mM and further diluted with 4X MEM and H2O to 40 mM, pH 7.4. The Ab(1–42) sample was further diluted with 1X MEM to the desired concentration and supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin (Gibco), 1% MEM non-essential amino acid solution (Gibco), 1% MEM vitamins solution (Gibco), and 1% MEM amino acids solution (Gibco). Ab(1–42) was allowed to incubate with SH-SY5Y cells in the absence or presence of OMAB. Cell viability was measured after 48 hours using a resazurin reduction test, a test where blue non-fluorescent resazurin is reduced to pink highly fluorescent resorufin by living cells [25]. Fluorescence, directly proportional to cell viability, was measured in each well with a Tecan Infinite plate reader set to 535 nm excitation and 595 nm emission. Cytotoxicity is presented as a percentage of the fluorescence of control cells incubated with MEM only. Statistical analysis was performed using GraphPad Prism version 5.01 for windows (GraphPad Software, San Diego, California, USA). Viability of cells incubated with Ab oligomers in the presence or absence of OMAB was analyzed using One-Way ANOVA with Dunnett’s post test. Statistical significance was set to a p-value #0.001. Oligomer preparation and size-exclusion chromatography (SEC) Due to the labile nature of Ab oligomers their preparation must be carefully monitored. Dissolution of the peptide is perhaps the most critical step in the procedure where it is of paramount importance to achieve an effective monomerisation. Presence of fibrillar seeds otherwise results in an accelerated conversion into amyloid. There are several methods described in the literature, where the peptide is dissolved using organic solvents followed by dilution. However, in our hands these methods frequently result in impurities where fibrillar seeds prevent population of oligomers. We have therefore focused on a commonly used alternative where the peptide is dissolved at high pH followed by adjustment of the pH using the buffer of choice. Using the cytotoxic potential as PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13928