readout, we have optimized the procedure for oligomer prepara- tion under cell-culture conditions with respect to Ab concentration and time of incubation. Ab(1–42) (AlexoTech AB, Umea˚, Sweden) was dissolved in 10 mM NaOH to a concentration of 160 mM. The Ab(1–42) sample was further diluted to 10 mM or 50 mM (the higher concentration was used regarding subsequent screen for human auto-antibodies) in PBS and the pH was adjusted to 7.4. Following incubation for 1 hour at RT (OMAB) or 2 hours at 4uC (auto-antibodies), the samples were separated by size-exclusion chromatography (SEC) (GE Superdex-G75 10/30) at 4uC. To determine the molecular weight of the Ab species, a gel filtration protein standard (Bio-Rad) was separated by SEC under the same conditions as the Ab samples. Indirect ELISA using OMAB The OMAB antibody, diluted to 10 mM/ml in PBS, was adsorbed to Nunc-Immuno MaxiSorp plates (Nunc, Roskilde, Denmark). Ab-fractions collected from the SEC were allowed to bind to OMAB plates for 20 minutes at 0uC. Bound Ab was detected using a polyclonal rabbit anti-Ab antibody (AS08 328, Agrisera AB, Va¨nna¨s, Sweden) at a 1:1000 dilution followed by an anti-rabbit HRP-conjugated secondary antibody at a 1:5000 dilution (GE healthcare). EC-Blue (Medicago, Uppsala, Sweden) was used as a substrate for HRP and the signal was detected by measuring the absorbance at 450 nm. The signal of EC-Blue can be compared within, but not between plates. Blocking solution and antibody-dilutions were made with 5% Non-fat dry milk in PBST and all washes were performed with PBS containing 0.1% Tween- 20 (PBST). Indirect ELISA using anti-human IgM The anti-human IgM antibody (Thermo scientific, Rockford, USA), diluted to 10 mg/ml in PBS, was adsorbed to Nunc- Immuno MaxiSorp plates (Nunc, Roskilde, Denmark). Human plasma from eight anonymous healthy donors was diluted 4X in PBS, added to the wells, and incubated for 1 hour at RT. During this incubation step, IgM auto-antibodies present in the plasma were allowed to bind to the anti-IgM antibodies previously adsorbed to the plate. Ab-fractions collected from the SEC were thereafter allowed to bind to the IgM auto-antibodies for 20 minutes at 0uC. Detection, blocking, and washes were performed as described previously. Epitope mapping and determination of affinity using surface plasmon resonance (SPR) The OMAB antibody was immobilized at a density of 10 000–15 000 RU to a CM5 chip (GE Healthcare) using standard amine- coupling chemistry at pH 6. Analysis of Ab binding was performed at a flow rate of 50 ml/min in PBS at 25uC. Sensograms were corrected for non-specific interactions to a reference surface, and by double referencing [26]. For the determination of affinity constants, Ab(1–16) was injected at concentrations ranging from 125 nM to 6 mM. The dissociation constant for Ab(1–16) was determined by fitting the response at the end of each of the association phases to a single-site binding isotherm. Epitope mapping of OMAB was performed at a peptide concentration of 5 mM. Results Characterization of OMAB Monoclonal antibodies obtained from hybridomas were screened for reactivity against different forms of Ab using dot blots and indirect ELISA (data not shown). A particular clone, denoted OMAB, fulfilled the requirements of both being of the Targeting Ab Oligomers IgM isotype and exhibit Ab binding and was therefore characterized further. Using SPR, a technique that facilitates detailed monitoring of binding properties, the affinities between different monomeric Ab fragments and the OMAB antibody were studied. An interaction was detected between OMAB and Ab(1– 16), Ab(1–22), and Ab(1–40), while no reactivity was seen between OMAB and Ab(26–40) (Fig. 1A). Similar results were obtained using ELISA (data not shown). The dissociation rates were essentially the same for all interactions (except for OMAB with Ab(26–40)), with a T1/2 corresponding to about 75 s, which implies a similar, monovalent binding of OMAB to the different Ab fragments. The reactivity pattern of OMAB suggests that the Figure 1. OMAB binds to the N-terminus of Ab with an affinity of 0.5 mM. OMAB was immobilized to a CM5 chip and tested for biding to (A) Ab peptides of different lengths at a concentration of 5 mM. The responses were normalized by the molecular weight of each peptide. (B) Ab(1–16) ranging in concentration from 125 nM to 6 mM. All experiments were performed in PBS at 25 &degC. The dissociation constant for Ab(1–16) was determined by fitting the response at the end of each of the association phases to a single-site binding isotherm. doi:10.1371/journal.pone.0013928.g001 PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13928