Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide
The antibody binding curves are for Aβ fibril (A) and LC fibril (B) fractionated IVIg against plate-immobilized Aβ fibrils, and for Aβ fibril- (C) and LC fibril- (D) isolated IVIg IgGs against plate-immobilized LC fibrils. Each data point represents the average value from three binding studies, which were carried out using assay buffer consisting of 1% BSA in PBS containing 0.05% tween 20, pH 7.4. (E) The % IgGs isolated for each of four sequential passages of a preparation of 10 mg/ml IVIg in PBS, pH 7.4, through a column consisting of Aβ or LC (○) fibrils covalently cross-linked to N-hydroxysuccinimide (NHS) Sepharose®4 fast-flow agarose matrix (Amersham Biosciences Corp.). The plots also show the % IgGs isolated from IVIg over a control unconjugated deactivated sepharose matrix (beads only). The concentration of antibody in eluant preparations was determined by absorbance at 280 nm using an extinction coefficient of 1.25 and a relative molecular mass of 150,000. (F) The % depletion of anti-amyloidogenic NAbs in IVIg flow through preparations from Aβ and LC fibril columns, respectively, compared to the unfractionated preparation. Each antibody flow through preparation was generated from the fourth passage of IVIg through a fibril column, as described in Panel E (IgG eluant and flow through was generated for each passage). The % depletion of Aβ conformer and LC fibril reactive NAbs in each antibody flow through was determined from the absorbances at 280 nm of antibody eluants generated by passing IVIg and the antibody flow through preparations through Aβ conformer or LC fibril columns.