Unfractionated, Aβ- and Cibacron blue-isolated human IgGs binding to plate-immobilized PFs.

<p>¹Mon stands for IgG monomers.</p><p>^2,3SEC-isolated IgG monomers (mon), dimers, and HMW aggregates as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g002" target="_blank">2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a>.</p><p>Each value for EC₅₀ and maximum signal amplitude was determined from the average of two to three sigmoidal fitted antibody binding curves, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g008" target="_blank">8</a>.</p

IgG aggregates are primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated pAb IgGs.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~0.5 mg/mL of Aβ-isolated IVIg IgGs, and for IVIg, untreated, or diluted into column elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound IVIg IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: IgG binding curves against plate-immobilized PFs for untreated IVIg, and for Aβ column-isolated IVIg IgGs that were used unfractionated (Unfrac) or as SEC-isolated monomers (SEC Mon) or aggregates (SEC Aggs). SEC Aggs consisted of a pool of IgG conformers (dimers and HMW species) that eluted before the monomeric antibody. (<b>B</b>) Left panel: SEC chromatograms for ~0.5 mg/mL dye-isolated IVIg IgGs, and for unfractionated IVIg that was untreated or diluted into column elution buffer (PBS containing 1.5 M NaCl, pH 7.4) that was used to elute dye-bound IVIg IgGs. Right panel: IgG binding curves against plate-immobilized PFs for unfractionated and SEC-isolated conformers of dye-isolated IVIg IgGs, and for untreated IVIg.</p

Dimeric pAbs bind more tightly to PFs than their monomeric form.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~1 mg/mL of protein A-purified pAbs and IVIg in PBS, pH 7.4. SEC was carried out using a Hiprep16/60 Sephacryl S300 HR column (GE Healthcare) that was equilibrated in PBS, pH 7.4. Arrows indicate the elution of protein molecular weight standards. Right panel: Antibody binding curves against PFs for unfractionated (Unfrac) and SEC-isolated monomeric pAbs. (<b>B</b>) Left panel: Antibody binding curves against PFs for unfractionated and SEC-isolated monomeric and dimeric IVIg IgGs. Right panel: SEC chromatographs for ~0.2 mg/ml IVIg dimers and monomers in PBS, pH 7.4, after incubation under ELISA-like conditions (4 h at 37°C). (<b>C</b>) Dynamic light scattering for 0.8 mg/mL and 0.06 mg/mL of SEC-isolated IVIg monomers and (<b>D</b>) dimers, respectively. Dynamic light scattering was determined at room temperature immediately after SEC-isolation of each IgG conformer in PBS, pH 7.4. <i>R</i>_<i>h</i> and pd are abbreviations for hydrodynamic radius and polydispersity, respectively.</p

A human mAb generated against a non-amyloid target binds aggregated Aβ.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~15 mg/mL of mAb Avastin (anti-VEGF) and IVIg. SEC was carried out using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for unfractionated (Unfrac) IVIg, and for Avastin used unfractionated or as SEC-isolated monomers and dimers. (<b>B</b>) The top Western blots show immunoprecipitation (IP) of synthetic Aβ conformers (monomers (Mon), dimers, and PFs) by 100 μg/mL of Avastin and IVIg, and by 200 μg/mL of a pan-Aβ reactive polyclonal antibody, AW8. The blots were probed for Aβ using an Aβ N-terminal reactive mAb, 6E10 (Signet Laboratories). The lower Western blots show 20 μg/mL mAb Avastin's ability to IP 5 μg/mL of Aβ dimers and PFs in the presence of a 5-molar excess (with respect to Avastin) of a N-terminal 165-amino acid fragment of its immunogen VEGF (VEGF-165). Control IP experiments (Ctls) were carried out using 5 μg/mL mAb 6E10 and a mixture of Aβ dimers and PFs, or with 20 μg/mL Avastin and 1 μg/mL VEGF-165. The blots were probed for Aβ and VEGF-165 using mAb 6E10 and Avastin, respectively. In IPs carried out in the absence of VEGF-165, cross-reactivity of the secondary antibody, goat anti-human IgG (heavy and light, Jackson Immunoresearch Laboratories Inc), with Avastin’s Ig light chain caused a faint band that migrated near VEGF-165. (<b>C</b>) Avastin IgG conformers binding curves against plate-immobilized PFs in the presence or absence of a 1:10 dilution of IgG-depleted normal human sera. (<b>D</b>) Bar charts for solution-phase PF's, Aβ monomers, and non-amyloid native and aggregated molecule's inhibition of Avastin monomers an dimers binding to plate-immobilized PFs. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of Avastin conformers that were equivalent to their EC₅₀ values for PFs: 500 nM IgG Mon, and 200 nM IgG dimer. Each competition curve was carried out in duplicate, and bars represent the standard error.</p

IgG F(ab)ˈs but not Fc mediate IgG binding to PFs.

<p>Antibody binding curves are shown against plate-immobilized PFs for intact and fragmented IgGs from preparations of unfractionated IVIg (<b>A</b>) and for Cibacron blue-isolated IVIg IgGs (<b>B</b>). Antibody binding studies were carried out in triplicate and bars represent the standard error.</p

Aβ-isolated but not heat-induced Avastin aggregates have enhanced avidity for PFs.

<p>(<b>A</b>) Left panel: SEC chromatograms for 0.3 mg/mL of Aβ-isolated Avastin IgGs, untreated Avastin, and for the antibody diluted into elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound Avastin IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: Antibody binding curves against PFs for unfractionated IVIg and Avastin, and for Aβ-isolated Avastin IgGs. (<b>B</b>) Left panel: SEC chromatograms for ~5 mg/mL of unfractionated Avastin in PBS, pH 7.4, and for IgG conformers contained in supernatant of 71°C heated Avastin monomers (A_400nm 0.5 sup) in PBS, pH 7.4. Right panel: Antibody binding curves against PFs for soluble (A_400nm 0.5 sup) and insoluble (A_400nm 0.5 pellet) IgG conformers of heat-treated Avastin monomers, and for untreated Avastin and IVIg.</p

IVIg and Protein A-purified human and murine pAbs have similar anti-amyloid activities.

<p>(<b>A</b>) IgG binding curves against plate-immobilized PFs for IVIg and protein A-purified human (Hu) and murine (Mu) pAbs from pooled normal plasmas. Antibody binding curves are also shown for pAbs present in or dosed back into plasma. (<b>B</b>) Hybrid capture/competition ELISA curves for pAb's and IVIg's dose-dependent inhibition of PFs binding by plate-immobilized IVIg F(ab') fragments. The assay was carried out using 8 μg/ml solution-phase PFs. (<b>C</b>) Antibody binding curves for Hu and Mu pAb's nM cross-reactivity with plate-immobilized Aβ and TTR fibrils. (<b>D</b>) Left panel: Competition curves for solution-phase PF's and Aβ monomer's (Mon) inhibition of pAbs and IVIg binding to plate-immobilized PFs. Right panel: SAgg's and native TTR's (Nat) inhibition of pAbs and IVIg binding to plate-immobilized TTR fibrils. Competition studies were carried out using IgG concentrations, ~500 nM, which were equivalent to their EC₅₀ values for binding to PFs or TTR fibrils. Each binding or competition curve was carried out in duplicate, and bars represent the standard errors.</p

PAb aggregates have diverse avidities for Aβ.

<p>(<b>A</b>) Left panel: SEC chromatograms for untreated IVIg and for supernatants of ~4 mg/mL of SEC-isolated monomeric IVIg in PBS, pH 7.4, which was heated at 71°C until light scattering at A_400nm was 0.6 or 3.9, respectively. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, Aβ-isolated IVIg IgGs, and for IgG supernatants (sup) and PBS resuspendend pellets (pellet) of heat-treated IVIg. <b>(B</b>) Left panel: SEC chromatograms for ~4 mg/mL of untreated IVIg and for IVIg that was buffer exchanged at room temperature from gentle elution buffer (Pierce), pH 6.6, into PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, buffered exchanged IVIg that was used unfractionated or as SEC-isolated monomers, and for Aβ-isolated IVIg IgGs.</p

IVIg IgG conformers retain binding to Aβ in the presence of normal human sera and non-amyloid molecules.

<p>(<b>A</b>) Antibody binding curves against plate-immobilized PFs for SEC-isolated IVIg monomers (SEC Mon), dimers (SEC Dimer), and for Aβ-isolated IVIg IgGs with or without a 1:10 dilution of IgG-depleted normal human sera. Preparations of Aβ-isolated IVIg IgGs contained HMW, dimeric, and monomeric species (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">Fig 3A</a>). (<b>B</b>) Bar charts for solution-phase PFs, monomeric Aβ and for non-amyloid molecules inhibition of IVIg IgG conformers binding to plate-immobilized PFs. Non-amyloid molecules were chosen based on their: 1) Abundance <i>in vivo</i> (extracellular matrix and elastin fibrils), 2) Association with polyreactive autoantibodies (DNA), 3) High hydrophobicity (maize protein zein), and 4) Non-amyloid aggregate state [amorphous aggregated carboxymethylated ovalbumin (CM-Oval)]. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of IgG conformers that were equivalent to their EC₅₀ values for PFs: 400 nM IgG Monomers; 50 nM IgG dimers, and 20 nM Aβ-isolated IgGs. Each competition curve was carried out in duplicate, and bars represent the standard error. (<b>C</b>) IVIg IgG conformer binding curves against PFs and non-amyloid molecules, murine extracellular matrix gel (ECM) and human DNA.</p

Aβ and LC fibril fractionated NAbs binding to amyloid fibrils.

<p>The antibody binding curves are for Aβ fibril (<b>A</b>) and LC fibril (<b>B</b>) fractionated IVIg against plate-immobilized Aβ fibrils, and for Aβ fibril- (<b>C</b>) and LC fibril- (<b>D</b>) 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. (<b>E</b>) 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. (<b>F</b>) 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.</p