Peptides, Proteins, and Antibodies
Human 40- and 42-mer Aβ peptides were purchased from Quality Controlled Biochemicals (Hopkinton, MA, USA); mass spectrometric analysis showed them to be >90% pure. Before using, the lyophilized Aβ40 peptide was disaggregated by sequential exposure to trifluoroacetic acid, hexafluoroisopropanol (HFIP; Pierce, Rockford, IL, USA), and 2 mM NaOH, followed by 2× PBS (1× final). The ultracentrifuged sample yielded a final peptide concentration of ~0.2 mg/mL [12
]. The soluble 42-mer peptide was prepared at ~0.04 mg/mL in PBS by pretreatment with HFIP/NaOH [12
]. The peptide concentrations were determined at A215 nm
by reverse-phase HPLC using an Aβ 40 standard curve or by the MicroBCA assay (Pierce).Recombinant immunoglobulin light chain (LC) λ
6 variable domain, Jto, was produced in an Escherichia coli
expression system and purified using Amberlite XAD-7 (Sigma-Aldrich, St. Louis, MO, USA) [13
]. The soluble LC was sterile-filtered using a 0.22-µm polyvinylidene fluoride 25-mm Millex-GV syringe-driven filter unit (Millipore, Bedford, MA, USA). SDS-PAGE analyses confirmed that the protein was >90% pure, and protein concentration was determined by the MicroBCA assay.Normal donor plasma, coagulation reference plasma, which consisted of pooled plasma from healthy donors aged 20 to 60, and IVIG (Gammagard liquid) were provided by Baxter BioScience (Vienna, Austria). The blocking agent, essentially fatty-acid-free bovine serum albumin, was purchased from Sigma. All other reagents were of analytical grade.
Preparation of Peptide and Protein Aggregates
Soluble CAPS was prepared from the synthetic 40- or 42-mer Aβ peptides by incubation with 1.1 µM horseradish peroxidase and 250 µM H2
in PBS at 37°C for 3 h, and then purified using copper (CuSO4
) precipitation [12
]. CAPS was quantified using SDS PAGE (4–12% Bis Tris precast gels; Invitrogen, Carlsbad, CA, USA) and the MicroBCA assay. Electrospray ionization mass spectrometry (Applied Biosystems, Foster City, CA, USA) and dityrosine fluorescence (excitation at 320 nm and emission between 350 and 550 nm) confirmed that the aggregates consisted of low molecular weight (<38 kDa), cross-linked SDS stable species.Aβ40 and LC fibrils were grown from the soluble precursor proteins in PBS containing 0.02% sodium azide (PBSA). The reaction was monitored by thioflavin T fluorescence [13
]. Fibrils were harvested by centrifugation at 20,200
for 30 min at room temperature, then sonicated (2
30-s bursts) with a probe sonicator disruptor (Teledyne Tekmar, Mason, OH, USA), aliquoted, and stored at −20°C.
IgG Purification IgGs from donor plasma pools were isolated using a Melon Gel IgG Spin Purification Kit (Pierce). SDS-PAGE confirmed that the resultant samples were >95% pure. Antibody concentration was determined by absorbance at A280 nm with the use of a molar extinction coefficient of 210,000 M−1 cm−1.
Antibody Binding Assay
Antibody reactivity with amyloid fibrils, CAPS, and monomeric Aβ40 was determined at 37°C using the plate-immobilized conformers in our europium-linked immunosorbant assay (EuLISA) [13
]. All measurements were done in triplicate (error bars in the figures represent SD), and antibody concentrations that gave half-maximal binding (EC50
values) were determined from the sigmoidally fit binding curves (SigmaPlot 2000 ver. 6; Systat Software, Chicago, IL, USA).The EuLISA was performed with a 1:20 or serial dilution of plasma or purified IgGs in assay buffer (1% BSA in PBSA containing 0.05% Tween 20) in activated high-binding microtiter plate wells (COSTAR, Corning, NY, USA) coated with 400 ng of target protein and blocked with 1% BSA (Sigma-Aldrich) in PBSA. A biotinylated goat anti-human IgG (γ-chain specific, Sigma-Aldrich) served as the secondary antibody. After the addition of a Eu3+
streptavidin conjugate, followed by the releasing enhancement solution, Eu3+
time-resolved fluorescence was measured using a Victor2
1420 Multilabel Counter (Perkin Elmer, Waltham, MA, USA). The amount (fM) of lanthanide released was calculated from a standard curve using known concentrations of Eu3+
.To establish whether antibody binding was affected by a putative inhibitory plasma molecule, binding studies were done after pretreating the preparation with acid (0.1 M glycine HCl, pH 3.5), followed by dialysis against PBS (Amicon Ultra-4 filter unit, 30,000 Da m.w. cutoff, from Millipore) to dissociate antigen–antibody complexes [14
]. Assay signal was normalized for high-throughput plasma screening using a standard curve on each plate of coagulation reference plasma against the plate-immobilized amyloidogenic conformer.
Assurances Studies involving human specimens were in accordance with a protocol approved by the University of Tennessee Graduate School of Medicine’s Institutional Review Board.