The data were obtained from a screen of 260 normal plasma specimens as shown in Fig

The data were obtained from a screen of 260 normal plasma specimens as shown in Fig.?2. Our studies provide support for investigating the clinical significance and physiological function of this novel class of antibodies. Keywords: Natural, IgGs, amyloid, A, IVIG Introduction A pathological hallmark of Alzheimers disease (AD) and more than 25 other incurable protein misfolding diseases, called the amyloidoses, is the extracellular deposition of amyloid fibrils from unrelated proteins or peptides [1]. The primary culprits for AD are prefibrillar amyloidogenic assemblies of amyloid beta (A), a proteolyzed transmembrane 39C43 fragment of amyloid precursor protein [2C4]. However, fibril deposition may be the primary toxic event for several non-AD amyloidoses, including primary AL amyloidosis [5]. Currently, immunotherapy directed against amyloidogenic protein is the primary therapeutic approach for AD and is a recent strategy for other amyloid-associated disorders [6]. Active vaccination with A or passive administration of anti-A antibodies has shown promise in AD animal models and in some AD patients by inducing neuritic plaque clearance, neutralizing neurotoxic A oligomers, and/or improving cognitive functioning [7C9]. Antibodies that specifically react with pathogenic amyloidogenic aggregates that do not bind to the normally secreted precursor protein are less likely to have adverse effects. Presumably, such natural IgG antibodies contained in intravenous immunoglobulin (IVIG) are at least partly responsible for its promising clinical effects [10, 11]. We have recently shown that these antibodies cross-react with conformational epitopes on amyloid fibrils and oligomers, including binding to cross-linked -amyloid protein species (CAPS) [12]. These antibodies also inhibit in vitro amyloid fibril growth and expedite the clearance of patient-derived amyloid in mice [12, 13]. The results of our studies on a large number of plasma donor specimens provide new information on the extent and specificity of natural anti-amyloidogenic conformer-reactive IgGs, which are contained in presumably healthy individuals. Methods Peptides, Proteins, and Antibodies Human 40- and 42-mer A peptides were purchased from Quality Controlled Biochemicals (Hopkinton, MA, USA); Pikamilone mass spectrometric analysis showed them to be >90% pure. Before using, the lyophilized A40 peptide was disaggregated by sequential exposure to trifluoroacetic acid, hexafluoroisopropanol (HFIP; Pierce, Rockford, IL, USA), and 2?mM NaOH, followed by Pikamilone 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 A 40 standard curve or by the MicroBCA assay (Pierce).Recombinant immunoglobulin light chain (LC) 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-m polyvinylidene fluoride 25-mm Millex-GV syringe-driven filter Pikamilone 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 Pikamilone 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 Mouse monoclonal to Influenza A virus Nucleoprotein A peptides by incubation with 1.1?M horseradish peroxidase and 250?M H2O2 in PBS at 37C for 3?h, and then purified using copper (CuSO4) precipitation [12]. CAPS was quantified using SDS PAGE (4C12% 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.A40 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.