This information as well as the date and time of the phone call of the consent for the autopsy were noted in the hospital files according to a procedure approved by the Ethical Committee

This information as well as the date and time of the phone call of the consent for the autopsy were noted in the hospital files according to a procedure approved by the Ethical Committee. Supporting Information Figure S1 Atomic Force Analysis of A and -synuclein. pH 5. Determination of monomeric affinity constants for the anti-A Pyrithioxin dihydrochloride antibodies was performed using either A(1C40) or Pyrithioxin dihydrochloride A(1C16), in PBS buffer, at a flow rate of 50 l/min in at 25C. SPR sensograms were corrected for non-specific interactions to a reference surface, and by double referencing. The affinity constants for ASyM, and ASyO2 were performed in a similar manner using either full-length -synuclein or the monomeric peptide fragment covering the epitope of the specific antibody. The dissociation constant was determined by fitting the response at the end of each of the association phases to a single-site binding isotherm. SPR sensograms acquired through probing immobilised antibodies towards their corresponding monovalent antigens as Pyrithioxin dihydrochloride described within material and methods. (A) mAB-M. (B) mAB-O. (C) ASyM. (D) ASyO2. (E) ASyO5.(TIF) pone.0090857.s002.tif (2.1M) GUID:?91D5F8E2-4647-487F-9C1D-FDA613C00575 Figure S3: Size exclusion chromatography for isolation of -synuclein oligomers. Lyophilised -synuclein was dissolved at 10 mg/ml in 10 mM sodium phosphate buffer (pH 7.4). Dopamine was added to generate a final concentration of 73 M -synuclein and 1 mM dopamine. The sample was incubated for 24 hours at 37C with agitation and separated through size exclusion chromatography (GE Superdex-G200 10/30, Uppsala, Sweden) in PBS. The fractions within the borders separated by the striped lines where used.(TIF) pone.0090857.s003.tif (203K) GUID:?1E559B14-5208-40E4-BBFA-56556288464B Abstract Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the Pyrithioxin dihydrochloride antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the A peptide and -synuclein that are associated with Alzheimer’s and Parkinson’s diseases, respectively. The approach CYFIP1 is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies. Introduction The pathological self-assembly of proteins and peptides into amyloid fibrils is the defining characteristic of a group of more than twenty human diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [1]. Although amyloid fibrils are invariably present in the affected individuals, many studies have shown that soluble oligomeric assemblies, which can either precede amyloid formation or represent a stand-alone entity formed in parallel with the fibrils, exert the most potent detrimental physiological effects [2]C[11]. However, these oligomers are transient species and frequently only constitute a very minor fraction as compared to the amyloid and the non-aggregated native and precursor forms of Pyrithioxin dihydrochloride the specific protein or peptide. This significantly complicates characterization of oligomers and their selective therapeutic targeting. Intriguingly, antibodies that specifically target oligomeric species have been isolated [11]C[17]. However, the molecular properties of oligomer-specific antibodies are not well understood, which hinders both directed design as well as optimisation of such antibodies. There is, therefore, an urgent need for a method that can be used to consistently and reliably design oligomer-specific antibodies. Antibodies having the ability to identify structures exclusively present on oligomeric assemblies have previously been demonstrated [11], [13], [14],.