Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/552—Glass or silica
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• the invention relates to methods for the selective quantification of A-beta aggregates comprising the immobilization of A-beta capture molecules on a substrate, applying the sample to be examined to the substrate, adding probes labeled for detection, which are bound by specific binding to A- Beta aggregates mark this and detection of the labeled aggregates.
• AD Alzheimer's dementia
• Latin Morbus Alzheimer
• Age-related dementia is becoming an ever greater problem in today's society, as more life expectancy is affecting more and more people, and the disease is affecting social security systems and their affordability.
• amyloid-beta-peptide deposits are found in the brain and synuclein deposits in Parkinson's disease.
• the amyloid-beta-peptide deposits or peptide fibrils
• APP amyloid precursor protein
• AD Alzheimer's disease
• A-beta peptide consisting of the A-beta peptide
• additional neurofibrillary deposits from the tau protein.
• the precursor protein of the A- Beta-peptide, APP is located in the cell wall of neurons.
• A-beta fragments of different lengths and types such as A-beta 1 -40, A-beta 1 -42 or pGluA-beta 3-42.
• Monomeric A-beta peptides are also produced in the healthy organism throughout life.
• the plaque A-beta deposits are the cause of the disease symptoms.
• various studies indicate that especially the small, freely diffusing A-beta oligomers have the highest toxicity among all A-beta species and are responsible for the development and progression of AD.
• aggregates of the A-beta peptide are directly linked to AD pathogenesis.
• AD Alzheimer's disease
• biomarkers for AD.
• a starting point for such biomarkers has hitherto been the use of PET radio-tracers for imaging, based on the assumption that the radiolabelled substances bind amyloid plaques and thus could be a measure of plaque deposition after detection.
• PET radio-tracers for imaging, based on the assumption that the radiolabelled substances bind amyloid plaques and thus could be a measure of plaque deposition after detection.
• Disadvantages of this method are also the high costs and the necessary technical effort on devices that are not widely available.
• Another starting point will be z.Z. Examines the levels of various substances in the blood or cerebrospinal fluid (CSF) of patients and analyzes their utility as a biomarker.
• CSF cerebrospinal fluid
• One of these substances is the A beta peptide. So far, the most reliable way to determine the content of monomeric Abeta in the CSF of patients, possibly combined with the determination of the tau concentration. However, there is such a high variation of the values that reliable diagnosis by means of such biomarkers can not be made for an individual. The use of such a method is known from DE 69533623 T2. Despite these different approaches, no reliable biomarker has yet been established.
• Antibody is for example from WO2005 / 018424 A2. known.
• Another detection method is sandwich ELISA measurements.
• A-beta-specific antibodies are used to immobilize A-beta molecules.
• the same antibodies are subsequently used for detection.
• Monomers lead to no signal according to this method, since the antibody binding site is already occupied by the capture molecules.
• Specific signals are thus generated only by dimers or larger oligomers.
• such a method only allows the quantification of the sum of all aggregates present in a sample and not the characterization of individual aggregates.
• the ELISA-based method In order to reliably detect and quantify individual A-Beta aggregates, the ELISA-based method also lacks the necessary sensitivity.
• the use of such a sandwich ELISA method is known from WO2008 / 070229 A2.
• the object of the present invention was to provide a biomarker for protein aggregation diseases, in particular AD, as well as an ultra-sensitive method for the quantification and characterization of A-beta aggregates.
• a biomarker for protein aggregation diseases in particular AD
• an ultra-sensitive method for the quantification and characterization of A-beta aggregates By characterizing the biomarker, ie determining the number, amount and / or size of this substance (biomarker) in an endogenous fluid or tissue, a precise diagnosis of the disease and / or information about the course of the disease and the condition of the patient is to be made possible.
• Another object of the present invention was to provide a method for the selective quantification of pathogenic aggregates which cause and / or characterize a protein aggregation disease, in particular of A-Beta aggregates of any size and composition, A-beta oligomers and at the same time small, freely diffusing To provide A-beta oligomers.
• Step b) before step a) can be performed.
• Characterization of the A-beta aggregates or A-beta oligomers means determination of the shape, size and / or composition.
• a beta monomer refers to a peptide molecule that is part of the amyloid precursor protein APP, known by the name A beta.
• a beta amyloid precursor protein
• A-beta oligomers denotes both A-beta aggregates and A-beta oligomers and also small, freely diffusing A-beta oligomers.
• Oligomer according to the invention is a polymer formed from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomers or multiples from that. It may or may not be the case that all A-beta monomers in an A-beta oligomer are identical to one another.
• A-Beta aggregates are understood to mean both A-beta oligomers and small, freely diffusing A-beta oligomers. This also includes aggregates, such as fragments of fibrils, "protofibrils", “ADDLs", p56 * . It is essential for the present invention that the A-beta aggregates in terms of size are aggregates or polymers which can move in the body and not due to their size in the form of amyloid-beta-peptide-plaque Deposits are immobilized in the body.
• a material is selected which has the lowest possible unspecific binding capacity, in particular with respect to A-beta oligomers.
• a glass substrate is selected.
• the substrate can be coated with hydrophilic substances, preferably poly-D-lysine, polyethylene glycol (PEG) or dextran.
• the glass surface is hydroxylated and then activated with amino groups.
• CMD carboxymethyl-dextran
• the substrate is treated with an aqueous solution of CMD (at a concentration of 10 mg / ml or 20 mg / ml) and optionally N-ethyl-N- (3-dimethylaminopropyl) ) Carbodiimide (EDC), (200 mM) and N-hydroxysuccinimide (NHS), (50 mM) and then washed.
• EDC Carbodiimide
• NHS N-hydroxysuccinimide
• the carboxymethyl-dextran is covalently bound in a variant to the glass surface, which was first hydroxylated and then activated with amine groups, as described above.
• Microtiter plates preferably with a glass bottom, can also be used as the substrate. Since the use of concentrated sulfuric acid is not possible when polystyrene frames are used, the activation of the glass surface in one embodiment of the invention takes place analogously to Janissen et al. (Colloids Surf B BioInfaces, 2009, 71 (2), 200-207). In an alternative of the present invention, capture molecules are immobilized on the substrate to capture and fix the A-beta aggregates.
• the capture molecules are covalently bound to the substrate.
• the catcher molecules are covalently bonded to the coating, preferably the dextran layer.
• the anti-A-beta antibodies specifically bind an epitope of the A-beta aggregates.
• the epitope has an amino acid sequence of the amino-terminal portion of the A-beta peptide selected from subregions A-beta 1-8 (SEQ ID NO: 2), A-beta 1-11 (SEQ ID NO: 1).
• A-Beta 1-16 SEQ ID NO: 4
• A-Beta 3-11 SEQ ID NO: 5
• pyroGluA-Beta 3-11 SEQ ID NO: 6
• A-Beta 11 - 16 SEQ ID NO: 7
• pyroGluA-Beta 11 -16 SEQ ID NO: 8
• the capture molecules optionally after activation of the CMD-coated support by a mixture of EDC / NHS (200 or 50 mM), are immobilized on the substrate.
• ethanolamine in DMSO is used. Before applying the samples, the substrates or carriers are rinsed with PBS.
• the sample to be measured is incubated on the substrate thus prepared.
• the sample is applied directly to the substrate (uncoated substrate), optionally by covalent bonding on the optionally activated surface of the substrate.
• the sample is pretreated by one or more of the following methods: - heating (to a temperature up to the boiling point of the sample)
• enzymes for example proteases, nuclease, lipases, centrifuging,
• A-Beta aggregates are labeled by probes labeled for later detection.
• anti-A-beta antibodies are used as probes.
• Capture molecules and probes can be identical.
• capture molecules and probes differ.
• different anti-A-beta antibodies are used as catcher molecules and probes.
• capture molecules and probes are used, which are identical to each other, except for the possible dye marking.
• various probes are used which are identical to each other except for the eventual dye marking.
• at least 2 or more different capture molecules and / or probes are used which are made of different anti-A-beta antibodies and optionally also have different dye labeling.
• Capture molecules may be specific amino acid sequences of the A beta peptide, for example A-beta 1 -40 / 42, pyroGlu 3-40 / 42 or pyroGlu 11-40 / 42.
• probes several different molecules can be used, e.g. different anti-A-beta antibodies.
• uniformity of the Be Schweizer with catcher molecules, catcher molecules, labeled with fluorescent dyes can be used.
• a dye is preferably used which does not interfere with the detection.
• anti-A-beta antibodies that bind specifically to the N-terminal epitope of the A-beta peptide.
• the probes are characterized in that they emit an optically detectable signal selected from the group consisting of fluorescence, bioluminescence and chemiluminescence emission and absorption.
• the probes are labeled with dyes. These are preferably fluorescent dyes.
• the probes may differ both in their specific binding to the A-beta aggregates and in their different labeling, e.g. by fluorescent dyes. It is also possible to combine probes which are suitable for using FRET (fluorescence resonance energy transfer) as detection.
• FRET fluorescence resonance energy transfer
• probes specific for a particular A-beta aggregate species are used, such as A-beta (x-40,), A-beta (x-42), or pyro-glutamate A-beta (3x), pyro-glutamate A beta (11x).
• X is a whole, natural number between 1 and 40 or 42, where the One based on his knowledge of the sequence of the A-beta peptide determines the length of the sequence to be used.
• probes specific for particular A-beta aggregate forms may be used, such as the commercially available antibody "A-1" or "1-11".
• the present invention thus also relates to the use or use of A-beta-aggregate-specific or A-beta-oligomer-specific probes. These bind specifically to a specific A-beta aggregate, or A-beta oligomer, preferably for the species mentioned above. By specific binding to a specific A-beta aggregate or A-beta oligomer, the type and / or size as well as the structure of the A-Beta aggregate or A-beta oligomer can be determined.
• Another object of the present invention are thus also A-beta-aggregate-specific, or A-beta-oligomer-specific probes.
• A-beta peptides labeled with fluorescent dyes can be used as probes.
• the sample to be examined may be endogenous fluids or tissues.
• the sample is selected from CSF, blood, plasma and urine.
• the samples may undergo different processing steps known to those skilled in the art.
• the present invention is the possibility of determining A-Beta aggregates in untreated samples, preferably CSF.
• the present invention thus also provides a method for determining the composition, size and / or shape of A-beta aggregates.
• the detection of the labeled aggregates is done by scanning or other types of surface imaging.
• the detection is preferably carried out using confocal fluorescence microscopy, fluorescence correlation spectroscopy (FCS), in particular in combination with cross-correlation and single-particle immunosolvent Laser scanning assay and / or laser scanning microscope (LSM).
• the detection is performed in an alternative of the present invention with a confocal laser scanning microscope.
• a laser focus as e.g. used in laser scanning microscopy, or a FCS (Fluorescence Correlation Spectroscopy System), as well as the corresponding super-resolution variants such as STED or SIM.
• detection may be by a TIRF microscope, as well as the corresponding super-resolution variants thereof, such as STORM, dSTORM.
• detecting a high spatial resolution is advantageous.
• so many data points are collected that the detection of an aggregate in front of a background signal, which is e.g. is caused by device-specific noise, other nonspecific signals or nonspecifically bound probes.
• so many values are read out (readout values), such as location-resolved events, such as e.g. Pixels, are present.
• readout values such as location-resolved events, such as e.g. Pixels
• the information i. the values read out are multiplied, since separate information is obtained for each point, for each aggregate or for each detection event, depending on the particular probe which supplies the signal.
• This increases the specificity of the signal for each event.
• its composition can also be determined for each detected aggregate, i. the nature of the aggregate, that is the composition of Abeta species, e.g. A-Beta (1 - 40), A-Beta (1 - 42), Pyro-Glutamate-A-Beta (3 - 40/42, 11 -40/42) or mixtures thereof.
• the number of different probes is only due to the interference of limited use of fluorescent dyes. Thus, 1, 2, 3, 4 or more different probe-dye combinations can be used.
• Essential for the evaluation according to the method described above are spatially resolved information. This may be e.g. to be the type and / or intensity of fluorescence.
• the number of aggregates, their shape, size and / or their composition is determined according to the invention.
• information on the size of the oligomers can be obtained directly or indirectly, depending on whether the particles are smaller or larger than the spatial resolution of the imaging methods used, in one embodiment, algorithms for background minimization can be used and / or intensity thresholds can be applied.
• a fluorescent dye the dyes known to those skilled in the art can be used.
• GFP Green Fluorescence Protein
• conjugates and / or fusion proteins thereof, as well as quantum dots can be used.
• test results are objectively comparable and meaningful.
• an internal or external standard is used to quantify A-beta aggregates.
• the method according to the invention is a so-called surface FIDA (surface FIDA).
• the method according to the invention also shows a linearity with respect to the number of A-Beta aggregates over a large range.
• Another object of the present invention is the use of small, freely diffusible A-beta aggregates as biomarkers for the detection and detection of protein aggregation diseases, in particular AD.
• the invention also relates to a method for the detection and / or detection of protein aggregation diseases, in particular AD, characterized in that a sample of a body fluid of a patient, preferably CSF, is analyzed by the method according to the invention described above.
• Such standards for the quantification of oligomers or pathogenic aggregates which characterize a protein aggregation disease or an amyloid degeneration or protein misfolding disease are characterized in that a polymer is built up from polypeptide sequences which are identical in their respective sequence with the body's own proteins or have a homology of at least 50% over the corresponding portion of the body's own proteins that characterize a protein aggregation disease or an amyloid degeneration or protein misfolding disease, where the polymers do not aggregate.
• a standard in the sense of the present invention is a generally valid and accepted fixed reference used for comparing and determining properties and / or quantity, in particular for determining the size and amount of pathogenic aggregates from endogenous proteins.
• the standard in the sense of the present invention can be used for calibrating devices and / or measurements.
• the term "protein aggregation disease” may also include amyloid degenerations and protein misfolding diseases Examples of such diseases and the associated endogenous proteins are: A beta and tau protein for AD, alpha synuclein for Parkinson or prion Protein for Phon Diseases, for example human Creutzfeldt-Jakob Disease (CJD), sheep disease scrapie and bovine spongiform encephalopathy (BSE).
• “Homologous sequences” in the sense of the invention means that an amino acid sequence has an identity with an amino acid sequence from an endogenous pathogenic aggregate or oligomer causing a protein aggregation disease of at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%.
• identity the terms “homologous” or “homology” are used synonymously in the present description.
• the identity between two nucleic acid sequences or polypeptide sequences is calculated by comparison with the BESTFIT program based on the algorithm of Smith, TF and Waterman, MS (Adv. Appl. Math. 2: 482-489 (1981)) setting the following parameters for amino acids : Gap creation penalty: 8 and gap extension penalty: 2; and the following parameter for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3.
• the identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the entire sequence length, as determined by comparison with the aid of the GAP program based on the algorithm of Needleman, SB and Desire, CD. (J. Mol. Biol. 48: 443-453) is calculated by setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acid gap creation penalty: 50 and gap extension penalty: 3.
• amino acid sequences are identical for the purposes of the present invention, if they have the same amino acid sequence.
• corresponding subregion of the body's own proteins is to be understood as meaning those peptide sequences which, according to the definitions according to the invention, have an identical or with the stated percentage homologous peptide sequence of a monomer from which the standards according to the invention are built up.
• the standards do not aggregate, preferably by the use of monomeric sequences which do not aggregate, since the "corresponding subrange" of endogenous proteins is not responsible for the aggregation or which are responsible for blocking the aggregation Do not aggregate groups.
• the standards have a well-defined number of epitopes covalently linked (directly or via amino acids, spacers, and / or functional groups) for binding of the corresponding probes.
• Probes according to the invention are selected from the group consisting of: antibody, nanobody and affibody. Probes are also all molecules that have sufficient binding specificity for the aggregate to be detected, e.g. Dyes (thioflavin T, Congo red, etc.).
• the number of epitopes is determined by using a polypeptide sequence which is identical in its sequence to that subregion of the body's own proteins which forms an epitope or has a homology of at least 50% with this subregion, and thereby the biological Possesses activity of the epitope.
• a polypeptide sequence which is identical in its sequence to that subregion of the body's own proteins which forms an epitope or has a homology of at least 50% with this subregion, and thereby the biological Possesses activity of the epitope.
• Such a selected polypeptide sequence is incorporated in the desired number in the structure of the standards of the invention and / or linked together according to the invention.
• the standards according to the invention are polymers which are composed of the above-described polypeptide sequences, preferably epitopes, optionally containing further elements.
• above-described polypeptide sequences preferably epitopes, and / or their homologs having the biological activity of the corresponding epitope, the same or largest number of monomers based on the number of each one of the remaining monomer species of the standard and / or based on the number of all other monomers.
• the epitopes are epitopes of the A-beta peptide selected from subregions A-beta 1-8 (SEQ ID NO: 2), A-beta 1-11 (SEQ ID NO: 1).
• A-Beta 1-16 SEQ ID NO: 4
• A-Beta 3-11 SEQ ID NO: 5
• pyroGluA-Beta 3-11 SEQ ID NO: 6
• A-Beta 11 - 16 SEQ ID NO: 7
• pyroGluA-Beta 11 -16 SEQ ID NO: 8
• PyroGlu is the abbreviation for a pyroglutamate which is located at position 3 and / or 11 of the A beta peptide, preferably based on a cyclization of the N-terminal glutamate.
• the standard molecule according to the invention is a polymer of the above-defined polypeptide sequences.
• Oligomer according to the invention is a polymer formed from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomers (under Monomer is to be understood as the abovementioned polypeptide sequence), or multiples thereof, preferably 2-16, 4-16, 8-16, particularly preferably 8 or 16, or multiples thereof.
• the standards according to the invention are therefore oligomers or polymers according to the invention.
• the standards are water-soluble.
• the standards of the invention are constructed of different polypeptide sequences.
• such above-defined polypeptide sequences are aligned in a linear conformation.
• such above-defined polypeptide sequences are strung together to form a branched oligomer of the invention.
• such above-defined polypeptide sequences are strung together to form a cross-linked oligomer of the invention.
• Branched or cross-linked oligomers according to the invention can be prepared by linking individual building blocks by means of lysine or by click chemistry.
• the standards according to the invention ie the oligomers or polymers according to the invention, in addition to the polypeptide sequences present in a precisely defined number, preferably epitopes, may additionally contain additional amino acids, spacers and / or functional groups via which the polypeptide sequences, preferably epitopes are covalently linked together.
• the direct linkage of the polypeptide sequences, preferably epitopes with cysteine, in particular by means of disulfide bridging by cysteine is excluded (to avoid that reducing agents solve the bridging).
• an immediate linkage of the spacer with the polypeptide sequence on the one hand and with cysteine on the other hand excluded.
• the invention relates to a standard molecule comprising or constructed from copies of the amino-terminal portion of the A-beta peptide selected from subregions A-beta 1-8 (SEQ ID NO: 2), A-beta 1-11 (SEQ ID NO: 3), A-Beta 1-16 (SEQ ID NO: 4), A-Beta 3-11 (SEQ ID NO: 5) and pyroGluA-Beta 3-11 (SEQ ID NO: 6), A- Beta 11-16 (SEQ ID NO: 7) and pyroGluA-Beta 11 -16 (SEQ ID NO: 8), for example, the human N-terminal epitope (with the following sequence: DAEFRHDSGYE (1-11).
• the amplification of the epitopes by functional groups can be carried out before or after the synthesis of the individual building blocks.
• Characteristic of the standards according to the invention is the covalent linkage of the polypeptide sequences.
• polypeptide sequences to be used according to the invention may be identical to the sequence of the A-beta full-length peptide or show a homology of 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% having the sequence of A Beta full-length peptide.
• polypeptide sequences which are identical to a partial region of the A-beta full-length peptide or a homology of 50, 60, 65, 70, 71, 72, 73, 74 are also used to construct the standard molecules of the invention , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 , 100% with a portion of the A-beta full-length peptide.
• Essential for the sequences used according to the invention is their property not to aggregate (or controlled only under the conditions) and / or their activity as an epitope.
• the standards are constructed as dendrimers.
• the dendrimers according to the invention are composed of the above-described polypeptide sequences to be used according to the invention and may contain a central scaffold molecule.
• the scaffold molecule is a streptavidin monomer, more preferably a polymer, especially tetramer.
• the dendrimers of the invention contain in a variant polypeptide sequences which have a sequence which is identical to a portion of the A-beta peptide, or at least 50% homology to the corresponding Part area shows.
• the term at least 50% homology is also understood to mean a higher homology selected from the group consisting of 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%.
• Standards advantageously with higher aqueous solubility than pathogenic aggregates or oligomers from endogenous proteins, in one embodiment of the invention are formed from polypeptide sequences that are identical to the N-terminal region of the A beta peptide or at least 50% homology have to.
• the amino acid sequence A-beta 1 - 8 (SEQ ID NO: 2), A-beta 1 - 11 (SEQ ID NO: 3), A-beta 1 - is below the N-terminal region of an A-beta polypeptide.
• a standard molecule according to the invention may contain epitopes for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different probes.
• Epitopes characteristic of various probes can be incorporated into the standards of the invention by using polypeptide sequences that are identical to, or have at least 50% homology to, different regions of the A beta peptide, but the activity of the corresponding one Own epitops.
• polypeptide sequences that are identical or have 50% homology with the N-terminal region of the A-beta polypeptide, as well as polypeptide sequences that are identical or have at least 50% homology with the C-terminus, are used for this purpose of the A beta polypeptide.
• the standard molecules contain so-called spacers.
• a spacer is a molecule that has covalent bonds in it the standard molecule is incorporated and has certain physical and / or chemical properties that alter the properties of the standard molecule.
• hydrophilic or hydrophobic, preferably hydrophilic, spacers are used. Hydrophilic spacers are selected from the group of molecules formed from polyethylene glycol, sugar, glycerol, poly-L-lysine or beta-alanine.
• the standards according to the invention contain (further) functional groups in an alternative of the present invention.
• functional groups molecules that are covalently bound to the standard molecules.
• the functional groups contain biotin groups. This allows strong covalent attachment to streptavidin. Standard molecules containing biotin groups can thus be bound to molecules containing streptavidin groups. If the standard molecules of the invention contain biotin and / or streptavidin groups, then larger standards can be assembled or several, optionally different standard molecules, can be bound to a scaffold.
• the standard molecules contain dyes for spectrophotometric determination and / or aromatic amino acids.
• Aromatic amino acids are e.g. Tryptophan, tyrosine, phenylalanine or histidine, or selected from this group. The incorporation of tryptophan allows a spectrophotometric determination of the concentration of standards in solution.
• Another object of the present invention are dendrimers containing polypeptides that are identical in their sequence with the body's own proteins or have a homology of at least 50% over the corresponding portion with the body's own proteins that characterize a protein aggregation disease.
• the dendrimers of the invention may contain any of the above-described features of the standards or any combination thereof.
• An alternative of the present invention is:
• Dendrimers containing a well-defined number of epitopes for covalent binding of probes are well-defined numbers of epitopes for covalent binding of probes
• Dendrimer characterized in that it has a higher solubility in aqueous than the pathogenic aggregates of endogenous proteins that characterize a protein aggregation disease
• Dendrimer containing at least one spacer molecule and / or
• Dendrimer containing dyes for spectrophotometric determination and / or aromatic amino acids Dendrimer containing dyes for spectrophotometric determination and / or aromatic amino acids.
• the dendrimers have a radial symmetry.
• the branching of the first generation of the dendrimer is via lysine, in particular three Lys in amino acids.
• the polypeptide sequences in particular dendrimers, the polypeptide sequences, preferably epitopes, not via a bond to a sulfur atom, not via a thioether bond and / or not via cysteine (optionally by disulfide bridging Cysteine) with each other or with other elements of the standards such as amino acids, spacers and / or functional groups and / or other elements described above, in particular covalently bonded.
• the polypeptide sequences, preferably epitopes, and a spacer attached to the spacer are not linked to a sulfur atom, not via a thioether bond and / or via cysteine with each other or with other elements of the standards such as amino acids , further spacers and / or functional groups and / or other elements described above, in particular covalently bonded.
• the present invention further relates to a method for the preparation of a standard as described above.
• the standard of the invention is by means of peptide synthesis or recombinant processes known to those skilled in the art
• Another object of the present invention is the use of a standard or a dendrimer described above for the quantification of pathogenic aggregates or oligomers from the body's own proteins that characterize a protein aggregation disease.
• the standard is used to quantify A-beta oligomers.
• the oligomers or polymers according to the invention are used as standard in a method for the quantification of pathogenic aggregates or oligomers from endogenous proteins which characterize a protein aggregation disease or an amyloid degeneration or protein misfolding disease.
• the standards according to the invention are used in an embodiment of the present invention for calibration in the surface FIDA method, Elisa (sandwich Elisa) or FACS.
• the present invention relates to a kit comprising standard of the invention.
• the compounds and / or components of the kit of the present invention may be packaged in containers, optionally with / in buffers and / or solution. Alternatively, some components may be packaged in the same container. In addition or alternatively, one or more of the components could be attached to a solid support, such as a solid support. a glass plate, a chip or a nylon membrane or to the recess of a microtiter plate. Further, the kit may include instructions for using the kit for any of the embodiments.
• the standards are used to quantify pathogenic aggregates or oligomers from endogenous proteins by: in a first step, the standards or the dendrimers are labeled with probes and the number of probes bound to the standards or dendrimers is determined,
• pathogenic aggregates or oligomers from endogenous proteins which characterize a protein aggregation disease are labeled with probes, the number of probes binding to a respective pathogenic aggregate or oligomer is determined,
• step 2 the number of probes from step 1 binding to a standard or dendrimer is compared with that from step 2, and
• this determines the number and size of the oligomers from the body fluid.
• the standards according to the invention are used for the calibration of the surface FIDA method.
• the body's own pathogenic aggregates of body fluids eg A-beta aggregates are immobilized by a probe on a glass surface.
• an N-terminal capture probe can be used for this purpose.
• the aggregates are labeled by two different probes.
• A-beta aggregates for example, A-beta antibodies are used, which are both bound via an N-terminal binding epitope.
• the detection probes are labeled with, preferably different fluorescent dyes.
• monomer detection of endogenous polypeptides is ruled out by using three different or three differently labeled probes which bind to a similar or identical epitope in the test system.
• the detection of monomers can be excluded by not evaluating signals with a lower intensity by an intensity cut-off. Since larger aggregates have multiple binding sites for the two with different labeled dyes, monomer detection can alternatively or additionally be precluded by cross-correlation of these signals.
• the standards of the invention can be used as internal or external standards in the measurement.
• the present invention also provides a kit for the selective quantification of A-beta aggregates according to the method described above.
• a kit may contain one or more of the following components:
• Glass substrate coated with a hydrophobic substance preferably dextran, preferably carboxymethyl-dextran
• the compounds and / or components of the kit of the present invention may be packaged in containers, optionally with / in buffers and / or solution. Alternatively, some components may be packaged in the same container. In addition or alternatively, one or more of the components could be attached to a solid support, such as a solid support. a glass plate, a chip or a nylon membrane or to the recess of a microtiter plate. Further, the kit may include instructions for using the kit for any of the embodiments.
• the above-described catcher molecules are immobilized on the substrate.
• the KIT may contain solutions and / or buffers. To protect the dextran surface and / or the capture molecules immobilized thereon, they can be overcoated with a solution or a buffer.
• Another object of the present invention is the use of the method according to the invention for the diagnosis, early diagnosis and / or prognosis of AD.
• Another object of the present invention is the use of the method according to the invention for the monitoring of therapies of AD and Monitoring and / or reviewing the efficacy of drugs and / or therapeutics. This can be used in clinical trials, trials and therapy monitoring. For this purpose, samples are measured according to the method according to the invention and the results compared.
• Another object of the present invention is the use of the method according to the invention and the biomarker to decide whether a person is included in clinical trial.
• samples are measured in accordance with the method according to the invention and the decision is made with respect to a limit value.
• Another object of the present invention is a method for determining the effectiveness of active ingredients and / or healing methods by the method according to the invention, in which the results of samples are compared.
• the samples are body fluids, taken before or after, or at different times after administration of the active ingredients or implementation of the healing process.
• active substances and / or curative methods are selected, by means of which the A-Beta aggregates were reduced.
• the results are compared with a control which has not been subjected to the active ingredient and / or healing process.
• Preparation Substrate Glass supports were cleaned in an ultrasonic bath for 15 minutes. The surface was rinsed three times with water and dried in a nitrogen gas stream. The purified carriers were placed in a mixture of concentrated sulfuric acid and hydrogen peroxide of 3: 1 (VA /) for at least 30 minutes dipped to activate the hydroxyl groups. Subsequently, it was rinsed with water until the rinse water had a neutral pH. In a second rinsing step, 99% ethanol was used and then the carriers were dried in a nitrogen gas stream. The glass slides were immersed in a solution of 1-7% 3-aminopropyltriethoxysilane (APTES) in dry toluene for 1 to 4 hours.
• APTES 3-aminopropyltriethoxysilane
• CMD Carboxymethyl-dextran
• EDC N-ethyl-N- (3-dimethylaminopropyl) carbodiimide
• NHS N-hydroxysuccinimide
• a second activation of the surface was carried out with a solution of EDC / NHS (200 or 50 mM) for 5 minutes.
• the solution of the antibodies was added and incubated for 2 hours at 4 ° C. This covalently bound the antibodies to the CMD coated glass surface.
• the sample to be measured was incubated on the substrate for 1 hour, this became then washed twice with TBST (0.1%) (w / w), Tween-20 in TBS buffer, TBS: 50 nM Tris-HCl, 0.15 M nacl, pH 7.4).
• Nab 228, anti-mouse Alexa 633 and 6 E10 Alexa 488 antibodies were used.
• the Nab 228 antibodies were labeled with a KIT (Fluorescence Labeling KIT Alexa-647, Molecular Probes, Düsseldorf, Germany) according to the manufacturer's instructions.
• the labeled antibodies were stored in PBS with 2 mM sodium azide at 4 degrees in the dark.
• the amount of antibody used was dependent on the desired level of label.
• the probes were added and incubated for 1 hour at room temperature, then washed five times with TBST and twice with TBS.
• the measurement was carried out using a confocal laser scanning microscope LSM 710 (Carl Zeiss, Jena, Germany).
• the microscope was equipped with an argon ion laser and three helium neon lasers.
• the laser beams were focused on a diffraction-limited spot of 0.25 femtoliter volume.
• the fluorescence intensity of an area of 1000 ⁇ 1000 pixels was determined. Since different probes were used, a colocalization analysis was performed. In order to obtain representative values, this area was measured at several points of the carrier.
• an A-beta oligomer standard was constructed having 16 epitopes for N-terminal binding A beta antibodies (epitope corresponds to A beta (1-11), sequence: DAEFRHDSGYE).
• MAP multiple antigen antigen peptide
• A-beta1-11 N-terminal A beta epitopes A-beta1-11. These were coupled in accordance with FIG. 3A to a triple lysine core which contained two tryptophans for the exact determination of the MAP concentration by means of UVA / IS spectroscopy.
• a biotin tag was added N-terminally. This served to couple four 4-MAP units to a streptavidin tetramer, shown as B in FIG.
• To Incubation of 4-MAP and streptavidin was 16-MAP as shown in Figure 3 under C. 16-MAP was separated from other components of the incubation approach by size exclusion chromatography.
• MAP-16 was serially diluted in PBS and used in the sFIDA assay for the detection of A-beta oligomers. .Preparation glass plate
• Glass microtiter plates were cleaned in an ultrasonic bath for 15 minutes and then treated with a plasma cleaner for 10 minutes.
• the wells were incubated in 5 M NaOH for at least 3 hours, rinsed with water and then dried in a nitrogen gas stream.
• the glass surface was hydroxylated and then activated with amino groups.
• the glass plates were incubated in a solution of 5 M ethanolamine in DMSO overnight. Subsequently, the glass plates were rinsed with water and dried in a nitrogen gas stream.
• Carboxymethyl-dextran was dissolved in water at a concentration of 20 mg to ml and treated with N-ethyl-N- (3-dimethylaminopropyl) carbodiimide (EDC), (200 mM) and N-hydroxysuccinimide (NHS), (50 mM). After a pre-incubation of 10 minutes, the solution was incubated for a further 2 hours at room temperature. Subsequently, the glass plates were washed with water.
• EDC N-ethyl-N- (3-dimethylaminopropyl) carbodiimide
• NHS N-hydroxysuccinimide
• a second activation was carried out with a solution of EDC / NHS (200 or 50 mM) for 5 minutes.
• the solution of the antibodies was added and incubated for 2 hours at 4 ° C. This covalently bound the antibodies to the CMD-activated glass surface.
• the ⁇ -16-containing sample to be measured was incubated on the glass for 1 hour, then washed three times with TBST (0.1%) (w / w), Tween-20 in TBS buffer, TBS: 50 nM Tris-HCl, 0.15 M NaCl, pH 7.4).
• Alexa 488 antibodies and IC-16 antibodies were used.
• the IC16 antibodies were labeled with a kit (fluorescence labeling KIT Alexa-647, Molecular Probes, Düsseldorf, Germany) according to the manufacturer's instructions.
• the labeled antibodies were stored in PBS with 2 mM sodium azide at 4 ° C in the dark.
• the probes were added and incubated for 1 hour at room temperature, then washed five times with TBST and twice with water.
• TIRF total internal reflection
• Figure 4 shows the results of the measurements. It can be clearly seen that the sFIDA signal, i. the amount of colocalized pixels correlates with the concentration of MAP-16 molecules.
• A-beta monomers and oligomers consisting of synthetic A-beta, according to a protocol of Johannson et al., FEBS J. 2006, 273, pages 2618-2630, prepared and assayed with the system.
• the A beta oligomers were serially diluted in PBS, and in a concentration series, the linearity of the assay was checked.
• FRET means Förster resonance energy transfer. In FRET, the energy of an excited fluorochrome is transferred to a second fluorochrome. The FRET intensity depends, inter alia, on the distance between donor and acceptor and can be observed in the range of up to 10 nm. Thus, FRET in sFIDA should be able to be used to distinguish A beta monomers from A beta oligomers.
• a beta monomers and A beta oligomers were prepared by size exclusion chromatography and immobilized for sFIDA measurements as described above.
• the fluorochromes were excited at a wavelength of 488 nm and the FRET emission was detected at a wavelength of 705 nm.
• As controls also two samples were measured, in each of which only one fluorescent dye-coupled antibody was added.
• sFIDA is also suitable for detecting A-beta aggregates in the CSF of Alzheimer's mouse models and, if so, at what dilution.
• the CSF of APP / Ps1 mice and non-transgenic control animals was diluted 1: 10, 1:50 and 1: 250 in PBS buffer and measured by means of sFIDA.
• a ⁇ oligomer standard with 16 epitopes for N-terminal A ⁇ binding antibodies corresponding to the first 11 amino acids of A ⁇ (sequence: DAEFRHDSGYE).
• FIG. 4 Size exclusion chromatography separated from other components of the incubation approach.
• sFIDA measurements of MAP-16 at various concentrations diluted in PBS buffer PBS buffer without MAP-16 served as a negative control.
• FIG. 5 is a diagrammatic representation of FIG. 5
• A) sFIDA is non-sensitive to A ⁇ monomers, but B) detects A ⁇ oligomers concentration-dependently linearly and with high sensitivity.
• a ⁇ monomers and oligomers were prepared from synthetic A ⁇ by size exclusion chromatography and diluted in PBS buffer.
• FIG. 6 is a diagrammatic representation of FIG. 6

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