Method for detecting multimeric, preferably dimeric peptides using single-domain antibodies

Abstract

The invention relates to a method for detecting a multimeric, preferably dimeric peptide, containing a first and a second peptide monomer in a sample, comprising the following method steps: A) preparing a carrier material antibody conjugate, comprising a carrier material to which a single-domain carrier antibody specific to the first peptide monomer is bound; B) bringing the sample into contact with the carrier material antibody conjugate, wherein a complex is formed between the carrier material antibody conjugate and the dimeric peptide; C) bringing the complex into contact with a single-domain detection antibody specific to the second peptide monomer, which single-domain detection antibody comprises a detection marker, wherein the single-domain detection antibody binds to the complex, wherein the single-domain carrier antibody and the single-domain detection antibody identify the same epitope on the first peptide monomer and on the second peptide monomer, respectively; and D) detecting the dimeric peptide by means of the detection marker. A method of this type allows targeted detection of multimeric or dimeric peptides in a sample.

Description

[0001] Method for the detection of multimeric, preferably dimeric peptides using single-domain antibodies

[0002] Description

[0003] In many neurodegenerative diseases, such as Alzheimer's or Parkinson's, protein aggregates can be detected in the brain or neurons. Many of these protein aggregates are difficult to characterize, frequently consisting of poorly defined multimers, such as dimers or oligomers of proteins that are often at least partially unfolded, making detection by antibodies difficult. Detection of neurodegenerative diseases by antibodies is also complicated by heterophilic antibodies, which, as nonspecific antibodies, can cross-react with antigens of a different type and thus trigger false-positive test results. Immunoassays in which an analyte is detected by antibodies are often sensitive to cross-reacting substances, which can produce false test results.Another problem is that analytical antibodies are often very sensitive, so incorrect handling of an immunoassay can produce inaccurate test results.

[0004] An object of the present invention is to provide a method for detecting multimers, preferably dimeric peptides, that allows for more reliable detection of multimeric peptides. A further object of the present invention is to provide a method for detecting multimers, preferably dimeric peptides, in which the probability of cross-reacting heterophilic antibodies is reduced.

[0005] The object of the present invention is to provide a method for detecting multimeric proteins that is improved with respect to the aforementioned disadvantages. The invention furthermore relates to a method for detecting alpha-synuclein, which is also improved with respect to the aforementioned disadvantages. The invention furthermore relates to a kit for carrying out the method, expression constructs for expressing the specific antibodies, a positive control for carrying out the method, and certain carrier-antibody conjugates that can be used in the method.

[0006] The present invention relates to a method for detecting a multimeric peptide, preferably a dimeric peptide, containing a first and a second peptide monomer in a sample, comprising the method steps:

[0007] A) Providing a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for the first peptide monomer is bound,

[0008] B) contacting the sample with the carrier-antibody conjugate, whereby a complex is formed between the carrier-antibody conjugate and the multimeric peptide,

[0009] C) contacting the complex with a single-domain detection antibody specific for the second peptide monomer, which comprises a detection label, wherein the single-domain detection antibody binds to the complex, wherein the single-domain carrier antibody on the first peptide monomer and the single-domain detection antibody on the second peptide monomer recognize the same epitope, and

[0010] D) Detection of the multimeric peptide using the detection label.

[0011] In contrast to conventional antibodies, the method according to the invention uses single-domain antibodies. These are also referred to as "nanoantibodies" or "nanobodies" and are individual, monomeric antibody fragments of a monomeric variable domain of an antibody. These single-domain antibodies can, for example, be produced from the monomeric variable antigen-binding domains of heavy-chain antibodies produced by cartilaginous fish such as sharks or camels. The single-domain antibodies are thus antigen-binding antibody fragments that are monomeric, unlike conventional antibodies, which are composed of two heavy chains and two light chains. These single-domain antibodies are often considerably more stable than conventional antibodies, so that methods for detecting analytes based on single-domain antibodies can be carried out more reliably.Furthermore, the use of single-domain antibodies significantly reduces the occurrence of heterophilic, cross-reacting antibodies compared to conventional antibodies. Thus, detection methods based on single-domain antibodies are significantly more reliable.

[0012] During process step B), the carrier-antibody conjugate recognizes the epitope on a first peptide monomer of the multimeric peptide via the single-domain carrier antibody and binds the multimeric peptide to the carrier material via the first peptide monomer through complexation. The multimeric peptide immobilized on the carrier material can then be detected using a specific single-domain detection antibody. A conjugate is formed from the single-domain detection antibody bound to the multimeric peptide and the multimeric peptide bound to the carrier material. The multimeric peptide is then detected via the detection marker linked to the single-domain detection antibody.

[0013] The single-domain carrier antibody recognizes the same epitope on the first peptide monomer as the single-domain detection antibody recognizes on the second peptide monomer. Since the epitope is already occupied by the binding of the multimeric peptide to the single-domain carrier antibody, a second peptide monomer is required within the multimeric peptide to provide the same epitope for binding to the single-domain carrier antibody. The method according to the invention is thus capable of detecting dimeric peptides within multimeric protein agglomerates. The multimeric peptide can have any number of monomers, for example, it can be a trimer or a tetramer with three or four monomeric peptides. The detection of a dimeric peptide is preferred in the method according to the invention.Particularly preferably, the method according to the invention can be used to detect a homo-dimer in which two identical peptide monomers form the dimer.

[0014] In process step B), the single-domain carrier antibody binds to the first peptide monomer, blocking the epitope recognized by the single-domain detection antibody on the first peptide monomer. The single-domain detection antibody can therefore only bind the peptide on the second peptide monomer. Thus, the method according to the invention is particularly suitable for detecting dimeric peptides, even in protein agglomerates or protein oligomers. The method according to the invention can preferably be used to detect dimers selected from: beta-amyloid peptide dimer, tau dimer, phosphorylated tau dimer, or alpha-synuclein dimer. The method is preferably used to detect alpha-synuclein dimer.

[0015] In particular, the method according to the invention can be used to detect dimers in multimeric protein aggregates of beta amyloid, tau protein or alpha synuclein.

[0016] Beta-amyloid dimers, or aggregates of beta-amyloid, are formed by the cleavage of the amyloid precursor protein by enzymes such as beta-secretase or gamma-secretase and are found in senile plaques in patients with Alzheimer's disease, for example. Deposits of tau protein, which may contain the tau dimer, are also found in neurodegenerative diseases such as Alzheimer's disease or chronic traumatic encephalopathy. These diseases are also known as tauopathies and are characterized, among other things, by the accumulation of aggregates of tau protein in the brains of patients.

[0017] Alpha-synuclein is a small, soluble protein that, among other things, controls dopamine release in vertebrates. In neurodegenerative diseases such as Parkinson's disease or Lewvy body dementia, deposits of alpha-synuclein aggregates can be detected in the brain tissue of patients. The method according to the invention can thus also be used to detect synucleinopathies characterized by the accumulation of alpha-synuclein aggregates in the brain of patients.

[0018] The epitope recognized by the single-domain carrier antibody and the single-domain detection antibody on the first peptide monomer and the second peptide monomer can, for example, be a protein sequence of a length between 10 and 40 amino acids, preferably between 15 and 30 amino acids, to which the single-domain antibodies bind.

[0019] The multimeric peptide can, in particular, be a dimeric peptide. The dimeric peptide can have a monomer that has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to the amino acid sequence of SEQ. ID No. 1 (human alpha-synuclein), or SEQ. ID No. 2 (human tau protein), or SEQ. ID No. 3 (human beta-amyloid).

[0020] The percentage identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994 Nucleic Acid Research 22: 4673-4, 680). A nucleotide sequence or an amino acid sequence can also be used as a so-called "query sequence" to perform a search in public nucleic acid or protein sequence databases, for example, to identify further unknown homologous sequences that can also be used in embodiments of this invention. Such searches can be performed using the algorithm of Karlin and Altschul (1999 Proceedings of the National Academy of Sciences USA 87: 2264 to 2268), modified as in Karlin and Altschul (1993 Proceedings of the National Academy of Sciences USA 90: 5873 to 5877). Such an algorithm is included in the programs NBLAST and XBLAST by Altschul et al. (1999 Journal of Molecular Biology 215: 403 to 410).The BLAST program can also be used (Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) J. Mol. Biol. 215:403-410). If there are gaps between two sequences, gapped BLAST can be used, as described in Altschul et al. (1997 Nucleic Acid Research, 25: 3389 to 3402).

[0021] It is known to those skilled in the art that large plasmids can be produced not only using conventional cloning techniques, but also using techniques such as those described in U.S. patents Nos. 6,472,184 B1, entitled "Method for Producing Nucleic Acid Polymers," and 5,750,380, entitled "DNA Polymerase Mediated Synthesis of Double Stranded Nucleic Acid Molecules," which are hereby incorporated by reference in their entirety. These techniques allow the synthesis of large plasmids using solid-phase synthesis without the need for amplification of the plasmids in cell culture.

[0022] In the method, a carrier material-antibody conjugate can be used in which the single-domain carrier antibody is bound to the carrier material via a peptide linker. The peptide linker has a length of at least 20 amino acids and comprises an alpha-helical region. Such a peptide linker is particularly suitable for binding the single-domain carrier antibody to the carrier material without impairing its biological activity. The peptide linker preferably has a length of between 30 and 130 amino acids, more preferably a length of between 50 and 120 amino acids. Such lengths are particularly suitable for mobilizing the single-domain carrier antibody to the carrier material without significantly impairing the biological activity. The peptide linker can, for example,have an amino acid sequence which has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, particularly preferably at least 98%, further particularly preferably at least 99% to the amino acid sequence of a SUMO protein, such as a human SUMO protein and / or a mouse SUMO protein and / or a yeast SUMO protein, such as the yeast SUMO protein ubiquitin-like protein SMT3, the amino acid sequence of which is described under the identification number Q12306 (SMT3_YEAST) in the UniProt protein knowledgebase (UniProtKB). In particular, for example, the proteins deposited under the following identification numbers in the UniProt protein knowledgebase are also “SUMO proteins” within the meaning of the present invention: P63165 (SUMO1_HUMAN), P61956 (SUMO2_HUMAN), P55854 (SUMO3_HUMAN), Q6EEV6 (SUMO4_HUMAN), G2XKQ0 (SUMO5_HUMAN), P63166 (SUMO1_MOUSE), P61957 (SUMO2_MOUSE), Q9Z172 (SUMO3_MOUSE) and 014399 (UBL1_SCHPO).

[0023] Preferably, the peptide linker has an alpha-helical region selected from the alpha-helical region of a SUMO peptide (small ubiquitin-like modifier peptide) or an Fe region of an antibody. Preferably, the peptide linker has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ ID No. 4 (human SUMO1), SEQ ID No. 5 (mouse SUMO), SEQ ID No. 6 (human SUMO2), SEQ ID No. 7 (human SUMO3), SEQ ID No. 8 (yeast SUMO), or SEQ ID No. 9 (F c-region of a human antibody). These are fragments of the human SUMO sequences 1 to 3, a fragment of a mouse SUMO sequence, and a fragment of a yeast SUMO sequence. The inventors of the present method have found that these SUMO fragments, as well as the Fc region of a human antibody, are particularly suitable for immobilizing single-domain antibodies to a carrier material without loss of biological activity. The peptide linker can be bound to the carrier material via a tag sequence. Such a tag sequence enables the single-domain carrier antibody to be linked to the carrier material particularly reliably via the peptide linker. The tag sequence is preferably a His-tag sequence, which furthermore preferably has a length of between three and 20 histidines. For example, a tag sequence with six histidines or a tag sequence with 14 histidines can be used for linking to the carrier material.Furthermore, it is also possible to use a GST tag, which is a glutathione S-transferase. Such a GST tag can also bind well to support materials and thus enable the binding of the single-domain carrier antibody to the support material via the peptide linker. The tag sequence with the peptide linker can be bound via the N-terminus or the C-terminus of the single-domain carrier antibody. Preferably, the tag sequence and the peptide linker are bound to the N-terminus of the single-domain carrier antibody. Binding via the N-terminus of the single-domain carrier antibody particularly enables immobilization of the single-domain antibody to the support material without loss of biological activity. The tag sequence can also ensure correct orientation of the single-domain carrier antibody on the support material.This makes it possible, in particular, to ensure that the single-domain carrier antibody is immobilized to the carrier material on the one hand, but is still able to bind the epitope of the first peptide monomer on the other.

[0024] According to a further embodiment of a method according to the invention, the single-domain carrier antibody and the single-domain detection antibody can be the same single-domain antibody. This enables particularly simple detection of the same epitope on the first peptide monomer and the second peptide monomer of the multimeric peptide.

[0025] According to a further embodiment of a detection method according to the invention, the single-domain carrier antibody and the single-domain detection antibody are specific for alpha-synuclein. Preferably, the single-domain carrier antibody and the single-domain detection antibody have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (single-domain antibody NbSyn2), or to the amino acid sequence of SEQ. ID. No. 11 (single-domain antibody NbSyn87), particularly preferably they are identical to the amino acid sequence of SEQ. ID. No. 10 (single-domain antibody NbSyn2), or to the amino acid sequence of SEQ. ID. No. 11 (single-domain antibody NbSyn87). The single-domain antibody with SEQ. ID. No. 10 , the single-domain antibody “NbSyn2” can bind particularly to the C-terminus of alpha-synuclein, which is particularly conserved between alpha-synuclein from different species."NbSyn2" can recognize alpha-synuclein from a variety of species, including Homo sapiens and the mouse (mus musculus). In contrast, the single-domain antibody with SEQ ID No. 11, known as "Nbsyn87," binds to a less conserved region of human alpha-synuclein and is therefore suitable for the detection of primarily human alpha-synuclein.

[0026] The epitope on the first peptide monomer and the epitope on the second peptide monomer can be selected from an amino acid sequence having SEQ. ID. No.

[0027] 12 (amino acids 118-131 of alpha-synuclein recognized by NbSyn87) or SEQ. ID No.

[0028] 13 (amino acids 137-140 of alpha-synuclein recognized by NbSyn2). The epitope with SEQ ID No. 13 represents a highly conserved epitope of alpha-synuclein from a wide variety of species and can thus be used to target alpha-synuclein from a variety of sources. In contrast, the epitope with SEQ ID No. 12 is primarily suitable for the detection of human alpha-synuclein.

[0029] In process step A), a carrier material-antibody conjugate can be provided, the carrier material of which comprises a plastic. The plastic is preferably selected from a group consisting of: polyolefin, polystyrene, polyamide, polyurethane, polyvinyl chloride, phenolic polymers, nitrocellulose, latex, polysaccharide, composite material, ceramic, silicon dioxide, graphene, and metal. Silicon dioxide can comprise silicon wafer, silicon nitride, or glass as materials.

[0030] The carrier material preferably comprises a microtiter plate, microspheres, a microfluidic system (lab-on-a-chip system), or a microneedle patch comprising one of the above-mentioned materials. Microtiter plates, microspheres, or a microneedle patch are particularly well suited for immobilizing the single-domain carrier antibody and for subsequent binding with the single-domain detection antibody in process step C) and subsequent detection in process step D). The material of the microtiter plates, microspheres, or the microneedle patch is preferably plastic, selected from the group consisting of: polyolefin, polystyrene, polyvinyl chloride, polyamide, particularly preferably polystyrene or polyvinyl chloride. Other preferred materials are graphene or silicon dioxide.

[0031] For example, a microtiter plate with isolated wells can be used. In particular, a microtiter plate with 96 wells can be used. Such a microtiter plate can be used for an immunoassay according to a method of the invention in which single-domain antibodies are used.

[0032] In process step B) of a process according to the invention, the single-domain carrier antibody of the carrier material-antibody conjugate can bind to the dimeric peptide in a solution having a pH between 6.9 and 8, preferably a pH between 7.2 and 7.6 and an alkali metal salt concentration between 120 mM and 140 mM and a phosphate ion concentration between 10 mM and 15 mM.

[0033] In process step C) of a process according to the invention, the single-domain detection antibody can bind to the dimeric peptide in a solution having a pH between 6.9 and 8, preferably a pH between 7.2 and 7.6 and an alkali metal salt concentration between 120 mM and 140 mM and a phosphate ion concentration between 10 mM and 15 mM.

[0034] These conditions correspond to physiological conditions and enable particularly good binding of both the single-domain carrier antibody and the single-domain detection antibody to the epitopes of the dimeric peptide. A phosphate-buffered saline solution (PBS) is particularly suitable for performing steps B) and C). The solution contains 137 mM sodium chloride, 2.7 mM potassium chloride as alkali metal salts, and 12 mM total phosphate (in the form of HPO4). 2" and H2PO4"). The pH of the adjusted buffer solution is 7.4. The phosphate-buffered saline solution corresponds to the osmotic pressure of the human organism.

[0035] In one embodiment of a method according to the invention, a single-domain detection antibody comprising a detection marker selected from a group consisting of: biotin, biotinylatable peptide sequence, preferably Avitag, chemiluminescence marker, and fluorescence marker can be used in method step C).

[0036] Biotin, as a detection marker, binds strongly to streptavidin and avidin. These molecules can be coupled with enzymes such as horseradish oxidase, alkaline phosphatase, or glucose peroxidase, which allow detection of the multimeric peptide, especially the dimeric peptide, by means of a color reaction. Alkaline phosphatase, for example, can convert colorless p-nitrophenyl phosphate as a dye substrate, which is converted into the pale yellow p-nitrophenol. Peroxidase uses o-phenylenediamine as the dye substrate.

[0037] Instead of dye reactions, chemiluminescence reactions can also be used to detect the multimeric peptide. For example, the MSD (Meso Scale Discovery) reaction uses a single-domain detection antibody coupled to a metal, such as ruthenium. The microtiter plate has an electrode at the bottom of the wells. The binding of the single-domain detection antibody coupled to ruthenium enables a redox reaction with the electrode in the wells, producing light that can be detected by a CCD camera. This type of MSD detection method can be more sensitive than a dye reaction.It is also possible to use single-domain detection antibodies bound to fluorescent molecules as detection markers, whereby detection is carried out particularly sensitively using a laser, whereby the detection of individual molecules is also possible (Single Molecule Counting (SMC™) immunoassay technology).

[0038] Alternatively, a single-domain detection antibody can be used that has an Avi tag at the C-terminus, a sequence that can be specifically labeled with biotin using the enzyme biotin ligase (BirA).

[0039] In a further embodiment of a method according to the invention, a positive control reaction can be carried out, wherein a homodimer is used as the dimeric peptide for the positive control reaction, the monomer of which has a sequence identity of at least 80%, preferably 90%, more preferably at least 90% with the amino acid sequence of SEQ. ID. No. 14 (amino acid sequence of Ser87Cys), with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID. No. 14 remains unchanged. Such a homodimer has a disulfide bridge due to the amino acid cysteine ​​at position 87, which stabilizes the homodimer. Such a stabilized homodimer is particularly suitable as a positive control for a detection reaction according to the invention.

[0040] In step B) of the method according to the invention, a sample of cerebrospinal fluid, spinal fluid, blood, serum, plasma, a saliva sample, a tissue biopsy, a throat swab, a nasal swab, urine, or exosome diagnostics from a patient can be used. Such samples are particularly well suited for detecting a dimeric protein, in particular alpha-synuclein.

[0041] The method according to the invention can be used, in particular, to determine whether a symptom-free patient is at risk of developing Parkinson's disease. In particular, alpha-synuclein dimers are detected in polymorphic peptide oligomers. The polymorphic peptide oligomers can comprise a variety of poorly characterized protein aggregates, for example, dimers, trimers, tetramers, or multimers.

[0042] The present invention also relates to a method for detecting alpha-synuclein in a patient sample, comprising the method steps:

[0043] A) Providing a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for alpha-synuclein is bound,

[0044] B) contacting the sample comprising alpha-synuclein with the carrier-antibody conjugate, whereby a complex is formed between the carrier-antibody conjugate and the alpha-synuclein,

[0045] C) contacting the complex with a single-domain detection antibody specific for alpha-synuclein comprising a detection marker, wherein the single-domain detection antibody binds to the complex and

[0046] D) Detection of alpha-synuclein using the detection marker. This method enables particularly reliable detection of alpha-synuclein due to the single-domain carrier antibodies and the single-domain detection antibodies.

[0047] Such a method can also detect alpha-synuclein monomers. Various single-domain antibodies can be used as single-domain carrier antibodies and as single-domain detection antibodies, which recognize different epitopes of alpha-synuclein.

[0048] Such a method makes it particularly easy to detect alpha-synuclein monomers, as different epitopes are recognized on a monomeric alpha-synuclein. Such methods can be used in combination with methods for detecting dimeric alpha-synuclein in alpha-synuclein agglomerates to determine the ratio of dimeric alpha-synuclein to monomeric alpha-synuclein. In neurodegenerative diseases such as Alzheimer's disease, an increase in the ratio of dimeric alpha-synuclein to monomeric alpha-synuclein is expected.

[0049] According to a further embodiment of a method according to the invention, the method serves to detect Parkinson's disease in humans or to determine whether a symptom-free human patient is at risk of developing Parkinson's disease. A single-domain antibody is used as the single-domain carrier antibody that has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 11 (NbSyn87). Particularly preferably, the single-domain carrier antibody can be identical to the amino acid sequence of SEQ. ID. No. 11. The single-domain antibody "NbSyn87" can, as already explained above, specifically detect a C-terminal end of human alpha-synuclein and is thus particularly suitable for the determination of Parkinson's disease in humans.

[0050] A further embodiment of a method according to the invention serves to detect alpha-synuclein in vertebrates selected from a group consisting of: humans, mice, and monkeys, wherein the single-domain carrier antibody used is a single-domain antibody that has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2). Particularly preferably, the single-domain carrier antibody can be identical to the amino acid sequence of SEQ. ID. No. 10. As already described above, the antibody "NbSyn2" can detect alpha-synuclein from various species, including humans, mice, and monkeys.

[0051] The present invention also relates to a kit for carrying out a method as described above, wherein a multimeric peptide containing a first and a second peptide monomer can be detected in a sample within a protein agglomerate. The kit comprises the following components: a carrier material-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for the first peptide monomer is bound; a single-domain detection antibody specific for the second peptide monomer, which comprises a detection marker; and reagents for detecting the multimeric peptide using the detection marker.

[0052] Such a kit can be used for the detection of multimeric peptides containing a first and a second peptide monomer. The kit is particularly suitable for the detection of dimeric peptides containing a first and a second peptide monomer. The kit can contain the above-mentioned reagents, in particular the carrier-antibody conjugates, the single-domain detection antibodies with the detection marker, and suitable detection reagents as described above.

[0053] The kit may further contain instructions for performing the methods described herein. In particular, the instructions may include step-by-step instructions for performing a method for detecting a multimeric peptide using the method steps A) to D) as described above.

[0054] The kit may contain the single-domain detection antibody and the single-domain carrier antibody in a storage-stable form, for example, in lyophilized form. Furthermore, buffer solutions for carrying out the method, such as a PBS solution, may also be present in the kit. A washing solution for washing off unbound sample components may also be present, enabling the removal of unbound components of a sample after the complex between the carrier material-antibody conjugate and the multimeric, preferably dimeric, peptide has been formed. Likewise, the carrier material suitable for binding the single-domain carrier antibody may already be present in the kit. This can be, in particular, microtiter plates, microspheres, or a microneedle patch.

[0055] The single-domain carrier antibody and / or the single-domain detection antibody of the kit can have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2) or the amino acid sequence of SEQ. ID. No. 11 (NbSyn87). Particularly preferably, the single-domain carrier antibody and / or the single-domain detection antibody can be identical to the amino acid sequences of SEQ. ID. No. 11 or the amino acid sequence of SEQ. ID. No. 10.

[0056] The kit can further serve to detect an alpha-synuclein dimer and comprise a homodimer of alpha-synuclein as a standard for a positive control reaction, wherein the monomer has a sequence identity of at least 80%, preferably 90%, more preferably at least 90% with the amino acid sequence of SEQ. ID. No. 14 (alpha-synuclein Ser87Cys), with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID. No. 14 remains unchanged. Such a homodimer has a disulfide bridge due to the amino acid cysteine ​​at position 87, which stabilizes the homodimer. Therefore, this protein is particularly well suited as a standard.

[0057] Alternatively or additionally, the kit can also be used to detect alpha-synuclein in a patient's sample, in which case monomeric alpha-synuclein can preferably be used as a standard in the kit.

[0058] In particular, the kit may be suitable for detecting alpha-synuclein in a sample, so it further includes:

[0059] - a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for alpha-synuclein is bound,

[0060] - a single-domain detection antibody specific for alpha-synuclein comprising a detection marker, and - reagents for detecting alpha-synuclein using the detection marker.

[0061] The kit may also contain instructions for performing a method for detecting alpha-synuclein in a patient sample. This particularly applies to steps A) to D) as described above for detecting alpha-synuclein.

[0062] The kit can also be used to detect alpha-synuclein, where the single-domain carrier antibody and the single-domain detection antibody are different single-domain antibodies that recognize different epitopes of alpha-synuclein. Using such a kit, for example, a single-domain carrier antibody immobilized on a carrier material can recognize a first epitope of alpha-synuclein in process step B), and then, in process step C), the single-domain detection antibody recognizes a second epitope of alpha-synuclein on the bound alpha-synuclein that is different from the first epitope. Such a kit can therefore generally detect alpha-synuclein, regardless of its aggregation state. The alpha-synuclein can exist as a monomer, dimer, or multimer.

[0063] In particular, one of the single-domain antibodies can have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10. Preferably, one of the single-domain antibodies can be identical to the amino acid sequence of SEQ. ID. No. 10. In this case, the other single-domain antibody can have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 11. Preferably, the other single-domain antibody can be identical to the amino acid sequence of SEQ. ID. No. 11.

[0064] To detect human alpha-synuclein, a single-domain carrier antibody is preferably used which has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 11. The single-domain carrier antibody is preferably identical to the amino acid sequence of SEQ. ID. No. 11. A single-domain antibody is then used as the single-domain detection antibody, which preferably has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10. The single-domain detection antibody can preferably be identical to the amino acid sequence of SEQ. ID. No. 10.

[0065] As already explained above, the single-domain antibody "NbSyn87" recognizes a region of human alpha-synuclein that is poorly conserved among different alpha-synuclein proteins and is therefore specific for human alpha-synuclein. In contrast, the single-domain antibody "NbSyn2" recognizes a region that is conserved among different alpha-synuclein proteins from different species and thus allows the recognition of alpha-synuclein proteins from a wide variety of species, for example, Homo sapiens or the mouse (mus musculus).

[0066] “NbSyn87” as a single-domain carrier antibody thus only allows the binding of human alpha-synuclein, which can be bound by the single-domain detection antibody “NbSyn2” in the subsequent process step C) and detected in process step D), so that such a kit allows a detection specific for human alpha-synuclein.

[0067] To detect alpha-synuclein from a wide variety of species, a single-domain carrier antibody is preferably used which has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2). The single-domain carrier antibody is preferably identical to the amino acid sequence of SEQ. ID. No. 10. The same single-domain antibody is then used as the single-domain detection antibody, which preferably has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2). One of the single-domain detection antibodies can preferably be identical to the amino acid sequence of SEQ. ID. No. 10.

[0068] The single-domain carrier antibody having a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ ID. No. 10, can be used to detect alpha-synuclein from species such as Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Gorilla gorilla gorilla (western lowland gorilla), Macaca fascicularis (cynomolgus macaque), Pan paniscus (bonobo), Pan troglodytes (chimpanzee), Bos taurus (cattle), Serinus canaria (Canary Island chaffinch), Macaca mulatta (rhesus macaque), Sus scrofa (pig), Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii), Ateles geoffroyi (Geoffroy's spider monkey), Erythrocebus patas (red monkeys) (Cercopithecus patas), Lagothrix lagotricha (brown woolly monkey) (Humboldt's woolly monkey), Saguinus labiatus (red-bellied tamarin) and Xenopus laevis (African clawed frog).

[0069] A kit containing such a single-domain carrier antibody and single-domain detection antibody allows the binding of alpha-synuclein from a wide variety of species to the single-domain carrier antibody and thus allows the detection of a wide variety of alpha-synuclein proteins.

[0070] The present invention also relates to an alpha-synuclein homodimer, wherein the monomers have a sequence identity of at least 95%, more preferably identical to the amino acid sequence with the amino acid sequence of SEQ. ID. No. 14, with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID. No. 14 remains unchanged.

[0071] Such an alpha-synuclein homodimer is particularly stable due to the disulfide bridge between the cysteines of two monomers and is therefore particularly well suited as a positive control for the methods according to the invention.

[0072] The present invention also relates to an expression construct comprising a gene encoding an alpha-synuclein polypeptide, wherein the alpha-synuclein polypeptide has a sequence identity of at least 95%, more preferably identical to the amino acid sequence of SEQ. ID. No. 14, with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID. No. 14 remains unchanged. The expression construct can, for example, comprise the expression plasmid "pSumol" from the non-profit plasmid archive "Addgene," into which the gene encoding the above-mentioned alpha-synuclein polypeptide has been inserted. The nucleotide sequence of pSUMOI is the nucleotide sequence with SEQ ID No. 15. The expression construct can then be introduced into expression-competent host cells, for example, E. coli cells such as Rosetta™ 2 (pLysS) from Novagen.

[0073] A further object of the present invention is an expression construct comprising a gene encoding a single-domain antibody specific for a peptide monomer with a peptide linker bound to the single-domain antibody, wherein the peptide linker has a length of at least 20 amino acids and comprises an alpha-helical region.

[0074] The single-domain antibody encoded by the expression construct can be specific for alpha-synuclein, the tau protein, or beta-amyloid. The specific single-domain antibody encoded by the gene can be specific for a peptide monomer that has a sequence identity of at least 80%, preferably 90%, more preferably 95%, to an amino acid sequence of the amino acid sequence of SEQ ID NO: 1 (alpha-synuclein). The single-domain antibody can also be specific for beta-amyloid. Such single-domain antibodies are described in the publication Drews, A. et al. Sci. Rep. 6, 31910. The single-domain antibody can also be specific for the tau protein. Such single-domain antibodies are described in the publication Li et al. Journal of Controlled Release, 243, 2016, pages 1-10.

[0075] The single-domain antibody encoded by the expression construct may have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2) or SEQ. ID. No. 11 (NbSyn87).

[0076] Particularly preferably, the single-domain antibody may be identical to the amino acid sequence of SEQ. ID. No. 10 (NbSyn2) or SEQ. ID. No. 11 (NbSyn87).

[0077] The expression construct can encode a peptide linker having an alpha-helical region selected from the alpha-helical region of a SUMO peptide (small ubiquitin modifier peptide) or an Fe region of an antibody. The peptide linker preferably has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, to the amino acid sequence of SEQ. ID No. 4, 5, 6, 7, 8, or 9. The expression construct can, in particular, comprise an expression plasmid containing a gene encoding the single-domain antibody. In particular, the plasmid "pSUMOI" (SEQ. ID. No. 15) is considered as an expression plasmid, which allows the expression of the single-domain antibody with a 6 His-SUMO tag at the N-terminus of the single-domain antibody.

[0078] The invention also relates to a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody is bound, wherein the single-domain carrier antibody is bound to the carrier material via a peptide linker. The peptide linker has a length of at least 20 amino acids and includes an alpha-helical region. The peptide linker preferably has a length between 30 and 130 amino acids, more preferably between 50 and 120 amino acids.

[0079] The peptide linker in the carrier material-antibody conjugate may have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ ID NOS. 4, 5, 6, 7, 8, or 9.

[0080] In the carrier material-antibody conjugate, the single-domain carrier antibody can have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID. No. 10 or SEQ. ID. No. 11. Preferably, the single-domain carrier antibody is identical to the amino acid sequence of SEQ. ID. No. 10 or SEQ. ID. No. 11.

[0081] The single-domain carrier antibody can be bound to the carrier material via a tag, preferably via a His-tag or a GST-tag. The single-domain carrier antibody preferably has a His-tag.

[0082] The carrier material of the carrier-antibody conjugate can preferably be a microtiter plate, microspheres, or a microneedle patch. Due to the peptide linker and the tag, the single-domain carrier antibody can be immobilized on the carrier material in the correct orientation, allowing binding to the multimeric peptide, in particular to the dimeric alpha-synuclein. The present invention also relates to a single-domain antibody specific for a peptide monomer with a peptide linker bound to the single-domain antibody, wherein the peptide linker has a length of at least 20 amino acids and comprises an alpha-helical region.

[0083] The single-domain antibody may have a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to that of the amino acid sequence of SEQ ID NO: 11. The single-domain antibody may have a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to that of the amino acid sequence of SEQ ID NO: 10.

[0084] The peptide linker in the single-domain antibody may have a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ ID NOS. 4, 5, 6, 7, 8, or 9.

[0085] The explanations and disclosures relating to an inventive subject matter also apply mutatis mutandis to all other inventive subject matters, provided they do not contradict the specific explanations and disclosures of the other inventive subject matters. For example, the explanations and disclosures relating to the method of the invention also apply mutatis mutandis to the expression construct of the invention, the carrier material-antibody conjugate of the invention, the kit of the invention, the alpha-synuclein homodimer of the invention, the peptide monomer of the invention, and the single-domain antibody of the invention, and vice versa, provided they do not contradict the specific explanations and disclosures made with respect to the respective inventive subject matters.For example, the specific embodiments of the peptide linker disclosed in connection with the method according to the invention also apply with regard to the other subject matters according to the invention, provided that these explanations and disclosures do not contradict the specific explanations and disclosures made with regard to these subject matters according to the invention.

[0086] Further features and advantages of the invention will become apparent from the following specific description and the drawings. Like reference numerals in the figures indicate like or similar elements.

[0087] Of course, the embodiments discussed in the specific description and shown in the figures are only illustrative embodiments of the present

[0088] In the light of the present disclosure, the person skilled in the art will have a broad

[0089] A spectrum of possible variations is provided.

[0090] They show:

[0091] Figure 1 is a schematic representation of a method for detecting a multimeric peptide, in particular a dimeric peptide, using specific single-domain antibodies;

[0092] Figure 2 shows a plasmid map of a plasmid having a sequence encoding the single-domain carrier antibody “NbSyn2” tethered to a peptide linker having a human Sumo sequence;

[0093] Figure 3 shows the nucleic acid sequence of the single-domain carrier antibody “NbSyn2”, which has a human Sumo sequence and a His 6 tag at the N-terminus (SEQ ID No. 16);

[0094] Figure 4 shows the amino acid sequence of the single-domain carrier antibody “NbSyn2” whose nucleic acid sequence is shown in Figure 3 (SEQ ID No. 17);

[0095] Figure 5 is a plasmid map of a plasmid having a sequence encoding the single-domain carrier antibody “NbSyn87” tethered to a peptide linker having a yeast Sumo sequence;

[0096] Figure 6 shows a nucleic acid sequence of the single-domain carrier antibody “NbSyn87” which has a yeast Sumo sequence as well as a Z domain and a His 14 tag at the N-terminus (SEQ ID No. 18);

[0097] Figure 7 shows the corresponding amino acid sequence of the single-domain carrier antibody “NbSyn87” whose nucleic acid sequence is shown in Figure 6 (SEQ ID No. 19);

[0098] Figure 8 shows the luminescence at a wavelength of 650 nm as a function of different concentrations of the sample of a brain lysate at a concentration of a single-domain carrier antibody of 0.1 pg / ml;

[0099] Figure 9 shows a plasmid map of the plasmid "aSynS87C", whose nucleotide sequence is described in SEQ ID No. 21. Examples of embodiments

[0100] Figure 1 shows a schematic representation of a method according to the invention for detecting a multimeric peptide, in particular a dimeric peptide, using single-domain antibodies. A single-domain carrier antibody 10 is immobilized on a carrier material 16 by means of a His tag 10B via a peptide linker 10A. This single-domain carrier antibody 10 recognizes an epitope 14A' on a first peptide monomer of the multimeric peptide. The multimeric peptide can comprise a dimeric peptide 14 that contains a first peptide monomer 14A and a second peptide monomer 14B. The first peptide monomer 14A and the second peptide monomer 14B can, in particular, be identical, so that the dimeric peptide 14 is a homodimer. A single-domain detection antibody 12 recognizes the epitope 14B' on the second peptide monomer 14B. The epitope 14B' of the second peptide monomer 14B is the same epitope as the epitope 14A' of the first peptide monomer 14A.The single-domain detection antibody 12 can thus only bind to the dimeric peptide 14 if the dimeric peptide has two identical epitopes 14A' and 14B'. The single-domain detection antibody 12 has a detection marker 12A that can be bound by a reporter enzyme 12B. The reporter enzyme then enables the detection of the complex between the carrier-antibody conjugate and the dimeric peptide via a color reaction 18. The left side of the schematic representation in Figure 1 shows the detection of the dimeric peptide 14, while the right side of the schematic representation in Figure 1 shows how the carrier-antibody conjugate binds a first peptide monomer 14A.Since this first peptide monomer 14A has only one epitope 14A', but no further epitope 14B', the single-domain detection antibody cannot bind to the peptide monomer 14A, as its epitope is already bound and thus blocked by the single-domain detection antibody 10. Therefore, the peptide monomer cannot be detected if only peptide monomers are present in the sample to be analyzed. The method according to the invention is thus suitable for detecting dimeric peptides even within multimeric protein aggregates without detecting the corresponding monomeric peptides.

[0101] Figure 2 shows a plasmid map of the pSumol plasmid, which contains a gene sequence encoding the single-domain antibody "NbSyn2," designated "Syn2." The plasmid map shows that the Sumo sequence is present as a peptide linker at the N-terminus of the single-domain antibody. A 6His tag is located at the N-terminus of the Sumo sequence, which serves to attach the single-domain antibody "Syn2" via the peptide linker to a carrier material, such as an ELISA plate. This plasmid can be used to express the single-domain carrier antibody "Syn2" in, for example, E. coli. The nucleic acid sequence of the pSumol plasmid is described in SEQ ID No. 15.

[0102] Figure 3 shows the nucleic acid sequence of the single-domain carrier antibody "NbSyn2," with a human SUMO peptide sequence present as a peptide linker at its N-terminus (shown underlined in Fig. 3). In this case, the human SUMO peptide sequence is a peptide sequence of the human SUMO1 protein (small ubiquitin-related modifier 1), which is listed, for example, in the UniProt protein knowledge base (UniProtKB) under the identification number P63165. A 6His tag is present at this peptide linker (His tag shown in lowercase). At the C-terminus of the single-domain carrier antibody, whose nucleic acid sequence is shown underlined, there is an additional sequence that was introduced into the single-domain carrier antibody "NbSyn2" by cloning, but which has no effect on its activity.

[0103] Figure 4 shows the amino acid sequence of the single-domain carrier antibody "NbSyn2," whose nucleic acid sequence is shown in Figure 3. The amino acid sequence of the peptide linker Sumo is shown in bold. The amino acid sequence of the single-domain carrier antibody "NbSyn2" has 5 lysines that can be used as detection markers for biotinylation. The biotins can be recognized by streptavidin-labeled reporter enzymes and used to detect the multimeric peptide using a color reaction. In addition, Figure 4 also shows 10 amino acids of the additional sequence at the C-terminus of the single-domain carrier antibody. For clarity, this sequence is shown in abbreviated form in Figure 4.

[0104] Figure 5 shows the plasmid map of the plasmid "K63 GGGG SynNb87 cys," which contains a segment encoding the single-domain antibody "NbSyn87." At the 5' end, this segment is linked to a gene segment encoding part of a yeast SUMO peptide sequence. The yeast SUMO peptide sequence (scSUMO) in this case is a peptide sequence of the yeast SUMO protein ubiquitin-like protein SMT3, which is listed, for example, in the UniProt protein knowledge base (UniProtKB) under the identification number Q12306. Furthermore, the Z domains 1 and 2 are present, as well as a 14 His tag for attaching the single-domain carrier antibody to a support material. This plasmid can also be used to express the single-domain carrier antibody "NbSyn87" in E. coli. The nucleic acid sequence of the plasmid “K63 GGGG SynNb87 cys” is described in SEQ ID No. 20.

[0105] Figure 6 shows the nucleic acid sequence of the single-domain carrier antibody "NbSyn87" (bold sequence "NbSyn87"), at whose 5'-end the SMT3 sumo peptide sequence is present (underlined sequence), followed by Z-domains 1 and 2 (bold sequence). Furthermore, a 14His tag is present for attachment to the carrier material (shown in lowercase in Figure 6).

[0106] Figure 7 shows the amino acid sequence of the nucleic acid sequence shown in Figure 6. The bold amino acid sequence of "NbSyn87" contains 5 lysines for biotinylation as a detection marker. The SMT3 sumo peptide sequence is underlined, and the 14His tag is shown in italics.

[0107] Introduction of a point mutation Ser87Cvs in alpha-Svnuclein

[0108] The inventors introduced a point mutation into human alpha-synuclein to ensure that pure homodimers, rather than a mixture of higher-order oligomers, are produced under redox conditions. Alpha-synuclein has four serines: serine 9, 42, 87, and 129. Serine 129 was excluded as a possible mutation site because of its important function as a phosphorylation site in vivo. Serine 9 and 42 are both located in the N-terminal region of alpha-synuclein and would therefore most likely not form stable synthetic homodimers. Therefore, the inventors introduced a point mutation of serine 87 cysteine. Measuring alpha-synuclein as units of "dimer equivalents" using these stoichiometric, well-characterized αSynSer87Cys dimers as assay standards will enable a reproducible measurement.

[0109] The plasmid "aSynS87C" was used to express the point mutation of alpha-synuclein in E. coli. Figure 9 shows the plasmid map of the plasmid "aSynS87C," which was obtained by cloning an insert encoding the point mutant of alpha-synuclein S87C into the vector pSUMOI in the region described in the plasmid map as the "customer insert." A His-tag and the SUMO1 sequence were fused to the N-terminus of the point mutant.

[0110] Preparation of synthetic dimer standards

[0111] Purified recombinant monomeric aSyn with the point mutation S87C was dimerized according to a protocol adapted from O'Nuallain et al., 2010 (O'Nuallain et al., J Neurosci 30(43), 2010, 14411-14419). Briefly, 40 μM aSynS87C was diluted 1:1 with 20 mM ammonium bicarbonate (pH 8.2) and incubated for five days at 25°C and 300 rpm in a thermomixer. Larger aggregates were dissolved by lyophilizing the dimer reaction and resuspending it in 50 mM Tris-HCl with 5 M guanidine (pH 8.0) overnight. The next day, the samples were subjected to size-exclusion chromatography. The same procedure was repeated for human beta-synuclein S72C, human gamma-synuclein S92C, and mouse aSynS42C, except that they were first precipitated with the he2D Clean-Up Kit (GE Healthcare, Piscataway, NJ) as previously described (Wildburger et al., 2015, Mol Cell Proteomics 14(5), 1288-1300) to remove excess imidazole.

[0112] Expression of the alpha-Svnuclein1-140Ser87Cvs dimer or the monomeric alpha-Svnuclein

[0113] All required synucleins were expressed in Rosetta™ 2(DE3)plysS Competent Cells (Novagen). This BL21-derived E. coli strain enables enhanced expression of eukaryotic proteins through a chloramphenicol resistance (CamR)-containing plasmid that delivers the tRNAs for rare codons. Rare codons are codons that are more common in the transcription of human genes than in bacterial genes and can therefore cause problems in the transcription of proteins of human origin in other E. coli. The CamR gene enables growth in the presence of 34 pg / ml chloramphenicol (CAMP).

[0114] Transformation was performed by heat shock for 30 seconds at 42°C. To achieve better transformation efficiency, 10 pg of the respective subcloned plasmid was added to a 20 pL aliquot of E. coli 30 min before heat shock, followed by SOC medium (Novagen). After 1 hour of incubation at 37°C, the E. coli were plated on an LB agarose plate containing 34 pg / ml CAMP and 0.025 mg / ml Strep to create a selection pressure for clones containing the rare codon plasmid and the transformed plasmid. The plate was incubated overnight at 37°C to allow colony formation. Single colonies were selected for inoculation into a liquid LB culture containing the same concentrations of CAMP and Strep and also incubated overnight. Glycerol stocks were prepared by adding 700 pL of the liquid culture to 300 pL of glycerol and stored at -80°C.The liquid culture was induced with 1 mM IPTG for 3 hours and harvested by centrifugation at 5500 x g for 20 minutes. The supernatant was removed before freezing at -20°C.

[0115] Expression of single domain antibodies:

[0116] NbSyn2 was expressed in Rosetta-gami™ B(DE3)plysS Competent Cells (Novagen). This cell line has the advantage over the Rosetta line used for synucleins in that it also encodes proteins that promote the formation of disulfide bonds. Heat shock was performed as described above. Induction with IPTG was performed at 30°C with 0.5 mM IPTG overnight, and the cells were harvested by centrifugation for 20 min at 5500 xg. The supernatant was removed before freezing at -20°C. Induction of NbSyn87 was performed in the same way.

[0117] Purification of alpha-synucleins and single-domain antibodies

[0118] Lysis of the E. coli cells was performed on ice by sonication after resuspension in 5 ml of buffer per gram of E. coli; high-salt buffer with 20 mM imidazole. 10 μL of DNAsei and PI were used in a ratio of 1:100. Sonication was performed in 10-second pulses with 30-second pauses. The whole-cell lysate was pre-purified by centrifugation at 10,000 xg for 10 minutes at room temperature in a benchtop centrifuge.

[0119] A His GraviTrap (GE Healthcare) was used to purify the His-tagged protein from the pre-cleared cell lysate (PCL). After equilibrating the column with native high-salt buffer containing 20 mM imidazole, the sample was added. Washing the column with one volume of native high-salt buffer containing 20 mM imidazole removed non-ionic interactions. To remove the salt, a wash with native low-salt buffer was performed. To achieve optimal elution, an imidazole series (50, 100, 150, 200, 300, 500 mM) in low-salt buffer was used. Only fractions containing the desired protein were collected (see the table below, where collected fractions are marked with an "X").

[0120] To clean the column after use, 10 ml of denaturation buffer was added. After washing twice with high-salt buffer containing 20 mM imidazole, the column was stored in 20% ethanol. The collected fractions were pooled in a 3 kDa Amicon (regenerated cellulose) molecular weight cutoff filter and exchanged with 20 ml of low-salt buffer containing 20 mM imidazole. The filter was stored in PBS containing 0.02% sodium azide.

[0121] Removal of the tag from alpha-synucleins and single-domain detection antibodies

[0122] The buffer-exchanged sample was made up to 1 ml, and 40 U of His-tagged SUMO protease (SAE0067, Sigma / Merck) were added along with 1 mM DTT. To allow SUMO cleavage of the alpha-synucleins and the single-domain detection antibodies, the samples were incubated overnight at 4°C. Upon cleavage by the SUMO protease, the respective His-SUMO tag was cleaved. After cleavage, the sample was reapplied to the His-Gravitrap to remove the cleaved HIS-SUMO tag and the HIS-tagged SUMO protease. The column was then re-equilibrated, and the flowthrough was collected with the now tagless alpha-synucleins or single-domain detection antibodies.

[0123] Biotinylation of the purified single-domain detection antibodies

[0124] For biotinylation of the single-domain detection antibodies, EZ-Link™ NHS Biotin (ThermoFisher) was used in a 20-fold molar excess. The biotin stock solution was prepared by dissolving 2 mg of biotin in 590 μl of dimethyl sulfoxide (DMSO). The required amount of biotin was calculated according to the manufacturer's protocol and added to the single-domain detection antibody. They were incubated on top of each other for 30 minutes and then buffered in a 3 kDa Amicon (regenerated cellulose) molecular weight cutoff filter with 20 ml of PBS (NbSyn2) or subjected to size-exclusion chromatography (SEC) on a Superdex™ 10 / 300 GL (GE Healthcare) (NbSyn87) to remove excess biotin.

[0125] Biotinylated single-domain detection antibodies can be particularly easily detected using enzymes coupled to streptavidin, such as streptavidin-horseradish peroxidase.

[0126] Method for detecting dimeric alpha-synuclein:

[0127] His6-SUMO-NbSyn87 (oligomer assay, human) or His6-SUMO-NbSyn2 (total, pan-assay) was used to coat black 384-well Nunc MaxiSorp plates (#460518, Nalge Nunc, Rochester, NY) at 20 pg / ml in a carbonate buffer (35 mM sodium bicarbonate, 16 mM sodium carbonate, 3 mM sodium azide, pH 9.6) at 20 μl / well overnight at 4 °C. Plates were washed 5x between steps with PBS containing 0.005% Tween-20 in a BioTek EXL405 plate washer (BioTek, Winooski, VT). The sample plates were blocked with 0.2 μm filtered 4% BSA (bovine serum albumin) (#7030, Sigma-Aldrich, St. Louis, MO) in PBS for 1 hour at room temperature. The samples or the dimer standard, respectively, were blocked.The alpha-synuclein monomer (for the total assay) was analyzed either neat or diluted in standard dilution (0.2 μm filtered 0.25% BSA, 0.005% Tween-20, 3 mM sodium azide, 2 pg / mL aprotinin (EMD Chemicals, Gibbstown, NJ), 1 pg / mL leupeptin (EMD Chemicals), in PBS) to a final volume of 20 μL and loaded onto plates coated with His6-SUMO-NbSyn87 or His6-SUMO-NbSyn2, respectively. Brain lysates from 9-week-old or 6-month-old mice of the Thy1 mouse model were used as samples (Chesselet et al., Neurotherapeutics. 2012 Apr; 9(2): 297-314). Transgenic mice of this model express human alpha-synuclein under the control of a Thy-1 promoter. A 9-point standard curve was generated using 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 pg / ml of alpha-synuclein 140Ser87Cys dimer or monomeric alpha-synuclein (without mutations) and loaded in triplicate in all experiments.All samples and the standard were stored on ice during processing. The plates were centrifuged at 1000xg shortly after adding the samples and standards to ensure that the liquid sat properly at the bottom of each well and to remove air bubbles. The samples and standards were equilibrated overnight at 4°C on the plates with the single-domain carrier antibodies bound to them. After a further wash step to remove unbound solutes, alpha-synuclein oligomers or total alpha-synuclein were each detected using labeled (biotinylated) NbSyn87 as a single-domain detection antibody at a concentration of 100 ng / ml in PBS containing 0.2 μm filtered 0.1 mg / ml nonfat dry milk plus 0.005% Tween-20. Binding of the single-domain detection antibodies to exposed alpha-synuclein oligomers or to total alpha-synuclein was allowed for 1 hour at room temperature.Poly-Streptavidin HRP-20 (65R-S103PHRP, Fitzgerald, Acton, MA) was then incubated at 30 ng / mL in PBS with 0.2 μm of filtered 0.5% BSA plus 0.005% Tween-20 for one hour at room temperature with gentle stirring. After a final wash, the assay was developed by adding SuperSignal™ West Femto Maximum Sensitivity Substrate (34094, Thermofisher), and the luminescence was read on a Tecan Infinite M Plex plate reader. The standard curve was then used to calculate the concentration of dimeric alpha-synuclein in the samples in units of "dimer equivalents" for the oligomer assay. Figure 8 shows the luminescence at 650 nm of a method according to the invention for detecting dimeric alpha-synuclein at a concentration of 0.1 pg / ml of a single-domain carrier antibody His6-SUMO-NbSyn2 at different concentrations of a single-domain detection antibody NbSyn2 plotted on the x-axis.At all concentrations of the single-domain detection antibody, either 10 pg of brain lysate (bars labeled 20), 2 ng of brain lysate (bars labeled 22), or no sample, negative control (bars labeled 24) were used. It is clearly evident that at all concentrations of the single-domain detection antibody, the sample containing 10 pg of brain lysate exhibits the highest luminescence, while the luminescence of the sample containing a small amount of brain lysate and no brain lysate exhibits approximately the same luminescence. Therefore, the method according to the invention can detect the dimeric human alpha-synuclein contained in the brain lysates with high sensitivity. The comparable luminescence in the samples with a small amount of brain lysate and the negative control is partly due to nonspecific binding of the single-domain detection antibody to the support material.

[0128] Method for detecting dimeric tau protein:

[0129] Dimeric tau protein can be detected in the same way as described above for dimeric alpha-synuclein using the aforementioned tau-specific antibodies. The same tau-specific antibody can be used as the single-domain carrier antibody and the single-domain detection antibody, so that a signal from the single-domain detection antibody can only be generated if dimeric tau proteins or multimeric tau proteins are present in the sample, which have two identical epitopes for binding to both the single-domain carrier antibody and the single-domain detection antibody.

[0130] Method for detecting dimeric beta-amyloid:

[0131] Dimeric beta-amyloid can be determined analogously to dimeric tau protein.

Claims

Patent claims 1. Method for detecting a multimeric, preferably dimeric, peptide containing a first and a second peptide monomer in a sample, comprising the following process steps: A) Providing a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for the first peptide monomer is bound, B) Contacting the sample with the carrier material-antibody conjugate, whereby a complex is formed between the carrier material-antibody conjugate and the dimeric peptide, C) Contacting the complex with a single-domain detection antibody specific for the second peptide monomer, which includes a detection marker, wherein the single-domain detection antibody binds to the complex, wherein the single-domain carrier antibody on the first peptide monomer and the single-domain detection antibody on the second peptide monomer recognize the same epitope, and D) Detection of the dimeric peptide using the detection marker.

2. A method according to the preceding claim, wherein a carrier material-antibody conjugate is used, in which the single-domain carrier antibody is bound to the carrier material via a peptide linker, wherein the peptide linker has a length of at least 20 amino acids and comprises an alpha-helical region, preferably wherein the length is between 30 and 130 amino acids, more preferably between 50 and 120 amino acids.

3. A method according to the preceding claim, wherein the peptide linker comprises an alpha-helical region of a SUMO peptide (small ubiquitin-like modifier peptide), further preferably wherein the peptide linker has a sequence identity of at least 80%, preferably 90%, further preferably at least 95% to the amino acid sequence of SEQ. ID No. 4, 5, 6, 7, 8 or 9, preferably SEQ. ID No.

4.

4. A method according to one of the preceding claims 2 or 3, wherein the peptide linker is bound to the carrier material via a tag sequence, preferably wherein the tag sequence is selected from a group consisting of: a His tag, preferably a His-tag having between 3 and 20 histidines, or a GST-tag, further preferably wherein the peptide linker with the tag sequence is bound to the N-terminus of the single-domain carrier antibody.

5. Method according to any of the preceding claims, wherein the single-domain carrier antibody and the single-domain detection antibody are the same single-domain antibody.

6. Method according to any of the preceding claims, wherein the dimeric peptide is a dimeric oligo- or polypeptide, preferably wherein the dimeric peptide comprises or is a homodimer.

7. Method according to any of the preceding claims for the detection of neurodegenerative diseases, such as Parkinson's disease, wherein the dimeric peptide is selected from: beta amyloid peptide dimer, tau dimer, phosphorylated tau dimer, or alpha synuclein dimer, preferably alpha synuclein dimer.

8. Method according to the preceding claim for the detection of Parkinson's disease, wherein the dimeric peptide comprises a monomer that has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to the amino acid sequence of SEQ. ID No. 1 or Tau protein or Beta-amyloid protein.

9. A method according to the preceding claim for the detection of Parkinson's disease, wherein the single-domain carrier antibody and the single-domain detection antibody are specific for alpha-synuclein, preferably wherein the single-domain carrier antibody and the single-domain detection antibody have a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to the amino acid sequence of SEQ. ID No. 10 or to the amino acid sequence of SEQ. ID No.

11.

10. Method according to any of the preceding claims, wherein in process step B) the single-domain carrier antibody binds to the first peptide monomer, blocking the epitope recognized by the single-domain detection antibody on the first peptide monomer.

11. A method according to any of the preceding claims, wherein a positive control reaction is additionally carried out, wherein a homodimer is used as the dimeric peptide for the positive control reaction, the monomer of which has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to the amino acid sequence of SEQ. ID No. 14, with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID No. 14 remains unchanged.

12. Method according to one of the preceding claims, wherein in method step B) a sample selected from: cerebrospinal fluid, blood, serum, blood plasma, saliva, tissue biopsy, throat swab, nasal swab, urine, exosome diagnostics of a patient is used.

13. Method according to any of the preceding claims for determining whether a symptom-free patient is at risk of developing Parkinson's disease, wherein alpha-synuclein dimer is detected as a dimeric peptide.

14. Method for the detection of alpha-synuclein in a patient sample, comprising the following procedural steps: A) Providing a carrier-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for alpha-synuclein is bound, B) Contacting the sample comprising alpha-synuclein with the carrier material-antibody conjugate, whereby a complex is formed between the carrier material-antibody conjugate and the alpha-synuclein, C) Contacting the complex with a single-domain detection antibody specific for alpha-synuclein, which includes a detection marker, wherein the single-domain detection antibody binds to the complex, and D) Detection of alpha-synuclein using the detection marker.

15. Method according to the preceding claim for the detection of monomeric alpha-synuclein, wherein various single-domain antibodies are used as single-domain Carrier antibodies and can be used as single-domain detection antibodies that recognize different epitopes of alpha-synuclein.

16. A method according to one of the preceding claims 14 or 15 for detecting Parkinson's disease in humans or for determining whether a symptom-free human patient is at risk of developing Parkinson's disease, wherein a single-domain antibody is used as the single-domain carrier antibody which has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to that of the amino acid sequence of SEQ. ID No.

11.

17. A method according to one of the preceding claims 14 or 15 for the detection of alpha-synuclein in vertebrates selected from the group consisting of: human, mouse, rat, gorilla, cattle, wherein a single-domain antibody is used as the single-domain carrier antibody which has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to that of the amino acid sequence of SEQ. ID No.

10.

18. Kit for carrying out a method according to any one of the preceding claims 1 to 13 for detecting a dimeric peptide, containing a first and a second peptide monomer in a sample, comprising: - a carrier material-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for the first peptide monomer is bound - a single-domain detection antibody specific for the second peptide monomer, comprising a detection marker, - Reagents for the detection of the dimeric peptide using the detection marker.

19. Kit according to the preceding claim, further comprising instructions for carrying out the method according to any one of the preceding claims 1 to 13.

20. Kit for carrying out a method according to any one of the preceding claims 14 to 17 for detecting alpha-synuclein in a sample, comprising: - a carrier material-antibody conjugate comprising a carrier material to which a single-domain carrier antibody specific for alpha-synuclein is bound - a single-domain detection antibody specific for alpha-synuclein, comprising a detection marker, - Reagents for the detection of alpha-synuclein using the detection marker.

21. Kit according to the preceding claim, further comprising instructions for carrying out the method according to any one of the preceding claims 14 to 17.

22. Alpha-synuclein homodimer, wherein the monomers have a sequence identity of at least 95%, further preferably identical to the amino acid sequence of SEQ. ID No. 14, with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID No. 14 remains unchanged.

23. Expression construct comprising a gene encoding an alpha-synuclein polypeptide, wherein the alpha-synuclein polypeptide has a sequence identity of at least 95%, further preferably identical to the amino acid sequence of SEQ. ID No. 14, with the proviso that the amino acid cysteine ​​at position 87 of SEQ. ID No. 14 remains unchanged.

24. Expression construct comprising a gene encoding a single-domain antibody specific for a peptide monomer with a peptide linker bound to the single-domain antibody, wherein the peptide linker has a length of at least 20 amino acids and includes an alpha-helical region.

25. Expression construct according to the preceding claim, wherein the peptide monomer has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to the amino acid sequence of SEQ. ID No.

1.

26. Expression construct according to one of the preceding claims 24 or 25, wherein the single-domain antibody has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to that of the amino acid sequence of SEQ. ID No. 11, or wherein the single-domain antibody has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to that of the amino acid sequence of SEQ. ID No.

10. I. Expression construct according to any one of the preceding claims 24 to 26, wherein the peptide linker comprises an alpha-helical region of a SUMO peptide (small ubiquitin-like modifier peptide), further preferably wherein the peptide linker has a sequence identity of at least 80%, preferably 90%, further preferably at least 95% to the amino acid sequence of SEQ. ID No. 4, 5, 6, 7, 8 or 9, preferably SEQ. ID No.

4.

28. Carrier material-antibody conjugate comprising a carrier material to which a single-domain carrier antibody is bound, wherein the single-domain carrier antibody is bound to the carrier material via a peptide linker, wherein the peptide linker has a length of at least 20 amino acids and comprises an alpha-helical region, preferably wherein the length is between 30 and 130 amino acids, more preferably between 50 and 120 amino acids.

29. Carrier material-antibody conjugate according to the preceding claim, wherein the peptide linker has a sequence identity of at least 80%, preferably 90%, further preferably at least 95% to the amino acid sequence of SEQ. ID No. 4, 5, 6, 7, 8 or 9, preferably SEQ. ID No.

4.

30. Carrier material-antibody conjugate according to one of the preceding claims 28 or 29, wherein the single-domain carrier antibody has a sequence identity of at least 80%, preferably 90%, more preferably at least 95%, more preferably identical to the amino acid sequence of SEQ. ID No. 10 or to the amino acid sequence of SEQ. ID No.

11.

31. For a peptide monomer specific single-domain antibody with a peptide linker bound to the single-domain antibody, wherein the peptide linker has a length of at least 20 amino acids and includes an alpha-helical region.

32. Single-domain antibody according to the preceding claim, wherein the single-domain antibody has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to that of the amino acid sequence of SEQ. ID No. 11, or wherein the single-domain antibody has a sequence identity of at least 80%, preferably 90%, further preferably at least 95%, further preferably identical to that of the amino acid sequence of SEQ. ID No.

10.

33. Single-domain antibody according to one of the preceding claims 31 or 32, wherein the peptide linker has a sequence identity of at least 80%, preferably 90%, more preferably at least 95% to the amino acid sequence of SEQ. ID No. 4, 5, 6, 7, 8 or 9, preferably SEQ. ID No. 4.

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