RU2807994C2 - Cellular vamp cleavage assay - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a method for producing a polyclonal antibody (embodiments). In one embodiment, the method includes isolating and further purifying a polyclonal antibody capable of binding to full-length VAMP and a C-terminal VAMP cleavage product in a cell lysate, wherein the C-terminal VAMP cleavage product is derived from VAMP cleavage by a clostridial neurotoxin from blood obtained from an animal.

EFFECT: invention expands the arsenal of methods for producing polyclonal antibodies that bind to full-length VAMP and the C-terminal VAMP cleavage product in cell lysate.

10 cl, 7 dwg, 2 tbl, 3 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to cell-based assays for VAMP-degrading clostridial neurotoxins.

BACKGROUND OF THE ART

Bacteria of the genus Clostridia produce highly active and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include those produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes AG, and those produced by C. baratii and C. butyricum .

Clostridial neurotoxins act by inhibiting cholinergic transmission in the peripheral nervous system, particularly at the neuromuscular junction. In nature, clostridial neurotoxins are synthesized as a single-chain polypeptide that is modified post-translationally through a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as an activation site, which is located between the cysteine residues that provide the interchain disulfide bond. This double-chain form is the active form of the toxin. The two chains are called the heavy chain (H-chain), having a molecular weight of approximately 100 kDa, and the light chain (L-chain), having a molecular weight of approximately 50 kDa. The H chain contains an N-terminal component for translocation ( _HN domain) and a C-terminal component for targeting ( _HC domain). The cleavage site is localized between the L chain and the H _N domain. Following binding of the H _C domain to its target neuron and internalization of the bound toxin into the cell via the endosome, the H _N domain translocates the L chain across the endosomal membrane and into the cytosol, and the L chain provides protease function (also known as a noncytotoxic protease).

Noncytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (eg, SNAP25, VAMP, or syntaxin) - see Gerald K (2002) Cell and Molecular Biology (4th edition) John Wiley & Sons, Inc. The acronym SNARE comes from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. The acronym SNAP25 comes from the term synaptosome associated protein (25 kilodalton). The acronym VAMP comes from the term Vesicle Associated Membrane Protein. SNARE proteins are integral to the fusion with an intracellular vesicle, and thus the secretion of molecules through the transport of vesicles out of the cell. The protease function is a zinc-dependent endopeptidase activity and has high substrate specificity for SNARE proteins. Accordingly, once delivered to the desired target cell, the noncytotoxic protease is capable of inhibiting cellular secretion from the target cell. Clostridia neurotoxin L-chain proteases are noncytotoxic proteases that cleave SNARE proteins. The L chain proteases BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, and TeNT cleave VAMP (also designated synaptobrevins), the L chain proteases BoNT/A and BoNT/E cleave SNAP25, and L -chain protease BoNT/C cleaves both SNAP25 and syntaxin, resulting in inhibition of neurotransmitter release and consequent neuroparalysis (Rossetto, O. et al., “Botulinum neurotoxins: genetic, structural and mechanistic insights.” Nature Reviews Microbiology 12.8 (2014 ): 535-549) (Zhang et al., “Identification and characterization of a novel botulinum neurotoxin”; Nature Communications, 2017, 8:14130).

Clostridial neurotoxins target and enter neurons by binding to their specific receptors through their receptor binding domains (H _C ), well defined in the literature (Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis , Physiol Rev , 2000, 80, 717-766). Receptor binding determines the efficiency and specificity of BoNT for neuronal recognition. BoNT/B, DC and G share two homologous synaptic vesicle proteins synaptotagmin I and II (Syt I/II) as their receptors, while BoNT/A, E, D, F and TeNT use another synaptic vesicle protein SV2. In addition to protein receptors, all BoNTs require lipid coreceptor gangliosides, which are widely distributed on neuronal surfaces.

Clostridia neurotoxins are used in therapy to treat motor and autonomic disorders. Several BoNT/A products (including Botox®, Dysport® and Xeomin®) and one BoNT/B product (Neurobloc®/Myobloc®) are approved by regulatory authorities for use in humans.

Typically, the activity of pharmaceutical BoNT products is quantified in MLD50 units (lethal dose for 50% of mice), where one unit corresponds to the median intraperitoneal lethal dose in mice. However, the MLD50 unit for botulinum toxins is not a standardized unit. Indeed, the assays used by various manufacturers of marketed toxins differ, in part, in the choice of dilution buffer (Straughan, D. W., 2006, ATLA 34(3), 305-313; Hambleton and Pickett, Hambleton, P., and A. M. Pickett ., 1994, Journal of the Royal Society of Medicine 87.11: 719). In addition, due to ethical considerations and recent regulations, it is currently preferable to avoid the use of activity assays using animals. Cell-based potency assays eliminate the need for animal testing and associated ethical concerns. Following cell intoxication, clostridial neurotoxin activity can be measured by assessing the extent of SNARE cleavage in the target cell, for example by Western blotting. Alternatively, SNARE cleavage can be detected and quantified using a sandwich ELISA method. Such methods work well for cleavage of SNAP25 and syntaxin (see, e.g., Pellett, Sabine, et al. “Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type A potency determination.” Journal of pharmacological and toxicological methods 61.3 (2010):304-310; Fernández-Salas, Ester, et al. “Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.” PLoS One 7.11 (2012): e49516 ; Kalandakanond S et al. "Cleavage of intracellular substrates of botulinum toxins A, C and D in mammalian target tissue" The Journal of Pharmacology and Experimental Therapeutics (2001):749-755; Peng L et al. "Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP25 in neuron survival." Nature Communications (2013): 4:1472). However, the VAMP cleavage product in cell lysates has so far been shown to be unusually difficult to detect. Indeed, although VAMP cleavage-specific antibodies that recognize cleaved VAMP are available and useful for detecting VAMP cleavage in extracellular or cellular fraction assays (Hallis, Bassam, B. A. James, and Clifford C. Shone, “Development of novel "Assays for botulinum type A and B neurotoxins based on their endopeptidase activities." Journal of clinical microbiology 34.8 (1996): 1934-1938; Kegel, B., et al. "An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product." Toxicology in Vitro 21.8 (2007): 1641-1649; Fujita-Yoshigaki, Junko, et al. "Vesicle-associated Membrane Protein 2 Is Essential for cAMP-regulated Exocytosis in Rat Parotid Acinar Cells The Inhibition of cAMP-dependent Amylase Release by Botulinum Neurotoxin B." Journal of Biological Chemistry 271.22 (1996): 13130-13134), these antibodies do not detect cleaved VAMP in cell assays.

The consensus view in the field to date has been that the cleaved VAMP product should be degraded very quickly in the cell and thus does not contribute to the duration of action of BoNT (Foran, Patrick G., et al. "Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A Basis for Distinct Durations of Inhibition of Exocytosis in Central Neurons." Journal of biological chemistry 278.2 (2003): 1363-1371). However, Schiavo et al. showed that both VAMP cleavage products were present in small synaptic vesicle fractions obtained from rat cerebral cortex treated with BoNT/B and TeNT using Coomassie blue staining (Schiavo G., et al (1992), Tetanus and Botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359 p832-835). This suggests that VAMP products from a cellular source may be present, although the synaptosome preparation may not contain all of the proteases present in the total cell lysate. Dong et al (2004) described that in PC12 cells expressing YFP-Syb(FL)-CFP, signals from both VAMP products were detectable after cleavage by BoNT/B, and that the YFP-N-terminal VAMP cleavage product was distributed in the cytosol and redistribute to the nucleus, while the CFP-C-terminal product remains localized in the vesicle (Dong M., et al (2004) Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells. PNAS 101 (41) p14701 -14706). This evidence suggests that both VAMP products may be present, but as yet unknown cellular processes interfere with antibody recognition of the N-terminal product. Thus, standard practice is to measure VAMP cleavage only by the disappearance of the full-length band (see eg. Pellett, Sabine, et al. “A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies.” FEBS letters 581 , 25 (2007): 4803-4808.; Whitemarsh, Regina CM, et al. "Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection Biological Sciences: Applied Biological Sciences." Toxicological Sciences, 2012, 126(2):426-435). However, assays based on signal loss come with a risk of error, as discrepancies in overall protein loading may be present, which can then lead to either an overestimation or an underestimation of VAMP disappearance. A housekeeping protein that remains unchanged during BoNT treatment can be used to normalize VAMP disappearance to density for a control protein. The disadvantage here is that the signals must be consistent between antibodies and within a linear range to detect any differences for normalization purposes. Although qualitatively this may be a useful measure of BoNT activity, it is not suitable for more detailed quantitation and in particular for determining the activity of BoNT pharmaceutical formulations.

Thus, there is a need for cellular VAMP cleavage assays based on signal amplification.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an antigenic polypeptide containing a VAMP epitope, wherein said antigenic polypeptide consists of 10-65 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridia neurotoxin cleavage site in said VAMP.

In another aspect, the invention provides a polypeptide comprising an antigenic polypeptide of the invention, wherein the polypeptide does not comprise a region of more than 17, preferably 16, more preferably 15 consecutive amino acids having 100% sequence identity with the amino acid sequence of natural VAMP.

In another aspect, the invention provides an antigenic protein comprising a polypeptide of the invention covalently linked to a carrier.

In another aspect, the invention relates to the use of an antigenic polypeptide or protein of the invention to produce antibodies to the C-terminal cleavage product of VAMP. In one embodiment, an epitope of the invention is used to produce a polyclonal antibody against the C-terminal cleavage product of VAMP. In another embodiment, an epitope of the invention is used to produce a monoclonal antibody against the C-terminal cleavage product of VAMP.

In another aspect, the invention provides an antibody that binds to an antigenic polypeptide or protein of the invention.

In another aspect, the invention relates to the use of an antibody of the invention in a cell-based signal-enhancing assay for VAMP cleavage via a clostridial VAMP-cleaving neurotoxin.

In another aspect, the invention provides a method for determining VAMP cleavage by a VAMP-cleaving clostridia neurotoxin in a cell, comprising:

a) bringing the cell into contact with the clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

b) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following cleavage of the VAMP by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody according to the invention; And

c) detecting by suitable methods the binding of said first detection antibody to the C-terminal cleavage product of VAMP.

In another aspect, the invention relates to a method for determining immune resistance to a VAMP-degrading clostridial neurotoxin in a patient, comprising:

a) adding a VAMP-cleaving clostridial neurotoxin to the test sample obtained from the patient;

b) bringing the cell into contact with the test sample from step a) under conditions suitable for the activity of the clostridia neurotoxin;

c) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following VAMP cleavage by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody according to the invention;

d) detecting by suitable methods the binding of said first detection antibody to the C-terminal cleavage product of VAMP;

e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

f) repeating steps a) to e) with the negative control sample instead of the test sample; And

g) comparing the amount of C-terminal VAMP cleavage product bound to said first detection antibody in steps (e) and (f), wherein detecting a lower amount of C-terminal VAMP cleavage product bound to said first detection antibody in step (e) compared to the amount of C-terminal VAMP cleavage product bound to said first detection antibody in step (f), is indicative of the presence of neutralizing antibodies against the VAMP-cleaving clostridial neurotoxin.

In another aspect, the invention provides a kit comprising a cell susceptible to intoxication by a VAMP-degrading neurotoxin; and a first detection antibody against cleaved VAMP, wherein said first detection antibody is an antibody of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the inventors' discovery that it is possible to obtain antibodies suitable for use in a cellular VAMP cleavage assay based on signal amplification.

In particular, we showed that for detection of VAMP cleavage in vitro , detection of epitopes located C-terminal to the BoNT cleavage site is key. Indeed, we have demonstrated herein the successful detection of a neuronal VAMP2 cleavage product by Western blotting (WB) using antibodies that bind epitopes located C-terminal to the BoNT/F and/or BoNT/D cleavage sites and/or BoNT/B, which are adjacent to the cleavage sites of BoNT/D and/or BoNT/F, and/or BoNT/B. In particular, such antibodies are capable of detecting both full-length VAMP and the cleavage product in cell lysate. This tool allows the quantification of BoNT activity in a cell-based signal amplification assay by monitoring the appearance of the VAMP cleavage product.

In a first aspect, the invention relates to an antigenic polypeptide containing a VAMP epitope, wherein said antigenic polypeptide consists of 10-65 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridia neurotoxin cleavage site in said VAMP.

The term "clostridium neurotoxin", as used herein, means any polypeptide that enters a neuron and inhibits the release of neurotransmitters. This process involves binding of the neurotoxin to a low- or high-affinity receptor, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "clostridium neurotoxin" includes any polypeptide produced by Clostridium bacteria that enters a neuron and inhibits the release of neurotransmitters, and such polypeptides produced by recombinant technologies or chemical methods. The double-chain form is the active form of the neurotoxin. These two chains are called the heavy chain (H chain), having a molecular weight of approximately 100 kDa, and the light chain (L chain), having a molecular weight of approximately 50 kDa. Clostridia neurotoxins include botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT). AG BoNT serotypes can be distinguished based on inactivation by specific neutralizing antiserum, where such serotype classification correlates with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further classified into different subtypes based on percentage amino acid sequence identity.

One example amino acid sequence of the BoNT/A neurotoxin is provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9). One example amino acid sequence of the BoNT/B neurotoxin is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). One example amino acid sequence of the BoNT/C neurotoxin is provided as SEQ ID NO: 3 (UniProt accession number P18640). One example amino acid sequence of the BoNT/D neurotoxin is provided as SEQ ID NO: 4 (UniProt accession number P19321). One example amino acid sequence of the BoNT/E neurotoxin is provided as SEQ ID NO: 5 (NCBI RefSeq accession number WP_003372387). One example amino acid sequence of the BoNT/F neurotoxin is provided as SEQ ID NO: 6 (UniProt accession number Q57236). One example amino acid sequence of the neurotoxin BoNT/G is provided as SEQ ID NO: 7 (NCBI RefSeq accession number WP_039635782). One example amino acid sequence of the BoNT/X neurotoxin is provided as SEQ ID NO: 41 (Genbank accession number BAQ12790.1). One example amino acid sequence of the TeNT neurotoxin is provided as SEQ ID NO: 8 (UniProt accession number P04958).

The term “ _HC domain”, as used herein, refers to a functionally distinct region of the heavy chain of a neurotoxin with a molecular weight of approximately 50 kDa, which mediates the binding of the neurotoxin to a receptor located on the surface of the target cell. The _HC domain consists of two structurally distinct subdomains, the "H _CN subdomain" (the N-terminal portion of the _HC domain) and the "H _CC subdomain" (the C-terminal portion of the _HC domain), each having a molecular mass of approximately 25 kDa.

The term “LH _N domain”, as used herein, means a neurotoxin that lacks an _HC domain and consists of an endopeptidase domain (“L” or “light chain”) and a domain responsible for translocation of endopeptidase into the cytoplasm (heavy chain H _N domain) .

Exemplary L, _HN , _HCN , and _HCC domains are shown in Table 1.

Table 1 - Exemplary L, _HN , _HCN , and _HCC domains

Clostridium neurotoxin Access number SEQ ID NO L H _N H _CN H _CC BoNT/A1 A5HZZ9 1 1-448 449-872 873-1094 1095-1296 BoNT/B1 B1INP5 2 1-441 442-859 860-1081 1082-1291 BoNT/C1 P18640 3 1-449 450-867 868-1095 1096-1291 BoNT/D P19321 4 1-442 443-863 864-1082 1083-1276 BoNT/E1 WP_003372387 5 1-423 424-846 847-1069 1070 -1252 BoNT/F1 Q57236 6 1-439 440-865 866-1087 1088-1278 BoNT/G WP_039635782 7 1-446 447-864 865-1089 1090-1297 BoNT/X BAQ12790.1 41 1-439 440-891 892-1105 1106-1306 TeNT P04958 8 1-456 457-880 881-1111 1112-1315

The above reference sequences should be considered as a guide as slight variations according to serosubtypes may be present. As an example, slightly different clostridia sequences are cited in US 2007/0166332 (the entire contents of which are therefore incorporated by reference).

Vesicle-associated membrane proteins (VAMPs) are a family of SNARE proteins that have a similar structure and are involved in vesicle fusion and exocytosis, particularly the release of neurotransmitters. VAMPs are members of a family of SNARE proteins called the synaptobrevin family and including members such as VAMP1, VAMP2 (both also known as synaptobrevin), VAMP3 (also known as cellubrevin), VAMP4, VAMP5, VAMP7 (also known as SYBL1 or tetanus toxin insensitive VAMP), VAMP8 (also known as endobrevin), YKT6, SEC22A and others. VAMP1, VAMP2 and VAMP3 are cleaved by the light chains BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X and TeNT. BoNT/X also cleaves VAMP4, VAMP5 and YKT6.

The term “VAMP epitope”, as used herein, refers to the portion of the VAMP protein to which an antibody binds.

In a preferred embodiment, the antigenic polypeptide of the invention consists of 10-65, 10-60, 10-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20 , 10-19, 10-18, 10-17, 10-16 or 10-15 amino acid residues, preferably 10-15 amino acid residues. For example, an antigenic polypeptide of the invention may consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acid residues.

In a preferred embodiment, the antigenic polypeptide of the invention contains or consists of a VAMP epitope containing an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VAMP sequence containing at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid residues located immediately C-terminal to the clostridial neurotoxin cleavage site in specified VAMP.

The amino acid sequences of natural VAMPs, particularly rat and human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and YKT6, and their corresponding clostridia neurotoxin VAMP cleavage sites are shown in Table 2 and Figure 1.

Table 2 - Sites of VAMP cleavage by clostridial neurotoxin

VAMP SEQ ID NO BoNT/F5 and BoNT/FA Other BoNT/F BoNT/D and BoNT/DC BoNT/B and TeNT BoNT/G BoNT/X VAMP1_rats (Q63666) 9 Leu56-Glu57 Gln60-Lys61 Lys61-Leu62 No splitting Ala83-Ala84 Arg68-Ala69 VAMP1_human (P23763) 10 Leu56-Glu57 Gln60-Lys61 Lys61-Leu62 Gln78-Phe79 Ala83-Ala84 Arg68-Ala69 VAMP2_rats (P63045) eleven Leu54-Glu55 Gln58-Lys59 Lys59-Leu60 Gln76-Phe77 Ala81-Ala82 Arg66-Ala67 VAMP2_human (P63027) 12 Leu54-Glu55 Gln58-Lys59 Lys59-Leu60 Gln76-Phe77 Ala81-Ala82 Arg66-Ala67 VAMP3_rats (P63025) 13 Leu41-Glu42 Gln45-Lys46 Lys46-Leu47 Gln63-Phe64 Ala68-Ala69 Arg53-Ala54 VAMP3_human (Q15836) 14 Leu37-Glu38 Gln41-Lys42 Lys42-Leu43 Gln59-Phe60 Ala64-Ala65 Arg49-Ala50 VAMP4_rats (D4A560) 42 No splitting No splitting No splitting No splitting No splitting Lys87-Ser88 VAMP4_human (O75379) 43 No splitting No splitting No splitting No splitting No splitting Lys87-Ser88 VAMP5_rats (Q9Z2J5) 44 No splitting No splitting No splitting No splitting No splitting Arg40-Ser41 VAMP5_human (O95183) 45 No splitting No splitting No splitting No splitting No splitting Arg40-Ser41 YKT6_rats (Q5EGY4) 46 No splitting No splitting No splitting No splitting No splitting Lys173-Ser174 YKT6_human (O15498) 47 No splitting No splitting No splitting No splitting No splitting Lys173-Ser174

In one embodiment, the VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6.

In one embodiment, the VAMP is selected from VAMP1, VAMP2 and/or VAMP3.

In one embodiment, the VAMP is selected from VAMP4, VAMP5 and/or YKT6.

In a preferred embodiment, the VAMP is a human VAMP, more preferably a human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6.

In one embodiment, the VAMP is selected from human VAMP1, VAMP2 and/or VAMP3.

In one embodiment, the VAMP is selected from human VAMP4, VAMP5 and/or YKT6.

In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a VAMP cleavable epitope for BoNT/F, wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/F cleavage site of VAMP.

Examples of VAMP epitopes for BoNT/F, more specifically VAMP1, VAMP2 and/or VAMP3 epitopes for BoNT/F, include:

• KLSELDDRADALQ (SEQ ID NO: 15)

• QKLSELDDRADALQ (SEQ ID NO: 16)

• KLSELDDRAD (SEQ ID NO: 17)

• KLSELDDRADALQAGAS (SEQ ID NO: 18)

• DQKLSELDDRADALQ (SEQ ID NO: 31).

In one embodiment, the VAMP epitope for BoNT/F, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/F, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 15 - SEQ ID NO: 18 and SEQ ID NO: 31. In a preferred embodiment , the VAMP epitope for BoNT/F contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the VAMP epitope for BoNT/F contains or consists of KLSELDDRADALQ (SEQ ID NO: 15).

In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a VAMP epitope for BoNT/D, wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/D cleavage site of VAMP.

Examples of BoNT/D VAMP epitopes, more specifically the BoNT/D VAMP1, VAMP2 and/or VAMP3 epitopes, include:

• KLSELDDRADALQ (SEQ ID NO: 15)

• LSELDDRADALQ (SEQ ID NO: 19)

• LSELDDRADA (SEQ ID NO: 20)

• LSELDDRADALQAGAS (SEQ ID NO: 21).

In one embodiment, the VAMP epitope for BoNT/D, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/D, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 15, and SEQ ID NO: 19 - SEQ ID NO: 21. In a preferred embodiment , the VAMP epitope for BoNT/D contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to KLSELDDRADALQ (SEQ ID NO:15). In a more preferred embodiment, the VAMP epitope for BoNT/D contains or consists of KLSELDDRADALQ (SEQ ID NO: 15).

In one embodiment of an antigenic polypeptide of the invention, the VAMP epitope is a BoNT/F5 or BoNT/FA cleavable VAMP epitope, wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/F5 cleavage site or BoNT/FA cleavage site in VAMP .

Examples of VAMP epitopes for BoNT/F5 or BoNT/FA, more specifically VAMP1, VAMP2 and/or VAMP3 epitopes for BoNT/F5 or BoNT/FA, include:

• ERDQKLSELDDRA (SEQ ID NO: 32)

• LERDQKLSELDDRA (SEQ ID NO: 33)

• VLERDQKLSELDDRA (SEQ ID NO: 34).

In one embodiment, the VAMP epitope for BoNT/F5 or BoNT/FA, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/F5 or BoNT/FA, contains or consists of at least 90% amino acid sequence 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 32 - SEQ ID NO: 34. In a preferred embodiment implementation, the VAMP epitope for BoNT/F5 or BoNT/FA contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to ERDQKLSELDDRA (SEQ ID NO: 32). In a more preferred embodiment, the VAMP epitope for BoNT/F5 or BoNT/FA contains or consists of ERDQKLSELDDRA (SEQ ID NO: 32).

In one embodiment of an antigenic polypeptide of the invention, the VAMP epitope is a VAMP epitope for BoNT/B or TeNT, wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/B cleavage site or TeNT cleavage site of VAMP.

Examples of VAMP epitopes for BoNT/B or TeNT, more specifically VAMP1, VAMP2 and/or VAMP3 epitopes for BoNT/B or TeNT, include:

• FETSAAKLKRKYW (SEQ ID NO: 22)

• FESSAAKLKRKYW (SEQ ID NO: 23)

• QFETSAAKLKRKYW (SEQ ID NO: 24)

• FETSAAKLKR (SEQ ID NO: 25)

• FETSAAKLKRKYWWKN (SEQ ID NO: 26)

• ETSAAKLKRKYWWK (SEQ ID NO: 48)

• FETSAAKLKRKYWWK (SEQ ID NO: 49)

• QFESSAAKLKRKYW (SEQ ID NO: 50)

• FESSAAKLKR (SEQ ID NO: 51)

• FESSAAKLKRKYWWK (SEQ ID NO: 52).

In one embodiment, the VAMP epitope for BoNT/B or TeNT, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/B or TeNT, contains or consists of an amino acid sequence of at least 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 22 - SEQ ID NO: 26, and SEQ ID NO: 48 - SEQ ID NO: 52. In a preferred embodiment, the VAMP epitope for BoNT/B or TeNT contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100% identical to FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49). In a more preferred embodiment, the VAMP epitope for BoNT/B or TeNT contains or consists of FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49). Surprisingly, antibodies binding the latter epitope allow not only detection of VAMP cleavage by BoNT/B, but also VAMP cleavage by BoNT/F.

In one embodiment of an antigenic polypeptide of the invention, the VAMP epitope is a VAMP epitope for BoNT/G wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/G cleavage site of VAMP.

Examples of VAMP epitopes for BoNT/G, more specifically VAMP1, VAMP2 and/or VAMP3 epitopes for BoNT/G, include:

• AKLKRKYWWKN (SEQ ID NO: 27)

• AAKLKRKYWWKN (SEQ ID NO: 28)

• AKLKRKYWWKNCKM (SEQ ID NO: 29)

• AKLKRKYWWKNLKM (SEQ ID NO: 30).

In one embodiment, the VAMP epitope for BoNT/G, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/G, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 27 - SEQ ID NO: 30. In a preferred embodiment, the VAMP epitope for BoNT/G contains or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AKLKRKYWWKN (SEQ ID NO: 27). In a more preferred embodiment, the VAMP epitope for BoNT/G contains or consists of AKLKRKYWWKN (SEQ ID NO: 27).

In one embodiment of the antigenic polypeptide of the invention, the VAMP epitope is a VAMP epitope for BoNT/X, wherein at least 8 amino acid residues are immediately C-terminal to the BoNT/X cleavage site of VAMP.

Examples of BoNT/X VAMP epitopes, more specifically BoNT/X VAMP1, VAMP2 and/or VAMP3 epitopes, include:

• ADALQAGASQF (SEQ ID NO: 53)

• ADALQAGASQ (SEQ ID NO: 54)

• RADALQAGASQF (SEQ ID NO: 55)

• ADALQAGASQFE (SEQ ID NO: 56)

• ADALQAGASVF (SEQ ID NO: 57)

• ADALQAGASV (SEQ ID NO: 58)

• ADALQAGASVFE (SEQ ID NO: 59)

• RADALQAGASVF (SEQ ID NO: 60)

• RADALQAGAS (SEQ ID NO: 61).

In one embodiment, the VAMP epitope for BoNT/X, particularly the VAMP1, VAMP2 and/or VAMP3 epitope for BoNT/X, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 53 - SEQ ID NO: 61. In a preferred embodiment, the VAMP epitope for BoNT/X contains or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ADALQAGASQF (SEQ ID NO: 53). In a more preferred embodiment, the VAMP epitope for BoNT/X contains or consists of ADALQAGASQF (SEQ ID NO: 53).

Other examples of VAMP epitopes for BoNT/X, and more specifically VAMP4 epitopes for BoNT/X, include:

• SESLSDNATAF (SEQ ID NO: 62)

• SESLSDNATA (SEQ ID NO: 63)

• KSESLSDNATAF (SEQ ID NO: 64)

• SESLSDNATAFS (SEQ ID NO: 65).

In one embodiment, the VAMP epitope for BoNT/X, particularly the VAMP4 epitope for BoNT/X, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 62 - SEQ ID NO: 65. In a preferred embodiment, the VAMP epitope for BoNT/X contains or consists of an amino acid sequence , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SESLSDNATAF (SEQ ID NO: 62). In a more preferred embodiment, the VAMP epitope for BoNT/X contains or consists of SESLSDNATAF (SEQ ID NO: 62).

Other examples of VAMP epitopes for BoNT/X, and more specifically VAMP5 epitopes for BoNT/X, include:

• SDQLLDMSSTF (SEQ ID NO: 66)

• SDQLLDMSST (SEQ ID NO: 67)

• RSDQLLDMSSTF (SEQ ID NO: 68)

• SDQLLDMSSTFN (SEQ ID NO: 69)

• SDQLLDMSSAF (SEQ ID NO: 70)

• SDQLLDMSSA (SEQ ID NO: 71)

• RSDQLLDMSSAF (SEQ ID NO: 72)

• SDQLLDMSSAFS (SEQ ID NO: 73)

• RSDQLLDMSS (SEQ ID NO: 74).

In one embodiment, the VAMP epitope for BoNT/X, in particular the VAMP5 epitope for BoNT/X, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 66 - SEQ ID NO: 74. In a preferred embodiment, the VAMP epitope for BoNT/X contains or consists of an amino acid sequence , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SDQLLDMSSTF (SEQ ID NO: 66). In a more preferred embodiment, the VAMP epitope for BoNT/X contains or consists of SDQLLDMSSTF (SEQ ID NO: 66).

Other examples of VAMP epitopes for BoNT/X, and more specifically, YKT6 epitopes for BoNT/X, include:

• SEVLGTQSKAF (SEQ ID NO: 75)

• SEVLGTQSKA (SEQ ID NO: 76)

• KSEVLGTQSKAF (SEQ ID NO: 77)

• SEVLGTQSKAFY (SEQ ID NO: 78).

In one embodiment, the VAMP epitope for BoNT/X, particularly the YKT6 epitope for BoNT/X, contains or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from: SEQ ID NO: 75 - SEQ ID NO: 78. In a preferred embodiment, the VAMP epitope for BoNT/X contains or consists of an amino acid sequence , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEVLGTQSKAF (SEQ ID NO: 75). In a more preferred embodiment, the VAMP epitope for BoNT/X contains or consists of SEVLGTQSKAF (SEQ ID NO: 75).

As used herein, the "percentage of sequence identity" between two or more nucleic acid sequences or amino acid sequences is a function of the number of identical nucleotides or amino acids at identical positions separated by the aligned sequences. Thus, % identity can be calculated as the number of identical nucleotides or amino acids at each position in the alignment divided by the total number of nucleotides or amino acids in the aligned sequence multiplied by 100. Calculation of % sequence identity can also take into account the number of gaps, and the length of each gaps that must be introduced to optimize the alignment of two or more sequences. Sequence comparisons and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms known to one skilled in the art, for example, a global alignment mathematical algorithm (such as described in Needleman and Wunsch, J. Mol. Biol. 48(3) , 443-453, 1972).

In another aspect, the invention provides a polypeptide comprising an antigenic polypeptide of the invention, wherein the polypeptide does not comprise a region of more than 17, 16, 15, 14, 13, 12, 11, 10, preferably 16, more preferably 15, consecutive amino acids having 100% sequence identity with the amino acid sequence of natural VAMP. One skilled in the art will appreciate that such a polypeptide is also antigenic.

In a preferred embodiment, the polypeptide contains a covalent linker, preferably at the N-terminus and/or at the C-terminus. Examples of covalent linkers that are suitable for the invention are presented below.

In another aspect, the invention provides an antigenic protein comprising a polypeptide of the invention covalently linked to a carrier.

Preferably, the carrier is a non-immunogenic or weakly immunogenic protein. Examples of suitable carriers include limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple binding peptide (MAP).

In one embodiment, the antigenic protein contains a covalent linker between the polypeptide of the invention (which may already contain a linker as described above) and the carrier. Said linker may be one or more amino acids, natural or non-natural, which are well known in the art to form covalent bonds with other amino acids (of the polypeptide and/or carrier) due to the presence of reactive groups present at their N-terminus, C -end and/or side chains. Notably, an amino acid having a primary amino group (-NH2) at the N-terminus and/or side chain (such as lysine) can react with an amino acid having a carboxyl (-COOH) group at the C-terminus and/or side chain (such as aspartic acid or glutamic acid), to form a covalent bond; an amino acid having a sulfhydryl (-SH) group on the side chain (such as cysteine or selenocysteine) can react with an amino acid having a sulfhydryl (-SH) group on the side chain (such as cysteine or selenocysteine) to form a covalent bond. For example, the covalent linker may be a cysteine added to the C-terminus or N-terminus of a polypeptide of the invention, wherein said cysteine forms a disulfide bridge with another cysteine added to or present in the carrier. The covalent linker may alternatively or additionally be in the form of multiple amino acids forming a spacer, for example the linker may be a peptide containing uncharged amino acids with small R side groups, such as, for example, glycine, alanine, valine, leucine or serine. Examples of suitable spacers according to the invention include G-spacers such as GGG, GGGG and GGGGS, or A-spacers such as AAA, AAAA and AAAAV. In one embodiment, the linker consists of from about 1 to about 30 amino acid residues, preferably from about 2 to about 25 amino acid residues, more preferably from about 3 to about 20 amino acid residues, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

In another aspect, the invention relates to the use of an antigenic polypeptide or protein of the invention to produce antibodies to the C-terminal cleavage product of VAMP. In one embodiment, an epitope of the invention is used to produce a polyclonal antibody against the C-terminal cleavage product of VAMP. In another embodiment, an epitope of the invention is used to produce a monoclonal antibody against the C-terminal cleavage product of VAMP.

Methods for producing antibodies are well known in the art, see, for example, Greenfield, Edward A., ed. Antibodies: a laboratory manual . Cold Spring Harbor Laboratory Press, 2014; Leenaars, Marlies, and Coenraad F. M. Hendriksen. “Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations.” Ilar Journal 46.3 (2005): 269-279.

Polyclonal antibodies that bind a VAMP epitope as described herein can be produced by injecting an animal, e.g., a mammal, such as a rabbit, goat, mouse, hamster, or monkey, or an egg, e.g., a chicken egg, with an antigenic polypeptide or protein of the invention . Polyclonal antibodies for the VAMP epitope, as described herein, can be isolated from the animal (e.g., blood) or egg and further purified by well-known methods, such as protein affinity chromatography to obtain an IgG fraction, or through affinity purification against the VAMP epitope. used to produce antibodies. Several contract research organizations provide on-demand antibody collection services, for example Eurogentec provides a “28-day rapid program” that provides immunization on day 0, followed by 3 booster injections on days 7, 10 and 18. Interim blood collection on day 21 and the final blood draw on day 28. This is one example of a general method for producing polyclonal antibodies well known in the art.

Monoclonal antibodies that bind the VAMP epitope as described herein can be produced using the hybridoma method. See, for example, Chapter 7, Greenfield, Edward A., ed. Antibodies: a laboratory manual . Cold Spring Harbor Laboratory Press, 2014. Briefly, a host animal, e.g., a mammal such as a rabbit, goat, mouse, hamster or monkey, is subjected to one or more injections of an antigenic polypeptide or protein of the invention to stimulate the formation of lymphocytes that produce or capable of producing antibodies that specifically bind to cleaved VAMP. The antibody titer in an immunized animal can be monitored over time by standard methods such as ELISA (enzyme-linked immunosorbent assay). Alternatively, lymphocytes can be immunized in vitro using a suitable cell line culture. At an appropriate time after immunization, for example when antibody titers are highest, antibody-producing cells are isolated from the animal. Typically, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The isolated antibody-producing cells are fused to an immortalized cell line using a suitable fusion agent such as polyethylene glycol to produce a hybridoma cell. Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine or human myeloma cells. Typically, a mouse myeloma cell line is fused with splenocytes collected from a suitably immunized mouse to produce a hybridoma. Preferred immortalized cell lines are mouse myeloma cell lines that are sensitive to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT). Any of a number of myeloma cell lines can be used as a fusion partner according to standard methods, for example the myeloma lines P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14. The hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after a few days in culture because they are not transformed). The culture medium in which the hybridoma cells are grown can then be assayed for the presence of monoclonal antibodies that bind the VAMP epitope as described herein. For example, hybridoma supernatants can be screened using cleaved-VAMP positive media in an immunoprecipitation assay, an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA), or a cell-based activity assay. The binding affinity of a monoclonal antibody can also be determined, for example, by Scatchard assay. See, for example, Peter J. Munson and David Rodbard, Ligand: A Versatile Computerized Approach For Characterization of Ligand-Binding Systems, 107(1) Anal. Biochem. 220-239 (1980). Once the desired hybridoma cells have been identified, limiting dilution techniques are used to isolate clones derived from a single cell until a clonal cell line expressing the desired monoclonal antibody is obtained. Alternatively, monoclonal antibodies that bind a VAMP epitope as described herein can be generated by screening a recombinant combinatorial immunoglobulin library, such as, for example, a phage display antibody library, using an antigenic polypeptide, protein or peptide of the invention. Kits for generating and screening phage display libraries are commercially available, such as the Recombinant Phage Antibody System (Amersham GE Healthcare, Piscataway, NJ); and SurfZAP™ Phage Display Kit (Stratagene, La JoIIa, CA). In addition, examples of methods and reagents that can be used to prepare and screen an antibody display library can be found, for example, in Ladner et al. US Patent 5223409; Borrebaeck et al. US Patent 5,712,089; Griffiths et al. US Patent 5885793; Griffiths et al. US Patent 5,962,255; McCafferty et al. US Patent 5969108; Griffiths et al. US Patent 6,010,884; Jespers et al. US Patent 6017732; Borrebaeck et al. US Patent 6,027,930; Johnson et al. US Patent 6140471; McCafferty et al. US Patent 6,172,197, the entire contents of each of which are hereby incorporated by reference.

In another aspect, the invention relates to an antibody that binds an antigenic polypeptide or protein of the invention.

In one embodiment, the antibody is a polyclonal antibody.

In one embodiment, the antibody is a monoclonal antibody.

The binding affinity between an antibody and an antigenic polypeptide or protein can be assessed by determining the equilibrium dissociation constant (K _D ), which measures the rate at which new antibody-antigen complexes form compared to the rate at which antibody-antigen complexes dissociate at equilibrium. The equilibrium dissociation constant is expressed in M and determined by the ratio Kd/Ka at equilibrium, where Ka is the antibody binding rate constant and Kd is the antibody dissociation rate constant. K _D= [Ab] x [Ag]/[Ab+Ag], where [Ab] is the molar concentration of the antibody, [Ag] is the molar concentration of the antigen, and [Ab+Ag] is the molar concentration of the antibody-antigen complex , where all concentrations represent the concentrations of the components when the system is in equilibrium. The lower the equilibrium dissociation constant, the more tightly the antibody is bound to its antigen, or the higher the binding affinity between the antibody and the antigen.

In one embodiment, the K _D between the antibody of the invention and an epitope of the antigenic polypeptide or protein is lower than 10 ^-6 M. In a preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-7 M. in a more preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-8 M. In a more preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-9 M. in a more preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-10 M. In a more preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-11 M. in a more preferred embodiment, the K _D between the antibody of the invention and the antigenic polypeptide or protein is lower than 10 ^-12 M.

In another aspect, the invention relates to the use of an antibody of the invention in a cell-based signal amplification assay for VAMP cleavage via a clostridial VAMP-cleaving neurotoxin.

In one embodiment, the use is in vitro or ex vivo use.

In another aspect, the invention provides a method for determining VAMP cleavage by a VAMP-cleaving clostridia neurotoxin in a cell, comprising:

a) bringing the cell into contact with the clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

b) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following cleavage of the VAMP by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody according to the invention; And

c) detecting by suitable methods the binding of said first detection antibody to the C-terminal cleavage product of VAMP.

In one embodiment, the method of the invention further comprises d) quantifying, by suitable methods, the amount of C-terminal VAMP cleavage product associated with said first detection antibody.

In one embodiment of the method of the invention, step b) comprises contacting the cytoplasmic contents of said cell with a second detection antibody against full-length VAMP under conditions suitable for binding said second detection antibody against full-length VAMP; step c) involves detecting, by suitable means, the binding of the second detection antibody to full-length VAMP, and step d) involves quantifying, by suitable means, the amount of full-length VAMP bound to said second detection antibody.

In one embodiment, the method is an in vitro or ex vivo method.

One skilled in the art will appreciate that an increase in the amount of C-terminal VAMP cleavage product bound to the first antibody and/or a decrease in the amount of full-length VAMP bound to the second detection antibody are indicative of increased VAMP cleavage by the VAMP-cleaving clostridial neurotoxin.

In one embodiment, the second detection antibody is the same as the first detection antibody and binds to the C-terminal cleavage product of VAMP and to full-length VAMP.

In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP, but not to the C-terminal cleavage product of VAMP. Suitably, the second detection antibody binds to a VAMP epitope located N-terminal to the clostridial neurotoxin cleavage site. Examples of useful antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

In a specific embodiment, a method for determining VAMP cleavage by a VAMP-cleaving clostridia neurotoxin in a cell comprises:

a) bringing the cell into contact with the clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

b) bringing the cytoplasmic contents of said cell into contact with

• a first detection antibody to the C-terminal cleavage product of VAMP following cleavage of the VAMP by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding of the first detection antibody to the C-terminal cleavage product of VAMP, wherein said first detection antibody is an antibody of the invention, which binds to the C-terminal cleavage product of VAMP and to full-length VAMP; and with

• a second detection antibody that binds to full-length VAMP but not to the C-terminal cleavage product of VAMP;

c) detection by suitable means

• binding of the first antibody to the C-terminal cleavage product of VAMP and to full-length VAMP; And

• binding of the second detection antibody to full-length VAMP; And

d) quantification by suitable means:

• the combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody; And

• the amount of full-length VAMP bound to the second detection antibody.

One skilled in the art will appreciate that a decrease in the amount of full-length VAMP bound to the second detection antibody and no change in the combined amount of full-length VAMP and C-terminal VAMP cleavage product bound to the first detection antibody are indicative of cleavage of VAMP by the VAMP-cleaving clostridial neurotoxin .

In another specific embodiment, a method for determining VAMP cleavage by a VAMP-cleaving clostridia neurotoxin in a cell comprises:

a) bringing the cell into contact with the clostridial neurotoxin under conditions suitable for the activity of the clostridial neurotoxin;

b) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following cleavage of the VAMP by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody of the invention that binds to the C-terminal cleavage product of VAMP and to full-length VAMP;

c) detection by suitable means

• binding of the first antibody to the C-terminal cleavage product of VAMP; And

• binding of the first detection antibody to full-length VAMP;

wherein the signal resulting from binding of the first detection antibody to the C-terminal cleavage product of VAMP can be distinguished from the signal resulting from binding of the first detection antibody to full-length VAMP; And

d) quantification by suitable means:

• the amount of C-terminal VAMP cleavage product bound to the first detection antibody; And

• the amount of full-length VAMP bound to the first detection antibody.

One skilled in the art will appreciate that an increase in the amount of C-terminal VAMP cleavage bound to the first antibody and a decrease in the amount of full-length VAMP bound to the first detection antibody are indicative of increased VAMP cleavage by the VAMP-cleaving clostridial neurotoxin.

In another aspect, the invention relates to a method for determining immune resistance to a VAMP-degrading clostridial neurotoxin in a patient, comprising:

a) adding a VAMP-cleaving clostridial neurotoxin to the test sample obtained from the patient;

b) bringing the cell into contact with the test sample from step a) under conditions suitable for the activity of the clostridia neurotoxin;

c) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following VAMP cleavage by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody according to the invention;

d) detecting by suitable methods the binding of said first detection antibody to the C-terminal cleavage product of VAMP;

e) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

f) repeating steps a) to e) with the negative control sample instead of the test sample; And

g) comparing the amount of C-terminal VAMP cleavage product bound to said first detection antibody in steps (e) and (f), wherein detecting a lower amount of C-terminal VAMP cleavage product bound to said first detection antibody at step (e) compared to the amount of C-terminal VAMP cleavage product bound to said first detection antibody in step (f) is indicative of the presence of neutralizing antibodies against the VAMP-cleaving clostridial neurotoxin.

In one embodiment, step f) further includes repeating steps a) - e) with a positive control sample.

As used herein, the term “anti-VAMP-cleaving clostridial neurotoxin neutralizing antibodies” means any antibody that can, under physiological conditions, bind to the region of the VAMP-cleaving clostridial neurotoxin in such a way as to reduce or prevent the VAMP-cleaving clostridial neurotoxin from exerting its therapeutic effect in a patient.

In one embodiment, the patient is a mammal. In a preferred embodiment, the patient is a human.

In one embodiment, the sample is selected from blood, plasma, serum and lymph fluid obtained from the patient.

The test sample may be obtained from the patient prior to exposure to a VAMP-cleaving clostridial neurotoxin, after a single treatment with a VAMP-cleaving clostridial neurotoxin, or after multiple treatments with a VAMP-cleaving clostridial neurotoxin. In a specific embodiment, the test sample comes from a patient who is resistant to treatment with the VAMP-degrading clostridium neurotoxin.

As used herein, the term "control sample" means any sample in which the presence or absence of the test sample is known, and includes both negative and positive control samples. For neutralizing antibodies against a VAMP-degrading clostridial neurotoxin, a negative control sample may be obtained from an individual who has never been exposed to a VAMP-degrading clostridial neurotoxin and may include, without limitation, a sample from the same individual who provided the test sample but obtained before exposure to treatment with a VAMP-cleaving clostridial neurotoxin; a sample obtained from another individual who had never been exposed to the VAMP-degrading clostridial neurotoxin; a pooled sample obtained from many different individuals who had never been exposed to the VAMP-degrading clostridial neurotoxin.

For neutralizing antibodies against a VAMP-degrading clostridial neurotoxin, a positive control sample may be obtained from an individual who exhibits immune resistance to a VAMP-degrading clostridial neurotoxin, and includes, without limitation, an individual tested positive in patient-based assays; an individual tested positive in an in vivo biological assay; and an individual for whom hyperimmunity is indicated, for example, a patient vaccinated against the VAMP-degrading clostridial neurotoxin.

In one embodiment, the method is an in vitro or ex vivo method.

In one embodiment of a method for determining immune resistance, step c) comprises contacting the cytoplasmic contents of said cell with a second detection antibody against full-length VAMP under conditions suitable for binding said second detection antibody against full-length VAMP; step d) involves detecting, by suitable means, the binding of the second detection antibody to full-length VAMP, and step e) involves quantifying the amount of full-length VAMP bound to said second detection antibody.

In one embodiment, the second detection antibody is the same as the first detection antibody and binds to the C-terminal cleavage product of VAMP and to full-length VAMP.

In an alternative embodiment, the second detection antibody is different from the first detection antibody and binds to full-length VAMP, but not to the C-terminal cleavage product of VAMP. Suitably, the second detection antibody binds to a VAMP epitope that is N-terminal to the clostridial neurotoxin cleavage site. Examples of useful antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

In a specific embodiment, a method for determining immune resistance to a VAMP-degrading clostridial neurotoxin in a patient includes:

a) adding a VAMP-cleaving clostridial neurotoxin to the test sample obtained from the patient;

b) bringing the cell into contact with the test sample from step a) under conditions suitable for the activity of the clostridia neurotoxin;

c) bringing the cytoplasmic contents of said cell into contact with

• a first detection antibody to the C-terminal cleavage product of VAMP following cleavage of the VAMP by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding of the first detection antibody to the C-terminal cleavage product of VAMP, wherein said first detection antibody is an antibody of the invention, which binds to the C-terminal cleavage product of VAMP and to full-length VAMP; and with

• a second detection antibody that binds to full-length VAMP but not to the C-terminal cleavage product of VAMP;

d) detection by suitable means

• binding of the first antibody to the C-terminal cleavage product of VAMP and to full-length VAMP; And

• binding of the second detection antibody to full-length VAMP;

e) quantification

• a combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody; And

• the amount of full-length VAMP bound to the second detection antibody;

f) repeating steps a) to e) with the negative control sample instead of the test sample; And

g) comparing the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein detecting a lower combined amount of C-terminal VAMP cleavage product and full-length VAMP bound to the first detection antibody detection, and/or a higher amount of full-length VAMP bound to the second detection antibody in step (e), compared to the corresponding amounts in step (f), is indicative of the presence of neutralizing antibodies against the VAMP-cleaving clostridial neurotoxin.

In another specific embodiment, a method for determining immune resistance to a VAMP-degrading clostridial neurotoxin in a patient comprises:

a) adding a VAMP-cleaving clostridial neurotoxin to the test sample obtained from the patient;

b) bringing the cell into contact with the test sample from step a) under conditions suitable for the activity of the clostridia neurotoxin;

c) contacting the cytoplasmic contents of said cell with a first detection antibody to the C-terminal VAMP cleavage product following VAMP cleavage by a VAMP-cleaving clostridial neurotoxin under conditions suitable for binding the first detection antibody to the C-terminal VAMP cleavage product, wherein said first the detection antibody is an antibody of the invention that binds to the C-terminal cleavage product of VAMP and to full-length VAMP;

d) detection by suitable means

• binding of the first antibody to the C-terminal cleavage product of VAMP and to full-length VAMP; And

• binding of the first detection antibody to full-length VAMP;

wherein the signal resulting from binding of the first detection antibody to the C-terminal cleavage product of VAMP can be distinguished from the signal resulting from binding of the first detection antibody to full-length VAMP;

e) quantification

• the amount of C-terminal VAMP cleavage product bound to the first detection antibody; And

• the amount of full-length VAMP bound to the first detection antibody;

f) repeating steps a) to e) with the negative control sample instead of the test sample; And

g) comparing the amount of C-terminal VAMP cleavage product bound to said first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage product bound to said first detection antibody is detected, and /or a higher amount of full-length VAMP bound to the first detection antibody in step (e) compared to the corresponding amounts in step (f) is indicative of the presence of neutralizing antibodies against the VAMP-cleaving clostridial neurotoxin.

As used herein, "VAMP-cleaving clostridial neurotoxin" means a clostridial neurotoxin that binds to a receptor on a target cell, translocates the clostridial light chain (L) into the cytosol, which in turn proteolytically cleaves VAMP, thereby impairing the secretion of molecules by vesicular transport carried out by the cell.

Preferably, in the methods or uses of the invention, the VAMP-cleaving clostridium neurotoxin contains a BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT light chain. Suitably, the light chain of BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT contains a sequence selected from:

- amino acid residues 1-441 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1-442 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1-439 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1-446 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1-439 of SEQ ID NO: 41 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity therewith, and

- amino acid residues 1-456 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them.

It is understood that the light chain of BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT, as described herein, has the ability to cleave VAMP.

In one embodiment of the methods or uses of the invention, the VAMP-cleaving clostridium neurotoxin is selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X and TeNT. Suitably, BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT contains a sequence selected from:

- SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

- SEQ ID NO: 4, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

- SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

- SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

- SEQ ID NO: 41 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it, and

- SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

It is understood that the clostridial neurotoxin BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT, as described herein, has the ability to bind to a receptor on the target cell, translocate the clostridial light chain into the cytosol and break down VAMP.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a mosaic neurotoxin. The term "mosaic neurotoxin" as used in this context refers to a naturally occurring clostridial neurotoxin that contains at least one functional domain from another type of clostridial neurotoxin (e.g., a clostridial neurotoxin of another serotype), wherein the clostridial neurotoxin does not typically contain said at least one functional domain. Examples of natural VAMP-cleaving mosaic neurotoxins are BoNT/DC and BoNT/FA. BoNT/DC contains an L chain and a serotype D H _N domain, and a serotype C H _C domain Nakamura K, et al. "Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan." Vet. Microbiol. (2010): 140:147-154., while BoNT/FA consists of a BoNT/F5 light chain, an H _N domain closely related to the F1 subtype, and a BoNT/A1 H _C domain (Pellett, Sabine, et al. "Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain." mSphere 1.1 (2016): e00100-15).

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a mosaic neurotoxin selected from BoNT/DC and BoNT/FA.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a chimeric neurotoxin. The term "chimeric neurotoxin", as used herein, means a neurotoxin containing one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may comprise an LH _N domain derived from a first neurotoxin and an _HC domain derived from a second neurotoxin. Another example of a chimeric neurotoxin is a neurotoxin containing an LH _N H _CN domain derived from a first neurotoxin and an H _CC domain derived from a second neurotoxin. Examples of chimeric neurotoxins are presented in GB1607901.4 (not yet published), the contents of which are incorporated herein by reference.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a chimeric neurotoxin comprising:

- light chain (L) from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT,

- H _N domain from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT,

- H _CN domain from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT,

- H _CC domain from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT, and

at least two of the domains are derived from different clostridial neurotoxins.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a chimeric neurotoxin comprising:

- LH _N domain from a first clostridial neurotoxin selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT,

- domain H _CN H _CC from a second clostridial neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT,

wherein the first and second clostridial neurotoxins are different.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a chimeric neurotoxin comprising:

- LH _N H _CN domain from a first clostridial neurotoxin selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT,

- H _CC domain from a second clostridial neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT

wherein the first and second clostridial neurotoxins are different.

The VAMP-cleaving clostridium neurotoxin may be a modified neurotoxin or a derivative thereof, including, but not limited to, the neurotoxins described below. A modified neurotoxin or derivative thereof may contain one or more amino acids that are modified from the native (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the toxin. As an example, a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the neurotoxin, such as biological activity or persistence.

The modified VAMP-cleaving clostridium neurotoxin, as described herein, retains the ability to bind to a receptor on a target cell to translocate the light chain into the cell cytoplasm and cleave VAMP.

The modified VAMP-cleaving clostridium neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified H _C domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) neurotoxin . Such modifications in the H _C domain may include modification of residues in the ganglioside binding site or in the protein receptor binding site of the H _{C C} domain that alter binding to the ganglioside receptor and/or protein receptor in the target nerve cell. Examples of such modified neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are therefore incorporated by reference in their entirety. For example, the H _CC domain of the BoNT/B neurotoxin contains at least one amino acid residue substitution, addition or deletion that has the effect of increasing the binding affinity of the BoNT/B H _CC domain for human Syt II compared to the native BoNT/B H _CC sequence. Suitable substitution, addition or deletion of an amino acid residue in the H _CC subdomain of BoNT/B is described in WO2013/180799 and PCT/US2016/024211, which have not yet been published (both of which are incorporated by reference). Suitable substitution, addition or deletion of an amino acid residue in the H _CC subdomain includes substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and their combinations.

In one embodiment, the VAMP-cleaving clostridium neurotoxin is a retargeted neurotoxin. The term "retargeted neurotoxin" (also referred to as "targeted secretion inhibitors", "TSI", "TVEMP" or "TEM"), as used herein, refers to a clostridium neurotoxin containing a targeting moiety (TM) that binds to an unrelated clostridia receptor. TM can replace, partially or completely, the H _C or H _CC domain of the heavy chain of a clostridial neurotoxin. Examples of retargeted neurotoxins are described in WO96/33273, WO98/07864, WO00/10598, WO01/21213, WO01/53336; WO02/07759 WO2005/023309, WO2006/026780, WO2006/099590, WO2006/056093, WO2006/059105, WO2006/059113, WO2007/138339, WO2007/106115, WO2007/106799, WO2009/150469, WO2009/150470, WO2010/055358 , WO2010/020811, WO2010/138379, WO2010/138395, WO2010/138382, WO2011/020052, WO2011/020056, WO2011/020114, WO2011/020117, WO2011/2 0119, WO2012/156743, WO2012/134900, WO2012/134897, WO2012 /134904, WO2012/134902, WO2012/135343, WO2012/135448, WO2012/135304, WO2012/134902, WO2014/033441, WO2014/128497, WO2014/053651, WO 2015/004464, all of which are incorporated herein by reference. .

Examples of cells suitable for use in the methods or applications of the invention include a prokaryotic cell, for example, an E. coli cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, a human cell, a mouse cell, a primate cell, and/or a neuronal cell. Preferably, the cell is a neuronal cell, in particular a cell with high sensitivity to BoNT,

A highly BoNT sensitive cell is a cell that is sensitive to BoNT intoxication. In some embodiments, a cell with high sensitivity to BoNT is a cell that is sensitive to intoxication by, for example, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less less, about 10 pM or less, about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, about 1 pM or less, about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less than, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM, about 0.1 pM or less, about 90 fM or less, about 80 fM or less, about 70 fM or less, about 60 fM or less, about 50 fM or less, about 40 fM or less, about 30 fM or less, about 20 fM or less, or about 10 fM or less BoNT.

Preferably, the cell has a high sensitivity (as defined above) to the VAMP-cleaving BoNT.

In one embodiment, the cell is a primary neuronal cell with high sensitivity to BoNT, for example, cortical neurons, hippocampal neurons and/or spinal cord neurons. For example, the cell is a rat cortical neuron.

In one embodiment, the cell is derived from a neuronal cell line with high sensitivity to BoNT, for example, BE(2)-M17, Kelly, LA1-55n, N1 E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12 , SH-SY5Y, SiMa and/or SK-N-BE(2)-C.

In one embodiment, the cell is a neuronal cell derived from a stem cell, in particular an induced pluripotent stem cell (iPS cell), e.g. i-Cell® Neurons, i-Cell® DopaNeurons, iCell Glutamatergic Neurons Neurons, motor neurons iCell MotoNeurons (Cellular dynamics Inc), cortical neurons of the brain, neural stem cells (Axol Biosciences), neurons Peri.4U neurons, CNS.4U neurons, Dopa.4UNeurons (Axiogenesis), MNP cells (Lonza), cortical neurons, motor neurons (iStem) and/or iPSC-derived nerve cells (MTI-GlobalStem).

In one embodiment, a cell can be modified through recombinant technology to express high levels of VAMPs, such as VAMP1, VAMP2 VAMP3, VAMP4, VAMP5 and/or YKT6, more preferably VAMP1, VAMP2 and/or VAMP3.

In one embodiment, in which the VAMP-cleaving neurotoxin is BoNT/B, BoNT/DC or BoNT/G, the cell expresses high levels of synaptotagmin I and/or synaptotagmin II (Syt I/Syt II). In one embodiment, in which the VAMP-cleaving neurotoxin is BoNT/B, BoNT/D-C, or BoNT/G, the cell is modified by recombinant technology to express high levels of synaptotagmin I and/or synaptotagmin II (Syt I/Syt II).

In one embodiment, in which the VAMP-cleaving neurotoxin is BoNT/FA, BoNT/F, BoNT/D or TeNT, the cell expresses high levels of synaptic vesicle protein (SV2). In one embodiment, in which the VAMP-cleaving neurotoxin is BoNT/FA, BoNT/F, BoNT/D or TeNT, the cell is modified through recombinant technology to express high levels of synaptic vesicle protein (SV2).

As used herein, "conditions suitable for clostridial neurotoxin activity" refer to the conditions (e.g., temperature, pH, cofactors, etc.) under which the clostridial neurotoxin can bind to the clostridial neurotoxin receptor present on the cell membrane, translocating lung the clostridia neurotoxin chain into the cell cytoplasm and cleave VAMP.

In one embodiment of the methods of the invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for a period of from about 1 hour to about 48 hours. In one embodiment of the method of the invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for a period of from about 2 hours to about 36 hours. In one embodiment of the method of the invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for a period of from about 4 hours to about 24 hours.

For example, conditions suitable for clostridial neurotoxin activity may include incubation at 37°C for 24 hours.

As used herein, "conditions suitable for binding a first detection antibody to cleaved VAMP" and "conditions suitable for binding a second detection antibody to full-length VAMP" refer to conditions (e.g. temperature, pH, cofactors, etc. .), in which the first and/or second detection antibody can bind to cleaved VAMP and/or full-length VAMP.

In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 4°C for a period of from about 8 hours to about 48 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 4°C for a period of from about 10 hours to about 24 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 4°C for a period of from about 12 hours to about 16 hours.

In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25°C for a period of from about 30 minutes to about 8 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25°C for a period of from about 1 hour to about 4 hours. In one embodiment of the method of the invention, conditions suitable for antibody binding may include incubation at about 25°C for a period of from about 1.5 hours to about 3 hours.

Methods suitable for detecting and quantifying the binding of a detection antibody to cleaved or full-length VAMP are well known in the art. For example, the binding of a detection antibody to cleaved or full-length VAMP can be detected and quantified by Western blotting. Because each protein is promoted according to a specific molecular weight on SDS-PAGE, cleaved VAMP can be detected at lower molecular weights than full-length VAMP. Band analysis by densitometry allows the percentage of cleavage to be read using both the full-length band and the cleaved band within the same lane in the gel. Alternatively, VAMP cleavage can be detected and quantified using an enzyme-linked immunosorbent assay (ELISA), such as a sandwich ELISA.

In one embodiment of the methods of the invention, the first detection antibody is a polyclonal antibody, and the binding of the first detection antibody to the C-terminal cleavage product of VAMP is detected and quantified in an enzyme-linked immunosorbent assay.

In one embodiment of the methods of the invention, the first detection antibody is a polyclonal antibody, and the binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot analysis.

In one embodiment of the methods of the invention, the first detection antibody is a monoclonal antibody, and the binding of the first detection antibody to the C-terminal cleavage product of VAMP is detected and quantified in an enzyme-linked immunosorbent assay.

In one embodiment of the methods of the invention, the first detection antibody is a monoclonal antibody, and the binding of the first detection antibody to the C-terminal cleavage product of VAMP is detected and quantified in a Western blot analysis.

In one embodiment of the methods of the invention, the cell is lysed before its cytoplasmic contents are brought into contact with the detection antibody(ies).

In an alternative embodiment of the methods of the invention, the cell is permeabilized before bringing its cytoplasmic contents into contact with the detection antibody(ies).

In another aspect, the invention provides a kit comprising a cell that is susceptible to intoxication by a VAMP-degrading neurotoxin; and a first detection antibody against cleaved VAMP, wherein said first detection antibody is an antibody of the invention.

In one embodiment, the kit further contains a second detection antibody that binds to full-length VAMP, but not to the C-terminal cleavage product of VAMP. Suitably, the second detection antibody binds to a VAMP epitope that is N-terminal to the clostridial neurotoxin cleavage site. Examples of useful antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

This disclosure is not limited to the illustrative methods and materials described herein, and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numeric ranges include numbers that define a range. Unless otherwise indicated, any nucleic acid sequences are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in amino- to carboxy-terminal orientation, respectively.

When a range of values is presented, it will be understood that each intermediate value, up to tenths of one lower limit, unless the context clearly requires otherwise, between the upper and lower limits of that range is also specifically described. Each lesser range between any specified value or intermediate value within a specified range and any other specified or intermediate value within that specified range is included in this specification. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range in which either, neither, or both limits are included in the smaller ranges is also included in this specification, subject to any specifically excluded limit within said range. When a specified range includes one or both of the limits, ranges excluding either one or both of those included limits are also included in this description.

It should be noted that, within the scope of the invention and in the accompanying claims, the unspecified and qualified singular forms include references to the plural unless the context clearly requires otherwise. Thus, for example, a reference to “clostridium neurotoxin” includes a plurality of such drug candidates, and a reference to “clostridium neurotoxin” includes a reference to one or more clostridial neurotoxins and equivalents known to those skilled in the art, etc.

The invention has now been described, by way of example only, in relation to the following figures and examples.

FIGURES

Figure 1 - VAMP sequences with clostridial neurotoxin cleavage sites. (A) sequences of human and rat VAMP1, VAMP2 and VAMP3 with cleavage sites BoNT/F5 and BoNT/FA, BoNT/F, BoNT/D and BoNT/DC, BoNT/B, BoNT/G, TeNT and BoNT/X. (B) human and rat VAMP4, VAMP5 and YKT6 sequences with BoNT/X cleavage sites.

Figure 2 - VAMP sequences with Ab epitopes (i.e. immunogenic epitope regions) and BoNT/F, BoNT/D and BoN/B cleavage sites. Sequences of human and rat VAMP1, VAMP2, and VAMP3 are shown for comparison. Human and rat VAMP2 sequences are identical in selected epitope regions. Cleavage sites are indicated by arrows: the VAMP2 cleavage site for BoNT/F and BoNT/D is located at adjacent amino acids, Q58-K59 and K59-L60, respectively, while the cleavage site for BoNT/B is localized at amino acids Q76-F77 (based on position amino acids in the human VAMP2 sequence).

Figure 3 - Cell-free cleavage of recombinant VAMP2-GFP using MBP-LF and LH_ND. Recombinant VAMP2-GFP incubated with 0.01 μg/μl LH_ND or MBP-LF for 1 hour at 37°C. Equal volumes of sample buffer were added, and 0.5 μg (Coomassie) and 0.3 μg (blots) of protein were separated by SDS-PAGE and either stained using Coomassie or blotted using various anti-VAMP2 antibodies. The figure shows the localization of antibody epitopes. Recombinant protein representation and linear length of epitopes are not to scale. 1 - BSA, 2 - VAMP2-GFP, 3 - Cleaved VAMP2-GFP (aa.59/60-end), 4 - Cleaved VAMP2-GFP (aa.1-58/59).

Figure 4 - VAMP cleavagein vitro after treatment with BoNT/F and BoNT/D. Rat cortical neurons grown in 96-well plates until DIV18-21 were treated for 24 hours with either BoNT/F (A), or BoNT/D (B). Lysates were separated by SDS-PAGE and blotted using custom-made anti-VAMP2 antibodies: anti-Pep1, anti-Pep2 or anti-Pep3, or using the commercial antibody ab181869. 1 - full-length VAMP2, 2 - split VAMP2. Data for the anti-pep 2 antibody show a dose-dependent disappearance of full-length VAMP2 and the appearance of a lower molecular weight cleaved fragment. The signals from both bands were used to quantify the dose-dependent percentage of VAMP2 cleavage by BoNT/F (C) and BoNT/D (D).

Figure 5 - (A) Rat cortical neurons were treated with natural BoNT/F1 (≤), natural BoNT/A1 (•) or recombinant BoNT/FA (Δ) for 24 hours. Cells were lysed, separated by SDS-PAGE, and blotted for VAMP-2 or SNAP-25 digestion. The percentage of SNARE cleavage was determined by the ratio of full-length to cleaved protein by densitometric analysis. Data fit was determined using a four-parameter logistic equation, and the concentration of BoNT required to achieve 50% of the maximum SNARE cleavage (pEC50) was determined (B). Data represent mean±s.e.m. (n=3 (BoNT/F1 and BoNT/A1) or 4 (BoNT/FA) independent experiments in triplicate).

Figure 6 - VAMP cleavagein vitro after treatment with BoNT/B and BoNT/F. Rat cortical neurons grown to DIV18-21 were treated for 24 hours with either BoNT/F or BoNT/B. Lysates were separated by SDS-PAGE and blotted using a new custom-made anti-VAMP2 antibody (anti-Pep4 antibody), BoNT/B cleavage-specific anti-VAMP2 antibody, or anti-Pep1, anti-Pep2, or anti-Pep3 antibodies.

EXAMPLES

Example 1: Detection of VAMP proteolytic cleavage by BoNT/D and BoNT/F

A - Methods

1. Obtaining antibodies

Antibodies were obtained from Eurogentec using the manufacturer's 28-day speedy program (https://secure.eurogentec.com/product/research-anti-protein-28-day-speedy-polyclonal-packages.html?country=gbr). Two rabbits were immunized per peptide using the following peptides:

- VAMP PEP1: H2N- SNR RLQ QTQ AQV DEC -CONH2 (SEQ ID NO:39);

- VAMP PEP2: AcNH - KLS ELD DRA DAL Q - CONH2 (SEQ ID NO:15); or

- VAMP PEP3: H2N - CLQ AGA SQ - CONH2 (SEQ ID NO:40).

Animals were given a first immunization and three subsequent booster immunizations. Blood sampling was carried out before immunization, intermediate sampling and final sampling.

2. Cleavage of recombinant protein

Active constructs containing the light chain domain and the BoNT/D translocation domain, or equivalent BoNT/F domains fused to maltose binding protein (MPB), were prepared as previously described (Masuyer et al., “Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B. J Struct Biol", 174, p52-57, 2011; Sutton et al., "Preparation of specifically activated endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications." Protein Expression and Purification 40:31-41, 2005). Briefly, either LH _N D (SEQ ID NO: 35) or a fusion protein called MBP-LF (SEQ ID NO: 36) (the latter is an MBP fusion protein with a BoNT/F1 light chain and a C-terminal 6-histidine motif ; MPB and 6-histidine motif are well-known affinity tags), diluted to 0.01 μg/μl in assay buffer (50 mM HEPES pH7.2, 200 μM ZnCl2, 1 μg/μl BSA, 10 mM DTT). VAMP2-GFP (SEQ ID NO: 37) (a fusion protein of amino acids 2-94 of human VAMP2 and a detectable green fluorescent protein (GFP) marker) was diluted to 8 μM in assay buffer (50 mM HEPES pH7.2, 200 μM ZnCl2 , 1 µg/µl BSA, 10 mM DTT). Equal volumes of LH _N D or MBP-LF and VAMP2-GFP (SEQ ID NO: 37) (8 μM) were combined and incubated at 37°C for 1 hour. Reactions were stopped by adding 2x reducing sample buffer (LDS NuPage sample buffer, 100 mM DTT).

3. Culture of rat cortical neurons

Rat cortical neurons were obtained from CD E17-E18 rat embryos. Sectioned cortical tissue was collected in ice-cold Hank's balanced salt solution (HBSS) without Ca2+ or Mg2+ and then dissociated in papain solution for 40 min at 37°C following the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded into poly-L-ornithine (PLO)-coated 96-well plates at a density of 20,000 cells/well in 125 μl Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS), and 100 units/ml penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. An additional 125 μl of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added at DIV (day in vitro ) 4. Cells were maintained by replacing half of the medium twice a week. At DIV 11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the medium to prevent proliferation of non-neuronal cells.

4. BoNT processing

Rat cortical neurons at DIV 18-21 were treated with a range of concentrations of native BoNT/F1 (Metabiologics, US) (1 nM - 0.1 pM), or BoNT/D (Metabiologics, US) (10 nM - 1 pM) per well. in triplicate for 24 hours at 37°C. The media was removed and the cells were washed once with PBS. Cells were lysed in 40 μl of LDS sample buffer (LDS NuPage buffer, 1 mM DTT, 1:500 benzonase) for 10 min at room temperature.

5. SDS-PAGE and Western blotting

Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysates were loaded onto a lane of 12% Bis-Tris gels and separated in MES buffer at 200 V for 50 min. Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using the low MW program. Membranes were blocked for 1 hour at room temperature using 5% skim milk/PBS-Tween and then incubated with custom-made anti-VAMP2 primary antibodies: anti-Pep1, anti-Pep2, or anti-Pep3, or with commercial anti-VAMP2 antibodies (Abcam ab3347 and ab181869), overnight at 4°C. Membranes were washed 3 times in PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. Membranes were washed 3×5 min in PBS-Tween, then developed using SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

B - Results

Evaluation of recombinant protein detection

The regions of three selected peptide epitopes from VAMP2 relative to BoNT cleavage sites are shown in Figure 2. The sequences of human and rat VAMP1, VAMP2 and VAMP3 are shown for comparison. Human and rat VAMP2 sequences are identical in selected epitope regions. The cleavage sites for BoNT/B and BoNT/D are located on adjacent amino acids.

Initially, antibodies were tested in a cell-free assay using recombinant VAMP2-GFP. Substitutes BoNT/F and BoNT/D (MBP-LF and LH _N D) containing the toxin light chain enzymatic domains were used to cleave the VAMP protein (Figure 3). In addition, two other commercially available anti-VAMP2 antibodies were used as comparison; ab3347 (epitope aa.1-18) and ab181869 (epitope within aa.1-100). Figure 3 shows that the anti-Pep1 antibody detected full-length VAMP2 and the N-terminal cleaved portion (aa1-58/59) with greatly reduced signal. As expected, there was no detection of the C-terminal cleavage product by this antibody because its epitope is not localized to this part. Anti-Pep2 and anti-Pep3 antibodies detected both the full-length protein and the C-terminal cleavage products of VAMP2-GFP. Ab3347 detected only full-length VAMP2 and not the N-terminal cleavage fragment, while ab181869 detected both.

These initial results indicate that the antibodies were capable of detecting full-length recombinant VAMP2 and its expected cleavage products. The exception was ab3347, which detected only full-length VAMP2 and not the N-terminal cleavage fragment.

Evaluation of endogenous protein detection

The next question was whether these antibodies could detect any degradation products in the assay of neuronal cells, in which endogenous proteases may be present. Primary rat cortical neurons were treated with either BoNT/F or BoNT/D and lysed for WB analysis (Figure 4). The anti-Pep1 antibody recognized only the full-length protein, and no detectable cleavage product was present. The anti-Pep 2 antibody detected both the full-length and the C-terminal cleavage product. The anti-Pep3 antibody showed a weak signal with very poor affinity for the VAMP monomer in cell lysates and detected higher molecular weight molecules that were most likely dimers and other proteins (data not shown). The signal for the full-length monomer was very low, however, a band for the cleaved BoNT/F and BoNT/D C-terminal product was present. In other words, the anti-Pep3 antibody did not detect full-length VAMP, but weakly detected the cleaved BoNT/F and BoNT/D C-terminal fragment. This was in contrast to earlier results from the cell-free system, which showed a strong signal for full-length and cleaved recombinant VAMP. The commercial antibody Ab3347 was not tested in vitro due to lack of detection of cleaved protein in the cell-free assay. Despite positive binding to the N-terminal cleaved recombinant fragment in a cell-free assay, the commercial antibody ab181869 detected full-length VAMP2 in cortical lysates but not the cleaved fragment in cortical lysates. Data for Pep 2 were used to quantify the dose-dependent cleavage of VAMP2 by BoNT/F (Figure 4C) and BoNT/D (Figure 4D).

We initially showed that both degradation products of recombinant VAMP could be detected in a cell-free system. However, when moving to cell lysate, the inventors also showed that the N-terminal product is not detectable, however, in this case, mechanisms other than degradation may be involved that are not yet known. In contrast, we showed that the C-terminal fragment of VAMP, which is still associated with the vesicle membrane, is not degraded or altered in a manner that would prevent antibody binding and detection by Western blotting. The Pep2 epitope is adjacent to the cleavage site of BoNT/D and BoNT/F, and an antibody raised against this peptide detects both full-length VAMP and the cleavage product. In contrast, anti-Pep3 antibody raised against a shorter epitope away from the BoNT F/D cleavage site also detects, albeit weakly, the cleavage product.

Example 2: Detection of VAMP Proteolytic Cleavage by BoNT/FA and BoNT/F1 in Rat Cortical Neurons

A - Methods

1. Culture of rat cortical neurons

Rat cortical neurons were prepared as detailed in Example 1.

2. BoNT processing

Rat cortical neurons at DIV 18-21 were treated with a concentration range (1 pM - 1 fM) of recombinant BoNT/FA (SEQ ID NO: 38) or a concentration range (1 nM - 1 pM) of native BoNT/F1 (Metabiologics, US) , or a range of concentrations (1 nM - 1 fM) of native BoNT/A1 (List Biological Laboratories Inc., US), in triplicate wells, for 24 hours, at 37°C. The media was removed and the cells were washed once with PBS. Cells were lysed in 40 μl of sample buffer (LDS NuPage buffer, 1 mM DTT, 1:500 benzonase) for 10 min at room temperature.

3. SDS-PAGE and Western blotting of rat cortical neurons

Rat cortical neurons were lysed in 40 μl of lysis buffer (LDS NuPage sample buffer, 1 mM DTT, and 1:500 benzonase) for 10 min at room temperature. Samples were boiled at 90°C for 5 minutes, and 15 μl of lysates were loaded onto a lane of 12% Bis-Tris gels and separated in either MOPS buffer at 200 V for 80 min (SNAP-25) or MES buffer at 200 B for 50 min (VAMP2). Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using mixed MW (SNAP25) or low MW (VAMP2) programs. Membranes were blocked for 1 hour at room temperature using 5% skim milk/PBS-Tween and then incubated with either anti-SNAP25 antibody (Sigma S9684 1:4000) or anti-Pep2 antibody (1:500), a custom-made antibody against VAMP2 (Eurogentec), as described in example 1; each primary antibody was incubated overnight at 4°C. Membranes were washed 3 times in PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. Membranes were washed 3×5 min in PBS-Tween, then developed using SuperSignal West Dura or West Femto chemiluminescent substrate and visualized using the Syngene Pxi system. Band densitometry was analyzed using Genetools software, and % protein degradation was determined using the ratio of full-length protein to degradation product for both SNAP-25 and VAMP2.

B - Results

After treatment with BoNT/F1, BoNT/A1 or BoNT/FA for 24 hours, rat cortical neurons were lysed, separated by SDS-PAGE and analyzed by Western blotting for VAMP-2 (BoNT/F1 and BoNT/FA) or SNAP-25 (BoNT/A1). The percentage of SNARE cleavage was determined from the ratio of full-length to cleaved protein by densitometric analysis.

The results are shown in Figure 5. Recombinant BoNT/FA cleaved VAMP-2 with activity pEC50=12.75±0.14, n=4. Natural BoNT/F1 cleaved VAMP-2 with activity pEC50=10.77±0.12, n=3. Natural BoNT/A1 cleaved SNAP-25 with activity pEC50=12.38±0.14, n=3.

Example 3: Detection of VAMP Proteolytic Cleavage by BoNT/B in Rat Cortical Neurons

A - Methods

1. Obtaining an antibody

The monoclonal antibody was produced at Abcam using rabbits immunized with the Pep4 peptide: FETSAAKLKRKYWWK (SEQ ID NO:49).

A BoNT/B cleavage-specific anti-VAMP2 antibody (Kegel et al., Toxicology in Vitro; 2007, 21: p1641-1649) was used for the comparative study.

2. Culture of rat cortical neurons

Rat cortical neurons were obtained from CD E17-E18 rat embryos. Sectioned cortical tissue was collected in ice-cold Hank's balanced salt solution (HBSS) without Ca2+ or Mg2+ and then dissociated in papain solution for 40 min at 37°C following the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded into poly-L-ornithine (PLO)-coated 96-well plates at a density of 20,000 cells/well in 125 μl Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS), and 100 units/ml penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. An additional 125 μl of Neurobasal medium containing 2% B27, 0.5 mM GlutaMAX was added at DIV (day in vitro ) 4. Cells were maintained by replacing half of the medium twice a week. At DIV 11, 1.5 μM cytosine β-D-arabinofuranoside (AraC) was added to the medium to prevent proliferation of non-neuronal cells.

3. BoNT processing

Rat cortical neurons were cultured in T25 flasks and treated with 1 nM and 10 pM BoNT/B (obtained from List Biological Laboratories, Inc.) (SEQ ID NO:2) for 24 hours at 37°C. The media was removed and the cells were washed once with PBS. Cells were lysed in 1.5 ml of NuPage sample buffer (LDS NuPage buffer, 1 mM DTT, 1:500 benzonase) for 10 min at room temperature.

4. SDS-PAGE and Western blotting

Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysates were loaded onto a lane of 12% Bis-Tris gels and separated in MES buffer at 200 V for 50 min. Proteins were transferred to nitrocellulose membranes by Transblot Turbo (Biorad) using the low MW program. Membranes were blocked for 1 hour at room temperature using 5% skim milk/PBS-Tween and then incubated with custom-made anti-Pep1, anti-Pep2, anti-Pep3, or anti-Pep4 antibodies, or a BoNT-B cleavage-specific antibody, in overnight at 4°C. Membranes were washed 3 times in PBS-Tween and incubated with anti-rabbit HRP secondary antibody for 1 hour at room temperature. Membranes were washed 3×5 min in PBS-Tween, then developed using SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

B - Results

Based on the results obtained in Examples 1 and 2 above, implying that epitope localization was key to the detection of cleaved VAMP in vitro , a new monoclonal antibody was generated against an epitope adjacent to the BoNT/B cleavage site located on the C-terminal side.

This antibody was tested in the same rat cerebral cortex assay after treatment with BoNT/B and BoNT/F, and compared with anti-Pep1, anti-Pep2, anti-Pep3, and BoNT/B cleavage-specific antibodies (Figure 6). The epitope regions for all antibodies compared were located N-terminal to the BoNT/B cleavage site. Figure 6 shows that epitope localization of the novel antibody targeting Pep4 allows detection of full-length VAMP2 as well as cleavage products for both BoNT/B and BoNT/F treatment. In contrast, anti-Pep2 and anti-Pep3 antibodies detected only the BoNT/F cleavage product and not the BoNT/B cleavage product. The anti-Pep1 antibody did not detect any cleavage products as expected. The BoNT/B cleavage-specific antibody also did not detect any BoNT/B cleavage products in these cell lysates.

Overall, the present data indicate that an important factor to consider for the detection of cleaved VAMP is the localization of the antibody epitope. Only antibodies raised against epitopes located in the membrane-bound VAMP fragment, after cleavage, were able to detect this fragment. By localizing the monoclonal antibody epitope to the C-terminus of VAMP, it was hypothesized that this region would be present in VAMP fragments produced by VAMP-cleaving neurotoxins serotypes B, D, and F. This has been shown to be a circumstance allowing the production of a single antibody (anti-Pep4 Mab ), providing positive results for BoNT/B and BoNT/F treated neurons. In addition, since TeNT shares the same cleavage site as the BoNT/B and BoNT/D cleavage site located in close proximity to the BoNT/F cleavage site, it is expected that this antibody can also be used in the cleavage of TeNT and BoNT/D.

An additional advantage of the Pep4 epitope region is that antibodies directed against this region can detect both full-length and cleaved VAMP with similar sensitivity. The ability to simultaneously detect both forms of a protein in the same sample provides a reliable tool for normalization, without the need to blot additional housekeeping proteins. This provides a very useful and direct signal accumulation Western blot assay for quantifying BoNT activity in cell models.

The present data also show differences in VAMP detection between cell-free recombinant protein assays and the whole-cell model. It is this inability to detect cellular degraded VAMP that formed the basis of the hypothesis that VAMP degradation in the cell occurs very quickly (Foran et al., “Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E and F compared with the long-lasting type A.” J Biol Chem 278 (2) pp1363-1371 2003). Unlike the antibodies of the present invention, most commercially available anti-VAMP antibodies are generated against epitopes in the N-terminal region of the protein, and thus the N-terminal fragment of VAMP was the focus of these earlier studies. Although the present description shows that the smaller C-terminal fragment of VAMP is not degraded in the cell, the larger N-terminal fragment was also not detected. However, it is interesting to note that our results in a cell-free system indicate that not all commercial antibodies are capable of detecting the expected N-terminal fragment, even when present in a cell-free system devoid of any proteases. From the present data, it can be concluded that the VAMP degradation hypothesis clearly applies only to the N-terminal fragment, and that the C-terminal VAMP fragment is not degraded and remains associated with the vesicle membrane.

SEQUENCE INFORMATION

• SEQ ID NO: 1 - BoNT/A1 - UniProtKB accession number P10845 ( Clostridium botulinum)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGF NLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVK KVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININK FLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNY GEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

• SEQ ID NO: 2 - BoNT/B1 - UniProtKB accession number P10844 ( Clostridium botulinum)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNN VQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGF NISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEA NKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNY IDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNN LNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLF LAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

• SEQ ID NO: 3 - BoNT/C1 - UniProtKB accession number P18640 ( Clostridium botulinum)

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKS EFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNL SRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRK DTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLID SHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIY INGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYY ASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE

• SEQ ID NO: 4 - BoNT/D - UniProtKB accession number P19321 ( Clostridium botulinum)

MTWPVKDFNYSDPVNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQ SSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNI ENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVE DFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDE LNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYT NKWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQ SNLGNYGIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE

• SEQ ID NO: 5 - BoNT/E - Accession number WP_003372387 ( Clostridium botulinum)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMH ELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPI TGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVP YIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYIIQHGSILG ESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDS KLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTM NFKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

• SEQ ID NO: 6 - BoNT/F - UniProtKB accession number YP_001390123 ( Clostridium botulinum)

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYTFND ISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGN LAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTT EATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVK EYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINK WIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQ IIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN

• SEQ ID NO: 7 - BoNT/G - UniProtKB accession number WP_039635782 ( Clostridium botulinum)

MPVNIKNFNYNDPINNNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCL NVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIAS KNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYNTQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKG VIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDF DDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISD YINKWFSITITNDRLGNANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQT QLFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

• SEQ ID NO: 8 - TeNT - UniProtKB accession number P04958 ( Clostridium tetani)

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDN VIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGF NIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKV NQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAMIN INIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKG NNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLY VSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

• SEQ ID NO: 9 - VAMP1_rat (Q63666)

MSAPAQPPAEGTEGAAPGGGPPGPPPNTTSNRRLQQTQAQVEEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYIFT

• SEQ ID NO: 10 - VAMP1_human (P23763)

MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT

• SEQ ID NO: 11 - VAMP2_rats (P63045)

MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

• SEQ ID NO: 12 - VAMP2_human (P63027)

MSATAATAAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

• SEQ ID NO: 13 - VAMP3_rats (P63025)

MSTGVPSGSSAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGISVLVIIVIIIIVWCVS

• SEQ ID NO: 14 - VAMP3_human (Q15836)

MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

• SEQ ID NO: 15 - VAMP epitope

KLSELDDRADALQ

• SEQ ID NO: 16 - VAMP epitope

QKLSELDDRADALQ

• SEQ ID NO: 17 - VAMP epitope

KLSELDDRAD

• SEQ ID NO: 18 - VAMP epitope

KLSELDDRADALQAGAS

• SEQ ID NO: 19 - VAMP epitope

LSELDDRADALQ

• SEQ ID NO: 20 - VAMP epitope

LSELDDRADA

• SEQ ID NO: 21 - VAMP epitope

LSELDDRADALQAGAS

• SEQ ID NO: 22 - VAMP epitope

FETSAAKLKRKYW

• SEQ ID NO: 23 - VAMP epitope

FESSAAKLKRKYW

• SEQ ID NO: 24 - VAMP epitope

QFETSAAKLKRKYW

• SEQ ID NO: 25 - VAMP epitope

FETSAAKLKR

• SEQ ID NO: 26 - VAMP epitope

FETSAAKLKRKYWWKN

• SEQ ID NO: 27 - VAMP epitope

AKLKRKYWWKN

• SEQ ID NO: 28 - VAMP epitope

AAKLKRKYWWKN

• SEQ ID NO: 29 - VAMP epitope

AKLKRKYWWKNCKM

• SEQ ID NO: 30 - VAMP epitope

AKLKRKYWWKNLKM

• SEQ ID NO: 31 - VAMP epitope

DQKLSELDDRADALQ

• SEQ ID NO: 32 - VAMP epitope

ERDQKLSELDDRA

• SEQ ID NO: 33 - VAMP epitope

LERDQKLSELDDRA

• SEQ ID NO: 34 - VAMP epitope

VLERDQKLSELDDRA

• SEQ ID NO: 35- LH _N D

MGSMTWPVKDFNYSDPVNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTS NQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNK GFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGV QAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECS VTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEAHHHHHHHHHH

• SEQ ID NO: 36 - MBP-LF

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGL TFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPVAINSFNYNDPV NDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYTFNDISGGHNSSTESFI ADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPK IIDSIPDKGLVEKIVKFAVDKLAAALEHHHHHH

• SEQ ID NO:37 (recombinant VAMP2-GFP)

GPLGSSATAATAPPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKLENVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

• SEQ ID NO: 38 - recombinant BoNT/FA

MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEY ATAFNDISIASHSPSLFIKDPALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDSNGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNI GALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCKSVIPRKGTKAPPRLCITVNNRDLFFIASQESYGENTINTYKEIDDTTTLDPSFEDILDKVILNFNEQVIPQMPNRNVSTDIQKDNYIPKYDYNRTDIIDSYEVGRNYNTFFYLNAQKFSPNESNITLTSSFDTGLLEGSKVYTFFSSDFINNINKPVQALLFIEWV KQVIRDFTTEATKTSTVDKLKDISLVVPYIGLALNIGDEIYKQHFAEAVELVGAGLLLEFSPEFLIPTLLIFTIKGYLTGSIRDKDKIIKTLDNALNVRDQKWKELYRWVVSKWLTTINTQFNKRKEQMYKALKNQATAIKKIIENKYNNYTTDEKSKIDSSYNINEIERTLNEKINLAMKNIEQF ITESSIAYLINIINNETIQKLKSYDDLVRRYLLGYIRNHSSILGNSVEELNSKVNNHLDNGIPFELSSYTNDSLLIRYFNKNYGELKYNCILNIKYEMDRDKLVDSSGYRSRINIGTGVKFSEIDKNQVQLSNLESSKIEVILNNGVIYNSMYENFSTSFWIRIPKYFRNINNEYKIISCMQNNSGWEVSLNFSNMNSKII WTLQDTEGIKKTVVFQYTQNINISDYINRWIFVTITNNNRLSNSKIYINGRLINEESISDLGNIHASNNIMFKLDGCRDPHRYIWIKYFNLFDKELNKKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYLDVNNVGIRGYMYLKGPRGRIVTTNIYLNSTLYMGTKFIIKKYASGNK DNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSAVEIPDVGNLSQVVVMKSENDQGIRNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIGKASRTFGCSWEFIPVDDGWGESSLHHHHHHHHHH

• SEQ ID NO: 39 - Pep1

SNRRLQQTQAQVDEC

• SEQ ID NO: 40 - Pep3

CLQAGASQ

• SEQ ID NO:41 - BoNT/X, Genbank accession number BAQ12790 ( Clostridium botulinum )

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNY RSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQ GQLLESSYFEKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPFKN MSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFM IKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIW YLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQ LEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

• SEQ ID NO: 42 - VAMP4_rats (D4A560)

MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALAAAILLLMIITQIILHLKK

• SEQ ID NO: 43 - VAMP4_human (O75379)

MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

• SEQ ID NO: 44 - VAMP5_rats (Q9Z2J5)

MAGKELERCQRQADQVTEIMLNNFDKVLERDGKLSELQQRSDQLLDMSSAFSKTTKTLAQQKRWENIRCRVYLGLAVAGGLLLILVVLLVIFLPSGEDSSKP

• SEQ ID NO: 45 - VAMP5_human (O95183)

MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN

• SEQ ID NO: 46 - YKT6_rats (Q5EGY4)

MKLYSLSVFYKGEPKAVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSAKGSRASVKEQEYLCHVYVRSDSLAGVVIADSEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYTALDGHLSRYQNPREADPMSKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQ NSCCAIM

• SEQ ID NO: 47 -YKT6_human (O15498)

MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARK QNSCCAIM

• SEQ ID NO: 48 - VAMP epitope

ETSAAKLKRKYWWK

• SEQ ID NO: 49 - VAMP epitope

FETSAAKLKRKYWWK

• SEQ ID NO: 50 -VAMP epitope

QFESSAAKLKRKYW

• SEQ ID NO: 51 -VAMP epitope

FESSAAKLKR

• SEQ ID NO: 52 -VAMP epitope

FESSAAKLKRKYWWK

• SEQ ID NO: 53 -VAMP epitope

ADALQAGASQF

• SEQ ID NO: 54 -VAMP epitope

ADALQAGASQ

• SEQ ID NO: 55 -VAMP epitope

RADALQAGASQF

• SEQ ID NO: VAMP 56-epitope

ADALQAGASQFE

• SEQ ID NO: VAMP 57 epitope

ADALQAGASVF

• SEQ ID NO: VAMP 58 epitope

ADALQAGASV

• SEQ ID NO: 59-epitope VAMP

ADALQAGASVFE

• SEQ ID NO: 60 -VAMP epitope

RADALQAGASVF

• SEQ ID NO: 61 -VAMP epitope

RADALQAGAS

• SEQ ID NO: 62 -VAMP epitope

SESLSDNATAF

• SEQ ID NO: 63 -VAMP epitope

SESLSDNATA

• SEQ ID NO: 64 -VAMP epitope

KSELSDNATAF

• SEQ ID NO: 65 -VAMP epitope

SESLSDNATAFS

• SEQ ID NO: 66 -VAMP epitope

SDQLLDMSSTF

• SEQ ID NO: 67 -VAMP epitope

SDQLLDMSST

• SEQ ID NO: 68 -VAMP epitope

RSDQLLDMSSTF

• SEQ ID NO: 69 -VAMP epitope

SDQLLDMSSTFN

• SEQ ID NO: 70 -VAMP epitope

SDQLLDMSSAF

• SEQ ID NO: 71 -VAMP epitope

SDQLLDMSSA

• SEQ ID NO: 72 -VAMP epitope

RSDQLLDMSSAF

• SEQ ID NO: 73 -VAMP epitope

SDQLLDMSSAFS

• SEQ ID NO: 74 -VAMP epitope

RSDQLLDMSS

• SEQ ID NO: 75 -VAMP epitope

SEVLGTQSKAF

• SEQ ID NO: 76 -VAMP epitope

SEVLGTQSKA

• SEQ ID NO: 77 -VAMP epitope

KSEVLGTQSKAF

• SEQ ID NO: 78 -VAMP epitope

SEVLGTQSKAFY

Claims (41)

1. A method for producing a polyclonal antibody, comprising isolating and further purifying a polyclonal antibody capable of binding to full-length VAMP and the C-terminal VAMP cleavage product in a cell lysate, where the C-terminal VAMP cleavage product is obtained by VAMP cleavage with a clostridial neurotoxin, from: blood obtained from an animal that was injected with: i. an antigenic polypeptide, wherein the antigenic polypeptide comprises a vesicle-associated membrane protein (VAMP) epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP-containing sequence at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in said VAMP; ii. a polypeptide comprising an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues residues that are immediately C-terminal to a clostridial neurotoxin cleavage site in said VAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; or iii. antigenic protein containing covalently bound to a carrier: a) an antigenic polypeptide, where the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues , which are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP; or a polypeptide comprising an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in said VAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP. 2. A method for producing a polyclonal antibody, comprising isolating and further purifying a polyclonal antibody capable of binding to full-length VAMP and a C-terminal VAMP cleavage product in a cell lysate, wherein the C-terminal VAMP cleavage product is obtained by cleaving VAMP with a clostridial neurotoxin from an egg obtained from an animal , and the following is introduced into the egg: i. an antigenic polypeptide, wherein the antigenic polypeptide comprises a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in said VAMP; ii. a polypeptide comprising an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in said VAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; or iii. antigenic protein containing covalently bound to a carrier: a) an antigenic polypeptide, where the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues , which are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP; or b) a polypeptide containing an antigenic polypeptide, wherein the antigenic polypeptide contains a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site of said VAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP. 3. A method for producing a monoclonal antibody, including: (a) fusing the antibody-producing cells with an immortal cell line using a suitable fusion agent to form hybridoma cells, wherein the antibody-producing cells: i. were isolated from a host animal that had received one or more injections: a) an antigenic polypeptide, wherein the antigenic polypeptide comprises a vesicle-associated membrane protein (VAMP) epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to the sequence of VAMP containing at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in said VAMP; b) a polypeptide containing an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to a clostridial neurotoxin cleavage site in said VAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; or c) an antigenic protein containing covalently bound to a carrier: A. an antigenic polypeptide, wherein the antigenic polypeptide comprises a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues , which are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP; or B. a polypeptide containing an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP, and where the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; or ii. are lymphocytes immunized in vitro : a) an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues , which are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP; b) a polypeptide containing an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP, and where the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; or c) an antigenic protein containing covalently bound to a carrier: A. an antigenic polypeptide, wherein the antigenic polypeptide comprises a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues , which are immediately C-terminal to the clostridial neurotoxin cleavage site in the specified VAMP; or B. a polypeptide containing an antigenic polypeptide, wherein the antigenic polypeptide includes a VAMP epitope, wherein said antigenic polypeptide consists of 10-17 amino acid residues, wherein said VAMP epitope contains an amino acid sequence that is at least 90% identical to a VAMP sequence containing at least 8 amino acid residues that are immediately C-terminal to a clostridial neurotoxin cleavage site in said BAMP, and wherein the polypeptide does not contain a region of more than 17 consecutive amino acids having 100% sequence identity with the natural amino acid sequence of VAMP; (b) identifying hybridoma cells that produce a monoclonal antibody capable of binding to full-length VAMP and the C-terminal VAMP cleavage product in a cell lysate, wherein the C-terminal VAMP cleavage product is produced by cleavage of VAMP with a clostridial neurotoxin; (c) isolating a clonal cell line expressing the monoclonal antibody using limiting dilution procedures; And (d) obtaining a monoclonal antibody. 4. Method according to any one of paragraphs. 1-3, where the specified antigenic polypeptide consists of 10-16 amino acid residues. 5. Method according to any one of paragraphs. 1-4, where the specified antigenic polypeptide consists of 10-15 amino acid residues. 6. Method according to any one of paragraphs. 1-5, wherein said VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6. 7. Method according to any one of paragraphs. 1-6, wherein said VAMP epitope contains or consists of an amino acid sequence at least 90% identical to a VAMP sequence selected from: SEQ ID NO: 15 - SEQ ID NO: 34, and SEQ ID NO: 48 - SEQ ID NO : 78. 8. Method according to any one of paragraphs. 1-7, wherein said VAMP epitope is an epitope of VAMP1, VAMP2 and/or VAMP3 and is selected from: -VAMP epitope for BoNT/F or BoNT/D containing or consisting of KLSELDDRADALQ (SEQ ID NO: 15); - a VAMP epitope for BoNT/F5 or BoNT/FA containing or consisting of ERDQKLSELDDRA (SEQ ID NO: 32); - a VAMP epitope for BoNT/B or TeNT containing or consisting of FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49); And - a VAMP epitope for BoNT/X containing or consisting of ADALQAGASQF (SEQ ID NO: 53). 9. Method according to any one of paragraphs. 1-8, wherein the polypeptide containing the antigenic polypeptide does not contain a region of more than 16 or 15 consecutive amino acids having 100% sequence identity with the amino acid sequence of natural VAMP. 10. Method according to any one of paragraphs. 1-9, where the K _D between said antibody and said epitope is lower than 10 ^-7 M.