A biosensor for real-time monitoring of clostridial neurotoxin production

Abstract

The present invention is a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process. The biosensor comprises a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.

Description

[0001] BIOSENSOR FIELD OF THE INVENTION The present invention relates to biosensors for monitoring clostridial neurotoxin production and methods thereof. BACKGROUND Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X (see WO 2018 / 009903 A2), as well as those produced by C. baratii and C. butyricum. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum neurotoxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system. In nature, clostridial neurotoxins (e.g. botulinum neurotoxins [BoNTs]) are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (HNdomain) and a C-terminal targeting component (HCdomain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the HCdomain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the HNdomain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non- cytotoxic protease). Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin). The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. The use of clostridial neurotoxins in therapeutic and cosmetic treatment of humans and other mammals is anticipated to expand to an ever-widening range of diseases and ailments that can benefit from the properties of these toxins. In view of this, there is an increasing demand for large-scale manufacture of clostridial neurotoxins and appropriate formulation thereof. Botulinum toxins, including the type-A toxins, are conventionally obtained through a culturing and fermentation process. The resulting fermentation solution typically contains whole bacteria, lysed bacteria, culture media nutrients, and fermentation by-products, in addition to botulinum toxin molecules. The fermentation solution is filtered to remove whole and / or lysed cellular components, and optionally other fermentation medium residues, to yield a clarified culture. The clarified culture solution contains botulinum toxin molecules and various impurities that can be removed to obtain concentrated, purified botulinum toxin (e.g., BoNT / A1), suitable for compounding into a botulinum toxin pharmaceutical composition. Monitoring the production of botulinum toxins produced during the culture and fermentation process typically involves the collection and off-line analysis of a sample from the fermentation solution. Off-line analysis may be performed hours or even days after a sample is collected and therefore does not allow for the real-time monitoring of botulinum toxin production. Further, the testing methods required for off-line monitoring are generally slow, laborious and expensive. For example, determining the concentration of botulinum toxin in a sample typically uses multiple steps (e.g., to separate the toxin complex from residual impurities from the fermentation process) and involves the manual handling of samples having an inherent biosafety risk. Further, processing of clostridial neurotoxins in batches comes with several drawbacks, including potential differences in sensitivity due to the use of batches of antibodies for immunoprecipitation and thus reduced reproducibility and robustness. Thus, there is a need in the art for alternative and / or improved methods for monitoring the production of botulinum toxins during the culture and fermentation process. Specifically, the present invention relates to biosensors and methods for monitoring the production of botulinum toxins in real-time. SUMMARY OF THE INVENTION The present inventors have developed novel biosensors, binding substrates and methods for the rapid, real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process. Advantageously, the biosensors, binding substrates and methods of the invention may be highly sensitive, thereby allowing for the detection of picomolar concentrations of clostridial neurotoxins in a composition. The biosensors and methods of the invention advantageously also allow for the detection of specific modified (e.g., oxidised), proteolytically activated or aggregated forms (for example, dimers and multimers, optionally having reduced potency) of clostridial neurotoxins. Thus, the biosensors may be used to determine whether the fermentation process produces a homogenous population of clostridial neurotoxin polypeptides and / or determine the proportion of clostridial neurotoxin variants produced. The biosensors, binding substrates and methods of the invention have been specifically designed to require minimal steps and equipment, meaning that they are suitable for use with agents that may cause serious and potentially lethal side effects in subjects exposed to said agents (e.g., in biosafety level 3 environments). In addition, the biosensors, binding substrates and methods of the invention overcome the challenges associated with variability in the sensitivity of batches of reagents (e.g., monoclonal antibodies) used to monitor clostridial neurotoxin production using conventional off-line approaches. The present invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. Also is a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. In some embodiments, the binding region comprises one or more paratopes that bind(s) clostridial neurotoxin. The term “paratope” is a well-defined term in the art that refers to the antigen-binding site of an antibody or an antibody fragment. In some embodiments, the binding region comprises one or more amino acid sequence(s) that specifically bind(s) to the clostridial neurotoxin, and wherein said one or more amino acid sequence(s) comprise the CDR sequences of one or more antibodies; preferably, wherein the antibodies are single-chain antibodies such as nanobodies, single-chain variable fragments (scFvs), single-chain Fab (scFab), minibodies or diabodies; further preferably wherein the single-chain antibodies are camelid or shark nanobodies. In some embodiments, said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA- H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA- H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. For example, said one or more amino acid sequence(s) may comprise a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. In some embodiments, the biosensor or binding substrate comprise one or more amino acid sequence comprises the CDR sequences from nanobodies capable of neutralising clostridial neurotoxin toxicity such as ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof. In some embodiments, the biosensor or substrate comprise one or more amino acid sequence comprising a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof. The nanobody of the invention may be multimer, such as a dimer, trimer or tetramer. In some embodiments, the binding region comprises one or more amino acid sequence(s) selected from (and optionally further comprises) a sequence that specifically binds to a clostridial neurotoxin selected from the one or more of: an oxidised form of clostridial neurotoxin (having reduced potency), an incorrect proteolytically activated form of a clostridial neurotoxin (having reduced potency), and / or aggregated forms of clostridial neurotoxin, including dimers and multimers (having reduced potency). The biosensor and / or binding substrate of the invention are advantageously highly sensitive. In some embodiments, the biosensor and / or binding substrate may be used to detect clostridial neurotoxins at a concentration of less than 1ng / ml, preferably less than 0.1ng / ml. Also provided is a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor of the invention; c. exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. Optionally, prior to contacting the liquid medium with a biosensor, the bacterial host cell may be lysed. Also provided is a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate of the invention; b. culturing the bacterial host cell in a liquid medium; c.exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. In some embodiments, step d comprises donor fluorescence intensity at said biosensor, wherein increased donor fluorescence intensity of at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. In some embodiments, step d comprises detecting acceptor fluorescence intensity at said biosensor, wherein decreased acceptor fluorescence intensity at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. In some embodiments, step d comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin. In some embodiments, step d comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin. DETAILED DESCRIPTION In one aspect, the invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. In one aspect, the invention provides a biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, wherein the surface is configured for immobilisation of a neurotoxin, and further comprising a binding substrate, wherein the binding substrate comprises: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. In one aspect, the invention provides a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. In one aspect, the invention provides a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor of the invention; c. exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. In one aspect, the invention provides a method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate of the invention; b. culturing the bacterial host cell in a liquid medium; c. exciting said donor fluorophore; and d. determining resonance transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. The biosensor of the invention enables rapid, real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process. The biosensor comprises a surface for performing the analysis of binding events and / or kinetics (e.g., a support) and a binding substrate. In other aspects, the invention provides a binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the binding substrate is not immobilised to a surface. For example, in some embodiments, the binding substrate may be expressed intracellularly in a host cell that is capable of producing a clostridial neurotoxin polypeptide. In some embodiments, the support is configured to capture or immobilise the binding substrate. In other embodiments, the support is configure to capture or immobilise clostridial neurotoxin polypeptides. In some embodiments, the binding substrate comprises a means for indicating the presence or absence of clostridial neurotoxin polypeptides in a composition. In particular, the binding substrate may comprise two different states, wherein one state indicates the presence of clostridial neurotoxin polypeptides (e.g., via the binding of clostridial neurotoxin polypeptides to the binding substrate) and another state indicating the absence of clostridial neurotoxin polypeptides (e.g., via the absence of binding of clostridial neurotoxin polypeptides to the binding substrate). The means for indicating the presence or absence of a clostridial neurotoxin may comprise one or more modules (e.g., 1, 2, 3, 4, 5 or more modules). Preferably, the one or more modules are genetically encoded. For example, in preferred embodiments, the one or more modules are expressed as a single-chain polypeptide. The term “single-chain” may refer to a single polypeptide molecule having a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In other words, each recited element of the single-chain polypeptide may be connected to the other element(s) by means of a peptide bond. Exemplary modules may be selected from detectable labels and clostridial neurotoxin binding regions. In preferred embodiments, the one or more comprise one or more detectable label. For example, the binding substrate may comprise 1, 2, 3, 4, 5 or more detectable labels, preferably 2 detectable labels. The detectable label may be a label that can be detected visually by way of the label’s optical properties. A detectable label may be a fluorescent label (e.g., a fluorophore). Such a label may be detected using any number of fluorescent techniques including fluorescent microscopy. In preferred embodiments, the binding substrate comprises one or more detectable label, wherein the detectable label is a fluorophore. As used herein, the term fluorophore encompasses fluorescent proteins, bioluminescent proteins, fluorescent dyes (e.g., non- protein organic dyes) and quantum dots. Preferably the fluorophore is a fluorescent protein. Preferably, the means for indicating the presence or absence of a clostridial neurotoxin comprises at least two detectable labels (e.g., two fluorophores). For example, in preferred embodiments, the binding substrate comprises a first and second detectable label (e.g., a first and second fluorophore). In other embodiments, the binding substrate may comprise a first detectable label (e.g., a fluorophore) and a moiety that reduces the fluorescence intensity of the fluorophore under certain conditions, such as a fluorescence quencher or Au-nano particle. In some embodiments, the binding substrate comprises a first (donor) fluorophore and a second (acceptor) fluorophore. In preferred embodiments, the donor fluorophore and acceptor fluorophore are positioned such that following activation of the donor fluorophore, resonance energy transfer (e.g., fluorescence (or Förster) resonance energy transfer, FRET) is exhibited between said donor fluorophore and said acceptor. As used herein, the term "FRET" refers to non-radiative energy transfer between two fluorophores having different emission wavelengths, in which the excitation energy of a donor fluorophore in an excited state is transferred to an acceptor, and thus emission from the fluorescence acceptor, or the quenching of the fluorescence donor is observed (Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 2nd ed., New York:Plenum Press, 1999). Thus, as used herein, the terms “donor” or "donor fluorophore" may refer to a fluorophore acting as a donor in the FRET phenomenon, and the terms "acceptor" or “acceptor fluorophore” may refer to a fluorophore acting as an acceptor in the FRET phenomenon. Preferably, the donor fluorophore and the acceptor fluorophore are functionally connected by one or more amino acid sequence that specifically binds to a clostridial neurotoxin. The term “functionally connect” refers to joining the donor fluorophore and the acceptor fluorophores in such a way that any intervening sequence (e.g., a binding region) does not prevent the primary amino acid sequence of each fluorophore forming a functional tertiary structure (e.g., a functional fluorophore). Methods for determining whether a given sequence functionally connects a donor and acceptor fluorophore are known in the art. For example, the skilled person will be able to determine whether a given binding region functionally connects a donor and acceptor fluorophore using for example, visual detection using fluorescent microscopy to measure FRET (e.g., by sensitized emission, acceptor photobleaching and / or FLIM-FRET methods) or solution-based methods to measure FRET (e.g using filter or monochromatic spectrophotometers). Preferably, the donor fluorophore and acceptor fluorophore are separated by a binding region comprising one or more sequences that bind to clostridial neurotoxin polypeptides. In other words, the binding region may comprise one or more sequences (e.g., 1, 2, 3, 4, 5 or more sequences) that bind to clostridial neurotoxin polypeptides. Preferably, the binding substrate is configured such that binding of a clostridial neurotoxin to the binding region increases the distance between the donor fluorophore and the acceptor fluorophore. Preferably, the binding substrate is configured such that binding of a clostridial neurotoxin to the binding region results in a change in the fluorescence intensity from the donor fluorophore and / or the acceptor fluorophore. For example, in some embodiments, the donor and acceptor fluorophores are separated by an amino acid sequence that binds to clostridial neurotoxin polypeptides in the presence of said clostridial neurotoxin. In some embodiments, in the absence of clostridial neurotoxin polypeptides, the binding region is unbound by clostridial neurotoxin polypeptides and the donor and acceptor fluorophores are in close proximity such that resonance energy transfer between said donor fluorophore and said acceptor occurs. However, in the presence of clostridial neurotoxin polypeptides, said clostridial neurotoxin polypeptides bind to the binding region, thereby increasing the distance between the donor and acceptor fluorophores such that resonance energy transfer between said donor fluorophore and said acceptor is reduced. Resonance energy transfer between the donor and the acceptor fluorophore may be detected as increased emission from the acceptor fluorophore and / or quenching from the donor fluorophore. Alternatively, absence of resonance energy transfer between the donor and the acceptor fluorophore may be detected as reduced emission from the acceptor, or increased emission from the donor fluorophore. In preferred embodiments, a biosensor of the invention comprises a plurality of binding substrates. In some embodiments, the binding substrates are identical. In other embodiments, the biosensor comprises two or more different binding substrates. The different binding substrates may comprise different regions such that a first plurality of binding substrates binds to a first population of clostridial neurotoxin polypeptides and a second plurality of binding substrates binds to a second population of clostridial neurotoxin polypeptides. In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of oxidised clostridial neurotoxin production during a bacterial fermentation process. For example, in some embodiments, a biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to an oxidised form of clostridial neurotoxin. In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to a non-oxidised form of clostridial neurotoxin and a second plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to an oxidised form of clostridial neurotoxin (having reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, such that the plurality of binding substrates may be used to distinguish between the binding of a first and second population of clostridial neurotoxins (e.g., oxidised and non-oxidised clostridial neurotoxins). In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of incorrectly proteolytically activated clostridial neurotoxin production during a bacterial fermentation process. For example, in some embodiments, the biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to an incorrect proteolytically activated form of a clostridial neurotoxin (e.g., having reduced potency). In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to a proteolytically active clostridial neurotoxin and a second plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to an incorrect proteolytically activated form of a clostridial neurotoxin (e.g., having reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, such that the plurality of binding substrates may be used to distinguish between the binding of a first and second population of clostridial neurotoxins (e.g., proteolytically active clostridial neurotoxins and incorrect proteolytically activated form of a clostridial neurotoxins). In some embodiments, a biosensor comprising one or more different binding substrates may be used in a method for real-time monitoring of the production of aggregated forms of clostridial neurotoxins during a bacterial fermentation process. For example, in some embodiments, the biosensor may comprise a plurality of binding substrates, wherein at least a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to aggregated forms of clostridial neurotoxin, including dimers and multimers. In some embodiments, the biosensor may comprise a plurality of binding substrates, wherein a first plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to a clostridial neurotoxin monomer and a second plurality of binding substrates comprise a binding region comprising a sequence that specifically binds to aggregated forms of clostridial neurotoxin, including dimers and multimers (having reduced potency). Each plurality of binding substrates may further comprise different donor and acceptor fluorophores, such that the plurality of binding substrates may be used to distinguish between the binding of a first and second population of clostridial neurotoxins (e.g., monomers and multimers). Preferably, binding of clostridial neurotoxin polypeptides to the binding substrates increases donor fluorophore emission and / or reduces acceptor fluorophore emission in a concentration dependent manner. In other words, the biosensors may be used to quantify the concentration of clostridial neurotoxin polypeptides in a composition. In preferred embodiments, the donor and acceptor fluorophores are fluorescent proteins. A suitable fluorescent protein may be selected by the skilled person. For example, one or more fluorescent protein may be selected from the list comprising: green fluorescent proteins (e.g., GFP, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and derivatives and variants thereof), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mTagBFP and derivatives and variants thereof), cyan fluorescent proteins (e.g., ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal) and derivatives and variants thereof), yellow fluorescent proteins (e.g., EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana and derivatives and variants thereof), orange fluorescent proteins (e.g., Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine and derivatives and variants thereof), and red fluorescent proteins (e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143 and derivatives and variants thereof). fluorescent proteins can be found at https: / / www.fpbase.org / table / . The FRET donor and acceptor pairs are not particularly limited, and the skilled person will be able to select suitable FRET donor and acceptor pairs. For example, in most embodiments, the donor and acceptor fluorophores will be different (e.g., have different excitation and emission wavelengths). Typically, FRET occurs between two fluorophores in close proximity with substantial overlap (>30%) between the donor’s emission and acceptor’s absorption spectra. Further, the skilled person will appreciate that to maximize the FRET signal, the donor may be selected based on its quantum yield (e.g., by selecting a donor with a suitably high quantum yield calculated as the number of emitted photons per absorbed photons) and / or an acceptor with a suitably high extinction coefficient (e.g., linking the quantity of absorbed light, at a given wavelength, to the concentration of fluorophore in solution). Commonly used donor and acceptor pairs may be selected from a cyan donor fluorophore and a yellow acceptor fluorophore or a green donor fluorophore and a red acceptor fluorophore. In some embodiments, the donor fluorophore may be a green fluorescent protein (e.g., selected from mClover3, GFP, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and derivatives and variants thereof) and the acceptor fluorophore may be a red fluorescent protein (e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143 and derivatives and variants thereof). In some embodiments, the donor fluorophore may be a cyan fluorescent protein (e.g., selected from ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal) and derivatives and variants thereof) and the acceptor fluorophore may be a yellow fluorescent protein (e.g., selected from EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana and derivatives and variants thereof). Exemplary, non-limiting examples of FRET pairs may be selected from the list comprising: ECFP-EYFP, mTurquoise2-sEYFP, mTurquoise2-mVenus, EGFP-mCherry, Clover-mRuby2, mClover3-mRuby3, mNeonGreen-mRuby3, eqFP650-iRFP, mAmetrine-tdTomato, LSSmOrange-mKate2, EGFP-sREACh, EGFP-ShadowG, EGFP-activated PA-GFP, EGFP- Phanta, mTagBFP-sfGFP, mVenus-mKOκ or CyOFP1-mCardinal. Alternatively, suitable FRET pairs may be selected using one or several online tools, including FRbase FRET Calculator (https: / / www.fpbase.org / fret / ). Preferably, the donor fluorophore is mClover3. Preferably, the acceptor fluorophore is Preferably, the donor fluorophore is mClover3 and the acceptor fluorophore is mRuby3. In some embodiments, increased donor fluorescence intensity (e.g., compared to a negative control comprising no clostridial neurotoxin) at said biosensor is indicative of the presence of clostridial neurotoxin. In some embodiments, decreased acceptor fluorescence intensity at said biosensor (e.g., compared to a negative control comprising no clostridial neurotoxin) is indicative of the presence of clostridial neurotoxin. The donor fluorophore is preferably N-terminal to the clostridial neurotoxin binding region. In some embodiments, the donor fluorophore is the most N-terminal element of the binding substrate. A donor fluorophore may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45. In some embodiments, a donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45. In some embodiments, a donor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength within the range of from 499 to 510 nm, preferably about 506 nm and / or a peak emission wavelength within the range of from 512 nm to 522 nm, preferably 518 nm. A donor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45. In some embodiments, a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45. In some embodiments, a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45, wherein the donor fluorophore has a peak excitation wavelength within the range of from 499 to 510 nm, preferably about 506 nm and / or a peak emission wavelength within the range of from 512 nm to 522 nm, preferably 518 nm. The acceptor fluorophore (e.g., having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore) is preferably C-terminal to the clostridial neurotoxin binding region. In some embodiments, the acceptor is the most C-terminal element of the binding substrate. An acceptor fluorophore may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46. In one embodiment, an acceptor fluorophore may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46. In some embodiments, a donor fluorophore may comprise a polypeptide sequence having least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46, wherein the acceptor fluorophore has a peak excitation wavelength within the range of from 522 to 564 nm, preferably about 558 nm and / or a peak emission wavelength within the range of from 579 nm to 609 nm, preferably 592 nm. An acceptor fluorophore may consist of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46. In one embodiment, an acceptor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46. In some embodiments, a donor fluorophore may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46, wherein the acceptor fluorophore has a peak excitation wavelength within the range of from 522 to 564 nm, preferably about 558 nm and / or a peak emission wavelength within the range of from 579 nm to 609 nm, preferably 592 nm. In some embodiments, the binding substrate may further comprise additional elements such as one or more purification tags. For example, the purification tag may be selected from an epitope tag selected from the list comprising: his, FLAG, HA, V5, Myc, and Strep. Alternatively (or additionally), the purification tag may be selected from a protein / domain tag selected from the list comprising GST, MBP, SUMO, CBP, Halo, Mus A and FATT. Alternatively (or additionally), the binding substrate may further comprise additional elements such as one or more spacer sequence. For example, in some embodiments, a spacer sequence may be a sequence positioned between the donor fluorophore and the binding region. In some embodiments, a spacer sequence may be positioned between the acceptor fluorophore and the binding region. In some embodiments, the binding substrate comprises a first spacer sequence positioned between the donor fluorophore and the binding region and a second spacer sequence positioned between the acceptor fluorophore and the binding region. Where more than one spacer is present, the spacers may have the same or different polypeptide sequences. It is well-within the skilled person’s capability to select an appropriate spacer sequence and size. A spacer may be of any suitable length, such as 3-20, 2-15, 5-15 or 4-8 amino acids in length. A spacer may comprise (or consist of) glycine and serine residues. The binding region may comprise (or consist of) one or more sequence that binds specifically to a clostridial neurotoxin polypeptide. For example, the binding region may comprise (or consist of) 1, 2, 3, 4, 5 or more sequences that bind specifically to a clostridial neurotoxin polypeptide. The sequences may be or different. Preferably, the sequences are different. As described herein, the binding region may comprise (or consist of) a multimer of sequences that bind to a single clostridial neurotoxin polypeptide. For example, when the binding region comprises a sequence comprising the CDR sequences of 2, 3 or 4 antibodies, the antibodies may bind to different epitopes on the same clostridial neurotoxin polypeptide. Use of a binding region comprising more than one sequence that binds specifically to a single clostridial neurotoxin polypeptide advantageously may increase the binding affinity of the interaction (e.g., as measured by KD). The clostridial neurotoxin polypeptide may comprise (or consist of) a sequence selected from any one of BoNT / A-G, BoNT / X or TeNT. The clostridial neurotoxin polypeptides are described herein and may be selected from the group comprising wild-type polypeptides, modified or chimeric polypeptide and fragments thereof. For example, the binding region may bind specifically to a polypeptide comprising a sequence having at least 70% sequence identity to any one of SEQ ID NO: 1 to 18. In some embodiments, the binding region may bind specifically to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NO: 1 to 18. Preferably, the binding region may bind specifically a polypeptide comprising any one of SEQ ID NO: 1 to 18. For example, the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to any one of SEQ ID NO: 1 to 18. In some embodiments, the binding region may bind specifically a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NO: 1 to 18. Preferably, the binding region may bind specifically to a polypeptide consisting of any one of SEQ ID NO: 1 to 18. For example, the binding region may bind specifically to a polypeptide comprising a sequence having at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the binding region may bind specifically to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 1. Preferably, the binding region may bind specifically a polypeptide comprising SEQ ID NO: 1. For example, the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the binding region may bind specifically a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 1. the binding region may bind specifically to a polypeptide consisting of SEQ ID NO: 1. For example, the binding region may bind specifically to a polypeptide comprising a sequence having at least 70% sequence identity to SEQ ID NO: 13. In some embodiments, the binding region may bind specifically to a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 13. Preferably, the binding region may bind specifically a polypeptide comprising SEQ ID NO: 13. For example, the binding region may bind specifically to a polypeptide consisting of a sequence having at least 70% sequence identity to SEQ ID NO: 13. In some embodiments, the binding region may bind specifically a polypeptide consisting of a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 13. Preferably, the binding region may bind specifically to a polypeptide consisting of SEQ ID NO: 13. In some embodiments, the binding region may comprise (or consist of) one or more sequence that binds specifically to a clostridial neurotoxin light chain (L)(e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin translocation domain (HN) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin HCNdomain (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a clostridial neurotoxin HCCdomain (e.g., as defined herein). For example, in some embodiments, the binding region may comprise (or consist of) one or more sequence that binds specifically to a BoNT / A light chain (L)(e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / A heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / A translocation domain (HN) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / A HCNdomain (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / A HCCdomain (e.g., as defined herein). For example, in some embodiments, the region may comprise (or consist of) one or more sequence that binds specifically to a BoNT / B light chain (L)(e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / B heavy chain (H) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / B translocation domain (HN) (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / B HCNdomain (e.g., as defined herein). In some embodiments, the binding region may comprise (or consist of) any sequence that binds specifically to a BoNT / B HCCdomain (e.g., as defined herein). In some embodiments, the binding region comprises one or more sequence(s) that bind specifically to a chimeric clostridial neurotoxin polypeptide (e.g., mrBoNT / AB). In some embodiments, the binding region comprises one or more sequence(s) that bind specifically to a modified clostridial neurotoxin polypeptide. In some embodiments, the modified clostridial neurotoxin polypeptide is an oxidised form of clostridial neurotoxin. In some embodiments, the binding region comprises one or more sequence(s) that bind specifically to an incorrect proteolytically activated form of a clostridial neurotoxin. In some embodiments, the binding region comprises one or more sequence(s) that bind specifically to aggregated forms of clostridial neurotoxin, including dimers and multimers. In some embodiments, the one or more amino acid sequence comprises the CDR sequences of one or more antibodies. The term "antibody", as used herein, broadly refers to any immunoglobulin (Ig) molecule such as full-length “conventional” antibodies comprising four polypeptide chains (e.g., two heavy (H) chains and two light (L) chains), and any functional fragment (e.g., antigen-binding fragment), mutant, variant, or derivation thereof which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody entities are known in the art, non-limiting embodiments of which are discussed below. In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLcomprises three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR2, FR3, CDR3, FR4. Antibodies may be polyclonal (pAb) or monoclonal (mAb). Alternatively, the term functional fragment (e.g., antigen-binding fragment) includes antibody fragments. Non-limiting examples of antibody fragments include Fab, Fv, scFv, dAb, Fd, Fab’ or F(ab’)2, tandem scFv and diabodies. Antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass or functional fragment (e.g., antigen- binding fragment), mutant, variant, or derivation thereof. Antibodies of the invention can be from any species (e.g., mouse, human, chicken, rat, rabbit, sheep, shark and camelid). Preferably, antibodies of the invention are camelid (e.g., from camels, dromedaries, alpacas, llamas, or vicuñas) or cartilaginous fish (e.g., from sharks or rays) antibodies or functional fragments (e.g., antigen-binding fragments), mutants, variants, or derivations thereof. For example, as used herein, a "human antibody” is defined as an antibody having variable and constant regions derived from human germline immunoglobulin sequences, but which may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. In some embodiments, an antibody of the invention may be a "CDR-grafted antibody"; defined as an antibody which comprises heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VHand / or VLare replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3 or all three CDRs) has been replaced with human CDR sequences. In some embodiments, an antibody of the invention may be a "humanized antibody"; defined as an antibody which comprise heavy and light chain variable region sequences from a non- human species (e.g., a mouse) but in which at least a portion of the VHand / or VLsequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human and VLsequences to replace the corresponding nonhuman CDR sequences. In some embodiments, an antibody of the invention may be a "chimeric antibody"; defined as an antibody which comprises heavy and light chain variable region sequences from one species and constant region sequences from another species. The present invention encompasses chimeric antibodies having, for example, murine heavy and light chain variable regions linked to human constant regions. Antibodies of the invention may be bispecific, dual specific, or multi-specific (e.g., binding to two or more different antigens). Preferably, antibodies of the invention will bind to the same antigen (e.g., bind to the same clostridial neurotoxin polypeptides). Also encompassed are antibody constructs, defined as a polypeptide comprising one or more the antigen binding fragment of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Preferably, the binding region comprises a single-chain antibody or multimer (e.g., dimer, trimer or tetramer) thereof. Suitable single-chain antibodies include the VHand VLdomains of conventional antibodies (e.g., full-length human antibodies), single-chain variable fragments (scFv), single-chain Fabs (scFab), minibodies, diabodies or single chain antibodies from camelids or cartilaginous fish and any functional fragment (e.g., antigen-binding fragment), mutant, variant, or derivation thereof which retains the essential epitope binding features of a single-chain antibody. Functional single-chain antibodies produced by camelids are known as HcAbs (heavy chain antibodies). HcAbs comprise only heavy chains (H) and no light chains (L). HcAbs also lack the first constant domain of the heavy chain (CH1). The N-terminal region comprises a variable domain, known as VHH, which binds to a specific antigen, and two constant domains. The VHH in an HcAb is the structural and functional equivalent of the Fab fragment of conventional antibodies. Therefore, the antigen-binding site of HcAbs consists only of a single domain that is directly linked through a hinge region to the Fc domain. In preferred embodiments, the binding region comprises (or consists of) a camelid VHH or a camelid HcAb. Functional single-chain antibodies by cartilaginous fish are known as Immunoglobulin Novel Antigen Receptors (IgNARs). IgNARs have a homodimeric structure comprising two heavy chain polypeptides, each comprising a single variable domain (VNAR) and five constant domains. In preferred embodiments, the binding region comprises (or consists of) an VNAR from cartilaginous fish (e.g., a shark IgNAR). VHHs and VNARs derived from camelid and cartilaginous fish, respectively, are also known as nanobodies or nanoantibodies (Nbs). Any single-chain antibody or functional fragment (e.g., VHH or VNAR), mutant, variant, or derivation thereof which retains the essential epitope binding features of said single-chain antibody may be used in the present invention, so long as said antibody binds to a clostridial neurotoxin. Suitable single-chain antibodies are known in the art. For example, single-chain antibodies may comprise the three CDR sequences (e.g., CDR1, CDR2 and CDR3) of one of the exemplary single-chain camelid antibodies shown in Table 1. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to any one of the single-chain camelid antibodies shown in Table 1; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of the single-chain camelid antibodies shown in Table 1; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention is selected from Table 1. Further, a person skilled in the art would be able to determine whether an alternative single- chain antibody binds to a clostridial neurotoxin using routine experimental methods. Table 1: Exemplary single-chain camelid antibodies Clone Protein Epitope Subunit Neutralisation Genbank JDY-33 ciA-H7 A1 Lc Strong HQ700708 JDT-2 ciA-D1 A1 Lc Strong JEC-3 ciA-H4 A1 Lc Not done JEC-11 ciA-H11 A1 Lc Not done JDY-46 ciA-C2 A2 Hc-RBD Strong HQ700705 JDY-9 ciA-B5 A3 Hc Strong HQ700704 JED-27 ciA-F12 A4 Lc None HQ700706 JDU-26 ciA-D12 A5 Lc None HQ700702 JDY-2 ciA-A5 A6 None Weak HQ700703 JDY-59 ciA-G5 A7 None Weak HQ700707 JFA-10 ciB-H11 B1 None Not done HQ700712 JFX-30 ciB-A11 B2 None Not done HQ700709 JFV-48 ciB-B5 B3 None Not done HQ700711 JFV-40 ciB-B9 B4 None Not done HQ700710 JEZ-2 ciA-H7 / B5 A1 / A3 Not done Strong JFK-21 ciA-F12 / D12 A4 / A5 Not done Not done JGA-3 ciB-A11 / B5 B2 / B3 Not done Not done In some embodiments, the binding region comprises the CDR sequences from nanobodies selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB- A11 / B5, or combinations thereof. Preferably, the binding region comprises three CDR sequences from at least one nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA- H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB- B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. In some embodiments, the binding region comprises a sequence having at least 70% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA- G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. For example, in some embodiments the binding region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to the full- length sequence of one or more nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5. Preferably, the binding region comprises the full-length sequence of one or more nanobody selected from the list comprising: ciA-H7, ciA- D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB- B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to any one of SEQ ID NO: 22 to 44; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NO: 22 to 44; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention is selected from any one of SEQ ID NO: 22 to 44. Further, a person skilled in the art would be able to determine whether an alternative single- chain antibody binds to a clostridial neurotoxin using routine experimental methods. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 22; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention comprises SEQ ID NO: 22. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 23; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 23; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention comprises SEQ ID NO: 23. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 24; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 24; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention comprises SEQ ID NO: 24. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 25; optionally wherein the antibody the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 25; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention comprises SEQ ID NO: 25. In some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 70% sequence identity to SEQ ID NO: 26; optionally wherein the antibody retains the binding specificity as shown in Table 1. For example, in some embodiments, a single-chain antibody of the invention comprises the three CDR sequences (e.g., CDR1, CDR2 and CDR3) and has at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 26; optionally wherein the antibody retains the binding specificity as shown in Table 1. Preferably, a single-chain antibody of the invention comprises SEQ ID NO: 26. In preferred embodiments, the antibodies (e.g., a VHH and VNAR derived from camelid or cartilaginous fish) are capable of neutralising clostridial neurotoxin toxicity. Suitable neutralising antibodies are known in the art. Further, a person skilled in the art would be able to determine whether an antibody is a neutralising antibody using routine experimental methods. In some embodiments, when the antibody is a multimer, only one of the antibodies may be neutralising. For example, a dimer may comprise a first neutralising antibody and a second non-neutralising antibody. In some embodiments, the potency of the antibodies may be enhanced by combining two or more antibodies (e.g., a neutralising antibody and a non- neutralising antibody, such as ciA-B5 and ciA-D12). In particularly preferred embodiments, the antibodies are selected from ciA-H7 (SEQ ID NO: 24 or 25), ciA-C2 (SEQ ID NO: 22 or 23), ciA-D12 (SEQ ID NO: 29), ciA-B5 (SEQ ID NO: 27) and multimers (e.g., dimers, trimers or tetramers) thereof. Of these, cia-H7 binds to BoNT / A light chain (L), ciA-B5 binds to the BoNT / A translocation domain (HN), and ciA-C2 binds to BoNT / A at a region between the HCCand HCNdomains. Alternatively, the antibody may be a dimer comprising ciA-B5 and ciA-D12 (e.g., ciA-B5-D12, SEQ ID NO: 26) wherein each single- chain antibody simultaneously binds to BoNT / A. Antibodies of the invention are not limited to a particular method of generation or production. Thus, the invention provides antibodies which have been manufactured from a hybridoma that secretes the antibody, as well as antibodies from a recombinantly produced cell that has been transformed or transfected with a nucleic acid or nucleic acids encoding the antibody. Preferably, the antibodies (e.g., single-chain antibodies such as camelid VHH or a camelid HcAbs and / or a cartilaginous fish VNAR or a cartilaginous fish IgNAR) are recombinantly produced. Such hybridomas, recombinantly produced cells, and nucleic acids form part of the invention. An antibody, or antigen-binding fragment thereof, of the invention is selective or specific for a clostridial neurotoxin polypeptide, or a particular epitope of a clostridial neurotoxin polypeptide as described herein. By specific, it will be understood that the antibody binds to the molecule of interest, in this case a clostridial neurotoxin polypeptide, with no significant cross-reactivity to non-clostridial neurotoxin polypeptides. In some embodiments, an antibody that is specific for a particular a clostridial neurotoxin epitope of the invention will show no significant cross- reactivity with other clostridial neurotoxin epitopes. For example, an antibody that is specific for BoNT / A heavy chain may show no significant cross-reactivity with BoNT / B heavy chain. Cross-reactivity may be assessed by any suitable method. Cross-reactivity of an antibody for a clostridial neurotoxin epitope with another a clostridial neurotoxin epitope or non-clostridial neurotoxin polypeptide may be considered significant if the antibody binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to the clostridial neurotoxin epitope. Binding affinity may be quantified in any suitable way, e.g. by KD. In some embodiments, the support is configured for immobilisation of binding substrates to said support. Immobilisation of the binding substrate on a support advantageously allows for convenient washing of any unbound complex and easy removal of supernatant while minimising loss of bound complexes. For example, immobilisation of a binding substrate on a support advantageously allows for convenient washing of any clostridial neurotoxin polypeptides (e.g., BoNT / A) that do not bind to the immobilised binding substrate and easy removal of supernatant while minimising loss of immobilised binding substrate-clostridial neurotoxin complexes. Immobilisation means are known in the art. In other embodiments, the support is configured for immobilisation of clostridial neurotoxin polypeptides to said support. Immobilisation of the clostridial neurotoxin polypeptides on a support advantageously allows for convenient washing of any unbound complex and easy removal of supernatant while minimising loss of bound complexes. For example, immobilisation of clostridial neurotoxin polypeptides on a support advantageously allows for convenient washing of any binding that do not bind to the immobilised clostridial neurotoxin polypeptides and easy removal of supernatant while minimising loss of immobilised clostridial neurotoxin-binding substrate complexes. Immobilisation means are known in the art. The support may take any number of forms. For example, any suitable surface for immobilising polypeptides (e.g., the binding substrate or clostridial neurotoxin) may be used. Non-limiting examples of suitable supports include beads, plates, columns, chips or reaction vessels. In preferred embodiments, the support is an amine reactive 2nd generation (AR2G) support. The skilled person will appreciate that AR2G supports comprise a high density of carboxylic acids that can be activated by reaction with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and s-NHS (N-hydroxysulfosuccinimide) to generate highly reactive NHS esters. The NHS esters rapidly react with the primary amines of biomolecules such as proteins and peptides to form highly stable amide bonds. The resulting immobilised biomolecules can be used for screening or kinetic experiments. Successful regeneration (e.g., using an acidic buffer, such as a buffer with a pH of from about 1 to about 2, preferably from about 1.0 to 1.7) will remove the analyte binding partner and leave the immobilised protein of interest intact on the biosensor surface. Thus, in some embodiments, the binding substrate is immobilised on an amine reactive 2nd generation (AR2G) support by the formation of stable amine bonds between the NHS esters on the support and primary amines in the binding substrate. Preferably, the binding substrate is immobilised on an amine reactive 2nd generation (AR2G) support. In some embodiments, the binding substrate is configured for binding to an AR2G support. Alternatively, the binding substrate is configured for binding to a support via a tag. In some embodiments, a tag, such as a GST tag, is present on the binding substrate and an appropriate binding partner (e.g. glutathione or a derivative thereof) present on the support. Alternatively, clostridial neurotoxin polypeptides may be immobilised on an amine reactive 2nd generation (AR2G) support by the formation of stable amine bonds between the NHS esters on the support and primary amines in the clostridial neurotoxin polypeptides. Preferably, the clostridial neurotoxin polypeptides are immobilised on an amine reactive 2nd generation (AR2G) support. In some embodiments, the AR2G support is configured for binding to clostridial neurotoxin polypeptides. Alternatively, the clostridial neurotoxin polypeptides are configured for binding to a support via a tag. In some embodiments, a tag, such as a GST tag, is present on the binding substrate and an binding partner (e.g. glutathione or a derivative thereof) present on the support. The biosensor of the invention may be used to monitor binding events and kinetics in real-time. Real-time monitoring facilitates the rapid optimisation and control of biological processes. In some embodiments, real-time monitoring may be either at-line, on-line or in-line. The biosensor of the invention may be used to monitor binding events and kinetics at-line. The biosensor of the invention may be used to monitor binding events and kinetics on-line. The biosensor of the invention may be used to monitor binding events and kinetics in-line. At-line measurement typically involve the removal of a sample, which is then analysed in close proximity to the production process, either manually or by using automated sampling devices. On-line measurement typically involves diverting a sample from the manufacturing process with a by-pass stream and may be returned to the bioreactor. On-line measurement enables constant monitoring and, therefore, control of a production process. Finally, in-line (or in-situ) measurements typically occur directly in the bioreactor with a process sensor. In contrast, an off-line measurement typically involves removing a sample from a bioreactor prior to analysing said sample. Often, the sample will require pretreatment (e.g., filtration and / or dilution) between the step of removal from the bioreactor and analysis. Together with the complexity involved in manual handling, a major disadvantage of off-line measurement is the time delay, which results in lower measurement frequency and possibly of lower product yields. A binding substrate of the invention may be produced by expressing a nucleic acid encoding said binding substrate in a suitable host cell. For example, the host cell may be a bacterial cell such as an E. coli cell (e.g., a DH5α cell or a BL21 cell) or bacteria in the genus Clostridia such as C. botulinum. The binding substrate may then be isolated from said host cell using standard techniques. Also described are nucleic acid molecules encoding the binding substrate of the invention. A nucleic acid encoding a binding substrate of the invention may encode: a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; and a polypeptide sequence having at 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most preferably, polypeptide sequence comprising SEQ ID NO: 46. A nucleic acid encoding a binding substrate of the invention may encode: a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22 or 23; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22 or 23; most preferably, polypeptide sequence comprising SEQ ID NO: 22 or 23; and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most preferably, polypeptide sequence comprising SEQ ID NO: 46. A nucleic acid encoding a binding substrate of the invention may encode: a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22 or 23, wherein the CDR sequences are as defined in SEQ ID NO: 23; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22 or 23, wherein the CDR sequences are as defined in SEQ ID NO: 23; most preferably, polypeptide sequence comprising SEQ ID NO: 22 or 23; and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most preferably, polypeptide sequence comprising SEQ ID NO: 46. A binding substrate of the invention may comprise: a polypeptide sequence having at 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most preferably, polypeptide sequence comprising SEQ ID NO: 46. A binding substrate of the invention may comprise: a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22 or 23; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22 or 23; most preferably, polypeptide sequence comprising SEQ ID NO: 22 or 23; and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most preferably, polypeptide sequence comprising SEQ ID NO: 46. A binding substrate of the invention may comprise: a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 45; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 45; most preferably, polypeptide sequence comprising SEQ ID NO: 45; a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 22 or 23, wherein the CDR sequences are as defined in SEQ ID NO: 23; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 22 or 23, wherein the CDR sequences are as defined in SEQ ID NO: 23; most preferably, polypeptide sequence comprising SEQ ID NO: 22 or 23; and a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 46; preferably a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 46; most polypeptide sequence comprising SEQ ID NO: 46. A method of the invention may be used to determine the concentration of clostridial neurotoxin polypeptides in a composition. In order to obtain said concentration, the method may be carried out using at least a second third and / or fourth composition comprising a different concentration of the clostridial neurotoxin polypeptides, and determining a resonance energy transfer value for the at least second, third, and / or fourth compositions. In some embodiments, the biosensor enables the detection of a clostridial neurotoxin at a concentration of less than 5ng / ml, less than 4 ng / ml, less than 3 ng / ml, less than 2 ng / ml, less than 1ng / ml, less than 0.9 ng / ml, less than 0.8 ng / ml, less than 0.7 ng / ml, less than 0.6 ng / ml, less than 0.5 ng / ml, less than 0.4 ng / ml, less than 0.3 ng / ml, less than 0.2 ng / ml, or less than 0.1 ng / ml. Preferably, the biosensor enables the detection of a clostridial neurotoxin at a concentration of less than 0.1ng / ml. In some instances, herein, “biosensors” are referred to. However, this is to indicate that more than one “biosensor” may be present when carrying out the method. It is not intended to necessarily indicate that two or more different types of biosensors are present, although this is encompassed. Thus, in some embodiments, the biosensors may be of one type (e.g. all the biosensors comprise a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a plurality of substantially identical binding substrates comprising: (i) a first donor fluorophore; (ii) a clostridial neurotoxin binding region; and (iii) an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore. In other embodiments, the biosensors may be of one type (e.g. all the biosensors comprise a surface for performing the analysis of binding events and kinetics, and onto which is immobilised clostridial neurotoxin polypeptides, and separately a plurality of substantially identical binding substrates comprising (i) a first donor fluorophore; (ii) a clostridial neurotoxin binding region; and (iii) an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore. Preferably, the biosensors are of one type. Similarly, in some instances herein, “binding substrates” are referred to. However, this is to indicate that more than one “binding substrate” may be present when carrying out the method. It is not intended to necessarily indicate that two or more different types of binding substrates are present, although this is encompassed. Thus, the binding substrates may be of one type (e.g. all the binding substrates are substantially identical and comprise: (i) a first donor fluorophore; (ii) a clostridial neurotoxin region; and (iii) an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore. Preferably, the binding substrates are of one type. In one aspect, the present invention is directed to a method for real-time (e.g., in-line, at-line or on-line) monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention is directed to a method for in-line monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention is directed to a method for at-line monitoring of clostridial neurotoxin production during a bacterial fermentation process. In some embodiments, the present invention is directed to a method for on-line monitoring of clostridial neurotoxin production during a bacterial fermentation process. The method of the invention may further comprise the step of culturing a host cell capable of producing a clostridial neurotoxin. Preferably, the host cell is a bacterial host cell (e.g., E. coli or bacteria in the genus Clostridia such as C. botulinum). In some embodiments, a nucleic acid sequence encoding the clostridial neurotoxin is modified for codon-biasing according to the ultimate host cell (e.g. E. coli or bacteria in the genus Clostridia such as C. botulinum) expression system that is to be employed. Preferably, the clostridial neurotoxin is produced by culturing a bacterial host cell (e.g., E. coli or bacteria in the genus Clostridia such as C. botulinum) capable of producing a clostridial neurotoxin in a liquid medium. In some embodiments, the method for real-time monitoring of clostridial neurotoxin production comprises the step of contacting the medium (e.g., liquid medium) with a biosensor of the invention. In some embodiments, the In some embodiments, the clostridial neurotoxin is secreted into the fermentation solution. In other embodiments, wherein the clostridial neurotoxin is not secreted into the fermentation solution, the method may further comprise a step of releasing the clostridial neurotoxins from host cells. Means for releasing the clostridial neurotoxins from host cells are known in the art, and include both mechanical and non-mechanical means of cell disruption or lysis. Also provided is a method for real-time (e.g., in-line, at-line or on-line) monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding of the invention. As such, the binding substrate is expressed in the host cell. The method of the invention may further comprise the step of culturing the transformed host cell. Preferably, the transformed host cell is a bacterial host cell (e.g., E. coli or bacteria in the genus Clostridia such as C. botulinum). In some embodiments, a nucleic acid sequence encoding the clostridial neurotoxin is modified for codon-biasing according to the transformed host cell (e.g. E. coli or bacteria in the genus Clostridia such as C. botulinum) expression system that is to be employed. The methods of the invention further comprise the step of exciting a donor fluorophore in said biosensor. In some embodiments, the exciting of the donor fluorophore requires the donor fluorophore to be exposed to radiation having a wavelength selected from the donor excitation radiation range. In some embodiments, the excitation wavelength is specifically chosen to avoid directly exciting the acceptor fluorophore and thereby reducing cross-talk and background signal. The methods of the invention may further comprise the step of measuring a property of the biosensors. In preferred embodiments, the method comprises the step of determining resonance energy transfer at said biosensor. As indicated above, this may be determined by measuring donor and / or acceptor fluorescence intensity. Preferably, this may be determined by measuring acceptor fluorescence intensity. Detection of a clostridial neurotoxin in a sample is performed by measuring emissions from the donor fluorophore and the acceptor using a fluorescence analysis system. The emissions from the fluorescence donor and the fluorescence acceptor are measured with a fluorescence analysis system or the like, and if a change in the amount or concentration of a clostridial neurotoxin occurs, a change in the emissions from the fluorescence donor and the fluorescence acceptors occurs. Thus, the present invention can be used to measure a change in the clostridial neurotoxin amount or concentration. FRET may be measured by various different means, each of which will be known to the skilled person. Exemplary, non-limiting means of measuring FRET include sensitized emission, acceptor photobleaching, fluorescent lifetime imaging microscopy FRET (FLIM FRET), fluorophore donor spectral imaging and homo-FRET and polarization anisotropy imaging. For example, sensitized emission typically involves exciting a donor fluorophore and collecting the signal from both the donor fluorophore and acceptor fluorophore using specifically selected emission filters. Using this method, the acceptor fluorescence increases in the presence of donor, whereas the donor fluorescence decreases in the presence of the acceptor. The ratiometric change of the fluorescence intensity can then be used to measure FRET. Alternatively, acceptor photobleaching is a technique based on the quenching of donor fluorescence during FRET when some of the donor fluorescence energy is transferred to the acceptor. In FLIM FRET, the donor fluorescence is quenched by the FRET interaction, and the amount of quenching can be calculated by measuring the decrease in fluorescence decay time of the donor molecule. Spectral imaging involves recording the entire emission spectrum of both donor and acceptor fluorescence upon excitation of the donor. Spectral imaging is based on the principle that overlapping spectra can be separated not just by their emission peaks but also their distinct overall shapes. Finally, like regular FRET, homo-FRET involves the transfer of excited-state energy between fluorophores. However, the selected fluorophores in homo- FRET are identical. Preferably, determining the relative resonance energy transfer at said biosensor comprises the step of comparing the resonance energy transfer to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. In some embodiments, a suitable control may be a negative control (e.g., a composition that does not comprise a clostridial neurotoxin polypeptide). The negative control may be a negative reference standard. The negative reference standard may correspond to a value that has been either theoretically or experimentally determined and which represents a negative result in a method of the invention. The value may have been determined prior to carrying out a method of the invention or may be determined simultaneously with, or subsequent to, carrying out a method of the invention. In some embodiments (e.g., when the control is a negative control), increased donor fluorescence intensity at said biosensor as compared to said control may be indicative of the presence of clostridial neurotoxin. In contrast, decreased donor fluorescence intensity at said biosensor as compared to said control may allow for a determination to be made that a composition comprises no (or undetectable amounts of) clostridial neurotoxin polypeptides. In some embodiments (e.g., when the control is a negative control), reduced acceptor fluorescence intensity at said biosensor as compared to said control may be indicative of the presence of clostridial neurotoxin. In when the control is a negative control, the same level of acceptor fluorescence intensity when compared to the negative control indicates that a composition tested does not comprise clostridial neurotoxin polypeptides. In such instances, this may allow for a determination to made that a composition does not comprise clostridial neurotoxin polypeptides. In some embodiments, a suitable control may be a positive control (e.g., a composition that comprises a known or pre-determined concentration of clostridial neurotoxin polypeptides (e.g., a clostridial neurotoxin polypeptide that is capable of binding to the binding region). In some embodiments (e.g., when the control is a positive control), increased donor fluorescence intensity at said biosensor as compared to said control may be indicative of higher amounts or concentration of clostridial neurotoxin. In contrast, decreased donor fluorescence intensity at said biosensor as compared to said control may be allow for a determination to be made that a composition comprises lower amounts or concentrations of clostridial neurotoxin polypeptides. In some embodiments, when the control is a positive control, reduced acceptor fluorescence intensity at said biosensor as compared to said control may be indicative of the higher amounts or concentration of clostridial neurotoxin. In some embodiments, when the control is a positive control, the same level of acceptor fluorescence intensity when compared to the positive control may allow for a determination to made that a composition tested comprises a known or pre-determined concentration of a clostridial neurotoxin polypeptides. In some embodiments, the method of the invention comprises the step of comparing the donor and / or acceptor fluorescence intensity of a test composition relative to the donor and / or acceptor fluorescence intensity of a control composition. The extent of the difference may be quantified to indicate the number of clostridial neurotoxin polypeptides. The extent of the difference may be quantified to indicate the concentration of clostridial neurotoxin polypeptides. For example, preferably the higher the donor fluorescence compared to a negative control, the higher the number of clostridial neurotoxin polypeptides. Preferably, the higher the donor fluorescence compared to a negative control, the higher the concentration of clostridial neurotoxin polypeptides. Alternatively, preferably, the lower the acceptor fluorescence compared to the negative control, the higher the number of clostridial neurotoxin polypeptides. Preferably, the the acceptor fluorescence compared to the negative control, the higher the concentration of clostridial neurotoxin polypeptides. In some embodiments, the step of determining resonance energy transfer at said biosensor (e.g., relative to a control) comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, optionally wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin. In some embodiments, the step of determining resonance energy transfer at said biosensor (e.g., relative to a control) comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, optionally wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin. The term “different” (and associated terms such as “change”, “changed”, and “difference”, and synonyms thereof) as used herein may mean a difference that is substantially different to a comparator (e.g. a control as described herein). A difference (and associated terms such as “change”, “changed”, and “difference”, and synonyms thereof) may mean a statistically- significant difference when compared to a comparator (e.g. a control as described herein). A “substantial difference” may be a difference of at least 5%, 10%, 15%, 20%, 25% or 30% when compared to a comparator (e.g. a control as described herein). The term “no difference” (and associated terms such as “unchanged” and “the same”, and synonyms thereof) may mean that there is no substantial difference when compared to a comparator (e.g. a control as described herein). No difference (and associated terms such as “unchanged” and “the same”, and synonyms thereof) may mean that there is no statistically-significant difference when compared to a comparator (e.g. a control as described herein). The term “lower” (and associated terms such as “less than”) as used herein may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% lower when compared to a comparator (e.g. a control as described herein). The term “lower” (and associated terms such as “less than”) may mean statistically- significantly lower when compared to a comparator (e.g. a control as described herein). The term “higher” (and associated terms such as “higher than”) as used herein may mean at least 10%, 25%, 20%, 50%, 75%, 100%, 150%, or 200% higher when compared to a comparator (e.g. a control as described herein). The term “higher” (and associated terms such as “higher than”) may mean statistically-significantly higher when compared to a comparator (e.g. a control as described herein). In some instances herein, “clostridial neurotoxin polypeptides” and “clostridial neurotoxin HCCdomains” (respectively) are referred to. However, this is to indicate that more than one clostridial neurotoxin polypeptide or clostridial neurotoxin HCCdomain (respectively) may be present (e.g. in a composition) when carrying out the method. It is not intended to necessarily indicate that two or more different types of clostridial neurotoxin polypeptides or clostridial neurotoxin HCCdomains (respectively) are present, although this is encompassed. Thus, the clostridial neurotoxin polypeptides or clostridial neurotoxin HCCdomains (respectively) may be of one type (e.g. all the clostridial neurotoxin polypeptides are BoNT / A polypeptides or all the clostridial neurotoxin HCCdomains are BoNT / A HCCdomains [respectively]) or multiple types (e.g. a portion of the clostridial neurotoxin polypeptides are BoNT / B polypeptides and a portion are BoNT / A polypeptides or a portion of the clostridial neurotoxin HCCdomains are BoNT / A HCCdomains and a portion are BoNT / B HCCdomains [respectively]). Preferably, the clostridial neurotoxin polypeptides are of one type. Preferably, the clostridial neurotoxin HCCdomains are of one type. The above applies analogously to some instances herein where “botulinum neurotoxin polypeptides” are referred to. A clostridial neurotoxin according to the invention may comprise a botulinum neurotoxin or a tetanus neurotoxin (TeNT) HCCdomain. A clostridial neurotoxin of the invention may comprise a BoNT / A HCCdomain, a BoNT / B HCCdomain, a BoNT / C1 HCCdomain, a BoNT / D HCCdomain, a BoNT / E HCCdomain, a BoNT / F HCCdomain, a BoNT / G HCCdomain, a BoNT / X HCCdomain, or TeNT HCCdomain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / B HCCdomain or a BoNT / A HCCdomain, more preferably a BoNT / B HCCdomain. A clostridial neurotoxin according to the invention may comprise a botulinum neurotoxin or a tetanus neurotoxin (TeNT) HCdomain. A clostridial neurotoxin of the invention may comprise a BoNT / A HCdomain, a BoNT / B HCdomain, a BoNT / C1 HCdomain, a BoNT / D HCdomain, a BoNT / E HCdomain, a BoNT / F HCdomain, a BoNT / G HCdomain, a BoNT / X HCdomain, or TeNT HCdomain. Preferably, a clostridial neurotoxin of the invention comprises a BoNT / B HCdomain or a BoNT / A HCdomain, more preferably a BoNT / B HCdomain. The term “clostridial neurotoxin” embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F). A reference BoNT / A sequence is shown as SEQ ID NO: 1. A reference BoNT / B sequence is shown as SEQ ID NO: 2. A reference BoNT / C1 (also referred to as BoNT / C herein) sequence is shown as SEQ ID NO: 3. A reference sequence is shown as SEQ ID NO: 4. A reference BoNT / E sequence is shown as SEQ ID NO: 5. A reference BoNT / F sequence is shown as SEQ ID NO: 6. A reference BoNT / G sequence is shown as SEQ ID NO: 7. A reference BoNT / X sequence is shown as SEQ ID NO: 8. A reference TeNT sequence is shown as SEQ ID NO: 9. The term “clostridial neurotoxin” may also embrace newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has closest sequence identity to BoNT / X, the Weissella oryzae encoded toxin called BoNT / Wo (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90, the Enterococcus faecium encoded toxin (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero encoded toxin (NCBI Ref.Seq: WP_034687872.1). Thus, a clostridial neurotoxin may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Thus, a composition of the invention may comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, or TeNT. Therefore, the clostridial neurotoxin polypeptides may be BoNT / A polypeptides, BoNT / B polypeptides, BoNT / C polypeptides, BoNT / D polypeptides, BoNT / E polypeptides, BoNT / F polypeptides, BoNT / G polypeptides, BoNT / X polypeptides, or TeNT polypeptides. Preferably, a clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. Thus, a composition of the invention may comprise BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X. Therefore, the clostridial neurotoxin polypeptides may be BoNT / A polypeptides, BoNT / B polypeptides, BoNT / C polypeptides, BoNT / D polypeptides, BoNT / E polypeptides, BoNT / F polypeptides, BoNT / G polypeptides, or BoNT / X polypeptides. Clostridial neurotoxins are formed from two polypeptide chains, the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises a C-terminal targeting component (receptor binding domain or HCdomain) and an N-terminal translocation component (HNdomain). Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself complexed to a number of accessory proteins. There are at present eight different classes of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity. BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A and BoNT / E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT / C1 cleaves syntaxin. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1. Tetanus toxin is produced in a single serotype by C. tetani. C. butyricum produces BoNT / E, while C. baratii produces BoNT / F. Examples of L-chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440 Botulinum type C1 neurotoxin: amino acid residues 1-441 Botulinum type D neurotoxin: amino acid residues 1-445 Botulinum type E neurotoxin: amino acid residues 1-422 Botulinum type F neurotoxin: amino acid residues 1-439 Botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457 For recently-identified BoNT / X, the L-chain has been reported as corresponding to amino acids 1-439 thereof, with the L-chain boundary potentially varying by approximately 25 amino acids (e.g.1-414 or 1-464). A BoNT / A L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-448 of SEQ ID NO: 1. A BoNT / B L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-440 of SEQ ID NO: 2. A BoNT / C1 L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-441 of SEQ ID NO: 3. A BoNT / D L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-445 of SEQ ID NO: 4. A BoNT / E L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-422 of SEQ ID NO: A BoNT / F L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-439 of SEQ ID NO: 6. A BoNT / G L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1- 441 of SEQ ID NO: 7. A BoNT / X L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-439 of SEQ ID NO: 8. A TeNT L-chain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1-457 of SEQ ID NO: 9. The above-identified reference sequences should be considered a guide, as slight variations may occur according to sub-serotypes. By way of example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different clostridial sequences: Botulinum type A neurotoxin: amino acid residues M1-K448 Botulinum type B neurotoxin: amino acid residues M1-K441 Botulinum type C1 neurotoxin: amino acid residues M1-K449 Botulinum type D neurotoxin: amino acid residues M1-R445 Botulinum type E neurotoxin: amino acid residues M1-R422 Botulinum type F neurotoxin: amino acid residues M1-K439 Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457 The translocation domain is a fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. In one embodiment the HCfunction of the H-chain may be removed by deletion of the HCamino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the HCfunction may be inactivated by chemical or biological treatment. Thus, in some embodiments the H-chain may be incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds. Examples of suitable (reference) Translocation Domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879) A BoNT / A HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 449-871 of SEQ ID NO: 1. A BoNT / B HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 441-858 of SEQ ID NO: 2. A BoNT / C1 HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-866 of SEQ ID NO: 3. A BoNT / D HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 446-862 of SEQ ID NO: 4. A BoNT / E HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 423-845 of SEQ ID NO: 5. A BoNT / F HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 440-864 of SEQ ID NO: 6. A BoNT / G HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 442-863 of SEQ ID NO: 7. A BoNT / X HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 440-892 of SEQ ID NO: 8. A TeNT HNdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 458-879 of SEQ ID NO: 9. The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007 / 0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences: Botulinum type A neurotoxin - amino acid residues (A449-K871) Botulinum type B neurotoxin - amino acid residues (A442-S858) Botulinum type C neurotoxin - amino acid residues (T450-N866) Botulinum type D neurotoxin - amino acid residues (D446-N862) Botulinum type E neurotoxin - amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440-K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - acid residues (S458-V879) In the context of the present invention, a variety of clostridial neurotoxin HNregions comprising a translocation domain can be useful in aspects of the present invention. The HNregions from the heavy chains of clostridial neurotoxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a HNregion from a clostridial neurotoxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial neurotoxin HNregions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial neurotoxin HNregions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids. For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press. The term HNembraces naturally-occurring neurotoxin HNportions, and modified HNportions having amino acid sequences that do not occur in nature and / or synthetic amino acid residues. In one embodiment said modified HNportions still demonstrate the above-mentioned translocation function. Examples of clostridial neurotoxin receptor binding domain (HC) reference sequences include: BoNT / A - N872-L1296 BoNT / B - E859-E1291 BoNT / C1 - N867-E1291 BoNT / D - S863-E1276 BoNT / E - R846-K1252 BoNT / F - K865-E1274 BoNT / G - N864-E1297 TeNT - I880-D1315 For recently-identified BoNT / X, the HChas been reported as corresponding to amino acids 893-1306 thereof, with the domain boundary potentially varying by approximately 25 amino acids (e.g.868-1306 or 918-1306). A BoNT / A HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 872-1296 of SEQ ID NO: 1. A BoNT / B HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 859-1291 of SEQ ID NO: 2. A BoNT / C1 HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 867-1291 of SEQ ID NO: 3. A BoNT / D HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 863-1276 of SEQ ID NO: 4. A BoNT / E HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 846-1252 of SEQ ID NO: 5. A BoNT / F HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 865-1274 of SEQ ID NO: 6. A BoNT / G HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 864-1297 of SEQ ID NO: 7. A BoNT / X HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 893-1306 of SEQ ID NO: 8. A TeNT HCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 880-1315 of SEQ ID NO: 9. A clostridial neurotoxin H-chain (e.g. the HCdomain portion) may further comprise a translocation facilitating domain (or a fragment thereof may be translocation facilitating domain fragment). Said domain facilitates delivery of the L-chain into the cytosol of the target cell and are described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is herein incorporated by reference thereto. By way of example, a translocation facilitating domain may comprise a clostridial neurotoxin HCNdomain or a fragment or variant thereof. In more detail, a clostridial neurotoxin HCNtranslocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a clostridial neurotoxin HCNtranslocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127) The above sequence positions may vary a little according to serotype / sub-type, and further examples of suitable (reference) clostridial neurotoxin HCNdomains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861-1097) Botulinum type C neurotoxin - amino acid residues (869-1111) Botulinum type D neurotoxin - amino acid residues (865-1098) Botulinum type E neurotoxin - amino acid residues (848-1085) Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127) Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with a non- clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCNtranslocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCNfacilitating domain may be combined with a clostridial translocation domain peptide, examples of which include: Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum type F neurotoxin - acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127) The HCpeptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25kDa each, namely the N-terminal region (commonly referred to as the HCNpeptide or domain) and the C- terminal region (commonly referred to as the HCCpeptide or domain). This fact is confirmed by the following publications, each of which is herein incorporated in its entirety by reference thereto: Umland TC (1997) Nat. Struct. Biol.4: 788-792; Herreros J (2000) Biochem. J.347: 199-204; Halpern J (1993) J. Biol. Chem.268: 15, pp.11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol.5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol.7: 1751- 1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Moreover, it has been well documented that the C-terminal region (HCC), which constitutes the C-terminal 160-200 amino acid residues, is responsible for binding of a clostridial neurotoxin to its natural cell receptors, namely to nerve terminals at the neuromuscular junction - this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy-chain lacking a functional heavy chain HCpeptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional HCCpeptide. In other words, the HCCpeptide region may be either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to reduce its native binding ability for nerve terminals at the neuromuscular junction. HCCreference sequences are presented below: Botulinum type A neurotoxin - amino acid residues (Y1111-L1296) Botulinum type B neurotoxin - amino acid residues (Y1098-E1291) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315). The above-identified reference sequences be considered a guide as slight variations may occur according to sub-serotypes. A BoNT / A HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1111-1296 of SEQ ID NO: 1. A BoNT / B HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1098-1291 of SEQ ID NO: 2. A BoNT / C1 HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1112-1291 of SEQ ID NO: 3. A BoNT / D HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1099-1276 of SEQ ID NO: 4. A BoNT / E HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1086-1252 of SEQ ID NO: 5. A BoNT / F HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1274 of SEQ ID NO: 6. A BoNT / G HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1106-1297 of SEQ ID NO: 7. A TeNT HCCdomain may comprise a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% to amino acid residues 1128-1315 of SEQ ID NO: 9. The term “clostridial neurotoxin” is also intended to embrace modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. By way of 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 toxin, for example biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative. A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified HCdomain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial neurotoxin. Such modifications in the HCdomain can include modifying residues in the ganglioside binding site of the HCin the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and / or the protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are hereby incorporated by reference in their entirety. Thus, a BoNT / A HCCdomain is preferably a modified BoNT / A HCCdomain, more preferably a modified BoNT / A HCdomain. Therefore, preferably, a clostridial neurotoxin in accordance with the present invention is a modified BoNT / A. Preferably, said modified clostridial neurotoxin comprises one or more modifications that increases the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications. Suitable modified clostridial neurotoxins are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 10-13 (preferably SEQ ID NO: 10 – mrBoNT / A) described herein. A modified BoNT / A may be one that comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. Such a modified BoNT / A may demonstrate a reduction in, or absence of, side effects compared to the use of known BoNT / A. Said modified BoNT / A may exhibit increased tissue retention properties, thereby providing increased potency and / or duration of action and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional adverse effects), thus providing further advantages. The modification may be a modification when compared to a BoNT / A shown as SEQ ID NO: 1, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 1. As the presence of a methionine residue at position 1 of SEQ ID NO: 1 (as well as the SEQ ID NOs corresponding to modified BoNT / A polypeptides described herein) is optional, the skilled person will take the presence / absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 1 includes a methionine, the position numbering will be as defined above (e.g. ASN 886 will be ASN 886 of SEQ ID NO: 1). Alternatively, where the methionine is absent from SEQ ID NO: 1 the amino acid residue numbering should be modified by -1 (e.g. ASN 886 will be ASN 885 of SEQ ID NO: 1). Similar considerations apply when methionine at position 1 of the other polypeptide sequences described herein is present / absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art. An alignment described herein for determining amino acid residue numbering may be carried out using any of the methods described herein for determining sequence homology and / or % sequence identity. The amino acid residue(s) indicated for modification above are surface exposed amino acid residue(s). A modified BoNT / A may comprise a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277. The term “one or more amino acid residue(s)” when used in the context of a modified BoNT / A preferably means at least 2, 3, 4, 5, 6 or 7 of the indicated amino acid residue(s). Thus, a modified BoNT / A may comprise at least 2, 3, 4, 5, 6 or 7 (preferably 7) modifications at the indicated amino acid residue(s). A modified BoNT / A may comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, the term “one or more amino acid residue(s)” when used in the context of a modified BoNT / A means all of the indicated amino acid residue(s). Preferably, beyond the one or more amino acid modification(s) at the indicated amino acid residue(s), the modified BoNT / A does not contain any further amino acid modifications when compared to SEQ ID NO: 1. The modification may be selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue. A modification as indicated above results in a modified BoNT / A that has an increased positive surface charge and increased isoelectric point when compared to the corresponding unmodified BoNT / A (e.g. SEQ ID NO: 1). Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and cell surface thus increasing retention at a site of administration and / or duration of action. The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made from a specific sequence of amino acids (also referred to when in a protein as amino acid residues). Each amino acid of the standard set of twenty has a different side chain (or R group), meaning that each amino acid residue in a protein displays different chemical properties such as charge and hydrophobicity. These properties may be influenced by the surrounding chemical environment, such as the temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors. Certain amino acid residues (detailed below) possess ionisable side chains that may display an electric charge depending on the surrounding pH. Whether such a side chain is charged or not at a given pH depends on the pKa of the relevant ionisable moiety, wherein pKa is the negative logarithm of the acid dissociation constant (Ka) for a specified proton from a conjugate base. For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values of approximately 4.1 (precise pKa values may depend on temperature, ionic strength and the microenvironment of the ionisable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as “physiological pH”). At low pH values, these side chains will become protonated and lose their charge. Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values of approximately 10-12. These side chains therefore exhibit a positive charge at a pH of 7.4. These side chains will become de-protonated and lose their charge at high pH values. The overall (net) charge of a protein molecule therefore depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the changes the overall charge on the protein. Accordingly, for every protein there is a given pH at which the number of positive and negative charges is equal and the protein displays no overall net charge. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry with which the skilled person would be familiar. The isoelectric point (pI) is therefore defined as the pH value at which a protein displays a net charge of zero. An increase in pI means that a higher pH value is required for the protein to display a net charge of zero. Thus, an increase in pI represents an increase in the net positive charge of a protein at a given pH. Conversely, a decrease in pI means that a lower pH value is required for the protein to display a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of a protein at a given pH. Methods of determining the pI of a protein are known in the art and would be familiar to a skilled person. By way of example, the pI of a protein can be calculated from the average pKa values of each amino acid present in the protein (“calculated pI”). Such calculations can be performed using computer programs known in the art, such as the Compute pI / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is the preferred method for calculating pI in accordance with the present invention. Comparisons of pI values between different molecules should be made using the same calculation technique / program. Where appropriate, the calculated pI of a protein can be confirmed experimentally using the technique of isoelectric focusing (“observed pI”). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel that has an immobilised pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pI of the protein. Results provided by isoelectric focusing are typically relatively low- resolution in nature, and thus the present inventors believe that results provided by calculated pI (as described above) are more appropriate to use. Throughout the present specification, “pI” means “calculated pI” unless otherwise stated. The pI of a protein may be increased or decreased by altering the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI may be provided by reducing the number of acidic residues, or by increasing the number of basic residues. A modified BoNT / A of the invention may have a pI value that is at least 0.2, 0.4, 0.5 or 1 pI units higher than that of BoNT / A (e.g. SEQ ID NO: 1). Preferably, a modified BoNT / A may have a pI of at least 6.6, e.g. at least 6.8. The properties of the 20 standard amino acids are indicated in the table below: Amino Acid Side Chain Aspartic acid Asp D Charged (acidic) Glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (basic) Lysine Lys K Charged (basic) Histidine His H Uncharged (polar) Asparagine Asn N Uncharged (polar) Glutamine Gln Q Uncharged (polar) Serine Ser S Uncharged (polar) Threonine Thr T Uncharged (polar) Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar) Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar) Alanine Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged (hydrophobic) Valine Val V Uncharged (hydrophobic) Leucine Leu L Uncharged (hydrophobic) Isoleucine Ile I Uncharged (hydrophobic) Proline Pro P Uncharged (hydrophobic) Phenylalanine Phe F Uncharged (hydrophobic) The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive). At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues. The following amino acids are considered polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan. The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine. In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into the BoNT / A polypeptide sequence, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from the clostridial toxin amino acid sequence, thus reducing the total number of amino acid residues in said sequence. Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard amino acids, as described above. Alternatively, the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above). By way of example, the replacement amino acid may be a basic non-standard amino acid, e.g. L-Ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts. In one embodiment, the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, wherein the substitution is a substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine). Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is substitution with lysine or arginine. Most preferably, the modification is substitution with lysine. Following modification in accordance with the invention, the modified BoNT / A is preferably capable of binding to the target cell receptors that unmodified BoNT / A (e.g. SEQ ID NO: 1) binds. Preferably, a modified BoNT / A for use in the invention comprises between 4 and 40 amino acid modifications located in the clostridial toxin HCNdomain. Said modified BoNT / A preferably also has pI of at least 6.6. Said modified BoNT / A preferably comprises modifications of at least 4 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue. For example, said modified BoNT / A may comprise modifications of at least 5 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue. Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of a DNA sequence encoding a polypeptide (e.g. encoding unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular cloning techniques, for example by site-directed mutagenesis where short strands of DNA (oligonucleotides) coding for the desired amino acid(s) are used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, a modified gene sequence can be chemically synthesised. A modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 10-13. In one embodiment, a modified BoNT / A may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 10-13. Preferably, a modified BoNT / A may comprise any one of SEQ ID NOs: 10-13. A modified BoNT / A may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 10-13. In one embodiment, a modified BoNT / A may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 10-13. Preferably, a modified BoNT / A may consist of any one of SEQ ID NOs: 10-13. Of the recited SEQ ID NOs, SEQ ID NO: 10 is most preferred. The skilled person will appreciate that where the polypeptide sequence of a modified varies compared to a given SEQ ID NO by way of % sequence identity, that the at least one of the modifications (e.g. that increase pI) are still present (e.g. unmodified) in the variant modified BoNT / A. Thus, a composition of the invention preferably comprises a modified BoNT / A, such as a modified BoNT / A as described above. In some embodiments, the clostridial neurotoxin polypeptides are modified BoNT / A polypeptides, the L-chain is a BoNT / A L-chain, the HNdomain is a BoNT / A HNdomain, and the HCCdomain (e.g. HCdomain) is a modified BoNT / A HCCdomain (e.g. a modified BoNT / A HCCdomain). A modified BoNT / A may comprise a substitution at one or more (preferably two or more, three or more, four or more, five or more or six or more, more preferably at all) of positions 930, 955, 991, 1026, 1052, 1229, and 886. Preferably, a modified BoNT / A comprises lysine or arginine (more preferably lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the modified BoNT / A comprises lysine or arginine (more preferably lysine) at least two, three, four, five, six or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the modified BoNT / A comprises lysine or arginine (more preferably lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886. A clostridial neurotoxin may comprise (or consist of) a hybrid or chimeric clostridial neurotoxin. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment the hybrid clostridial neurotoxin may comprise the entire light chain of a light chain from one clostridial neurotoxin subtype and the heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial neurotoxin subtype, with another portion of the heavy chain being from another clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to subjects who are immunologically resistant to a given clostridial neurotoxin subtype, to subjects who may have a lower than average concentration of receptors to a given clostridial neurotoxin heavy chain binding domain, or to subjects who may have a protease-resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in US which publication is hereby incorporated by reference in its entirety. A clostridial neurotoxin of the invention may be one comprising a BoNT / B HCCdomain, preferably a chimeric clostridial neurotoxin comprising a BoNT / B HCdomain. Thus, in a particularly preferred embodiment, a clostridial neurotoxin of the invention may be a chimeric clostridial neurotoxin comprising (preferably consisting of) a BoNT / A light-chain and translocation domain (LHNdomain), and a BoNT / B receptor binding domain (HCdomain). Most preferably, said BoNT / B HCdomain comprises the following substitutions E1191M and S1199Y. A suitable chimeric clostridial neurotoxin may be one taught in WO 2017 / 191315 A1, which is incorporated herein by reference. Such preferred sequences include SEQ ID NOs: 14-18, with SEQ ID NO: 14 being most preferred. The BoNT / A LHNdomain may be covalently linked to the BoNT / B HCdomain. Said chimeric BoNT / A is also referred to herein as “BoNT / AB” or a “BoNT / AB chimera”. The C-terminal amino acid residue of the LHNdomain may correspond to the first amino acid residue of the 310helix separating the LHNand HCdomains of BoNT / A, and the N-terminal amino acid residue of the HCdomain may correspond to the second amino acid residue of the 310helix separating the LHNand HCdomains in BoNT / B. Reference herein to the “first amino acid residue of the 310helix separating the LHNand HCdomains of BoNT / A” means the N-terminal residue of the 310helix separating the LHNand HCdomains. Reference herein to the “second amino acid residue of the 310helix separating the LHNand HCdomains of BoNT / B” means the amino acid residue following the N-terminal residue of the 310helix separating the LHNand HCdomains. A “310helix” is a type of secondary structure found in proteins and polypeptides, along with α- helices, β-sheets and reverse turns. The amino acids in a 310helix are arranged in a right- handed helical structure where each full turn is completed by three residues and ten atoms that separate the intramolecular hydrogen bond between them. Each amino acid corresponds to a 120° turn in the helix (i.e., the helix has three residues per turn), and a translation of 2.0 Å (= 0.2 nm) along the helical axis, and has 10 atoms in the ring formed by making the hydrogen bond. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C = O group of the amino acid three residues this repeated i + 3 → i hydrogen bonding defines a 310helix. A 310helix is a standard concept in structural biology with which the skilled person is familiar. This 310helix corresponds to four residues which form the actual helix and two cap (or transitional) residues, one at each end of these four residues. The term “310helix separating the LHNand HCdomains” as used herein consists of those 6 residues. Through carrying out structural analyses and sequence alignments, a 310helix separating the LHNand HCdomains was identified. This 310helix is surrounded by an α-helix at its N-terminus (i.e. at the C-terminal part of the LHNdomain) and by a β-strand at its C-terminus (i.e. at the N-terminal part of the HCdomain). The first (N-terminal) residue (cap or transitional residue) of the 310helix also corresponds to the C-terminal residue of this α-helix. The 310helix separating the LHNand HCdomains can be for example determined from publicly available crystal structures of botulinum neurotoxins, for example 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1 respectively. In silico modelling and alignment tools which are publicly available can also be used to determine the location of the 310helix separating the LHNand HCdomains in other neurotoxins, for example the homology modelling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and a number of other tools / services listed at the Internet Resources for Molecular and Cell Biologists (http: / / molbiol- tools.ca / ). In particular that the region around the “HN / HCN” junction is structurally highly conserved which renders it an ideal region to superimpose different serotypes. For example, the following methodology may be used to determine the sequence of this 310helix in other neurotoxins: 1. The structural homology modelling tool LOOP (http: / / loopp.org) was used to obtain a predicted structure of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. The structural (pdb) files thus were edited to include only the N-terminal end of the HCNdomain and about 80 residues before it (which are part of the HNdomain), thereby retaining the “HN / HCN” region which is structurally highly conserved; 3. The protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) was used to superpose each serotype onto the 3BTA.pdb structure; 4. The superposed pdb files were inspected to locate the 310helix at the start of the HCdomain of BoNT / A1, and corresponding residues in the other serotype were then identified; 5. The other BoNT serotype sequences were aligned with Clustal Omega in order to check that corresponding residues were correct. Examples of LHN, HCand 310helix domains determined by this method are presented below: Accession Number (Plus Sequence Neurotoxin LHNHC310helix Version after Decimal) BoNT / A1 (SEQ ID N / A 1-872 873-1296872NIINTS877NO: 1) BoNT / A2 X73423.3 1-872 873-1296872NIVNTS877DQ185900.1 (aka BoNT / A3 1-872 873-1292872NIVNTS877Q3LRX9.1) EU341307.1 (aka BoNT / A4 1-872 873-1296872NITNAS877Q3LRX8.1) EU679004.1 (aka BoNT / A5 1-872 873-1296872NIINTS877C1IPK2.1) BoNT / A6 FJ981696.1 1-872 873-1296872NIINTS877JQ954969.1 (aka BoNT / A7 1-872 873-1296872NIINTS877K4LN57.1) BoNT / A8 KM233166.1 1-872 873-1297872NITNTS877BoNT / B1 (a.k.a. SEQ B1INP5.1 1-859 860-1291859EILNNI864ID NO: 2) Accession (Plus Sequence Neurotoxin LHNHC310helix Version after Decimal) AB084152.1 (aka BoNT / B2 1-859 860-1291859EILNNI864Q8GR96.1) EF028400.1 (aka BoNT / B3 1-859 860-1291859EILNNI864A2I2S2.1) EF051570.1 (aka BoNT / B4 1-859 860-1291859EILNNI864A2I2W0.1) EF033130.1 (aka BoNT / B5 1-859 860-1291859DILNNI864A2I2U6.1) AB302852.1 (aka BoNT / B6 1-859 860-1291859EILNNI864A8R089.1) JQ354985.1 (aka BoNT / B7 1-859 860-1291859EILNNI864H9CNK9.1) JQ964806.1 (aka BoNT / B8 1-859 860-1292859EILNNI864I6Z8G9.1) Using structural analysis and sequence alignments, it was found that the β-strand following the 310helix separating the LHNand HCdomains is a conserved structure in all botulinum and tetanus neurotoxins and starts at the 8thresidue when starting from the first residue of the 310helix separating the LHNand HCdomains (e.g., at residue 879 for BoNT / A1). A BoNT / AB chimera may comprise an LHNdomain from BoNT / A covalently linked to a HCdomain from BoNT / B, • wherein the C-terminal amino acid residue of the LHNdomain corresponds to the eighth amino acid residue N-terminally to the β-strand located at the beginning (N-term) of the HCdomain of BoNT / A, and • wherein the N-terminal amino acid residue of the HCdomain corresponds to the seventh amino acid residue N-terminally to the β-strand located at the beginning (N- term) of the HCdomain of BoNT / B. A BoNT / AB chimera may comprise an LHNdomain from BoNT / A covalently linked to a HCdomain from BoNT / B, • wherein the C-terminal amino acid of the LHNdomain corresponds to the C- terminal amino acid residue of the α-helix located at the end (C-term) of LHNdomain of BoNT / A, and • wherein the N-terminal amino acid residue of the HCdomain corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-term) of LHNdomain of BoNT / B. The rationale of the design process of the BoNT / AB chimera was to try to ensure that the secondary structure was not compromised and thereby minimise any changes to the tertiary structure. Without wishing to be bound by theory, it is hypothesized that by not disrupting the four central amino acid residues of the 310helix in the BoNT / AB chimera ensures an optimal conformation for the chimeric neurotoxin. The LHNdomain from BoNT / A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 1, or a polypeptide sequence having at least 70% sequence identity thereto. The LHNdomain from BoNT / A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 1, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the LHNdomain from BoNT / A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 1. The HCdomain from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a polypeptide sequence having at least 70% sequence identity thereto. The HCdomain from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the HCdomain from BoNT / B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 2. Preferably, the LHNdomain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 1) and the HCdomain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 1). Preferably, a BoNT / B HCdomain further comprises at least one amino acid residue substitution, addition or deletion in the HCCdomain (e.g. subdomain) which has the effect of increasing the binding affinity of BoNT / B neurotoxin for human Syt II as compared to the natural BoNT / B sequence. Suitable amino acid residue substitution, addition or deletion in the BoNT / B HCCdomain have been disclosed in WO 2013 / 180799 and in WO 2016 / 154534 (both herein incorporated by reference). Suitable amino acid residue substitution, addition or deletion in the BoNT / B HCCdomain include 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 combinations thereof. Suitable amino acid residue substitution, addition or deletion in the BoNT / B HCCdomain further include combinations of two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q. Suitable amino acid residue substitution, addition or deletion in the BoNT / B HCCdomain also include a combination of three substitution mutations which are E1191M, S1199W and W1178Q. Preferably, the suitable amino acid residue substitution, addition or deletion in the BoNT / B HCCdomain includes a combination of two substitution mutations which are E1191M and S1199Y. The modification may be a modification when compared to unmodified BoNT / B shown as SEQ ID NO: 2, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 2. As the presence of a methionine residue at position 1 of SEQ ID NO: 2 is optional, the skilled person will take the presence / absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 2 includes a methionine, the position numbering will be as defined above (e.g. E1191 will be E1191 of SEQ ID NO: 2). Alternatively, where the methionine is absent from SEQ ID NO: 2 the amino acid residue numbering should be modified by -1 (e.g. E1191 will be E1190 of SEQ ID NO: 2). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present / absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art. A chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-18. In one embodiment, a chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of NOs: 14-18. Preferably, a chimeric clostridial neurotoxin may comprise any one of SEQ ID NOs: 14-18. A chimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 14-18. In one embodiment, a chimeric clostridial neurotoxin may consist of a polypeptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to any one of SEQ ID NOs: 14-18. Preferably, a chimeric clostridial neurotoxin may consist of any one of SEQ ID NOs: 14-18. Of the recited SEQ ID NOs, SEQ ID NO: 14 is most preferred. The skilled person will appreciate that where the polypeptide sequence of a chimeric clostridial neurotoxin comprising at least one BoNT / B HCCdomain mutation varies compared to a given SEQ ID NO by way of % sequence identity, that the at least one BoNT / B HCCdomain mutations (preferably E1191M and S1199Y) are present (e.g. are unmodified) in the variant chimeric clostridial neurotoxin. Thus, a composition of the invention most preferably comprises a chimeric clostridial neurotoxin, such as a chimeric clostridial neurotoxin as described above. In some embodiments, the clostridial neurotoxin polypeptides are chimeric clostridial neurotoxin polypeptides, the L-chain is a BoNT / A L-chain, the HNdomain is a BoNT / A HNdomain, and the HCCdomain (e.g. HCdomain) is a BoNT / B HCCdomain (e.g. a BoNT / B HCCdomain). In another embodiment, a clostridial neurotoxin of the invention may be a chimeric clostridial neurotoxin comprising a BoNT / X light-chain and translocation domain (LHNdomain), and a receptor binding domain (HCdomain) or a portion thereof from a different (i.e. non-BoNT / X) clostridial neurotoxin. A suitable chimeric and / or hybrid clostridial neurotoxin may be one taught in WO 2020 / 065336 A1, which is incorporated herein by reference. In embodiments where a clostridial neurotoxin described herein has a tag for purification (e.g. a His-tag) and / or a linker, said tag and / or linker are optional. The clostridial neurotoxins of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial neurotoxin complex. The clostridial neurotoxins of the present invention can be produced using recombinant nucleic acid technologies. Thus, in one embodiment, a clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin. In one embodiment a nucleic acid (for a DNA) comprising a nucleic acid sequence encoding a clostridial neurotoxin is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence may be selected from any of the nucleic acid sequences described herein. In a preferred embodiment, the vector has a promoter selected from: Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM) In another preferred embodiment, the vector has a promoter selected from: Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM) T5-lac operator IPTG 0.2 mM (0.05-2.0mM) The nucleic acid molecules may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques. The DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques. The above-mentioned nucleic acid sequence information is optionally modified for codon- biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed. The terms “nucleotide sequence” and “nucleic acid” are used synonymously herein. Preferably the nucleotide sequence is a DNA sequence. A clostridial neurotoxin of the invention is preferably present as a di-chain clostridial neurotoxin in which the L-chain is linked to the H-chain (or component thereof, e.g. the HNdomain) via a di-sulphide bond. Thus, a clostridial of the invention may be any clostridial neurotoxin or variant (expressed by way of % sequence identity to a given SEQ ID NO) herein that has been cleaved by a protease in its activation loop (at one or more sites). A clostridial neurotoxin preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 10 with a protease in its activation loop at one more sites. In one embodiment, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 10 with a protease in its activation loop at one more sites. Preferably, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 10 with a protease in its activation loop at one more sites. A clostridial neurotoxin most preferably comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 14 with a protease in its activation loop at one more sites. In one embodiment, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising at least 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to SEQ ID NO: 14 with a protease in its activation loop at one more sites. Preferably, a clostridial neurotoxin comprises an L-chain and H-chain, wherein the L-chain and H-chain are joined by a di-sulphide bond and are obtainable by (e.g. obtained by) cleaving a polypeptide comprising SEQ ID NO: 14 with a protease in its activation loop at one more sites. The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and method for cleaving activation loops to produce di-chain clostridial neurotoxins are taught in WO 2014 / 080206, WO2014 / 079495, and EP2677029A2, which are incorporated herein by reference. Suitable activation loop sequences are shown in the table below:Protein id aaaa startend BoNT / D_AB012112 437 450 BoNT / DC_AB745660 437 450 BoNT / C1_X62389 437 453 BoNT / CD_AB200360 437 453 BoNT / A4_EU341307 430 454 BoNT / A7_JQ954969 454 BoNT / A6_FJ981696 454 BoNT / A1_AF488749 454 BoNT / A5_EU679004 430 454 BoNT / A3_DQ185900 426 450 BoNT / A2_X73423 454 BoNT / A8_KM233166 430 454 BoNT / H_KGO15617 428 438 BoNT / E9_JX424534 414 429 BoNT / E12_KM370319 414 429 BoNT / E11_KF861875 411 426 BoNT / E10_KF861917 411 426 BoNT / E7_JN695729 411 426 BoNT / E8_JN695730 411 426 BoNT / E5_AB037711 411 426 BoNT / E6_AM695759 411 426 BoNT / E4_AB088207 411 426 BoNT / E3_EF028403 411 426 BoNT / E1_GQ244314 411 426 BoNT / E2_EF028404 411 426 BoNT / F7_GU213233 420 434 BoNT / F5_GU213211 428 442 BoNT / F1_GU213203 429 445 BoNT / F4_GU213214 429 445 BoNT / F2_GU213209 429 445 BoNT / F3_GU213227 429 BoNT / F6_M92906 445 TeNT_P04958 467 BoNT / G_X74162 450 BoNT / B4_EF051570 437 446 BoNT / B8_JQ964806 437 446 BoNT / B7_JQ354985 437 446 BoNT / B6_AB302852 437 446 BoNT / B2_AB084152 437 446 BoNT / B3_EF028400 437 446 Lys-C may cleave an activation loop C- to one or more of the lysine residues present therein. Where Lys-C cleaves the activation loop more than once, the skilled person will appreciate that a small peptide of the activation loop of a di-chain clostridial neurotoxin may be absent when compared to a SEQ ID NO shown herein. The invention provides a method of producing a single-chain clostridial neurotoxin having a light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain clostridial neurotoxin, and isolating the single-chain clostridial neurotoxin. In one aspect, the present invention provides a method of proteolytically processing a clostridial neurotoxin described herein, the method comprising contacting the clostridial neurotoxin with a protease that hydrolyses a peptide bond in the activation loop of the clostridial neurotoxin, thereby converting the (single-chain) clostridial neurotoxin into a corresponding di-chain clostridial neurotoxin (e.g. wherein the light chain and heavy chain are joined together by a disulphide bond). The present invention therefore provides a di-chain clostridial neurotoxin obtainable by a method of the invention. In one aspect, the invention provides a method for producing a therapeutic or cosmetic clostridial neurotoxin composition, the method comprising: (a) obtaining the results of the method according to the invention, and formulating and / or packaging the composition for therapeutic or cosmetic use when the clostridial neurotoxin concentration is the same as or higher than a positive control; or (b) subjecting the composition to further fermentation when the clostridial neurotoxin concentration is lower than a positive control, and formulating and / or packaging the further purified composition for therapeutic or cosmetic use. A composition of the invention may comprise a first clostridial neurotoxin formulation comprising one or more pharmaceutically acceptable carrier(s), excipient(s), adjuvant(s), propellant(s), and / or salt(s). In one aspect, there is provided a therapeutic or cosmetic clostridial neurotoxin composition obtainable by a method of the invention, optionally wherein the therapeutic or cosmetic clostridial neurotoxin composition is packaged. The term “obtainable” as used herein also the term “obtained”. In one aspect, the invention provides an isolated binding substrate of the present invention. In some embodiments, the isolated binding substrate is bound to a clostridial neurotoxin polypeptide. In one aspect, the invention provides an isolated complex comprising a clostridial neurotoxin bound to a binding substrate. In some embodiments, the binding substrate is immobilised to a support (e.g., a surface for performing the analysis of binding events and kinetics). In other embodiments, clostridial neurotoxin polypeptides are immobilised to a support (e.g., a surface for performing the analysis of binding events and kinetics). The isolated binding substrate may be complexed with a clostridial neurotoxin, wherein the clostridial neurotoxin is bound to the binding region. An isolated binding substrate may refer to a binding substrate that has been isolated from a cell. Such a binding substrate may have been produced recombinantly and isolated using standard techniques. Thus, in some embodiments, the term “isolated binding substrate” is intended to encompass binding substrates in an in vitro environment. Preferably, an isolated binding substrate is immobilised on a support as described herein for binding substrates generally. In one aspect, the invention provides the use of an isolated binding substrate for a clostridial neurotoxin for determining the presence or absence of clostridial neurotoxin polypeptides comprised in a composition, wherein the isolated binding substrate comprises a binding region. In one aspect, the invention provides a method for producing a therapeutic or cosmetic clostridial neurotoxin composition, the method comprising: (a) obtaining the results of a method according to the invention; and (b) formulating and / or packaging the composition for therapeutic or cosmetic use when the composition comprises clostridial neurotoxin polypeptides, optionally wherein the composition comprises at least a pre-determined concentration of clostridial neurotoxin polypeptides; or (c) subjecting the composition to further fermentation when it is determined that there are no clostridial neurotoxin polypeptides in the composition or when the concentration of clostridial neurotoxins is below a pre-determined value; and (d) formulating and / or further purified composition for therapeutic or cosmetic use. In some embodiments, the formulating may comprise the selection of preferred pharmaceutically acceptable carrier(s), excipient(s), adjuvant(s), propellant(s), and / or salt(s) for formulating a clostridial neurotoxin. In one embodiment, where the term “obtaining the result” or “obtaining the results” of a method or assay is recited herein, the method carried out to obtain said result(s) may be carried out as part of a method of the invention. In one aspect, the invention provides a kit comprising: (a) an isolated binding substrate; (b) a surface for performing the analysis of binding events and kinetics; and (b) optionally means for detecting binding of a botulinum neurotoxin to the binding substrate; and / or (c) optionally instructions for the use of the same. In one aspect, the invention provides a kit comprising: (a) an isolated binding substrate; and (b) optionally means for detecting binding of a botulinum neurotoxin to the binding substrate; and / or (c) optionally instructions for the use of the same. Embodiments related to the various methods of the invention are intended to be applied equally to alternative methods, products, and / or uses, and vice versa. SEQUENCE HOMOLOGY Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol.823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004). Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio.48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes); preferably this method is used to align a sequence with a SEQ ID NO described herein to define amino acid position numbering, as described herein. The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

[0002] ALIGNMENT SCORES FOR SEQUENCE IDENTITY A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -211 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 The percent identity is then calculated as: Total number of identical matches __________________________________________ x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker of up to about 20-25 residues, or an affinity tag. CONSERVATIVE AMINO ACID SUBSTITUTIONS Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and α -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo- threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non- occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol.202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem.33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention. Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett.309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention. Multiple amino acid substitutions can made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem.30:10832-7, 1991; Ladner et al., U.S. Patent No.5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of this disclosure. Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a clostridial neurotoxin” includes a plurality of such candidate agents and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. SEQUENCE LISTING Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional. SEQ ID NO: 1 - Polypeptide Sequence of BoNT / A (rBoNT / A) SEQ ID NO: 2 - Polypeptide Sequence of BoNT / B SEQ ID NO: 3 - Polypeptide Sequence of BoNT / C SEQ ID NO: 4 - Polypeptide Sequence of BoNT / D SEQ ID NO: 5 - Polypeptide Sequence of BoNT / E SEQ ID NO: 6 - Polypeptide Sequence of BoNT / F SEQ ID NO: 7 - Polypeptide Sequence of BoNT / G SEQ ID NO: 8 - Polypeptide Sequence of BoNT / X SEQ ID NO: 9 – Polypeptide Sequence of TeNT SEQ ID NO: 10 - Polypeptide Sequence of mrBoNT / A SEQ ID NO: 11 - Polypeptide Sequence of Cationic BoNT / A Variant 2 SEQ ID NO: 12 - Polypeptide Sequence of Cationic BoNT / A Variant 3 SEQ ID NO: 13 - Polypeptide Sequence of Cationic BoNT / A Variant 4 SEQ ID NO: 14 - Polypeptide Sequence of mrBoNT / AB SEQ ID NO: 15 - Polypeptide Sequence of BoNT / AB Variant 2 SEQ ID NO: 16 - Polypeptide Sequence of BoNT / AB Variant 3 SEQ ID NO: 17 - Polypeptide Sequence of BoNT / AB Variant 4 SEQ ID NO: 18 - Polypeptide Sequence of BoNT / AB Variant 5 SEQ ID NO: 19 – Polypeptide Sequence of Full-Length Human SV2a SEQ ID NO: 20 – Polypeptide Sequence of Full-Length Human SV2b SEQ ID NO: 21 – Polypeptide Sequence of Full-Length Human SV2c SEQ ID NO: 22 – Polypeptide sequence of Cia-C2 SEQ ID NO: 23 – Polypeptide sequence of alternative Cia-C2 (Genbank: HQ700705, AEJ91546.1) SEQ ID NO: 24 – Polypeptide sequence of CiaH7 SEQ ID NO: 25 – Polypeptide sequence of CiaH7 (Genbank: HQ700708, AEJ91549.1) SEQ ID NO: 26 – Polypeptide sequence of CiaD12 / B5 SEQ ID NO: 27 – Polypeptide sequence of ciA-B5 (Genbank: HQ700704, AEJ91545.1) SEQ ID NO: 28 – Polypeptide sequence of ciA-F12 (Genbank: HQ700706, AEJ91547.1) SEQ ID NO: 29 – Polypeptide sequence of ciA-D12 (Genbank: HQ700702, AEJ91543.1) SEQ ID NO: 30 – Polypeptide sequence of ciA-A5 (Genbank: HQ700703, AEJ91544.1) SEQ ID NO: 31 – Polypeptide sequence of ciA-G5 (Genbank: HQ700707, AEJ91548.1) SEQ ID NO: 32 – Polypeptide sequence of ciB-H11 (Genbank: HQ700712, AEJ91553.1) SEQ ID NO: 33 – Polypeptide sequence of ciB-A11 (Genbank: HQ700709, AEJ91550.1) SEQ ID NO: 34 – Polypeptide sequence of ciB-B5 (Genbank: HQ700711, AEJ91552.1) SEQ ID NO: 35 – Polypeptide sequence of ciB-B9 (Genbank: HQ700710, AEJ91551.1) SEQ ID NO: 36 – Polypeptide sequence of B5 (Genbank: HQ700704, AEJ91545.1) SEQ ID NO: 37 – Polypeptide sequence of ciA-F12 (Genbank: HQ700706, AEJ91547.1) SEQ ID NO: 38 – Polypeptide sequence of ciA-D12 (Genbank: HQ700702, AEJ91543.1) SEQ ID NO: 39 – Polypeptide sequence of ciA-A5 (Genbank: HQ700703, AEJ91544.1) SEQ ID NO: 40 – Polypeptide sequence of ciA-G5 (Genbank: HQ700707, AEJ91548.1) SEQ ID NO: 41 – Polypeptide sequence of ciB-H11 (Genbank: HQ700712, AEJ91553.1) SEQ ID NO: 42 – Polypeptide sequence of ciB-A11 (Genbank: HQ700709, AEJ91550.1) SEQ ID NO: 43 – Polypeptide sequence of ciB-B5 (Genbank: HQ700711, AEJ91552.1) SEQ ID NO: 44 – Polypeptide sequence of ciB-B9 (Genbank: HQ700710, AEJ91551.1) SEQ ID NO: 45 – Polypeptide sequence of mClover3 SEQ ID NO: 46 – Polypeptide sequence of mRuby3 SEQ ID NO: 1 - Polypeptide Sequence of Native BoNT / A (rBoNT / A) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWK VSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHAS NNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDV NNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSS RTLGCSWEFIPVDDGWGERPL SEQ ID NO: 2 - Polypeptide Sequence of BoNT / B MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE SEQ ID NO: 3 - Polypeptide Sequence of BoNT / C MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA LRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRK DINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN VDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLM WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNIN KFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSF QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNN DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYA IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYR HNYLVPTVKQGNYASLLESTSTHWGFVPVSE SEQ ID NO: 4 - Polypeptide Sequence of BoNT / D MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE VVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFP EFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIR ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS TSSFWKFISRDPGWVE SEQ ID NO: 5 - Polypeptide Sequence of BoNT / E MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK SEQ ID NO: 6 - Polypeptide Sequence of BoNT / F MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN EHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY DPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKF KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERF ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS SNGCFWSFISKEHGWQEN SEQ ID NO: 7 - Polypeptide Sequence of MPVNIKNFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSK DVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANV SINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFN NVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTF GGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVD KDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEF NGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAET IAYNTQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFE YLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQ KSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNK GHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSE EDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYID TNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSS GYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTY LQNEYTIISCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSS TNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKA SNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEK TTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKLRLGCNWQFIPVD EGWTE SEQ ID NO: 8 - Polypeptide Sequence of BoNT / X MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRD QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED SEQ ID NO: 9 – Polypeptide Sequence of TeNT MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFN PPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGN KNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLH GLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYK AIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTE IELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNT NAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAE KNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAP EYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVI SKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGN FIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYK LVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKN KLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIG ITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDIS GFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPK VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP DKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNN NQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDV QLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYV SYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDD KNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND SEQ ID NO: 10 - Polypeptide Sequence of mrBoNT / A MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWK VSLNYGEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDV NNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSS RTLGCSWEFIPVDDGWGERPL SEQ ID NO: 11 - Polypeptide Sequence of Cationic BoNT / A Variant 2 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFKKISLNNEYTIINCMENNSGWK VSLNYGEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSS RTLGCSWEFIPVDDGWGERPL SEQ ID NO: 12 - Polypeptide Sequence of Cationic BoNT / A Variant 3 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWK VSLNYGEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLKKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDV NNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSS RTLGCSWEFIPVDDGWGERPL SEQ ID NO: 13 - Polypeptide Sequence of Cationic BoNT / A Variant 4 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV LGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWK VSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHAS NNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDV NNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKRKEYRLATNASQA GVEKILSALEIPRVRRLSQVVVMKSKNDQGITNKCKMNLQDRRGNDIGFIGFHQFNNIAKLVASNWYNRQIERRS RRLGCSWEFIPVDDGWGERPL SEQ ID NO: 14 - Polypeptide Sequence of mrBoNT / AB MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDS TYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFK INIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPV YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYD NRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGW KISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIAN GEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKL KKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKW YLKEVKRKPYNLKLGCNWQFIPKDEGWTE SEQ ID NO: 15 - Polypeptide Sequence of BoNT / AB Variant 2 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH SEQ ID NO: 16 - Polypeptide Sequence of BoNT / AB Variant 3 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH SEQ ID NO: 17 - Polypeptide Sequence of BoNT / AB Variant 4 STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH SEQ ID NO: 18 - Polypeptide Sequence of BoNT / AB Variant 5 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE SEQ ID NO: 19 – Polypeptide Sequence of Full-Length Human SV2a MEEGFRDRAA YSRRSYSRFE EEDDDDDFPA 60 PSDGYYRGEG IPRAESGGKG ERMADGAPLA 120 GVRGGLSDGE GPPGGRGEAQ RRKEREELAQ QYEAILRECG HGRFQWTLYF VLGLALMADG 180 VEVFVVGFVL PSAEKDMCLS DSNKGMLGLI VYLGMMVGAF LWGGLADRLG RRQCLLISLS 240 VNSVFAFFSS FVQGYGTFLF CRLLSGVGIG GSIPIVFSYF SEFLAQEKRG EHLSWLCMFW 300 MIGGVYAAAM AWAIIPHYGW SFQMGSAYQF HSWRVFVLVC AFPSVFAIGA LTTQPESPRF 360 FLENGKHDEA WMVLKQVHDT NMRAKGHPER VFSVTHIKTI HQEDELIEIQ SDTGTWYQRW 420 GVRALSLGGQ VWGNFLSCFG PEYRRITLMM YYGLTVWFPD MIRHLQAVDY 480 ASRTKVFPGE RVEHVTFNFT LENQIHRGGQ YFNDKFIGLR LKSVSFEDSL FEECYFEDVT 540 SSNTFFRNCT FINTVFYNTD LFEYKFVNSR LINSTFLHNK EGCPLDVTGT GEGAYMVYFV 600 SFLGTLAVLP GNIVSALLMD KIGRLRMLAG SSVMSCVSCF FLSFGNSESA MIALLCLFGG 660 VSIASWNALD VLTVELYPSD KRTTAFGFLN ALCKLAAVLG ISIFTSFVGI TKAAPILFAS 720 AALALGSSLA LKLPETRGQV LQ 742 SEQ ID NO: 20 – Polypeptide Sequence of Full-Length Human SV2b MDDYKYQDNY GGYAPSDGYY RGNESNPEED AQSDVTEGHD EEDEIYEGEY QGIPHPDDVK 60 AKQAKMAPSR MDSLRGQTDL MAERLEDEEQ LAHQYETIMD ECGHGRFQWI LFFVLGLALM 120 ADGVEVFVVS FALPSAEKDM CLSSSKKGML GMIVYLGMMA GAFILGGLAD KLGRKRVLSM 180 SLAVNASFAS LSSFVQGYGA FLFCRLISGI GIGGALPIVF AYFSEFLSRE KRGEHLSWLG 240 IFWMTGGLYA SAMAWSIIPH YGWGFSMGTN YHFHSWRVFV IVCALPCTVS MVALKFMPES 300 PRFLLEMGKH DEAWMILKQV HDTNMRAKGT PEKVFTVSNI KTPKQMDEFI EIQSSTGTWY 360 QRWLVRFKTI FKQVWDNALY CVMGPYRMNT LILAVVWFAM AFSYYGLTVW FPDMIRYFQD 420 EEYKSKMKVF FGEHVYGATI NFTMENQIHQ HGKLVNDKFT RMYFKHVLFE DTFFDECYFE 480 DVTSTDTYFK NCTIESTIFY NTDLYEHKFI NCRFINSTFL EQKEGCHMDL EQDNDFLIYL 540 VSFLGSLSVL PGNIISALLM DRIGRLKMIG GSMLISAVCC FFLFFGNSES AMIGWQCLFC 600 GTSIAAWNAL DVITVELYPT NQRATAFGIL NGLCKFGAIL GNTIFASFVG ITKVVPILLA 660 AASLVGGGLI ALRLPETREQ VLM 683 SEQ ID NO: 21 – Polypeptide Sequence of Full-Length Human SV2c MEDSYKDRTS NQAVDRAQDE YTQRSYSRFQ DEEDDDDYYP 60 AGETYNGEAN GEYQGIPSMN QAKDSIVSVG QPKGDEYKDR 120 RELESERRAD EEELAQQYEL IIQECGHGRF QWALFFVLGM ALMADGVEVF VVGFVLPSAE 180 TDLCIPNSGS GWLGSIVYLG MMVGAFFWGG LADKVGRKQS LLICMSVNGF FAFLSSFVQG 240 YGFFLFCRLL SGFGIGGAIP TVFSYFAEVL AREKRGEHLS WLCMFWMIGG IYASAMAWAI 300 IPHYGWSFSM GSAYQFHSWR VFVIVCALPC VSSVVALTFM PESPRFLLEV GKHDEAWMIL 360 KLIHDTNMRA RGQPEKVFTV NKIKTPKQID ELIEIESDTG TWYRRCFVRI RTELYGIWLT 420 FMRCFNYPVR DNTIKLTIVW FTLSFGYYGL SVWFPDVIKP LQSDEYALLT RNVERDKYAN 480 FTINFTMENQ IHTGMEYDNG RFIGVKFKSV TFKDSVFKSC TFEDVTSVNT YFKNCTFIDT 540 VFDNTDFEPY KFIDSEFKNC SFFHNKTGCQ ITFDDDYSAY WIYFVNFLGT LAVLPGNIVS 600 ALLMDRIGRL TMLGGSMVLS GISCFFLWFG TSESMMIGML CLYNGLTISA WNSLDVVTVE 660 LYPTDRRATG FGFLNALCKA AAVLGNLIFG SLVSITKSIP ILLASTVLVC GGLVGLCLPD 720 TRTQVLM 727 SEQ ID NO: 22 – Polypeptide sequence of Cia-C2 QVQLVESGGG LVQVGGSLRL SCVVSGSDIS GIAMGWYRQA PGKRREMVAD IFSGGSTDYA 60 GSVKGRFTIS RDNAKKTSYL QMNNVKPEDT GVYYCRLYGS GDYWGQGTQV TVSS 114 SEQ ID NO: 23 – Polypeptide sequence of alternative Cia-C2 (Genbank: HQ700705, AEJ91546.1) QVQLVESGGG LAQPGGSLRL SCEASGFGTW FRFDENTVNW YRQPPGKSRE FDELVARYPK 60 SGIVTYLDSV KGRFTISRDN AKKMAFLQMD NLKPEDTAVY YCNVGEFWGQ GTQVTISSEP 120 KTPKPQ 126 Bold = CDR1 Underlined = CDR2 Italics = CDR3 SEQ ID NO: 24 – Polypeptide sequence of CiaH7 QVQLVESGGG LVQVGGSLRL SCVVSGSDIS GIAMGWYRQA PGKRREMVAD IFSGGSTDYA 60 P 121 SEQ ID NO: 26 – Polypeptide sequence of CiaD12 / B5 QVQLVESGGG LVQPGGSLRL SCVVSGSDFN TYIMGWYRQV PGKPRELVAD ITTEGKTNYG 60 GSVKGRFTIS RDNAKNTVYL QMFGLKPEDA GNYVCNADWK MGAWTAGDYG IDYWGKGTLV 120 TVSSGGGGSG GGGSGQVQLV ESGGGLVHPG GSLRLSCAPS ASLPSTPFNP FNNMVGWYRQ 180 APGKQREMVA SIGLRINYAD SVKGRFTISR DNAKNTVDLQ MDSLRPEDSA TYYCHIEYTH 240 YWGKGTLVTV SS 252 SEQ ID NO: 27 – Polypeptide sequence of ciA-B5 (Genbank: HQ700704, AEJ91545.1) QVQLVESGGG LVHPGGSLRL SCAPSASLPS TPFNPFNNMV GWYRQAPGKQ REMVASIGLR 60 INYADSVKGR FTISRDNAKN TVDLQMDSLR PEDSATYYCH IEYTHYWGKG TLVTVSSEPK 120 TPKPQ 125 SEQ ID NO: 28 – Polypeptide sequence of ciA-F12 (Genbank: HQ700706, AEJ91547.1) QVQLVESGGG LVQPGGSLRL SCAASGFTLG SRYMSWVRQA PGEGFEWVSS IEPSGTAWDG 60 DSAKGRFTTS RDDAKNTLYL QMSNLQPEDT GVYYCATGYR TDTRIPGGSW GQGTQVTVSS 120 EPKTPKPQ 128 SEQ ID NO: 29 – Polypeptide sequence of ciA-D12 (Genbank: HQ700702, AEJ91543.1) QVQLVESGGG LVQPGGSLRL SCVVSGSDFN TYIMGWYRQV PGKPRELVAD ITTEGKTNYG 60 GSVKGRFTIS RDNAKNTVYL QMFGLKPEDA GNYVCNADWK MGAWTAGDYG IDYWGKGTLV 120 TVSSGPKTPK PQ 132 SEQ ID NO: 30 – Polypeptide sequence of ciA-A5 (Genbank: HQ700703, AEJ91544.1) QLQLVESGGG YYAIGWFRQA PGKEREGVSC ISSSDGSTVY 60 TDSVKGRFTI TAVYYCATVV NYYCTAGGSI HASPYEIWGQ 120 GTQVTVSSAH HSEDP 135 SEQ ID NO: 31 – Polypeptide sequence of ciA-G5 (Genbank: HQ700707, AEJ91548.1) QVQLVESGGG DWVMSWFRQA PGKEREFVAS ITATSSLKYY 60 ADSVKGRFTI TAVYYCRSPN YWGQGTQVTV SAEPKTPKPQ 120 SEQ ID NO: 32 – Polypeptide sequence of ciB-H11 (Genbank: HQ700712, AEJ91553.1) QVQLVETGGG LVQAGGSLRL SCAGSGRSFS AAVMGWFRQA PGKEREFVAA LRQIIGSTHY 60 ADSVKGRFTI SRDNAKNMLY LDMNSLKPTD TAAYYCTAGP PTMLDVSTDR EYDTWGQGTQ 120 VTVSSAHHSE DPS 133 SEQ ID NO: 33 – Polypeptide sequence of ciB-A11 (Genbank: HQ700709, AEJ91550.1) QVQLVETGGG LVQPGGALRL SCAASVFGMD YYYIGWVRQA PGKEREGVSC ISNIGRTHYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT AVYYCAAAPL VGNYCPASYE YESWGQGTQV 120 TVSSAHHSED PS 132 GTQVTVSSAH HSEDPS 136 SEQ ID NO: 35 – Polypeptide sequence of ciB-B9 (Genbank: HQ700710, AEJ91551.1) QVQLVETGGG FYALAWFRQG PGKEREFVAA IGWIDGSTRY 60 TDSAKGRFTI TAVYSCTART QYGGSSADPK NYGYWGQGTQ 120 VTVSAEPKTP KPQ 133 SEQ ID NO: 36 – Polypeptide sequence of ciA-B5 (Genbank: HQ700704, AEJ91545.1) QVQLVESGGG TPFNPFNNMV GWYRQAPGKQ REMVASIGLR 60 INYADSVKGR PEDSATYYCH IEYTHYWGKG TLVTVSSEPK 120 TPKPQ 125 SEQ ID NO: 37 – Polypeptide sequence of ciA-F12 (Genbank: HQ700706, AEJ91547.1) QVQLVESGGG LVQPGGSLRL SCAASGFTLG SRYMSWVRQA PGEGFEWVSS IEPSGTAWDG 60 DSAKGRFTTS RDDAKNTLYL QMSNLQPEDT GVYYCATGYR TDTRIPGGSW GQGTQVTVSS 120 EPKTPKPQ 128 SEQ ID NO: 38 – Polypeptide sequence of ciA-D12 (Genbank: HQ700702, AEJ91543.1) QVQLVESGGG TYIMGWYRQV PGKPRELVAD ITTEGKTNYG GSVKGRFTIS GNYVCNADWK MGAWTAGDYG IDYWGKGTLV TVSSGPKTPK PQ 132 SEQ ID NO: 39 – Polypeptide sequence of ciA-A5 (Genbank: HQ700703, AEJ91544.1) QLQLVESGGG YYAIGWFRQA PGKEREGVSC ISSSDGSTVY 60 TDSVKGRFTI TAVYYCATVV NYYCTAGGSI HASPYEIWGQ 120 GTQVTVSSAH HSEDP 135 SEQ ID NO: 40 – Polypeptide sequence of ciA-G5 (Genbank: HQ700707, AEJ91548.1) QVQLVESGGG DWVMSWFRQA PGKEREFVAS ITATSSLKYY 60 ADSVKGRFTI TAVYYCRSPN YWGQGTQVTV SAEPKTPKPQ 120 SEQ ID NO: 41 – Polypeptide sequence of ciB-H11 (Genbank: HQ700712, AEJ91553.1) QVQLVETGGG AAVMGWFRQA PGKEREFVAA LRQIIGSTHY ADSVKGRFTI TAAYYCTAGP PTMLDVSTDR EYDTWGQGTQ VTVSSAHHSE DPS 133 SEQ ID NO: 42 – Polypeptide sequence of ciB-A11 (Genbank: HQ700709, AEJ91550.1) QVQLVETGGG YYYIGWVRQA PGKEREGVSC ISNIGRTHYA DSVKGRFTIS AVYYCAAAPL VGNYCPASYE YESWGQGTQV TVSSAHHSED PS 132 SEQ ID NO: 43 – Polypeptide sequence of ciB-B5 (Genbank: HQ700711, AEJ91552.1) QLQLVESGGG NYIIGWFRQA PGKEREGVSC IDRTGTVTHY 60 ADSVKGRFTI STDNVKNTVY LEMNDLKPED RWGVVSVCVI SDYYFDSWGQ 120 GTQVTVSSAH HSEDPS 136 SEQ ID NO: 44 – Polypeptide sequence of ciB-B9 (Genbank: HQ700710, AEJ91551.1) QVQLVETGGG LVQAGGSLRL SCTASGRTSS FYALAWFRQG PGKEREFVAA IGWIDGSTRY 60 TDSAKGRFTI SRDAAKNTMY LQMNSLKPED TAVYSCTART QYGGSSADPK NYGYWGQGTQ 120 VTVSAEPKTP KPQ 133 SEQ ID NO: 45 – Polypeptide sequence of mClover3 MVSKGEELFT GVVPILVELD GDVNGHKFSV RGEGEGDATN GKLTLKFICT TGKLPVPWPT 60 LVTTFGYGVA CFSRYPDHMK QHDFFKSAMP EGYVQERTIS FKDDGTYKTR AEVKFEGDTL 120 VNRIELKGID FKEDGNILGH KLEYNFNSHY VYITADKQKN CIKANFKIRH NVEDGSVQLA 180 DHYQQNTPIG DGPVLLPDNH YLSHQSKLSK DPNEKRDHMV LLEFVTAAGI THGMDELYK 239 SEQ ID NO: 46 – Polypeptide sequence of mRuby3 GVQTMRIKVI EGGPLPFAFD 60 EDGGVVTVTQ DTSLEDGELV 120 YNVKVRGVNF PSNGPVMQKK TKGWEPNTEM MYPADGGLRG YTDIALKVDG GGHLHCNFVT 180 TYRSKKTVGN IKMPGVHAVD HRLERIEESD NETYVVQREV AVAKYSNLGG GMDELYK 237 BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples. Figure 1 shows an exemplary expression construct. Figure 2 shows that FRET occurs in the absence of binding of BoNT / A to an exemplary binding substrate. Figure 3 shows the expression of binding substrates comprising camelid antibodies. Figure 4 is a schematic showing the immobilisation of proteins to an AR2G support. Figure 5 shows the binding kinetics for glycosylated SV2c. Figure 6 shows the binding kinetics for anti-BoNT / A (rabbit polyclonal antibody). Figure 7 shows the binding kinetics for mClover3-ciA-C2-mRuby3. Figure 8 shows the binding kinetics for mClover3-ciA-D12-ciA-B5-mRuby3. Figure 9 shows the binding kinetics for mClover3-ciA-H7-mRuby3. Figure 10 shows the binding kinetics for mClover3-SV2C 529-566-mRuby3. Figure 11 shows the binding kinetics for mClover3-SV2C 454-580-mRuby3. Figure 12 shows the binding kinetics for mClover3-SV2C 566-580-mRuby3. EXAMPLES Example 1: SV2 linker variants library for BoNT / A detection Materials & Methods Expression constructs encoding a binding substrate comprising a donor fluorophore (mClover3), a binding region and an acceptor fluorophore (mRuby3) were designed and cloned into pRSETa plasmids using Gibson Assembly (see, Fig. 1). The expression constructs comprise a binding region were selected from the following table. Table 2: SV2 binding region library Residues included (relative to full length SV2c, Design SEQ ID NO: 21) Length (AA) 1 454-580 126 2 454-519 65 3 454-529 75 4 454-566 112 5 519-566 47 6 519-580 61 7 519-529 10 0 (Original) 529-566 37 9 529-580 51 10 566-580 14 The resulting plasmids were used to transform DH5α cells. Colonies were picked and the presence of the plasmid confirmed by PCR and sequencing. Expression of the expression constructs was performed in BL21 cells and the resulting binding substrates were isolated. Design 0 (i.e., comprising amino acids 529-566 of full length SV2c, SEQ ID NO: 21) was selected for further analysis. As proof of concept, the mClover3-SV2c-mRuby3 binding substrate was assessed to determine whether fluorescent energy transfer occurs between the mClover3 donor and the mRuby3 acceptor in the presence and absence of BoNT / A. Specifically, in the presence of BoNT / A, no emission from the mRuby3 acceptor was detected. However, in the absence of BoNT / A, emission from mRuby3 peaked at a wavelength of 590 nm (see, Fig.2). were selected based on their ability to bind to BoNT / A. Three different expression constructs comprising a donor fluorophore (mClover3), a binding region and an acceptor fluorophore (mRuby3) was designed and cloned into a pRSETa plasmid using Gibson Assembly. In particular, three different constructs comprising binding regions comprising CiA-H7, ciA-C2, and a dimer comprising ciA-D12 and ciA-B5 (ciA-B5-D12) respectively were selected. The resulting plasmids were used to transform DH5α cells. Colonies were picked and the presence of the plasmid confirmed by PCR and sequencing. Expression of the expression constructs was performed in BL21 cells. The resulting binding substrates were isolated and separated using SDS-PAGE. Fig.3 shows the Coomassie blue staining of each construct. In particular, the expected bands were seen at 67.6 kD for ciA-C2, 66 kD for ciA-H7 and 81.2 kD for the ciA-B5-D12 dimer. Example 3: Analysis of binding kinetics Materials & Methods BoNT / A was immobilised to an Amine Reactive 2nd-Generation (AR2G) Bio-Layer Interferometry (BLI) (Octet® BLI platform) through EDC-catalysed amide bond formation. Specifically, a covalent bond is formed between a reactive amine on BoNT / A and the carboxy- terminated biosensor surface. Covalent immobilisation fastens the BoNT / A to the AR2G- biosensor surface for analysis of binding events and kinetic characterization. A schematic showing the immobilisation of BoNT / A an AR2G support with subsequent binding of a biosensor of the invention is shown in Fig.4. Binding of immobilised BoNT / A to each of the following analytes was measured using Bio- Layer Interferometry (BLI) (Octet® BLI platform): 1. glycosylated SV2c; 2. anti-BoNT / A (rabbit polyclonal antibody; 150 kDa; Stock conc: 2 mg / mL); 3. mClover3-ciA-C2-mRuby3 (67.6kDa; 1.5 mL; Stock conc: 1.7 mg / mL); 4. mClover3-ciA-D12-ciA-B5-mRuby3 300 µL; Stock conc: 10 mg / mL); 5. mClover3-ciA-H7-mRuby3 (66kDa; 1 mL; Stock conc: 6.8 mg / mL); 6. mClover3-SV2C 529-566-mRuby3 (59 kDa; 100 µL; Stock conc: 8.4 mg / mL); 7. mClover3-SV2C 454-580-mRuby3 (69 kDa; 200 µL; Stock conc: 0.2 mg / mL); and 8. mClover3-SV2C 566-580-mRuby3 (55.9 kDa; 220µL; Stock conc: 1.1 mg / mL). Results Glycosylated SV2c The binding kinetics between captured BoNT / A and SV2c are summarised in Table 3 and Fig. 5. In particular, binding of BoNT / A to glycosylated SV2C during the association phase was slow and gradual. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with clear signs of increased re-binding during the dissociation phase. Table 3: Binding kinetics of glycosylated SV2c KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 1.97x10-71.67x10-82.701x1032.46x1045.33x10-44.11x10-40.06 0.984 The above results confirm the validity of this method since the Kd during association is comparable to literature. Anti-BoNT / A (rabbit polyclonal antibody) The binding kinetics between captured BoNT / A and anti-BoNT / A (rabbit polyconal antibody) are summarised in Table 4 and Fig.6. In particular, binding of BoNT / A to anti-BoNT / A during the association phase was slow and gradual. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow. Table 4: Binding kinetics of anti-BoNT / A (rabbit polyclonal antibody) KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 2.71x10-75.15x10-85.85x1022.27x1031.58x10-41.17x10-40.62 0.975 The above results demonstrate that the BoNT / A folds into its native conformation, thereby allowing the anti-BoNTA antibody to bind to its BoNT / A epitope. mClover3-ciA-C2-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-C2 are summarised in Table 5 and Fig.7. In particular, binding of BoNT / A to VHH ciA-C2 during the association phase was initially fast, followed by a slow, gradual on-rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately 54% of the initial analyte / ligand interaction being detected. Table 5: Binding kinetics of mClover3-ciA-C2-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 7.26x10-75.27x10-83.29x1034.44x1032.39x10-32.34x10-41.88 0.993 A binding substrate comprising mClover3-CIaC2-mRuby3 shows an approximately 3-fold increase in affinity for BoNT / A compared to a binding substrate comprising an SV2 sequence. mClover3-ciA-D12-ciA-B5-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-D12 / B5 are summarised in Table 6 and Fig.8. In particular, binding of BoNT / A to VHH ciA-D12 / B5 during the association phase was initially fast, followed by a slow, gradual on-rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately 100% of the initial analyte / ligand interaction being detected. Table 6: Binding kinetics of mClover3-ciA-D12-ciA-B5-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 2.48x10-71.29x10-87.48x1031.28x1041.86x10-31.66x10-41.44 0.994 KD2 6.37x10-85.35x10-91.19x1043.7x1037.58x10-41.98x10-51.39 0.994 mClover3-ciA-H7-mRuby3 The binding kinetics between captured BoNT / A and VHH ciA-H7 re summarised in Table 7 and Fig.9. In particular, binding of BoNT / A to VHH ciA-H7 during the association phase was initially fast, followed by a slow, gradual on-rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately 50% of the initial analyte / ligand interaction being detected. Table 7: Binding kinetics of mClover3-ciA-H7-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 2.48x10-71.29x10-87.48x1031.28x1041.86x10-31.66x10-41.44 0.994 KD2 6.37x10-85.35x10-91.19x1043.7x1037.58x10-41.98x10-51.39 0.994 mClover3-SV2C 529-566-mRuby3 The binding kinetics between captured BoNT / A and mClover3-SV2C 529-566-mRuby3 are summarized in Table 8 and Fig.10. In particular, binding of BoNT / A to mClover3-SV2C 529- 566-mRuby3 during the association phase was initially fast, followed by a slow, gradual on- rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately 6.22% of the initial analyte / ligand interaction being detected. Table 8: Binding kinetics of mClover3-SV2C 529-566-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 2.78x10-71.13x10-85.85x1032.09x1041.63x10-32.36x10-40.28 0.983 mClover3-SV2C 454-580-mRuby3 The binding kinetics between captured BoNT / A and mClover3-SV2C 454-580-mRuby3 are summarized in Table 9 and Fig.11. In particular, binding of BoNT / A to mClover3-SV2C 454- 580-mRuby3 during the association phase was initially fast, followed by a slow, gradual on- rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately of the initial analyte / ligand interaction being detected. Table 9: Binding kinetics of mClover3-SV2C 454-580-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 1.58x10-77.06x10-91.44x1046.13x1042.27x10-34.33x10-40.23 0.989 mClover3-SV2C 566-580-mRuby3 The binding kinetics between captured BoNT / A and mClover3-SV2C 566-580-mRuby3 are summarized in Table 10 and Fig.12. In particular, binding of BoNT / A to mClover3-SV2C 566- 580-mRuby3 during the association phase was initially fast, followed by a slow, gradual on- rate. Dissociation from BoNT / A at pH 7.4, as assessed using a sensorgram, was found to be exceptionally slow, with approximately 12.3% of the initial analyte / ligand interaction being detected. Table 10: Binding kinetics of mClover3-SV2C 566-580-mRuby3 KD(M) Kon(M-1s-1) Koff(s-1) KD(M) Error Kon(M-1s-1) Error Koff(s-1) Error Bmax Full R2KD1 1.97x10-77.79x10-86.67x1021.02x1041.31x10-47.93x10-40.37 0.974 Conclusions Based on the above results, and particularly the above binding affinity (KD), mClover3-ciA-C2- mRuby3 was selected as the best performing binding substrate. Importantly, the inventors have shown that binding substrates comprising a donor and acceptor fluorophore and a binding region comprising a clostridial neurotoxin nanobody are able to bind to BoNT / A with high affinity. Specifically, the affinity is far greater than the binding affinity achieved using comparative binding substrates comprising an SV2c binding region. The resulting, highly sensitive, biosensors and binding substrates may thus be used to monitor clostridial neurotoxin production in a bacterial fermentation process in real time. All publications mentioned in the above are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0003] ASPECTS OF THE INVENTION A biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, wherein the surface is configured for immobilisation of a clostridial neurotoxin, and further comprising a binding substrate, wherein the binding substrate comprises: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. A binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor. The biosensor according to aspect 1 or the binding substrate according to aspect 2, wherein the binding region comprises one or more paratopes that bind(s) clostridial neurotoxin. The biosensor or the binding substrate according to aspect 3, wherein the paratopes(s) comprises one or more amino acid sequence(s) that specifically bind(s) to the clostridial neurotoxin, and wherein said one or more amino acid sequence(s) comprise the CDR sequences of one or more antibodies; preferably, wherein the antibodies are single- chain antibodies such as nanobodies, single-chain variable fragments (scFvs), single- chain Fab (scFab), minibodies or further preferably wherein the single-chain antibodies are camelid or shark nanobodies. 5. A biosensor or binding substrate according to aspect 4, wherein said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA- A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB- A11 / B5, or combinations thereof. 6. A biosensor or binding substrate according to aspect 5, wherein said one or more amino acid sequence(s) comprises a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof. 7. A biosensor or binding substrate according to aspect 4, wherein said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies capable of neutralising clostridial neurotoxin toxicity such as ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof. 8. A biosensor or binding substrate according to aspect 7, wherein said one or more amino acid sequence(s) comprises a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof. 9. A biosensor or binding substrate according to any one of aspects 5 to 8, wherein the nanobody is a dimer, trimer or tetramer; optionally, wherein the nanobody comprises ciA-B5-D12, or combinations thereof. 10. The biosensor according to any one of aspects 1 or 3-9 or the binding substrate according to any one of aspects 2-9, wherein said one or more amino acid sequence(s) is selected from (and optionally further comprises) a sequence that specifically binds to a clostridial neurotoxin selected from the one or more of: an oxidised form of clostridial neurotoxin (having reduced potency), an incorrect proteolytically activated form of a clostridial neurotoxin (having reduced potency), aggregated forms of neurotoxin, including dimers and multimers (having reduced potency). 11. The biosensor according to any one of aspects 1 or 3-10 or the binding substrate according to any one of aspects 2-10, wherein the clostridial neurotoxin is detectable at a concentration of less than 1ng / ml, preferably less than 0.1ng / ml. 12. A method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor according to any one or more of the preceding aspects; c. exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. 13. The method of aspect 12, wherein the clostridial neurotoxin is immobilised to the surface of the biosensor. 14. The method of aspect 13, wherein the clostridial neurotoxin is contacted with the binding substrate following immobilisation to the support. 15. The method of any one of aspects 12 to 14, wherein prior to contacting the liquid medium with a biosensor, the bacterial host cell is lysed. 16. A method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate according to any one of aspects 2-11; b. culturing the bacterial host cell in a liquid medium; c. exciting said donor fluorophore; and d. determining resonance transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. 17. The method of any one of aspects 12 to 16, wherein step d comprises detecting donor fluorescence intensity at said biosensor, wherein increased donor fluorescence intensity of at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. 18. The method of any one of aspects 12 to 16, wherein step d comprises detecting acceptor fluorescence intensity at said biosensor, wherein decreased acceptor fluorescence intensity at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin. 19. The method of any one of aspects 12 to 16, wherein step d comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin. 20. The method of any one of aspects 12 to 16, wherein step d comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin.

Claims

1. A biosensor for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said biosensor comprising a surface for performing the analysis of binding events and kinetics, and onto which is immobilised a binding substrate comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.

2. A binding substrate for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process comprising: a. a donor fluorophore; b. an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and c. a binding region that specifically binds to the clostridial neurotoxin; wherein said binding region being positioned between the donor fluorophore and the acceptor such that, following activation of the donor fluorophore, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.

3. The biosensor according to claim 1 or the binding substrate according to claim 2, wherein the binding region comprises one or more paratopes that bind(s) clostridial neurotoxin.

4. The biosensor or the binding substrate according to claim 3, wherein the paratope(s) comprises one or more amino acid sequence(s) that specifically bind(s) to the clostridial neurotoxin, and wherein said one or more amino acid sequence(s) comprise the CDR sequences of one or more antibodies; preferably, wherein the antibodies are single- chain antibodies such as nanobodies, single-chain variable fragments (scFvs), single- chain Fab (scFab), minibodies or diabodies; further preferably wherein the single-chain antibodies are camelid or shark nanobodies.

5. A biosensor or binding substrate to claim 4, wherein said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies selected from the list comprising: ciA-H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA- A5, ciA-G5, ciB-H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB- A11 / B5, or combinations thereof.

6. A biosensor or binding substrate according to claim 5, wherein said one or more amino acid sequence(s) comprises a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA- H7, ciA-D1, ciA-H4, ciA-H11, ciA-C2, ciA-B5, ciA-F12, ciA-D12, ciA-A5, ciA-G5, ciB- H11, ciB-A11, ciB-B5, ciB-B9, ciA-H7 / B5, ciA-F12 / D12 and ciB-A11 / B5, or combinations thereof.

7. A biosensor or binding substrate according to claim 4, wherein said one or more amino acid sequence(s) comprises the CDR sequences from nanobodies capable of neutralising clostridial neurotoxin toxicity such as ciA-C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof.

8. A biosensor or binding substrate according to claim 7, wherein said one or more amino acid sequence(s) comprises a sequence having at least 80% sequence identity to the full-length sequence of one or more nanobody selected from the list comprising: ciA- C2, ciA-H7, ciA-B5 and ciA-D1, or combinations thereof.

9. A biosensor or binding substrate according to any one of claims 5 to 8, wherein the nanobody is a dimer, trimer or tetramer; optionally, wherein the nanobody comprises ciA-B5-D12, or combinations thereof.

10. The biosensor according to any one of claims 1 or 3-9 or the binding substrate according to any one of claims 2-9, wherein said one or more amino acid sequence(s) is selected from (and optionally further comprises) a sequence that specifically binds to a clostridial neurotoxin selected from the one or more of: an oxidised form of clostridial neurotoxin (having reduced potency), an incorrect proteolytically activated form of a clostridial neurotoxin (having reduced potency), aggregated forms of clostridial neurotoxin, including dimers and multimers (having reduced potency).

11. The biosensor according to any of claims 1 or 3-10 or the binding substrate according to any one of claims 2-10, wherein the clostridial neurotoxin is detectable at a concentration of less than 1ng / ml, preferably less than 0.1ng / ml.

12. A method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. culturing a bacterial host cell capable of producing a clostridial neurotoxin in a liquid medium; b. contacting said liquid medium with a biosensor according to any one or more of the preceding claims; c. exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.

13. The method of claim 12, wherein prior to contacting the liquid medium with a biosensor, the bacterial host cell is lysed.

14. A method for real-time monitoring of clostridial neurotoxin production during a bacterial fermentation process, said method comprising: a. transforming a bacterial host cell capable of producing a clostridial neurotoxin with an expression construct encoding a binding substrate according to any one of claims 2-11; b. culturing the bacterial host cell in a liquid medium; c. exciting said donor fluorophore; and d. determining resonance energy transfer at said biosensor relative to a control, wherein a difference in resonance energy transfer at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.

15. The method of any one of claims 12 to 14, wherein step d comprises detecting donor fluorescence intensity at said biosensor, wherein increased donor fluorescence intensity of at said biosensor as compared to said control is indicative of the presence of clostridial neurotoxin.

16. The method of any one of claims 12 to 14, wherein step d comprises detecting acceptor fluorescence intensity at said biosensor, wherein decreased acceptor fluorescenceintensity at said biosensor as to said control is indicative of the presence of clostridial neurotoxin.

17. The method of any one of claims 12 to 14, wherein step d comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum at said biosensor, wherein a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum is indicative of the presence of clostridial neurotoxin.

18. The method of any one of claims 12 to 14, wherein step d comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum to the fluorescence amplitudes near a donor fluorophore emission maximum, wherein a decreased ratio at said biosensor as compared to the control is indicative of the presence of clostridial neurotoxin.

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