SEROTYPING OF BOTULINUM NEUROTOXINS

[0001] The present invention relates to novel methods of detecting Tetanus neurotoxin (TeNT) and C. botulinum neurotoxin activity in a test sample and to peptides or polypeptides used in said methods. The methods circumvent a previous requirement for neurotoxin serotyping based on antibodies.

[0002] Botulinum neurotoxins (BoNT) produced by the bacteria Clostridium botulinum, Clostridium butyricum and Clostridium baratii and TeNT produced by Clostridium tetani are the most poisonous bacterial protein toxins known [1]. BoNT cause the fatal disease botulism due to blockade of neurotransmitter release upon cleavage of soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE) proteins resulting in paralysis. The family of BoNT consists of the nine serotypes BoNT/A-H, X comprising more than 40 variants called subtypes [2, 110, 111]. Due to their low LD ₅₀ (<1 ng/kgbodyweight [3]) BoNT detection requires highly sensitive methods suitable for all known BoNT variants. [0003] The most widely established method for BoNT detection is still the lethal Mouse Bioassay (MBA) with a limit of detection of 10-100 pg/mL depending on the serotype [4-6]. However, the end point of this animal experiment is respiratory failure preceded by heavy suffering. Furthermore, the MBA requires large numbers of mice for serotyping and quantitation (> 100/sample), often displays high variations (20-40 % depending on the method used and the laboratory) and requires long readout times of up to four days. Neutralization of BoNT positive samples by preincubation with monovalent BoNT antisera prior to the MBA allows the determination of a known BoNT serotype.

[0004] The ex vivo mice phrenic nerve hemidiaphragm (MPN) assay replaces the MBA by closely reproducing in vivo respiratory failure thereby examining the full physiological pharmacodynamics of BoNT with similar sensitivity in a much shorter time (2-4 h), but clearly less animal consumption and hardly any animal suffering [7]. Analogously, preincubation of BoNT positive samples with monovalent BoNT antisera allows the determination of a known BoNT serotype by the MPN assay.

[0005] Neuronal cell-based assays for BoNT detection (NCBA) employ primary neuronal cell cultures, stem cell derived neurons and continuous cell lines like neuroblastoma cells and cover all steps of the cellular intoxication including cell surface binding, endocytosis, translocation of the LC into the cellular cytosol and enzymatic activity of the LC on SNARE substrates [8]. A very specific endpoint for BoNT activity is endogenous SNARE cleavage determined in cell lysates by Western blot [9-12] or by ELISA employing BoNT/A-cleavage sensitive antibodies [13-16], or in fixed cells by quantitative immunofluorescence methods also using BoNT/A- cleavage sensitive antibodies [12,17] or in live cells by Forster resonance energy transfer (FRET) detection of an exogenous SNARE-FRET reporter [18]. While BoNT/E causes a level of synaptic inhibition equivalent to the amount of cleaved SNAP-25, BoNT/A causes 50% inhibition of neurotransmitter release at 10-fold lower concentrations than required for 50% cleavage of SNAP-25 [19]. Therefore, measuring inhibition of neurotransmitter release e.g. by the release of pre-loaded, radioactively labelled neurotransmitter [8,20], pre-loaded fluorescent dyes [21,22] or ectopically expressed bioluminescent reporters [23] is a less specific, but more sensitive endpoint. This also accounts for the measurement of endogenous neurotransmitter release by high performance liquid chromatography (HPLC) or immunoassays, indirect measurement detecting enzymatic breakdown products or the postsynaptic currents by voltage clamping [24] or multi electrode arrays (MEAs) [25]. Prior neutralization of BoNT positive samples by monovalent BoNT antisera also allows the determination of a known BoNT serotype by the NCBA [26]. However, NCBA can only be used with highly purified BoNT samples and do not tolerate complex matrices like low pH orange juice or proteases-rich faeces etc. Their read out time ranges from 1 -3 days while cell culturing and differentiation of stem cells requires several weeks, skilled personnel and is very expensive. [0006] Immunoassays like ELISA include high sensitivity and sometimes too narrow specificity, but lack detection of active BoNT (i.e. false positive results) and require a large number of antibodies to reliably detect the more than 40 known subtypes of BoNT (i.e. high risk of false negative results) [27-29]. So far no single immunoassay has been produced to detect all BoNT subtypes in parallel. [0007] A newly identified BoNT serotype or subtype easily causes false-negative results in established in vitro assays due to lack of appropriate reagents. Knowledge of BoNT amino acid sequence, its receptors, protein substrate and its cleavage site constitute essential knowledge for safe detection of a BoNT by in vitro assays.

[0008] BoNT are 150 kDa AB-protein toxins comprising four domains which play individual roles in the intoxication mechanism [2,30]. Their heavy chain (HC) contains three domains and is responsible for neuronal binding, uptake and translocation of the 50 kDa light chain (LC), a Zn ²⁺ -dependent endoprotease [31], into the neuronal cytosol [2]. Upon reduction of the disulfide bridge connecting LC and HC the neuronal SNARE proteins are specifically hydrolysed at distinct peptide bonds. VAMP (vesicle associated membrane protein)/synaptobrevin-l/2 represent the substrate for BoNT/B, D, F, G, F5/H, X and TeNT [32-40, 111], whereas BoNT/A, C and E cleave SNAP-25 (synaptosome associated protein of 25 kDa) [41-45]. Except for BoNT/B and TeNT, which share the same cleavage site, hydrolysis occurs in unique positions (Figure 1). BoNT/C is in addition capable of hydrolyzing Syntaxin [42,46]. The LC do not recognize a consensus motif on the SNARE proteins, instead require long stretches of the substrates away from the scissile bond for optimal cleavage [36,43,47-53]. Accordingly, the SNAP-25 peptide was shown to wrap around the LC in a substrate cleft covered by the belt in the holotoxin [54]. [0009] The above described proteolytic specificity of BoNT is utilized by various in vitro endopeptidase assays which detect BoNT due to their catalytic activity by analysing SNARE cleavage products by sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot or HPLC [27]. In addition, SNARE proteins fused to fluorophores would release the fluorescent reporter upon cleavage [55,56] while SNARE peptides equipped with a chromophore and fluorophore exhibit FRET upon BoNT cleavage [18,57-63]. Depending on the SNARE-substrate offered one can distinguish between the group of serotypes cleaving VAMP (B, D, F, G, F5/H, X and TeNT) and that hydrolyzing SNAP-25 (BoNT/A, C and E), but not between serotypes within each group. Advances in mass spectrometry (MS) allowed the development of the Endopep-MS assay which combines the in vitro proteolytic assay employing short, SNARE protein based peptides whose serotype-specific cleavage products are unanimously detected by sophisticated MS equipment [64]. In combination with high-affinity antibody-mediated BoNT enrichment, 0.5 mLDso of BoNT/ A and 0.1 mLDso of BoNT/B can be detected [65]. The immunological specific recognition of non-cleaved SNARE proteins or the newly generated amino acid (aa) termini, the so-called neoepitopes, provides an alternative to the elaborate and expensive Endopep-MS. Polyclonal antibodies raised against residues 44- 94 of human VAMP -2 recognized only non-BoNT/B cleaved VAMP-2 [66] while polyclonal antibodies generated against aa 193-206 and aa 191-206 of SNAP-25 detected only non- BoNT/A cleaved SNAP-25 [13,67]. The first neoepitope antibodies were obtained from rabbits and specifically recognized the newly exposed N-terminus of the C-terminal BoNT/B cleavage product of VAMP-2 [68] and the newly exposed C-terminus of the N-terminal BoNT/A cleavage product of SNAP-25 [67]. Subsequently, polyclonal neoepitope antibodies against TeNT/BoNT/B-cleaved VAMP-2 [69], BoNT/C-cleaved SNAP-25 (aa 191-198) and Syntaxin (254-AVKYQSKA-261) [70] and BoNT/E-cleaved SNAP-25 (aa 173-180) [71 ] were used in endopeptidase immunoassays to quantify residual TeNT activity in TeNT toxoid vaccine or functionally active BoNT in pharmaceutical preparations, respectively. The first mouse monoclonal antibody (mAb) 4F3-2C1 was raised against the C-terminal BoNT/ A neoepitope of rat SNAP-25 (aa 183-197), is still the only commercially available neoepitope Ab (http://www.rdabs.com/files/datasheets/MC-6053.pdf) and was used in a protein chip membrane-capture assay [72] and quantitative immunofluorescence and Western blot methods [12] to detect BoNT/A. Subsequently, further mAb against BoNT/A-cleaved SNAP-25 [14,16,73], against the N-terminal BoNT/B neoepitope of rat VAMP-2 (77-FETSAA -83) [74], and against the C-terminal BoNT/E neoepitope of rat SNAP-25 (173-TQNRQIDR-180) [75] were generated for use in NCBA or in surface plasmon resonance based assays, respectively. However, endopeptidase immunoassays are hindered by multiple washing and binding steps, non-commercial availability of the required antibodies, possible lot-to-lot variability especially of polyclonal antibodies and unspecific recognition of peptides displaying epitopes similar to neoepitopes like e.g. in human serum albumin [76] or in complex matrices like serum [28,29]. [0010] Improved means and methods for testing biological samples for the presence of neurotoxin activity are highly desirable but not yet available. In particular, methods which avoid the current need for immunological reagents and have the potential for practical application are highly desirable. Thus, the technical problem underlying the present invention may be seen as the provision of means and methods for replacing existing methods by complying with the aforementioned needs. The technical problem is solved by the embodiments characterised in the claims and herein below.

[0011] Accordingly, the present invention relates to a method of detecting clostridial neurotoxin activity in a test sample, comprising (i) contacting a test sample comprising the neurotoxin with a peptide or polypeptide, said peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31, and 43-74; and (ii) detection of the presence or amount of at least one peptide or polypeptide or of at least one proteolytic cleavage product of said peptide or polypeptide; and (iii) concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin in the sample, wherein the neurotoxin is selected from the group consisting of C. tetani neurotoxin (TeNT) and C. botulinum neurotoxin (BoNT) of serotypes A, B, C, D, E, F, F5, X and H (also called BoNT/H or BoNT/FA, see Peck et al. 2017). [0012] Surprisingly the present inventors found that peptides or polypeptides comprising an amino acid sequence as shown in any one of SEQ ID NO: 1 to 31 and SEQ ID NO: 43 to 74 can be used for identifying a particular neurotoxin responsible for neurotoxin protease activity in a test sample. The aforementioned peptides and polypeptides allow detecting neurotoxin activity and, depending on the need, assigning the activity to either small groups of neurotoxin serotypes or to an individual serotype.

[0013] Summary of the invention

[0014] The present invention relates to a method of detecting clostridial neurotoxin activity in a test sample, comprising

(i) contacting a test sample comprising the neurotoxin with a peptide or polypeptide, wherein said peptide or polypeptide comprises an amino acid sequence according to SEQ ID NO: 75 or

SEQ ID NO: 76 or a variant thereof, wherein said variant of SEQ ID NO: 75 comprises a fragment of SEQ ID NO: 75 with at least one mutation at position 179, 180, 181, 183, 196, 197, 198, or 199 of SEQ ID NO: 75 and said variant of SEQ ID NO: 76 comprises a fragment of SEQ ID NO: 76 with at least one mutation at position 34, 43, 46, 54, 55, 58, 59, 60, 66, 67, 69, 72, 76, 77, 79, 81 or 82 of SEQ ID NO: 76, wherein said fragment comprises at least 20 consecutive amino acid residues of said amino acid sequence and wherein said mutation renders the peptide or polypeptide insusceptible of proteolytic cleavage by at least one neurotoxin;

(ii) detection of the presence or amount of at least one peptide or polypeptide or of at least one proteolytic cleavage product of said peptide or polypeptide; and

(iii) concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin in the sample,

wherein the neurotoxin is selected from the group consisting of C. tetani neurotoxin (TeNT) and C. botulinum neurotoxin (BoNT) of serotypes A, B, C, D, E, F, F5, H, X and variants thereof. [0015] In one embodiment said peptide or polypeptide comprises a fragment of SEQ ID NO: 75 with at least one mutation selected from the group consisting of D179 , R180W, II 8 IE, El 831, Nl 96Q, Q197N, R198K, and A199R, preferably a mutation or combination of mutations selected from the group consisting of (a) D179K and A199R, (b) D179K and N196Q and Q197N and R198K, (c) R180W and I181E and E183L (d) D179K, (e) A199R, and (f) N196Q and Q197N and R198 . [0016] In another embodiment said peptide or polypeptide comprises a fragment of SEQ ID NO: 76 with at least one mutation selected from the group consisting of Q34A, V43A, M46I, L54A, L54E, E55Q, E55L, 59R, A67R, A69N, A72D, Q76V, and T79S, preferably a mutation or combination of mutations selected from the group consisting of (a) T79S, (b) E55Q and K59R, (c) L54A and E55Q and K59R, (d) M46I and E55Q, (e) M46I and L54A and K59R, (f) M46I and E55Q and K59R and A72D, (g) M46I and L54A and E55Q and K59R and A72D, (h) M46I and L54A and E55Q and K59R, (i) K59R, (j) M46I, (k) E55Q, (1) A72D, (m) M46I, (n) 59R, (o) L54A and K59R, (p) E55Q, (q) A69N, (r) T79S, (s) A67R, (t) A67R and T79S, (u) E55Q and K59R and A67R, (v) L54A and E55Q and K59R and A67R, (w) L54E and E55L and K59R and A67R, (x) Q34A and K59R and A67R, (y) V43A and 59R and A67R, (z) M46I and E55Q and A67R, (aa) M46I and L54E and E55L and A67R, (bb) Q34A and M46I and A67R, (cc) V43A and M46I and A67R, (dd) M46I and L54A and K59R and A67R, (ee) M46I and K59R and A67R, (ff) M46I and E55Q and K59R and A67R and A72D, (gg) M46I and L54A and E55Q and K59R and A67R and A72D, (hh) M46I and L54A and E55Q and K59R and Q76V, (ii) L54E and E55L and K59R, (jj) Q34A and K59R, (kk) V43A and K59R, (11) M46I and L54E and E55L, (mm) Q34A and M46I, (nn) V43 A and M46I, (oo) K59R and A67R, (pp) L54A and K59R, (qq) E55Q and A67R, (rr) L54A and E55Q, (ss) Q76V, (tt) M46I and K59R, (uu) L54A and E55Q, (w) A67R, and (ww) M46I and L54A and E55Q and K59R.

[0017] In another embodiment the peptide or polypeptide is selected from the group consisting of SEQ ID NOs: 1-31, and 43-76. [0018] The present invention also relates to a method of detecting clostridial neurotoxin activity in a test sample, comprising (i) contacting a test sample comprising the neurotoxin with a peptide or polypeptide, said peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31, and 43-74; and (ii) detection of the presence or amount of at least one peptide or polypeptide or of at least one proteolytic cleavage product of said peptide or polypeptide; and (iii) concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin in the sample, wherein the neurotoxin is selected from the group consisting of C. tetani neurotoxin (TeNT) and C. botulinum neurotoxin (BoNT) of serotypes A, B, C, D, E, F, F5, X and H.

[0019] In one embodiment of the method of the invention, the detection of (a) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 1 is indicative of catalytic activity of BoNT/Al and serotype variants thereof; (b) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 43 or SEQ ID NO: 44 is indicative of catalytic activity of BoNT/Bl and serotype variants thereof;

(c) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 4 is indicative of catalytic activity of BoNT/C and serotype variants thereof;

(d) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID NO: 47 or SEQ ID NO: 48 or SEQ ID NO:

49 is indicative of catalytic activity of BoNT/D and serotype variants thereof;

(e) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 7 or SEQ ID NO: 8 is indicative of catalytic activity of BoNT/El and serotype variants thereof;

(f) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 9 or SEQ ID NO:

50 or SEQ ID NO: 51 or SEQ ID NO: 52 or SEQ ID NO: 53 is indicative of catalytic activity of BoNT/Fl and serotype variants thereof;

(g) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 10 or SEQ ID NO: 54 or SEQ ID NO: 55 is indicative of catalytic activity of BoNT/F5, BoNT/H (also called BoNT/HA or BoNT/FA) and serotype variants thereof;

(h) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 56 or SEQ ID NO: 57 is indicative of catalytic activity of TeNT and serotype variants thereof;

(i) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 58 is indicative of catalytic activity of BoNT/X and serotype variants thereof;

(j) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 13 or SEQ ID NO: 14 is indicative of catalytic activity of BoNT/Al and/or BoNT/C and serotype variants thereof; (k) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 15 is indicative of catalytic activity of BoNT/Al and/or BoNT/El and serotype variants thereof;

(1) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 16 is indicative of catalytic activity of BoNT/C and/or BoNT/El and serotype variants thereof;

(m) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 3 is indicative of catalytic activity of BoNT/B and/or BoNT/X and serotype variants thereof; (n) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 59 or SEQ ID NO: 60 or SEQ ID NO: 61 is indicative of catalytic activity of BoNT/D and/or BoNT/X and serotype variants thereof; (o) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: 62 or SEQ ID NO: 63 or SEQ ID NO: 64 is indicative of catalytic activity of BoNT/Fl and/or BoNT X and serotype variants thereof;

(p) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 10 is indicative of catalytic activity of BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof; (q) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 11 or SEQ ID NO: 12 is indicative of catalytic activity of TeNT and/or BoNT/X and serotype variants thereof; (r) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 17 or SEQ ID NO: 74 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/X and serotype variants thereof;

(s) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 65 is indicative of catalytic activity of BoNT/D and/or BoNT/F5 and/or BoNT/H and serotype variants thereof; (t) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 18 or SEQ ID NO: 66 is indicative of catalytic activity of BoNT/D and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(u) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 19 is indicative of catalytic activity of BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(v) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 67 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and serotype variants thereof;

(w) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 20 or SEQ ID NO: 68 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and/or BoNT/X and serotype variants thereof;

(x) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 21 or SEQ ID NO: 22 is indicative of catalytic activity of BoNT/Al and/or BoNT/C and/or BoNT/El and serotype variants thereof;

(y) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 23 or SEQ ID NO: 69 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(z) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 24 is indicative of catalytic activity of TeNT and/or BoNT/D and/or BoNT/F 1 and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof; (aa) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 25 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/Fl and/or BoNT/F5 and/or BoNT H and/or BoNT/X and serotype variants thereof;

(bb) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 26 or SEQ ID NO: 27 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(cc) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 71 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(dd) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 28 or SEQ ID NO: 72 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/X and serotype variants thereof;

(ee) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 29 is indicative of catalytic activity of BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(ff) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 73 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and serotype variants thereof; and

(gg) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 30 or SEQ ID NO: 31 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/F 1 and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof.

[0020] In another embodiment,

(a) the catalytic activity of BoNT/Al, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1 ; (b) the catalytic activity of BoNT/B, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 43 or 44;

(c) the catalytic activity of BoNT/C, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 4; (d) the catalytic activity of BoNT/D, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOs: 45-49;

(e) the catalytic activity of BoNT/E, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 7 or 8;

(f) the catalytic activity of BoNT/F, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOs: 50-53;

(g) the catalytic activity of BoNT/F5 and/or BoNT H, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 54 or 55;

(h) the catalytic activity of BoNT/X, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 58; and (i) the catalytic activity of TeNT, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 56 or 57.

[0021] In another embodiment, any of the methods described herein is performed by making use of a peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and 43-74. In a preferred embodiment said peptide or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 8, and 43-58.

[0022] In another embodiment, the peptide or polypeptide or cleavage product is coupled to a solid support. [0023] In another embodiment, the peptide, polypeptide or cleavage product comprises a detectable label.

[0024] In another embodiment, the detectable label is a luciferase, a green, red, cyan, yellow fluorescent protein, a peroxidase, a phosphatase, β-galactosidase, or a radioactive isotope. [0025] In another embodiment, the peptide, polypeptide or cleavage product comprises a tag for immobilization on a solid support.

[0026] In another embodiment, the tag is a strep-tag, a halo-tag (HA), a poly-his-tag, a poly- arginine-tag, a poly-HisAsn-tag, a HAT (natural histidine affinity)-tag, a S-tag, a flag-tag, a GST (glutathione-S-transferase)-tag, or a MBP (maltose-binding-protein)-tag. [0027] In another embodiment, said presence or amount of said peptide, polypeptide and/or cleavage product is determined from the detectable amount of cleavage product.

[0028] In another embodiment, said method comprises comparing the amount of peptide, polypeptide or cleavage product detectable in step (ii) of the method described herein above with the amount of peptide, polypeptide or cleavage product detectable in a reference sample. [0029] The present invention also relates to a peptide or polypeptide comprising (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and SEQ ID NOs: 43- 74, (b) an amino acid sequence variant of SEQ ID NO: 75, wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 75 with at least one mutation selected from the group consisting of D179K, R180W, I181E, E183I, N196Q, Q197N, R198 , and A199R or (c) an amino acid sequence variant of SEQ ID NO: 76 wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 76 with at least one mutation selected from the group consisting of Q34A, V43A, M46I, L54A, L54E, E55Q, E55L, K59R, A67R, A69N, A72D, Q76V, and T79S.

[0030] In one embodiment of the invention, the amino acid sequence variant of SEQ ID NO: 75 is selected from the group consisting of (a) D179K and A199R, (b) D179K and N196Q and Q197N and R198K, (c) R180W and I181E and E183I, (d) D179K, (e) A199R, and (f) N196Q and Q197 and R198 .

[0031] In one embodiment of the invention, the amino acid sequence variant of SEQ ID NO:

76 is selected from the group consisting of (a) T79S, (b) E55Q and K59R, (c) L54A and E55Q and K59R, (d) M46I and E55Q, (e) M46I and L54A and K59R, (f) M46I and E55Q and K59R and A72D, (g) M46I and L54A and E55Q and K59R and A72D, (h) M46I and L54A and E55Q and K59R, (i) K59R, (j) M46I, (k) E55Q, (1) A72D, (m) M46I, (n) K59R, (o) L54A and K59R,

(p) E55Q, (q) A69N, (r) T79S, (s) A67R, (t) A67R and T79S, (u) E55Q and K59R and A67R,

(v) L54A and E55Q and K59R and A67R, (w) L54E and E55L and K59R and A67R, (x) Q34A and 59R and A67R, (y) V43A and K59R and A67R, (z) M46I and E55Q and A67R, (aa) M46I and L54E and E55L and A67R, (bb) Q34A and M46I and A67R, (cc) V43A and M46I and A67R, (dd) M46I and L54A and 59R and A67R, (ee) M46I and K59R and A67R, (ff) M46I and E55Q and K59R and A67R and A72D, (gg) M46I and L54A and E55Q and 59R and A67R and A72D, (hh) M46I and L54A and E55Q and K59R and Q76V, (ii) L54E and E55L and K59R, (jj) Q34A and K59R, (kk) V43A and K59R, (11) M46I and L54E and E55L, (mm) Q34A and M46I, (nn) V43A and M46I, (oo) K59R and A67R, (pp) L54A and K59R, (qq) E55Q and A67R, (rr) L54A and E55Q, (ss) Q76V, (tt) M46I and K59R, (uu) L54A and E55Q, (w) A67R, and (ww) M46I and L54A and E55Q and K59R.

[0032] The present invention also relates to a peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and SEQ ID NOs: 43-74, preferably from the group consisting of SEQ ID NOs: 1-12 of the sequence listing or from the group consisting of SEQ ID NOs: l, 4, 7, 8, and 43-58.

[0033] In one embodiment, the peptide or polypeptide described according to the present teaching, comprises a tag for immobilization, preferably selected from the group consisting of a halo-tag (HA), strep-tag, a poly-his-tag, a poly-arginine-tag, a poly-HisAsn-tag, a HAT-tag, a S-tag, a flag-tag, a GST-tag, a MBP-tag.

[0034] In another embodiment the peptide or polypeptide comprises a detectable label, preferably selected from the group consisting of luciferase, a green fluorescent protein, a red fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, preferably GFP, mCherry, CFP, or YFP, or a peroxidase, a phosphatase, β-galactosidase, or a radioactive isotope.

[0035] In another embodiment, the peptide or polypeptide comprises at least one additional cleavage site for a protease, said protease being selected from the group consisting of TEV (cysteine protease from Tobacco Etch Virus), HRV (human rhino virus) 3C protease, Thrombin, Factor Xa, SUMO-protease (ULP1/2, Senpl-7). [0036] The present invention also relates to the use of the peptide or polypeptide of the present invention for specific detection of BoNT of serotype A, B, C, D, E, F, F5, H and X and variants thereof and TeNT.

[0037] Finally, the present invention also relates to a kit comprising the peptide or polypeptide of the present invention. Particularly preferred is a kit comprising a set of peptides or polypeptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 8, and 43-58 or a set of peptides or polypeptides consisting of an amino acid sequence according to SEQ ID NOs: 1, 4, 7, 8, and 43-58.

[0038J Detailed description of the invention

[0039] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these 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 limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0040] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. [0041] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0042] The following abbreviations are used in this specification:

[0043] aa: amino acid; BoNT, botulinum neurotoxin; BSA: bovine serum albumin; DTE: dithioerythritol; DTT: Dithiothreitol; EC ₅₀ , median effective concentration; ELISA: enzyme linked immunosorbent assay; FRET: Forster resonance energy transfer; GBS, ganglioside binding site; GFP, mCherry, CFP, YFP, green fluorescent protein, red fluorescent protein, cyan fluorescent protein, yellow fluorescent protein; GST, glutathion-S-transferase; h, human; HA, halo-tag; HAT: natural histidine affinity tag; HC, heavy chain; He, carboxyl-terminal half of HC; HcA, HcE etc., He fragment of BoNT serotype A, E etc, respectively; HCN and Hcc, 25 kDa halves of He; HN, amino-terminal half of HC; H6, hexahistidine-tag; 6xHN, (HisAsn)6 tag; HA, HaloTag; HPLC, high performance liquid chromatography; HRV, human rhinovirus 3C protease; LC, light chain; LD ₅₀ : median lethal dose; LD, luminal domain; mAb: monoclonal antibody; MBA/MLB, mouse lethality bioassay; MPN assay, mice phrenic nerve hemidiaphragm assay; MS: Mass spectrometry; MW, molecular weight; NCBA: neuronal cell based assay; NL: NanoLuc® Luciferase; S, C-terminal Streptag; SDS-PAGE, sodiumdodecyl- polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; SNAP-25: synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SV, synaptic vesicle; rSV2A-C, rat synaptic vesicle glycoprotein 2 isoform A, B or C; Syt, synaptotagmin; synaptobrevin, Syb; Syntaxin, Stx; TeNT: Tetanus Neurotoxin; TEV, cysteine protease from Tobacco Etch Virus; TMD, transmembrane domain; VAMP, vesicle associated membrane protein.

[0044] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0045] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0046] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0047] The present invention relates to a method of detecting clostridial neurotoxin activity in a test sample, comprising (i) contacting a test sample comprising the neurotoxin with a peptide or polypeptide, said peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31, and 43-74; and (ii) detection of the presence or amount of at least one peptide or polypeptide or of at least one proteolytic cleavage product of said peptide or polypeptide; and (iii) concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin in the sample, wherein the neurotoxin is selected from the group consisting of C. tetani neurotoxin (TeNT) and C. botulinum neurotoxin (BoNT) of serotypes A, B, C, D, E, F, F5, X and H.

[0048] The present invention also relates to a method of detecting clostridial neurotoxin activity in a test sample, comprising (i) contacting a test sample comprising the neurotoxin with a peptide or polypeptide, wherein said peptide or polypeptide comprises an amino acid sequence according to SEQ ID NO: 75 or SEQ ID NO: 76 or a variant thereof, wherein said variant of SEQ ID NO: 75 comprises a fragment of SEQ ID NO: 75 with at least one mutation at position 179, 180, 181, 183, 196, 197, 198, or 199 of SEQ ID NO: 75 and said variant of SEQ ID NO: 76 comprises a fragment of SEQ ID NO: 76 with at least one mutation at position 34, 43, 46, 54, 55, 58, 59, 60, 66, 67, 69, 72, 76, 77, 79, 81 or 82 of SEQ ID NO: 76, wherein said fragment comprises at least 20 consecutive amino acid residues of said amino acid sequence and wherein said mutation renders the peptide or polypeptide insusceptible of proteolytic cleavage by at least one neurotoxin; (ii) detection of the presence or amount of at least one peptide or polypeptide or of at least one proteolytic cleavage product of said peptide or polypeptide; and (iii) concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin in the sample, wherein the neurotoxin is selected from the group consisting of C. tetani neurotoxin (TeNT) and C. botulinum neurotoxin (BoNT) of serotypes A, B, C, D, E, F, F5, H, X and variants thereof. [0049] The term "variant" refers to peptides and polypeptides which differ in amino acid sequence from the reference amino acid sequence. A variant may differ by being an N-terminal and/or C-terminal deletion mutant of the reference sequence and/or by having at least one mutation in the amino acid sequence when compared to the reference sequence. Preferably the mutation is an amino acid substitution. The amino acid substitution may be for example a conservative amino acid substitution, a non-conservative amino acid substitution or a chemical modification of at least one amino acid residue, preferably a modification which is not observed in a eukaryotic or prokaryotic cell.

[0050] As used herein, the term "at least 20 consecutive amino acid residues" includes fragments of for example at least 20 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues. Such fragments may comprise the full amino acid sequence of the reference sequence or comprise an N-terminal and/or C-terminal deletion of said reference sequence. The amino acid sequence of SEQ ID NOs: 75 and 76 are examples of reference sequences. A deletion may comprise up to 10, 20, 30, 40, or 50 N-terminal and/ or C-terminal amino acid residues,

[0051] The term "consecutive amino acid residues" refers to a sequence of amino acids which is identical to the reference sequence. Regarding the reference sequence of SEQ ID NO: 75, the term also includes fragments of said sequence with one or more mutations at positions 179, 180, 181, 183, 196, 197, 198, or 199 of SEQ ID NO: 75. Regarding the reference sequence of SEQ ID NO: 76, the term also includes fragments of said sequence one or more mutations at positions 34, 43, 46, 54, 55, 58, 59, 60, 66, 67, 69, 72, 76, 77, 79, 81 or 82 of SEQ ID NO: 76.

[0052] As used herein, the term "detecting clostridial neurotoxin activity in a test sample" means demonstrating that the test sample comprises a protein having proteolytic activity that can be assigned to a neurotoxin of clostridial origin. Examples of neurotoxin of clostridial origin comprise neurotoxins such as BoNT/Al , BoNT/A2, BoNT/A3, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/H and BoNT/FA), BoNT/G , BoNT/X and TeNT. [0053] As used herein, the term "contacting a test sample comprising the neurotoxin with a peptide or polypeptide" means establishing conditions allowing a functional interaction between the neurotoxin and the peptide or polypeptide. Functional conditions are conditions allowing for the neurotoxin to be proteolytically active. In one embodiment, the peptide or polypeptide may be coupled to a solid support and the test sample is a liquid sample comprising the neurotoxin, wherein the liquid sample is applied to the solid support.

[0054] As used herein, the term "peptide" refers to an oligopeptide of up to 30 amino acids, whereas the term "polypeptide" refers to an amino acid chain of more than 31 amino acids. These terms also refer to peptides and polypeptides, respectively, which contain a modification including those required for coupling of the peptide or polypeptide to the solid support. Examples of modified amino acids comprise D-amino acids, selenocysteine, pyrrolysine, hydroxyproline, O-phosphoserine, O-phosphotyrosine, γ-carboxyglutamate, canavanine, azetidin-2-carbonic acid, ornithine, norleucine, norvaline, and homonorleucine. The "peptide or polypeptide" is the substrate of a clostridial neurotoxin, if it can be recognized and proteolytically cleaved by the neurotoxin. A peptide or polypeptide comprising an amino acid sequence selected from the group consisting of a particular amino acid sequence may be a peptide or polypeptide consisting of or comprising said amino acid sequence. Whereas the term "peptide or polypeptide" refers to the substrate of a neurotoxin, the term "cleavage product" refers to the product arising from the proteolytic activity of the neurotoxin. A cleavage product can be distinguished from the "peptide or polypeptide" by lacking at least one peptide bond. [0055] The term "peptide or polypeptide" also comprises detectably labelled peptides and polypeptides and peptides and polypeptides coupled to a solid support.

[0056] In one embodiment, the method described herein comprises the additional step of concluding from the particular neurotoxin identified in step (iii) on the presence of the particular Clostridium strain producing clostridial neurotoxin. Said strain is characterized by producing at least one of the following neurotoxins selected from the group consisting of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/H and BoNT/FA), BoNT/X and TeNT. [0057] As used herein, the term "test sample" refers to a sample comprising a biological sample to be tested, whereas the term "biological sample" refers to a specimen obtained from any source, comprising or suspected of comprising C. botulinum, C. butyricum, C. baratii or C. tetani or neurotoxin of C. botulinum, C. butyricum, C. baratii or C. tetani.

[0058] In one embodiment, the method described herein comprises prior to step (i) a step of obtaining a biological sample. The biological sample may be obtained from food such as milk, juices, water, meat, sausage, canned food, fish, sea food, honey, environmental samples such as soil, dust, air, and clinical samples such as tissue samples, blood, serum, feces.

[0059] In another embodiment, the biological sample or the test sample may be treated for example in order to stabilize the components of the biological sample or in order to establish the reaction conditions of the method described herein.

[0060] Such treatment may aim at lysing cells present in the sample. Accordingly, the method described herein may also comprise a step of adding to the biological sample or test sample a detergent. Preferably, the detergent is Polyoxyethylen(20)-sorbitan-monolaurat such as Tween- 20®, preferably added to a final concentration in the test sample of 0.5%. [0061] Such treatment may aim at stabilizing residual proteins of the sample or by providing conditions suitable for neurotoxin activity. Accordingly, the method described herein may also comprise a step of adding to the biological sample or test sample a salt such as NaCl, ZnCl ₂ , KC1, NaS0 ₄ , Na-glutamate, K-glutamate, CaCl ₂ , a reducing agent such as DTT (dithiothreitol), beta-mercaptoethanol, dithioerythritol (DTE), TCEP (tris(2-carboxyethyl)phosphine) or a buffer such as HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid ), MES (2-(N- morpholino)ethanesulfonic acid), glycine/hydrochloric acid, potassium hydrogen phthalate/hydrochloric acid, citric acid/sodium citrate, sodium acetate/acetic acid, potassium hydrogen phthalate/sodium hydroxide, disodium hydrogen phthalate/sodium dihydrogen orthophosphate, dipotassium hydrogen phthalate/potassium dihydrogen orthophospate, potassium dihydrogen orthophosphate/sodium hydroxide, barbitone sodium/hydrochloric acid, tris(hydroxylmethyl) aminomethane/hydrochloric acid, sodium tetraborate/hydrochloric acid, glycine/sodium hydroxide, sodium carbonate/sodium hydrogen carbonate, sodium tetraborate/sodium hydroxide, sodium bicarbonate/sodium hydroxide, sodium hydrogen orthophosphate/sodium hydroxide, or potassium chloride/sodium hydroxide. [0062] ZnCl ₂ may preferably be added to a final concentration in the test sample of up to 5 μΜ, up to 10 μΜ, up to 50 μΜ, up to 100 μΜ, up to 150 μΜ, up to 200 μΜ, up to 250 μΜ or up to

500 μΜ. Preferably, the test sample comprises at least 250 μΜ of ZnCl ₂ . [0063] DTT may preferably be added to a final concentration in the test sample of up to 1 mM, up to 2 mM, up to 5 mM, up to 10 mM, up to 15 mM up to 20mM, up to 25 mM, up to 50 mM or up to 100 mM. Preferably, the test sample comprises at least 20 mM of DTT.

[0064] HEPES pH 7.2 may preferably be added to a final concentration in the test sample of up to 1 mM, up to 2 mM, up to 5 mM, up to 10 mM, up to 15 mM, up to 20 mM, up to 50 mM or up to 100 mM. Preferably, the test sample comprises at least 50 mM of HEPES.

[0065] The method described herein may also comprise a step of adding a protease inhibitor not chelating Zn ²⁺ . Preferably, the protease inhibitor is selected from the group consisting of antipain-dihydrochloride, antithrombin III, aprotinin, APMSF (4-Amidino-phenyl) methane- sulfonyl fluoride, bestatin, Calpain Inhibitor- I/-II, chymostatin, dabigatran, danaparoid, 3,4- dichloroisocoumarin, E-64, hirudin, leupeptin, a2-macroglobulin, Pefabloc SC, pepstatin, phosphormidon, PMSF (Phenylmethylsulfonyl fluoride), rivaroxaban, TIMP-2 (Tissue Inhibitor of Metalloproteinase 2), TLCK/HCI (L-l-Chloro-3-[4-tosylamido]-7-amino-2- heptanone-HCI), TPCK (L-l-Chloro-3-[4-tosylamido]-4-phenyl-2-butanone), Trypsin Inhibitors from chicken egg white and soybean. [0066] As used herein, the term "detection of the presence or amount of at least one peptide or polypeptide or ... cleavage product" means assessing or monitoring the presence or amount of educt, product or both. It will be apparent to those of skilled in the art that, upon exposure of the peptide or polypeptide to the neurotoxin, i.e. after contacting the test sample with the peptide or polypeptide and depending on the particular neurotoxin activity, the peptide or polypeptide will be cleaved. Neurotoxin activity can be assessed by detecting the amount of educt, the amount of product or both, wherein said amount is preferably detected over time. The term "educt" refers to the peptide or polypeptide prior to any proteolytic cleavage by the neurotoxin, the term "product" refers to the cleaved product or cleavage product arising from the proteolytic cleavage of the peptide or polypeptide. The term "cleaved product" or "cleavage product" comprises the fragment arising from the amino acid sequence which is located in the uncleaved peptide or polypeptide N-terminal to the neurotoxin cleavage site and the fragment arising from the amino acid sequence which is located in the uncleaved peptide or polypeptide C-terminal to the neurotoxin cleavage site or both fragments. In other words, the term "cleaved product" or "cleavage product" covers an N-terminal cleavage product of the peptide or polypeptide, a C-terminal cleavage product of the peptide or polypeptide or both.

[0067] The amounts can be detected by any suitable methods, including SDS-PAGE, HPLC. A preferred method comprises measuring fluorescence or bioluminescence. In a preferred embodiment, neurotoxin activity results in the release of a labelled N- or C-terminal cleavage product of the peptide or polypeptide, wherein the released cleavage product is preferably labelled with a fluorophore or luminophore. Another preferred embodiment comprises measuring educts and/or products in a so-called Endopep-MS assay [64, 65].

[0068] The method described herein encompasses embodiments comprising a labelled peptide or polypeptide, which is coupled to a solid support via an amino acid residue located N-terminal to the cleavage site of a neurotoxin. In a preferred embodiment, said peptide or polypeptide is labelled at an amino acid residue located C-terminal to the cleavage site. The method described herein also encompasses embodiments comprising a labelled peptide or polypeptide, which is coupled to a solid support via an amino acid residue located C-terminal to the cleavage site of a neurotoxin. In a preferred embodiment, said latter peptide or polypeptide is labelled at an amino acid residue located N-terminal to the cleavage site. Since the neurotoxin activity of the aforementioned embodiments will result in release of a labelled fragment, the presence or amount of cleavage product may be detected by measuring the amount of detectable label in the supernatant of the solid support. [0069] A cleavage product of the present teaching comprises at least one amino acid residues. In other words, it may consist of a single amino acid residue or of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10, up to 16, up to 17, up to 18, up to 19, up to 22, up to 36, up to 40 or more amino acid residues. Longer cleavage products consist of up to 15, up to 20, up to 30, up to 40, up to 50, up to 51, up to 54, up to 57, up to 58, up to 59, up to 60, up to 76, up to 180, up to 197, up to 198 or up to 200 amino acid residues. Preferably, the cleavage product is detectably labelled.

[0070] The method of detecting neurotoxin activity described herein may also comprise a step of calculating a ratio of the amount of product over the amount of educt.

[0071] The term "concluding from the presence or amount of the peptide, polypeptide or cleavage product on the presence or amount of a neurotoxin" means drawing conclusions from detected amounts. Preferably, an increase in the amount of product, a decrease in the amount educt, or an increase of the ratio of amount of product over amount of educt are indicative for neurotoxin activity.

[0072] As used herein, the term "a neurotoxin of serotype A" comprises BoNT/Al according to SEQ ID NO:32 and serotype variants thereof, the term "a neurotoxin of serotype B" comprises BoNT/Bl according to SEQ ID NO:33 and serotype variants thereof, the term "a neurotoxin of serotype C" comprises BoNT/C according to SEQ ID NO:34 and serotype variants thereof, the term "a neurotoxin of serotype D" comprises BoNT/D according to SEQ ID NO:35 and serotype variants thereof, the term "a neurotoxin of serotype E" comprises BoNT/El according to SEQ ID NO:36 and serotype variants thereof, the term "a neurotoxin of serotype F" comprises BoNT/Fl according to SEQ ID NO:37, the term "a neurotoxin of serotype F5" comprises BoNT/F5 according to SEQ ID NO:38 and serotype variants thereof and the term "a neurotoxin of serotype G" comprises BoNT/G according to SEQ ID NO:39 and serotype variants thereof and the term "a neurotoxin of serotype H" comprises BoNT/H according to SEQ ID NO:40 and serotype variants thereof and the term "a neurotoxin of serotype X" comprises BoNT/X according to SEQ ID NO:42 and serotype variants thereof and the term "Tetanus neurotoxin" comprises TeNT according to SEQ ID NO:41 and serotype variants thereof. The term "serotype variant" is described herein below.

[0073] In one embodiment the detection of (a) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 1 is indicative of catalytic activity of BoNT/Al and serotype variants thereof;

(b) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 43 or SEQ ID NO: 44 is indicative of catalytic activity of BoNT/Bl and serotype variants thereof;

(c) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 4 is indicative of catalytic activity of BoNT/C and serotype variants thereof;

(d) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID NO: 47 or SEQ ID NO: 48 or SEQ ID NO:

49 is indicative of catalytic activity of BoNT/D and serotype variants thereof;

(e) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 7 or SEQ ID NO: 8 is indicative of catalytic activity of BoNT/El and serotype variants thereof;

(f) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: 50 or SEQ ID NO: 51 or SEQ ID NO: 52 or SEQ ID NO: 53 is indicative of catalytic activity of BoNT/Fl and serotype variants thereof;

(g) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 10 or SEQ ID NO: 54 or SEQ ID NO: 55 is indicative of catalytic activity of BoNT/F5, BoNT/H (also called BoNT/HA or BoNT/FA) and serotype variants thereof;

(h) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 1 1 or SEQ ID NO: 12 or SEQ ID NO: 56 or SEQ ID NO: 57 is indicative of catalytic activity of TeNT and serotype variants thereof; (i) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 58 is indicative of catalytic activity of BoNT/X and serotype variants thereof;

(j) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 13 or SEQ ID NO: 14 is indicative of catalytic activity of BoNT/Al and/or BoNT/C and serotype variants thereof; (k) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 15 is indicative of catalytic activity of BoNT/Al and/or BoNT/El and serotype variants thereof;

(1) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 16 is indicative of catalytic activity of BoNT/C and/or BoNT/El and serotype variants thereof;

(m) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 3 is indicative of catalytic activity of BoNT/B and/or BoNT/X and serotype variants thereof; (n) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 59 or SEQ ID NO: 60 or SEQ ID NO: 61 is indicative of catalytic activity of BoNT/D and/or BoNT/X and serotype variants thereof;

(o) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: 62 or SEQ ID NO: 63 or SEQ ID NO: 64 is indicative of catalytic activity of BoNT/Fl and/or BoNT/X and serotype variants thereof;

(p) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 10 is indicative of catalytic activity of BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof; (q) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 11 or SEQ ID NO: 12 is indicative of catalytic activity of TeNT and/or BoNT/X and serotype variants thereof; (r) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 17 or SEQ ID NO: 74 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/X and serotype variants thereof;

(s) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 65 is indicative of catalytic activity of BoNT/D and/or BoNT/F5 and/or BoNT/H and serotype variants thereof; (t) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 18 or SEQ ID NO: 66 is indicative of catalytic activity of BoNT/D and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(u) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 19 is indicative of catalytic activity of BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(v) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 67 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and serotype variants thereof; (w) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 20 or SEQ ID NO: 68 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and/or BoNT/X and serotype variants thereof;

(x) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 21 or SEQ ID NO: 22 is indicative of catalytic activity of BoNT/Al and/or BoNT/C and/or BoNT/El and serotype variants thereof;

(y) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 23 or SEQ ID NO: 69 is indicative of catalytic activity of BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(z) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 24 is indicative of catalytic activity of TeNT and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(aa) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 25 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(bb) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 26 or SEQ ID NO: 27 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(cc) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 71 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(dd) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 28 or SEQ ID NO: 72 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/X and serotype variants thereof;

(ee) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 29 is indicative of catalytic activity of BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof;

(ff) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 73 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and serotype variants thereof; and

(gg) a cleavage product of the peptide or polypeptide comprising SEQ ID NO: 30 or SEQ ID NO: 31 is indicative of catalytic activity of TeNT and/or BoNT/Bl and/or BoNT/D and/or BoNT/Fl and/or BoNT/F5 and/or BoNT/H and/or BoNT/X and serotype variants thereof. [0074] In a preferred embodiment, said method of the present teaching comprises a step of mass spectrometry or is an Endopep-MS assay.

[0075] In another embodiment the method of the present teaching is performed by making use of a peptide or polypeptide that can only be proteolytically cleaved by the protease of a single serotype so that the observation of a cleavage product is indicative of the catalytic activity of said protease. In this embodiment, (a) the catalytic activity of BoNT/Al , and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1; (b) the catalytic activity of BoNT/B, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 43 or 44; (c) the catalytic activity of BoNT/C, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 4; (d) the catalytic activity of BoNT/D, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOs: 45-49; (e) the catalytic activity of BoNT/E, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 7 or 8; (f) the catalytic activity of BoNT/F, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOs: 50-53; (g) the catalytic activity of BoNT/F5 and/or BoNT/H, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 54 or 55; (h) the catalytic activity of BoNT/X, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 58; and (i) the catalytic activity of TeNT, and serotype variants thereof, is established by detecting a cleavage product of a peptide or polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 56 or 57.

[0076] As used herein, the term "indicative of catalytic activity of BoNT/Al" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/Al . The term "indicative of catalytic activity of BoNT/B" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/B. The term "indicative of catalytic activity of BoNT/C" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/C. The term "indicative of catalytic activity of BoNT/D" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/D. The term "indicative of catalytic activity of BoNT/E" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/E. The term "indicative of catalytic activity of BoNT/F" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/F. The term "indicative of catalytic activity of BoNT/F 1 " means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/F5. the term "indicative of catalytic activity of BoNT/F5" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/F5. The term "indicative of catalytic activity of BoNT/H" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/H. The term "indicative of catalytic activity of BoNT/X" means that any of the cleavage products observed have been generated by the proteolytic activity of BoNT/X. The term "indicative of catalytic activity of TeNT" means that any of the cleavage products observed have been generated by the proteolytic activity of TeNT.

[0077] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/Al"also means that the peptide or polypeptide comprising SEQ ID NO: 1 of the sequence listing cannot be cleaved by any of BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/H and BoNT/FA), BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins.

[0078] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/B 1 " means that the peptide or polypeptide comprising SEQ ID NO: 43 or 44 of the sequence listing cannot be cleaved by any of BoNT/Al , BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, , BoNT/E9 BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/HA and BoNT/FA) , BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins. [0079] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/C" means that the peptide or polypeptide comprising SEQ ID NO: 4 of the sequence listing cannot be cleaved by any of BoNT/Al , BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/HA and BoNT/FA), BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins.

[0080] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/D" means that the peptide or polypeptide comprising any one of SEQ ID NOs: SEQ ID NOs: 45-49 of the sequence listing cannot be cleaved by any of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/HA and BoNT/FA), BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins. [0081] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/El " means that the peptide or polypeptide comprising SEQ ID NO: 7 or 8 of the sequence listing cannot be cleaved by any of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl , BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT DC, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/H (also called BoNT/HA and BoNT/FA), BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins.

[0082] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/Fl" means that the peptide or polypeptide comprising any one of SEQ ID NOs: 50-53 of the sequence listing cannot be cleaved by any of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l , BoNT/E12, BoNT/F5, BoNT/H (also called BoNT/HA and BoNT/FA), BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins. [0083] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/F5, BoNT/H" means that the peptide or polypeptide comprising SEQ ID NO: 54 or 55 of the sequence listing cannot be cleaved by any of BoNT/Al , BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9 BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl , BoNT/F2, BoNT/F3, BoNT/F4, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/G, BoNT/X and TeNT or the sequence variants of the aforementioned neurotoxins.

[0084] In a preferred embodiment, the term "indicative of catalytic activity of TeNT" means that the peptide or polypeptide comprising SEQ ID NO: 56 or 57 of the sequence listing cannot be cleaved by any of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El , BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl , BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/G, BoNT/X and BoNT/H (also called BoNT/HA and BoNT/FA) or the sequence variants of the aforementioned neurotoxins.

[0085] In a preferred embodiment, the term "indicative of catalytic activity of BoNT/X" means that the peptide or polypeptide comprising SEQ ID NO: 58 of the sequence listing cannot be cleaved by any of BoNT/Al, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7, B0NT/A8, BoNT/Bl, BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8, BoNT/B9, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/El, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El l, BoNT/E12, BoNT/Fl, BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8, BoNT/F9, BoNT/G, BoNT/H (also called BoNT/HA and BoNT/FA), TeNT or the sequence variants of the aforementioned neurotoxins.

[0086] As used herein, the term "serotype variant" comprises any variant of the neurotoxin within the same serotype. For example, a serotype variant of BoNT/Al comprises any of BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, B0NT/A6, BoNT/A7 and B0NT/A8. Preferably a serotype variant of BoNT/Al is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/Al . [0087] A serotype variant of BoNT/Bl comprises any BoNT/B2, BoNT/B3, BoNT/B4bv, BoNT/B5nP, B0NT/B6, BoNT/B7, B0NT/B8 and BoNT/B9. Preferably a serotype variant of BoNT/Bl is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/B 1. [0088] A serotype variant of BoNT/El comprises any of BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, B0NT/E6, BoNT/E7, B0NT/E8, BoNT/E9, BoNT/ElO, BoNT/El 1 and BoNT/E12. Preferably a serotype variant of BoNT/El is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/El . [0089] A serotype variant of BoNT/Fl comprises any of BoNT/F2, BoNT/F3, BoNT/F4, BoNT/F5, B0NT/F6, BoNT/F7, B0NT/F8 and BoNT/F9. Preferably a serotype variant of BoNT Fl is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/F 1. [0090] A serotype variant of BoNT/C is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/C. A serotype variant of BoNT/D is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/D. A serotype variant of BoNT/H is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/H. A serotype variant of BoNT/G is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/G. A serotype variant of BoNT/X is a variant BoNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85% in comparison to the amino acid sequence of BoNT/X. A serotype variant of TeNT is a variant TeNT polypeptide having an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80% or at least 85%) in comparison to the amino acid sequence of TeNT. [0091] In an alternative embodiment, the term "serotype variant" as used herein is characterized by the capability of cleaving the identical peptide bond of the peptide or polypeptide or yielding the identical cleavage products. [0092] In one embodiment, the peptide or polypeptide or cleavage product is coupled to a solid support. The peptide, polypeptide and/or cleavage product can be detectably labelled. The label can be attached within the peptide or polypeptide at an N-terminal or C-terminal amino acid residue with respect to the cleavage site or at an internal amino acid residue. The detectable label can be attached to an amino acid residue located N-terminal to a neurotoxin cleavage site or the detectable label can be attached to an amino acid residue located C-terminal said cleavage site. The present teaching, however, also encompasses peptides and polypeptides which are labelled at least one amino acid residue located N-terminal to the neurotoxin cleavage site and at least one amino acid residue located C-terminal to said cleavage site. In addition, the peptide or polypeptide described herein can be attached to a solid support. The peptide or polypeptide may be attached to a solid support via an amino acid residue located N-terminal to a neurotoxin cleavage site. However, the peptide or polypeptide may also be attached to a solid support via an amino acid residue located C-terminal to a neurotoxin cleavage site.

[0093] In other embodiments of the present teaching, the peptide or polypeptide described herein is attached to a solid support via an amino acid residue located N-terminal to a neurotoxin cleavage site and a detectable label is attached to an amino acid residue located C-terminal to said cleavage site. In another embodiment, the peptide or polypeptide is attached to a solid support via an amino acid residue located C-terminal to a neurotoxin cleavage site and a detectable label is attached to an amino acid residue located N-terminal to said cleavage site.

[0094] In one embodiment, the detectable label is a luciferase, a green, red, cyan, yellow fluorescent protein, in particular a superfolder green fluorescent protein (sfGFP) [77], mCherry [78], CFP, YFP [79] or a horseradish peroxidase [80], an alkaline phosphatase^ 1], β- galactosidase [82,83], or a radioactive isotope.

[0095] In another embodiment, the peptide, polypeptide or cleavage product comprises a tag for immobilization on a solid support. The tag for immobilization can be attached via an amino acid residue located N-terminal to a neurotoxin cleavage site or C-terminal to a neurotoxin cleavage site. Examples of tags comprise a strep-tag, a halo-tag (HA), a poly-his-tag, a poly- arginine-tag, a poly-HisAsn-tag, a HAT (natural histidine affinity)-tag, a S-tag, a flag-tag, a GST-tag, and a maltose-binding-protein-tag and derivatives of the aforementioned tags [84,85].

[0096] In one embodiment of the present teachings method, the presence or amount of peptide, polypeptide and/or cleavage product is detemiined from the detectable amount of cleavage product. The cleavage product may be the fragment arising from the amino acid sequence which is located in the uncleaved peptide or polypeptide N-terminal to the neurotoxin cleavage site and the fragment arising from the amino acid sequence which is located in the uncleaved peptide or polypeptide C-terminal to the neurotoxin cleavage site or both fragments. Preferably said fragment is labelled. If both fragments are labelled, it is preferred that the label of the fragments is different.

[0097] In another embodiment, the method described herein above comprises a step of comparing the amount of peptide, polypeptide or cleavage product detectable in step (ii) with the amount of peptide, polypeptide or cleavage product detectable in a reference sample. The reference sample preferably comprises the reference material of BoNT/Al , BoNT/B 1 , BoNT/C, BoNT/D, BoNT/El, BoNT/Fl, BoNT/F5, BoNT/H, BoNT/X or of TeNT [86].

[0098] The present invention also relates to a peptide or polypeptide comprising (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 and SEQ ID NOs: 43- 74, (b) an amino acid sequence variant of SEQ ID NO: 75, wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 75 with at least one mutation selected from the group consisting of Dl 79K, R180W, II 8 IE, El 831, N196Q, Q197N, R198K, and A199R or (c) an amino acid sequence variant of SEQ ID NO: 76 wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 76 with at least one mutation selected from the group consisting of Q34A, V43A, M46I, L54A, L54E, E55Q, E55L, K59R, A67R, A69N, A72D, Q76V, and T79S.

[0099] As used herein, the term "mutation" refers to an amino acid modification of at least one amino acid residue of a reference sequence. [00100] In one embodiment the peptide or polypeptide of the teaching comprises an amino acid sequence variant of SEQ ID NO: 75, wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 75 with a least one mutation selected from the group consisting of (a) D179K and A199R, (b) D179K and N196Q and Q197N and R198K, (c) R180W and I181E and E183I, (d) D179K, (e) A199R, and (f) N196Q and Q197N and R198K. [00101] In one embodiment the peptide or polypeptide of the teaching comprises an amino acid sequence variant of SEQ ID NO: 76, wherein said variant comprises a fragment of at least 20 consecutive amino acids of SEQ ID NO: 76 with a least one mutation selected from the group consisting of (a) T79S, (b) E55Q and K59R, (c) L54A and E55Q and 59R, (d) M46I and E55Q, (e) M46I and L54A and K59R, (f) M46I and E55Q and K59R and A72D, (g) M46I and L54A and E55Q and K59R and A72D, (h) M46I and L54A and E55Q and K59R, (i) K59R, (j)

M46I, (k) E55Q, (1) A72D, (m) M46I, (n) K59R, (o) L54A and K59R, (p) E55Q, (q) A69N, (r)

T79S, (s) A67R, (t) A67R and T79S, (u) E55Q and 59R and A67R, (v) L54A and E55Q and K59R and A67R, (w) L54E and E55L and K59R and A67R, (x) Q34A and 59R and A67R, (y) V43A and K59R and A67R, (z) M46I and E55Q and A67R, (aa) M46I and L54E and E55L and A67R, (bb) Q34A and M46I and A67R, (cc) V43A and M46I and A67R, (dd) M46I and L54A and K59R and A67R, (ee) M46I and K59R and A67R, (ff) M46I and E55Q and K59R and A67R and A72D, (gg) M46I and L54A and E55Q and K59R and A67R and A72D, (hh) M46I and L54A and E55Q and 59R and Q76V, (ii) L54E and E55L and K59R, (jj) Q34A and K59R, (kk) V43A and K59R, (11) M46I and L54E and E55L, (mm) Q34A and M46I, (nn) V43A and M46I, (oo) K59R and A67R, (pp) L54A and 59R, (qq) E55Q and A67R, (rr) L54A and E55Q, (ss) Q76V, (tt) M46I and K59R, (uu) L54A and E55Q, (w) A67R, and (ww) M46I and L54A and E55Q and K59R. [00102] The term "variant which comprises a fragment of at least 20 consecutive amino acids" refers to peptides and polypeptides of the teaching which comprise at least 20 amino acid residues of the reference sequence of SEQ ID NO: 75 or 76 with at least one mutation as identified above. Peptides and polypeptides of the teaching, which comprise a fragment of at least 20 consecutive amino acid residues of the reference sequence of SEQ ID NO: 75 or 76 include peptides and polypeptides which are variants of the full-length amino acid sequence of SEQ ID NOs: 75 and 76 with at least one mutation as identified above.

[00103] The present invention also relates to a peptide or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 or SEQ ID NOs: 43-74.

[00104] The present invention also relates to peptides or polypeptides comprising or consisting of the amino acid sequence of Synaptobrevin/VAMP-2 according to SEQ ID NO: 76 or of a fragment thereof. A fragment comprises at least 10, at least 20, at least 30, at least 40 at least 50 amino acid sequences. In a preferred embodiment, said fragment comprises up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90 amino acid sequences of Synaptobrevin VAMP-2. A fragment is preferably selected from the group consisting of amino acids 1-94, 2-94, 1-76, 2-76, 40-94, and 60-94. Preferably, said peptides and polypeptides comprise at least one mutation at a position of Synaptobrevin/VAMP-2 selected from the group consisting of 34, 43, 46, 54, 55, 58, 59, 60, 66, 67, 69, 72, 76, 77, 79, 81, 82. In a preferred embodiment, the mutation at said position is Q34A, V43A, M46I, L54A, E55Q, 59R, A67R, A69N, A72D, Q76V, and T79S. In another preferred embodiment the peptide or polypeptide comprises a mutation or combination of mutations selected from the group consisting of T79S, E55Q and K59R, L54A and E55Q and K59R, M46I and E55Q, M46I and L54A and 59R, M46I and E55Q and K59R and A72D, M46I and L54A and E55Q and K59R and A72D, M46I and L54A and E55Q and K59R, K59R, M46I, E55Q, A72D, M46I, K59R, L54A K59R, E55Q, A69N, T79S, A67R, A67R and T79S, E55Q and K59R and A67R, L54A and E55Q and K59R and A67R, L54E and E55L and 59R and A67R, Q34A and K59R and A67R, V43 A and 59R and A67R, M46I and E55Q and A67R, M46I and L54E and E55L and A67R, Q34A and M46I and A67R, V43 A and M46I and A67R, M46I and L54A and 59R and A67R, M46I and K59R and A67R, M46I and E55Q and K59R and A67R and A72D , M46I and L54A and E55Q and K59R and A67R and A72D, M46I and L54A and E55Q and 59R and Q76V, L54E and E55L and 59R, Q34A and K59R, V43A and K59R, M46I and L54E and E55L, Q34A and M46I, V43 A and M46I, K59R and A67R, L54A and K59R, E55Q and A67R, L54A and E55Q, Q76V, M46I and K59R, L54A and E55Q, A67R, M46I and L54A and E55Q and 59R. [00105] According to the teaching of the present application, the amino acid sequence of Synaptobrevin/VAMP-2 at position 66 can also be replaced with a mutation selected from the group consisting of R66A, R661, R66L, R66V and R66T which renders the peptide or polypeptide less susceptible to cleavage by BoNT/X. Wherein less susceptible is preferably a cleavage reduction of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. For example, R66A is typically associated with a 100 fold reduced cleavage. In other words, the aforementioned mutations reduce cleavage of up to 100 fold or more.

[00106] According to the teaching of the present application, the above mutation of Synaptobrevin/VAMP-2 at position 67 can also be replaced with a mutation selected from the group consisting of A67 , A67E, A67D, A67Q, A67 N, A67F and A67W which renders the peptide or polypeptide less susceptible to cleavage by BoNT/X.

[00107] The present invention also relates to peptides or polypeptides comprising or consisting of the amino acid sequence of SNAP-25 according to SEQ ID NO: 75 or of a fragment thereof. A fragment comprises at least 10, at least 20, at least 30, at least 40 at least 50 amino acid sequences. In a preferred embodiment, said fragment comprises up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90 amino acid sequences of SNAP-25. A fragment is preferably selected from the group consisting of amino acids 1-197, 2-197, 1-206, 2-206, 140- 197, 146-206 of SNAP-25. Preferably, said peptides and polypeptides comprise at least one mutation at a position of SNAP-25 selected from the group consisting of 179, 180, 181, 183, 196, 197, 198, 199 of SNAP-25. Preferably said mutation is D179 , R180W, I181E, E183I, N196Q, Q197N, R198 , and A199R. In another preferred embodiment the peptide or polypeptide comprises a mutation or combination of mutations selected from the group consisting of D179K and A199R, D179K and N196Q and Q197N and R198K, R180W and I181E and E183I, D179K, A199R, N196Q and Q197N and R198K.

[00108] In one embodiment, the peptide or polypeptide comprises a tag for immobilization, preferably selected from the group consisting of a halo-tag (HA), strep-tag, a poly-his-tag, a poly-arginine-tag, a poly-HisAsn-tag, a HAT (natural histidine affinity)-tag, a S-tag, a flag-tag, a GST-tag, a maltose-binding-protein-tag.

[00109] In another embodiment, the peptide or polypeptide described herein comprises a detectable label, preferably selected from the group consisting of luciferase, a green, red, cyan, yellow fluorescent protein (GFP, mCherry, CFP, YFP) or a peroxidase, a phosphatase, β- galactosidase, radioactive isotope. [00110] In another embodiment, the peptide or polypeptide described herein comprises at least one additional cleavage site for a protease, said protease being selected from the group consisting of TEV, Human rhino virus 3C protease, Thrombin, Faktor Xa, SUMO-protease (ULP1/2, Senpl -7).

[00111] The present invention also relates to the use of the peptide or polypeptide described herein above for specific detection of BoNT of serotype A, B, C, D, E, F, F5, H, X and variants thereof and TeNT.

[00112] Finally, the present invention also relates to a kit comprising the peptide or polypeptide described herein above. In a preferred embodiment, the kit comprises at least one peptide or polypeptide.

[00113] The figures show:

[00114] Figure 1: Overview of SNARE proteins SNAP-25 (A) and synaptobrevin/VAMP-2 (B) and derived generic peptidic substrates which are fused between an N-terminal His6- and Halotag (H6tHAtev) and a C-terminal reporter protein like luciferase (L). Serotype specific cleavage sites are indicated by black arrows.

[00115] Figure 2: Endopeptidase assay of rSNAP-25H6 wt (A) and H6tHAlS(146-206)L (B) with LC/A and H6trVAMP-2 2-97 (C) and H6tHAtevlV(2-94)LS (D) with LC/D for 1 h at 37°C in PBS pH 7.4. Analysis by SDS-PAGE, detection by Coomassie blue staining.

[00116] Figure 3: Generic and serotype-specific substrates (GS/X, SSS/X). (A) Generic substrates H6tHAtevlS(146-206)LS (top) cleavable by BoNT/A, C and E and H6tHAtevl V(2- 94)L (bottom) cleavable by BoNT/B, D, F, H, X and TeNT. H6tHAtevl S(146-206)LS D179KA199R is the serotype-specific substrate for BoNT/A (SSS/A), H6tHAtevlS(146- 206)LS D179KN196QQ197NR198K is preferably cleaved by BoNT/C (SSS/C) and H6tHAtevlS(140-197)L is preferably cleaved by BoNT/E (SSS/E). Analogous, H6tHAtevlV(60-94)L (SSS/B) is hydrolyzed only by BoNT/B and X, H6tHAtevlV(2-76)LS K59R (SSS/D) is cleaved by BoNT/D, F5/H and X, while H6tHAtevl V(2-76)LS M46I (SSS/F) is cleaved by BoNT/F, F5/H and X. (B) 12.5% SDS-PAGE analysis of generic and serotype- specific substrates, detection by Coomassie blue staining. (C) MW, yield, concentration and purity of GS/X and the six SSS/X. [00117] Figure 4: Determination of EC ₅ o for BoNT/A-F for the six serotype specific substrates (SSS/X) and the generic substrates H6tHAl S(146-206)LS and H6tHAlV(2-94)LS. Assay in JDZ-buffer for 1 h of incubation at 37°C (mean ± SD, n = 3-4).

[00118] Figure 5: Analysis of cleavage the generic substrate peptide H6tHAlS(146-206) or H6tHAlV(2-94), respectively, with three to four different detector molecules luciferase (L), NanoLuc® Luciferase (NL), superfolder green- fluorescent Protein (sfGFP) and mCherry by different concentrations of BoNT/A-F in JDZ-buffer after 1 h of incubation at 37°C (A-F). (G) Determination of the EC50 of the generic substrate peptides with different reporter molecules for BoNT/A-F. [00119] The following Examples illustrate the invention and shall, whatsoever, not be construed to limit its scope.

[00120] EXAMPLES

[00121] Example 1: Methods

[00122] Plasmid construction [00123] The genes encoding LC/A1 1-429, LC/B1 1-436, LC/C 1 -436, LC/D 1-436, LC/E1 1 - 411 and LC/F1 1-428, LC/G 1-435 and LC/T 1-438 fused to a C-terminal 6xHN-tag were inserted into the vector pPROTet.E133 (CLONTECH Laboratories, Inc.). The DNA fragments encoding LC/C-6xHN and LC/D-6xHN were also inserted into a pQe3 vector via EcoR I/Pst I sites. [00124] The plasmid pET-HAtev2SL was generated by inserting the fragment encoding the halo-tag (HA) and TEV cleavage site from pHA2SL into the plasmid pET-2SL [87]. Optionally, a StrepTag (S) was fused to the C-terminus of H6tHAtev2SL resulting in the plasmid pH6tHAtev2SLS. Restriction sites Kpn I and BamH I were used to insert different DNA fragments encoding the various SNAP-25 and VAMP -2 based peptides between TEV and luc. Single site mutations were introduced via PCR applying the GeneTailor™ site-directed mutagenesis system (Life Technologies, Darmstadt, Germany) and suitable primers (Eurofins, Ebersberg, Germany) using pH6tHAtevl SL(S) or pH6tHAtevlVL(S) as template DNA. PCR products encoding the different reporter molecules sfGFP, mCherry and NanoLuc® Luciferase were inserted via a BamH I and Nhe I restriction site. [00125] Nucleotide sequences of all newly generated constructs were verified by DNA sequencing (GATC Biotech, Konstanz, Germany).

[00126] Expression and purification of recombinant proteins

[00127] Recombinant LC/X-6xHN were expressed utilizing the E. coli strain TGI in 2YT medium supplemented with chloramphenicol (Cm, 34 mg/L) during 16 h of induction at 22°C in the presence of anhydrotetracyclin (aTC, 100 ng/mL) or in case of LC/C-6xHN and LC/D- 6xHN encoded in a pQe3 plasmid in E. coli M15 pRep4 strain as described previously [88]. LC/X-6xHN were isolated employing Co ²⁺ -Talon matrix (Takara Bio Europe S.A.S., Saint- Germain-en-Laye, France) and eluted in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 raM imidazole. Desired eluates were subjected to HPLC in 20 mM HEPES, pH 7.2, 150 niM - glutamate buffer (Superdex-200 16/60 column or HiPrep 16/10 Q FF, GE Healthcare, Germany) or dialyzed against 20 mM HEPES, pH 7.2, 150 mM K-glutamate, frozen in liquid nitrogen and kept at -70°C.

[00128] The generation of recombinant, activated full-length BoNT/A-F, BoNT/G, BoNT/X and TeNT was described previously [86,89,90,1 1 ].

[00129] Recombinant rat SNAP-25 aa 1-206 fused to a C-terminal His6tag (rSNAP-25H6) was expressed in E. coli M15 pRep4 strain as described previously [43]. The plasmid pET15b- VAMP-2 [52] encoding rat VAMP-2 aa 1 -97 fused to an N-terminal thrombin cleavable His6tag (H6trVAMP-2 1-97) was expressed in E. coli BL21DE3 strain upon induction by IPTG. Expression of all substrate constructs H6tHAtevXY(S) (X=SNARE peptide, Y=detector molecule) was done following a modified protocol of Stevens et al. [87]. The harvested cells were resuspended in 0.1 M Tris/HCl pH 7.6, 0.15 M NaCl, 5 mM MgCl ₂ supplemented with 1 mg/mL lysozyme. After centrifugation the clear cell lysate was applied to a Co ²⁺ -Talon matrix (Takara Bio Europe S.A.S., Saint-Germain-en-Laye, France). The matrix was washed first and finally with 50 mM Tris/HCl pH 7.6, 150 mM NaCl, 5 mM Imidazole, in between with washing buffer containing 1 M NaCl instead. The protein of interest was eluted by washing buffer supplemented with 100 mM imidazole. Desired eluates were pooled and subjected to gel filtration (Superdex-200 16/60 column, GE Healthcare, Germany) in PBS pH 7.4. The substrates comprising a C-terminal StrepTag were subsequently purified on Strep-Tactin® Superflow® matrix (IB A) according to the manufacturer's instructions and eluted in 0.1 M Tris/HCl pH 7.6, 0.15 m NaCl supplemented with 10 mM desthiobiotin. The desired eluates were dialyzed against PBS pH 7.4 or JDZ-buffer. All recombinant proteins were shock-frozen in liquid nitrogen, and kept at - 70°C.

[00130] Protein concentrations were determined subsequent to SDS-PAGE and Coomassie blue staining by densitometry using a LAS-3000 imaging system (Fuji Photo Film), the AIDA 3.51 software (Raytest, Straubenhardt, Germany) and BSA (100-1600 ng) as reference protein.

[00131] Endopeptidase assay

[00132] The endopeptidase assays were performed in PBS pH 7.4 (for LC) or JDZ-buffer (50 mM HEPES pH 7, 20 mM DTT, 250 μΜ ZnCl ₂ , 0.5 % Tween-20, for BoNT) unless otherwise stated in a total volume of 20 μΐ. The substrates (0.45 μΜ) and serially diluted LC or BoNT were incubated for 1 h at 37 °C. The reactions were stopped by the addition of 7 μΐ 4x Laemmli buffer. Samples were denatured at 99 °C for 2 min and then subjected to 10 % or 12.5 % SDS- PAGE. Protein bands were detected by Coomassie blue staining and subsequently quantified by densitometry using the software TINA (version 2.09f, Raytest, Straubenhardt, Germany).

[00133] Example 2: Development of substrates for detecting BoNT/ A, C and E

[00134] The SNARE protein SNAP-25 is exclusively cleaved by BoNT/A, C and E at unique peptide bonds. BoNT/A hydrolyses at Q197-R198 [41,43,91] whereas BoNT/C proteolyses SNAP-25 at the neighboring peptide bond R198-A199 [45] and BoNT/E further upstream between R180-1181 [41 ,43]. In the design of a generic substrate suitable for these three BoNT, a minimal substrate should include the SNARE motif S4 (residues 145-155 of SNAP-25) which is thought to be important for cleavage [53,92]. Subsequently, it was proposed that SNAP-25 146-202 is the minimum essential peptide for BoNT/A cleavage while BoNT/C needs the SNAP-25 peptide 93-202 [49,51]. We inserted the peptide 146-206 of SNAP-25 into the modified construct to serve as generic substrate for BoNT/A, C and E to yield H6tHAtevl S(146-206)L(S) in high yield and >99% purity (Figure 1 & 3).

[00135] To identify negative effects of the large N-terminal H6tHAtev-tag, the C-terminal reporter molecule luciferase and truncations of the SNAP-25 peptide on substrate hydrolysis the newly generated H6tHAtevl S(146-206)L substrate was compared with the full-length wild- type SNAP-25 (1 -206) substrate fused only C-terminal to a small His6Tag (rSNAP25-H6) [88] (Figure 1). In an endopeptidase assay H6tHAtevl S(146-206)L was incubated each with LC/A, LC/C and LC/E in PBS pH 7.4 for 1 hour at 37°C. The EC ₅ o values were determined for both substrate constructs by SDS-PAGE (Figure 2). As shown in Table 1, fusion of tags and reporter protein as well as truncations of SNAP-25 did not impair cleavage by LC/A or LC/E, but increased EC ₅₀ values two-fold. Only LC/C shows a very high EC ₅ o value for the H6tHAtevlS(146-206)L substrate, but it is known that SNAP-25 is a poor in vitro substrate for LC/C, as can be seen by the much higher EC50 values compared to those of LC/A and LC/E. We also analyzed the cleavage of H6tHAtevl S(146-206)LS by the full-length BoNT/A, C and E in an optimized cleavage buffer (JDZ-buffer, 50 mM HEPES pH 7.2, 20 mM DTT, 200 μΜ ZnCl ₂ , 0.5 % Tween-20) (Figure 4). Here, the EC50 values of BoNT/A, C and E were determined as 0.03 nM, 10 nM and 0.2 nM, respectively (Table 3). The cleavage rate of full- length BoNT in the optimized JDZ-buffer was clearly increased compared to LC in PBS buffer. All in all, H6tHAtevlS(146-206)L(S) can be used as a generic substrate to detect low concentrations of BoNT/ A, BoNT/C and BoNT/E.

[00136] Table 1 : Endopeptidase assay of H6tHAtevl S(146-206)L compared to rSNAP-25H6 wt by LC/A, LC/C and LC/E in PBS pH 7.4, for 1 h at 37°C (mean ± SD, n = 3).

Substrate ECso [nM]

LC/A LC/C LC/E

rSNAP-25H6 wt 1.67 + 0.58 2100 + 100 0.40 ± 0.17

H6tHAtevlS(146-206)L 0.87 ± 0.23 >3000 0.17 ± 0.06

[00137] Example 3: Development of substrates for detecting BoNT/B, D, F, F5/H, X and TeNT

[00138] BoNT/B, D, F, F5/H and G cleave VAMP-2 at different peptide bonds (BoNT/B: Q76- F77 [32], BoNT/D: K59-L60 [36], BoNT/F: Q58-K59 [33], BoNT/G: A81-A82 [37], BoNT/F5 and BoNT/H: L54-E55 [39,40], BoNT/X: R66-A67 [1 11]). Only TeNT cleaves VAMP-2 at the same site as BoNT/B (Q76-F77 [32,38]) (Figure 1).

[00139] It is known that BoNT/B needs at least 12 residues on both the N-terminal and C- terminal side of its cleavage site [93]. Schmidt et al. showed that the VAMP-2 peptide 35-75 is a suitable substrate for BoNT/D and BoNT/F [94] while BoNT/F7 produced by C. baratii requires residues 27-70 for optimal cleavage [95]. To cover also the substrate requirements of BoNT/G we choose a peptide covering aa 2-94 of VAMP-2 as the generic substrate for BoNT/B, D, F, F5/H, G, X and TeNT which was inserted into the modified construct mentioned above yielding H6tHAtevl V(2-94)L(S) in high yield and >95% purity (Figure 1 & 3).

[00140] Table 2: Endopeptidase assay of H6tHAtevlV(2-94)L(S) compared to H6trVAMP-2 2-97 in PBS pH 7.4, for 1 h at 37°C (mean ± SD; n.d., not determined).

Substrate EC50 [nM]

LC/B LC/D LC/F LC/F5 LC/G LC/T

H6trVAMP-22-97 (n=l) 60 1 2 n.d. >10,000 10

H6tHAtevlV(2-94)L (n=2-3) 160 ± 69 1.3 ± 0.35 6.0 + 2.65 n.d. >10,000 n.d.

H6tHAtevlV(2-94)LS (n=2-3) 80 ± 26 0.6 ± 0.07 2.3 ±0.35 30 ± 0 >10,000 83 ± 6 [00141] Cleavage of H6tHAtevlV(2-94)L(S) by LC/B, D, F, F5, G, and LC/T was compared with the cleavage of an N-terminal His6-tagged VAMP-2 lacking the C-terminal transmembrane domain (TMD) (H6trVAMP-2 21-97) in PBS pH 7.4. The EC ₅₀ of LC/B and LC/F for H6tHAtevl V(2-94)LS was factor 1.5-3 higher than for H6trVAMP-2 2-97 while the EC ₅₀ for LC/D was equal for both substrates. LC/F5 cleaved H6tHAtevlV(2-94)LS more efficient than LC/B and LC/T. The EC50 value for the LC/G was above the maximum concentration of LC/G. One can speculate that absence of TMD made VAMP-2 uncleavable by LC/G. The cleavage of H6tHAtevl V(2-94)LS by LC/T was decreased by factor 10 which might be attributed to the N-terminal halo-tag interfering with substrate recognition by LC/T. The order of LC activity in PBS employing H6trVAMP-2 2-97 as well as H6tHAtevlV(2-94)L is LC/D > LC/F > LC/F5 > LC/T » LC/B »> LC/G.

[00142] In conclusion, albeit cleavage of H6tHAtevlV(2-94)L(S) by LC/G is not detectable the most relevant serotypes LC/B, D, F, F5 and LC/T hydrolyze H6tHAtevlV(2-94)L(S) similarly to the H6trVAMP-2 2-97. We also analysed the cleavage of H6tHAtevlV(2-94)L(S) by the activated, full-length BoNT/B, BoNT/D and BoNT/F in the optimized JDZ-buffer (Figure 4). The EC50 values for BoNT/B, D and F are 0.07 nM, 0.2 nM and 0.03 nM (Table 3) and thus clearly in the picomolar concentration range which is required for sensitive detection methods. In different buffer conditions (PBS pH 7.4 supplemented with 20 μΜ ZnCl ₂ , 0.5 % Tween-20 and 5 mM DTT), the cleavage of H6tHAtevlV(2-94)L(S) yielded EC ₅₀ values of 2.5 nM, 30 nM, 20 nM and 0.2 nM for BoNT/Fl, LC/F 5, BoNT/H and BoNT/X, respectively (Table 4).

[00143] Example 4: Serotype specific substrates (SSS) for BoNT/A, C and E

[00144] Apart from highly sensitive detection of BoNT employing the generic substrates mentioned above, it is desirable to identify the serotype of BoNT to execute e.g. optimal medical treatment. The availability, characterisation and shelf life of immunological reagents for serotyping as well as the performance of the respective assays often leaves room for improvement. Therefore, we designed peptides that can only be cleaved by a single BoNT and called them serotype X specific substrates (SSS/X). To obtain hydrolysis by only one serotype, e.g. BoNT/A, the H6tHAtevl S(146-206)L has to be rendered insensitive to BoNT/C and BoNT/E by introducing point mutations and/or truncations either in the respective SNARE recognition motif or in the neighborhood of the scissile peptide bond. [00145] To obtain SSS/A, H6tHAtevl S(146-206)L already comprising the minimum essential peptide for BoNT/A cleavage had to be made uncleavable by BoNT/C and E. Truncating the 146-206 peptide to remove the BoNT/E cleavage site at Rl 80-1181 is not possible since BoNT/A would lose its SNARE motif. It was previously shown that the mutations D179A and D179V at the BoNT/E P2 site drastically reduces catalysis by BoNT/E [96], but the consequences on BoNT/A cleavage was not tested. Furthermore, D179 is considered as forming an important salt bridge with K224 of BoNT/E [97]. Consequently, we tried to destroy this salt bridge by charge reversal and tested the mutation D179K to prevent BoNT/E cleavage. While the cleavage rate of H6tHAtevlS( 46-206)L D179K by LC/A was marginally reduced (3 -fold) and unaltered for LC/C, the EC50 of LC/E increased -30,000-fold making D179 a suitable mutation to prevent BoNT/E cleavage. For BoNT/C, it has been shown that its PI ' residue Al 99 which also serves as P2' for BoNT/A renders SNAP-25 uncleavable by BoNT/C if mutated to arginine, respectively [98]. However, the effect of mutation A199R on BoNT/A and E cleavage has not investigated. On the other hand, a synthetic 15-mer peptide comprising the mutation A199L was not cleavable by BoNT/A [50] highlighting the critical importance of a small, aliphatic residue at position 199 for BoNT/A cleavage. We demonstrate that SNAP-25 A199R is equally well cleaved as SNAP-25 wild-type by BoNT/E and only 5-fold worse by BoNT/A. Subsequently, we inserted the mutation A199R in combination with D179K into H6tHAtevlS(146-206)L to abolish the cleavage by BoNT/C and E. The resulting substrate H6tHAtevl S(146-206)LS D179KA199R (Figure 3) turned out to be insensitive to BoNT/C and E while still being cleavable by BoNT/A (Figure 4). Compared to the generic substrate (H6tHAtevl S(146-206)LS) the EC50 of BoNT/A for SSS/A (H6tHAtevlS(146-206)LS D179KA199R, Figure 4) was increased to 0.4 nM while the EC50 of BoNT/C and E for SSS/A was increased to 200 nM and 1000 nM, respectively (Table 3). Thus, only 500-fold and 2500- fold higher concentrations of BoNT/C and E than BoNT/A would yield similar cleavage of SSS/A. The VAMP-2-cleaving serotypes do not exhibit any detectable cleavage of SSS/A at concentrations up to 1 μΜ. In conclusion, the SSS/A is able to specifically detect BoNT/A in concentrations from 0.01 nM to 10 nM.

[00146] Analogously to SSS/A the SSS/C was designed. Cleavage by BoNT/E is prevented by mutation D 179K. For BoNT/A, Vaidyanathan et al. demonstrated that mutant SNAP-25 R 198K retained full susceptibility to BoNT/C while cleavage by BoNT/A was reduced by factor 500 [51]. Previously, the Rl 98K mutation caused resistance of a synthetic 15-mer peptide towards BoNT/A. Furthermore, cleavage of the synthetic 15-mer by BoNT/A is strongly and slightly reduced due to mutation N196Q and Q197N, respectively [50]. Combining R198 and Q197A yielded a double mutant with 38,000-fold reduced EC50 of BoNT/A [99]. A recent study showed that the single mutation N196Q improved the cleavage of a synthetic 15-mer peptide by BoNT/C slightly, the mutation Q197N improved the cleavage by BoNT/C 5-fold and the mutation R198K 2.6-fold [100]. However, the corresponding triple mutant N196QQ197NR198K optimized for BoNT/C cleavage was not analyzed for cleavage by BoNT/A. Based on this data we generated the substrate H6tHAtevlS(146-206)LS D179KN196QQ197NR198 to serve as SSS/C (Figure 3) which should not be cleaved by BoNT/A and BoNT/E. Interestingly, SSS/C was cleaved better than the generic peptide H6tHAtevl S(146-206)LS (Figure 4). Its EC ₅₀ value decreased 16-fold (0.6 nM, Table 3). BoNT/A does not cleave the SSS/C at concentrations of up to 1 μΜ. Cleavage of SSS/C by BoNT/E occurred with an EC50 of ~1 μΜ, a ~1, 700-fold higher concentration than by BoNT/C. The VAMP-2-cleaving BoNTs do not cleave the SSS/C at concentrations up to 1 μΜ. In conclusion, the SSS/C is able to specifically detect BoNT/C in concentrations from 0.03 nM to 3 nM (Figure 4). [00147] BoNT/E requires the SNARE motif S4 spanning from aa 145-155 [53] for optimal cleavage of SNAP-25 [92]. The scissile bond for BoNT/E resides 17 and 18 residues upstream of those of BoNT/A and C, respectively. A C-terminal truncation clipping off the cleavage sites of BoNT/A and C should prevent cleavage by them. Accordingly, the SSS/E was generated by truncating SNAP-25 at the N- and C-terminus, resulting in the SNAP-25 based peptide 140- 197 and the SSS/E construct H6tHAtevlS(140-197)L (Figure 3). SSS/E was cleaved by BoNT/E with an EC ₅₀ of 0.2 nM which is identical to that of the generic substrate (Figure 4, Table 3). Cleavage of SSS/E by BoNT/A and BoNT/C was not detectable at concentrations up to 1 μΜ. Likewise, the cleavage of SSS/E by the VAMP-2-cleaving serotypes is absent up to concentrations of 1 μΜ (Figure 4). In conclusion, the SSS/E is able to specifically detect BoNT/E in concentrations from 0.003 nM to 1 μΜ.

[00148] Example 5: Serotype specific substrates for BoNT/B, D, F, F5/H, X and TeNT

[00149] Additionally to the generic substrate for the detection of the VAMP-2-cleaving BoNT (BoNT/B, D, F, F5/H, X) and TeNT, serotype-specific-substrates (SSS/X) susceptible only for a single serotype should be designed. Optimal proteolytic activity by BoNT/B was only observed with VAMP-2 based peptide substrates containing greater than 12 amino acid residues N-terminally and C-terminally of the cleavage site Q76-F77 [93]. The 35-residue VAMP-2 peptide 60-94 is cleaved by BoNT/B at the same rate as the larger VAMP-2 peptide 33-94 representing its helical domain [101]. The N-terminal truncation is intended to prevent the hydrolysis by BoNT/D, F and F5/H, which cleave VAMP-2 upstream of residue 60 and by TeNT, proteolysing the identical peptide bond as BoNT/B, but exhibiting the most demanding specificity by requiring the entire helical domain of VAMP-2 from aa 33-94 for optimal cleavage rates [48]. In view of that, SSS/B was obtained by inserting aa 60-94 of VAMP-2 into the modified construct yielding H6tHAtevl V(60-94)L (Figure 3). The BoNT/B EC ₅₀ for SSS/B was determined as 0.6 nM (Table 3) and thus 10-fold higher as for the generic substrate H6tHAtevlV(2-94)LS comprising the full luminal domain of VAMP-2. The SSS/B was not cleavable by BoNT/A, C, D, E, F5/H and TeNT at concentrations up to 1 μΜ while BoNT/F caused minor hydrolysis above 0.3 μΜ. In conclusion, the SSS/B is able to specifically detect BoNT/B in concentrations from 0.03 nM to 0.3 μΜ.

[00150] Contrary to SSS/B, the VAMP-2 peptide for BoNT/D as well as for BoNT/F were truncated at the C-Terminus to prevent cleavage by BoNT/B and TeNT, resulting in H6tHAtevl V(2-76)LS (Figure 3). The EC ₅₀ for LC/D, BoNT/F, LC/F5, BoNT/H and BoNT/X were determined as 6 nM, 3 nM, 30 nM, 21 nM and 1 nM, respectively, which is similar to the generic substrate H6tHAtevlV(2-94)LS. This result is congruent with data showing that the VAMP-2 peptide aa 35-75 is a suitable substrate for BoNT/D and F [94]. To obtain BoNT/D or BoNT/F specific substrates, additional mutations had to be inserted into H6tHAtevlV(2- 76)LS to prevent cleavage by BoNT/F or BoNT/D, respectively. In the case of SSS/D, residue K59 was mutated to arginine due to data of Sikorra et al. who observed no cleavage of the VAMP-2 K59R mutant by BoNT/F, but 85 % cleavage by BoNT/D [102], and of Chen et al. who described 100-fold decreased cleavage of VAMP-2 K59R by BoNT/F [103]. Cleavage of substrate H6tHAtevlV(2-76)LS K59R as SSS/D was analyzed for BoNT/A-F in JDZ-buffer (Figure 4). The cleavage of SSS/D by BoNT/D was only marginal decreased by factor 3 compared to its generic substrate. The EC50 values for cleavage by BoNT/D and F were determined as 0.6 nM and 6 nM (Table 3), a 10-fold difference between BoNT/D and F. BoNT/B as well as BoNT/A, C and E do not cleave the SSS/D at concentrations up to 1 μΜ. In conclusion, the SSS/D is able to specifically detect BoNT/D in concentrations from 0.01 nM to 1 nM.

[00151] VAMP-1 of human beings, chimpanzees, bonobos and certain rat species like Sprague- dawley is not cleavable by BoNT/D because of the mutation M48I [36,104,105]. Furthermore, Yamasaki et al. demonstrated that rat VAMP -2 M46I mutant was ~3, 500-fold less susceptible to cleavage by BoNT/D, but only 10-fold less sensitive to BoNT/F cleavage [36]. Accordingly, the homologous mutation M461 was inserted in the SSS for BoNT F. The substrate H6tHAtevl V(2-76)LS M46I to serve as SSS/F was generated and analyzed for BoNT cleavage (Figure 3). BoNT/F cleaves SSS/F with an ECso of 0.2 nM, which is 6-fold worse than the cleavage of the generic substrate H6tHAtevlV(2-94)LS (Table 3), but resembles the data obtained for rat VAMP -2 M46I. Likewise, BoNT D displays an EC50 of >1 μΜ, more than 5000-fold increased vs. the generic substrate H6tHAtevlV(2-94)LS (Figure 4) thereby confirming results of Yamasaki et al. [36]. Up to 1 μΜ of the other BoNT serotypes did not yield detectable cleavage of SSS/F. In conclusion, the SSS/F is able to specifically detect BoNT/F in concentrations from 0.03 nM to 0.3 μΜ.

[00152] Insertion of mutation A67R at the cleavage site of BoNT/X renders H6tHAtevlV(2- 94)LS A67R insensitive to BoNT/X (Table 4). Also mutation A67K, A67E, A67D, A67Q, A67 N, A67F and A67W render H6tHAtevl V(2-94)LS less susceptible to cleavage by BoNT/X. Similarly, other H6tHAtevlV(2-94)LS-derived peptides are rendered insensitive towards BoNT/X hydrolysis by mutation A67R. Also insertion of mutation R66A at the cleavage site of BoNT/X renders H6tHAtevlV(2-94)LS lOOx less susceptible to cleavage by BoNT/X. Similarly mutation R66I, R66L, R66V, R66T render H6tHAtevlV(2-94)LS less susceptible to cleavage by BoNT/X.

[00153] Table 3: EC ₅₀ of BoNT/A-F for the six serotype specific substrates (SSS/X) graphically determined from diagrams in Figure 4 (n.c. = not cleavable at concentrations up to 1 μΜ BoNT).

SSS/ GS/X n

ECso [nM]

A B C D E F

Al 0.4 n.c. n.c. n.c. n.c. n.c. 0.03 3-7

Bl n.c. 0.6 n.c. n.c. n.c. n.c. 0.07 3-7

C 200 n.c. 0.6 n.c. n.c. n.c. 10.00 3-7

BoNT/

n.c. n.c. n.c. 0.6 n.c. >1000 0.20 3-8

El 1000 n.c. 1000 n.c. 0.2 n.c. 0.20 3-8

Fl n.c. n.c. n.c. 6 n.c. 0.2 0.03 3-6 [00154] Table 4: Endopeptidase assay of different VAMP -based substrates by BoNT/H, BoNT/Fl, LC/F5 and BoNT/X in PBS pH 7.4 supplemented with 20 μΜ ZnCl ₂ , 0.5 % Tween-20 and 5 mM DTT, for 1 h at 37°C (mean ± SD, n = 1-3; n.d., not determined; n.c. = not cleavable at concentrations up to 1 μΜ BoNT).

ECso [nM]

Substrate

BoNT/H BoNT/Fl LC/F5 BoNT/X

H6tHAtevlV(2-94)LS 20 ± 0 2.5 ± 0.7 30 ± 0 0.2 ± 0.14

H6tHAtevlV(2-76)LS 21 ± 0 3.0 ± 0 30 ± 0 1.0 ± 0

H6tHAtevlV(60-94)L n.c. n.c. n.c. n.d.

H6tHAtevlV(2-94)LS A67R 10 ± 0 3.0 ± 0 n.d. n.c.

[00155] Example 6: Analysis of various reporter proteins fused to SSS/X

[00156] The generic and serotype-specific substrates were C-terminally fused to the firefly (Photinus pyralis) luciferase (luc, L; 60 kDa) acting as reporter molecule. Upon BoNT-specific cleavage of the peptide the released luc is transferred and bioluminescence is measured upon addition of luciferin, ATP and oxygen and Mg ²⁺ [87]. We also fused three other reporter molecules to the generic substrates: i) NanoLuc® Luciferase (NL) from the deep sea shrimp Oplophorus gracilirostris [106], ii) superfolder green fluorescent protein (sfGFP)[77] and iii) mCherry [107].

[00157] The NanoLuc is a small (19.1 kDa), ATP-independent luciferase that utilizes a coelenterazine substrate (furimazine) to produce bioluminescence [108]. It is much smaller while brighter than firefly luciferase and also exhibits greater physical stability.

[00158] In contrast, sfGFP does not catalyze a biochemical reaction, it just acts as fluorophore with an excitation wavelength of 485 nm and an emission wavelength of 510 nm. In comparison to GFP, sfGFP contains the 'cycle-3' mutations F99S, M153T, VI 6 A, the 'enhanced GFP' mutations F64L and S65T as well as the six novel mutations S30R, Y39N, N105T, Y145F, II 71V and A206V. sfGFP shows greater resistance to chemical denaturants and improved folding kinetics [77].

[00159] mCherry belongs to a series of mFruit fluorophores [107], mutants derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed [109]. The excitation and emission maxima of the mCherry protein are 587 nm and 610 nm, respectively, circumventing background fluorescence around 500 nm often observed in complex matrix samples. mCherry is monomelic, displays complete maturation yielding higher brightness (extinction coefficient x quantum yield), is more tolerant of N-terminal fusion proteins and much more photostable than DsRed [78].

[00160] The reporter molecules were fused to the generic substrates H6tHAlS(146-206) and H6tHAlV(2-94), respectively. Susceptibility of cleavage of H6tHAl S(146-206)LS, H6fHAlS(146-206)-sfGFP-S, H6tHAlS(146-206)-NL-S and H6tHAl S(146-206)-mCherry-S by BoNT/A, C and E was analyzed. Analogously, H6tHAl V(2-94)LS, H6tHAlV(2-94)- sfGFP-S and H6tHAlV(2-94)-mCherry-S were checked for cleavage by BoNT/B, D and F (Figure 5). BoNT/A cleavage of H6tHAl S(146-206)-NL-S is most efficient with an EC ₅₀ of 0.01 nM followed by H6tHAlS(146-206)LS, H6tHAlS(146-206)-sfGFP-S and H6tHAl S(146-206)-mCherry-S, the latter displaying factor 3 to 10 higher EC ₅₀ values than luc. With the exception of H6tHAl S(146-206)-mCherry-S, BoNT/E and C cleave all substrates similarly efficient. The cleavage of H6tHAlV(2-94)LS, H6tHAlV(2-94)-sfGFP-S and H6tHAlV(2-94)-mCherry-S by BoNT/B and F, respectively, deviates only by factor 2-4. In contrast, the fluorophores sfGFP and mCherry increase the EC50 of BoNT/D 10-fold compared to the H6tHAl V(2-94)LS (Figure 5; Table 5).

[00161] All in all, mCherry seems to slightly impair the cleavage by all six BoNTs, especially in the case of BoNT/A, C and E. In contrast, the difference in EC ₅₀ between the reporter molecules luc, NanoLuc or sfGFP is negligible compared to technical advantages provided by reporter molecules other than luc. [00162] Table 5 : Determination of the EC50 of the generic substrate peptides with different reporter molecules for BoNT/A-F.

EC50 gS/X + detector Y

[nM] luc NL sfGFP mCherry

BoNT A 0.03 0.01 0.03 0.10

B 0.07 - 0.02 0.03

C 10.00 10.00 20.00 40.00

D 0.20 - 1.00 2.00

E 0.20 0.20 0.10 0.60

F 0.03 - 0.06 0.06 Table 6: Peptides and polypeptides of the present teaching

YSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSS FNKYIGEY

KQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPV TA

NILDDNWDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDG RSLY

NKTLDCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSK NTSEHGQ

LDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQ LSDNVEDFTFTRSIEEALDN

SAKVYTYFPTLANKVNAGVQGGLFLMWANDWEDFTTNILRKDTLDKISDVSAIIPYI GPALNI

SNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLE QRIKRWKD

SYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQ VENLK

NSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHN IILVGEVD L AKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEV SE

EGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSN LPGYTIi

DSVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNM MGNM

KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFA KELDGKDINILF

NSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGY KIIIK

RIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTK DINDNII

FQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGN YASLL

ESTSTHWGFVPVSE

CAA38175 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK PPR

PTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTP EDTF

DFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFE GFGTLSIL

KVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRI RPQVSE

GFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHY DIAKRLNNINKTIPSSWISNI

DKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEWYSSQYNVKNRTHYFS RHYL

PVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESWDLFTKVCLR LTKNSR

DDSTCIKVKNNRLPYVADKDSiSQEIFENKIITDETNVQNYSDKFSLDESILDGQVP INPEIVDP

LLPNVNMEPLNLPGEEIVFYDDIT YVDYLNSYYYLESQ LSNNVENITLTTSVEEALGYSNKI

YTFLPSLAEKVNKGVQAGLFLNWANEWEDFTTNIMKKDTLDKISDVSVIIPYIGPAL NIGNSA

35 BoNT/D

LRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWK DSY

QWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVE NLKNS

LDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNII LVGEVDRLK

A VNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRV GD

NVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEY TIINSIEQN

SGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMK LYINGE

LKQSQKIEDLDEVKLD TIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKD

YWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKN PYSRILN

GDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIK NIVSKNK

YCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDP GWVE

CAA43999 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNG

36 BoNT/EI

DSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQF HIG

DASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGSIAIV TFSPEYS

FRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFG

GTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDA SGIYSVN

INKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNI NNLKVNF

RGQNANLNPRIITPITGRGLVK IIRFC NIVSVKGIR SICIEINNGELFFVASENSYNDDNINT

PKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTS DIEQHD

VNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAA LFVSWI

QQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALELLGAGIL LEFEPELL

IPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFN KRKEQMY

QALQNQVNAIKTIIESKYNSYTLEE NELTNKYDI QIENELNQKVSIAMNNIDRFLTESSISYL

MKIINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKL SSYTDDKI

LISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYN DKLSEV

NISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLN HNEIIWT

FEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILN LGNIHVSD

NILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDK EYYLLNVL

KPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKV IQRVNNSSTNDNLVRKNDQVYINFVASK

THLFPLYADTATTNKEKTIKISSSGNRFNQVWMNSVGNCTMNFKNNNGNNIGLLGFK ADTV

VASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

CAA57358 MPWINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNNA/VIIPERNTIGTDPSDF DPPA

SLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHT PINE

FHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSN DGFGSI

NIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKET IKVKQAPL

MIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYD INEYKDYF

QWKYGLDKNADGSYTVNENKFNEIY KLYSFTEIDLANKFKV CRNTYFIKYGFLKVPNLLD

DDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTK APPRLCIR

VNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISN QTLNTLV

QDDSYVPRYDSNGTSEIEEHNWDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQ VYTFF

SSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNI GNEVQKEN

37 BoNT/F1

FKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKE IYSWIVS

NWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIR EELNK VSLAMENIERFITESSIFYLMKLINEA VS LREYDEGVKEYLLDYISEHRSILGNSVQELNDL

VTSTLNNSIPFELSSYTNDKILILYFNKLY KIKDNSILDMRYENNKFIDISGYGSNISINGDWIY

STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTI IDCIRNN

NSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNS RIYINGNL

IDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPD PSILKDFW

GNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVII RKNGST

DISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQII VMDSIGNNC

TMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN

MPVEINSFNYDDLVNDNTILYIRPPYYERSNTYFKAFNIMENNA/VIIPERYRLGIE ASKFDPPDS

LKAGSDGYFDPNYLSTNTEKNRYLQIMIKLFKRINSNEAGKILLNQIKDAIPYLGNSYTA EDQF

TTNNRTISFNVRLANGTIEQEMANLIIWGPGPDLTTNRTGGTTYTPAQSLEAIPYKE GFGSIM

TIEFSPEYATAFNDISLTSHAPSLFIKDPALILMHELIHVLHGLYGTYTTGFKIKPN ITEPYMEVT

KPITSGEFLTFGGNDVN IPQLIQSQLRS VLDDYE IASRLN VNRATAEINIDKF YSYQL

YQFVKDSNGVYSVDLDKFNKLYDKIYSFTEFNLAHEFKIKTRNSYLAKNFGPFYLPN LLDNSI

YNEADGFNIGDLSVNYKGQVIGSDIDSIKKLEGQGVVSRVVRLCLNSSFKKNTKKPL CITVNN

GDLFFIASEDSYGEDTINTPKEIDDTTTLVPSFKNILDKVILDFNKQVTPQIPNRRI RTDIQEDN

YIPEYDSNGTSEIEEYNVVDLNAFFYLHAQKVPEGETNISLTSSIDTALSEESKVYT FFSSEF1

DTINEPVNAALFIDWISKVIRDFTTEATQKSTVDKIADISLIVPYVGLALNIVNETE GNFKEAFE

38 BoNT/F5 ADA79559

LLGAGILLEFVPELAIPVILVFTIKSYIDSYENKNKIIKAINNSLIEREAKWKEIYSWIV SNWLTRIN

TQFNKRKEQMYQALQNQVDAIKTAIEYKYNNYTSDEKNRLESEYNINNIEEELNKKV SLAMK

NIERFITESSISYLMKLINEAEVGKLKEYDKRVKRHLLEYIFDYRLILGEQGGELID LVTSTLNTS

IPFELSSYTNDKILIIYFNRLYKKIKDSSILDMRYENNKFIDISGYGSNISINGNVY IYSTNRNQF

GIYDDRLSEVNIAQNNDIIYNSRYQNFSISFWVRIPKHYRPMNHNREYTIINCMGNN NSGWKI

SLRTTGDCEIIWTLQDTSGNKKKLIFRYSQLGGISDYINKWIFVTITNNRLGNSRIY INGNLIVE

KSISNLGDIHVSDNILFKIVGCDDKMYVGIRYFKVFNTELDKTEIEILYSNEPDPSI LKDYWGNY

LLYN KYYLLNLLRNDKYITRNSDILNISHQRGVTKDLFIFSNYKLYEGVEVIIRKNGPIDISNT D

NFVRKNDLAYINWDHGVEYRLYADISITKPEKIIKLIRRSNPDDSLGQIIVMDSIGN NCTMNFQ

NNNGGNIGLLGFHSDNLVASSWYYNNIRRNTSSNGCFWSFISKEHGWQE

MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPD QFNAST

GVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG NASTPP

DKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPI SEGFGAR

MMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNL PITPNTK

EFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQG SGIDIS

LYKQIY NKYDFVEDPNGKYSVD DKFDKLY AL FGFTETNLAGEYGI TRYSYFSEYLPPI

KTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCK PVMYKN

TGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYNTQNNTIENNFSIDQLILDND LSSGIDLP

NENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQLTNSLN DALRNNN

39 BoNT/G Q60393 KVYTFFSTNLVEKANTWGASLFVNWVKGVIDDFTSESTQKSTIDKVSDVSIIIPYIGPAL NVG

NETA ENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNAL KRDQKWTD

MYGLIVSQWLSTVNTQFYTiKERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINID FNDIDFK

LNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELY LLDEVNILK

SKVNRHLKDSIPFDLSLYT DTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGS

DVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQT YLQNE

YTIISCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSIT ITNDRLGNAN

IYINGSLKKSEKILNLDRINSSNDIDF LINCTDTTKF\/WIKDFNIFGRELNATEVSSLYWIQSST

NTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLY LGLR

FIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRNA LVNSKEIQTQLFLAPINDDPTFYDVL

NKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVS IDKFRIF

CKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLT NAPS

YTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPK DGNAFNN

LDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGN DPNRD

ILIASNWYFNHL D ILGCDWYFVPTDEGWTND

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLN

IPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPL PLVSNGALT

LSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLSEVSF SPFYLKP

FDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFDTG KIETS

RQQNSLIFEELLTFGGIDS AISSLIIKKIIETA NNYTTLISERLNTVTVENDLLKYIKN IPVQG

RLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDN SYSTLEGF

NISSQGSNDFQGQLLESSYFEKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNNID LEDKKTT

SKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKDKLPPQ DITLSNYD

FTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESID SSKIRVELTD

SVDEALSNPNKVYSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVI DKSSDTL

WP_045538

42 BoNT/X AIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQV EAIVNNA

952

LDKRDQKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI D

DKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLE TKKTLDK

FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEV LNLGAEDG

KIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKP

TNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYIAFNGWNLI TITNNRSK

GSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQN EVVKLYN

YYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITF PNNIRY

GALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTT HDLTTD

FEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYEN LNLR

KHTKTNWYFIPKDEGWDED

MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQL ERI

AAH 18249. EEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPAR

75 SNAP-25

1 WDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALD GNEIDTQNRQIDRI

MEKADSNKTRIDEANQRAT MLGSG

NP_055047 MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKL

76 VAMP-2

.2 SELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

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