New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates

Journal of Immunological Methods 343 (2009) 21–27

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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

Research paper

New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates Russell G.A. Jones ⁎, Yvonne Liu, Dorothea Sesardic Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire. EN6 3QG, UK

a r t i c l e

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Article history: Received 15 October 2008 Received in revised form 7 January 2009 Accepted 8 January 2009 Available online 25 January 2009 Keywords: Enzyme Endopeptidase Endoprotease Botulinum toxin Light chain Neurotoxicity Neoepitope Trypsin Immunoassay

a b s t r a c t Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, C1 and E intra-cellularly cleave SNAP25 and/or Syntaxin (type C1 only) resulting in a flaccid paralysis. Although highly sensitive, robust in vitro endopeptidase immunoassays have been developed for some serotypes, an endopeptidase immunoassay for type C1 has not previously been described. The current studies utilised solid phase synthesized SNAP25(137–206) peptide substrate, and a new specific antibody to the SNAP25(191–198) octapeptide epitope that becomes exposed following cleavage by type C1 toxin. The highly specific nature of the detecting antibody was illustrated by the failure of antiSNAP25(191–198) to recognise the type A cleavage product which differs by just one amino acid residue. Conversely, anti-SNAP25(190–197), which recognises the type A cleavage product, fails to cross react with the type C1 toxin cleavage product. Utilising Syntaxin(232–266) peptide substrate, and a specific antibody to the cleavage product epitope, Syntaxin(254–261), it was also possible to develop an endopeptidase immunoassay. Assay sensitivities allowed the detection of less than 0.1 LD50/ml (25 pg/ml) of type C1 haemagglutinin-complexed toxin. The assay failed to detect toxin serotypes A, B, D, E, F or G and therefore also provides an alternative highly specific in vitro identity test. In the absence of trypsin inhibitors, the assay is also capable of detecting 2 pg/ml of trypsin activity, or trypsin like contaminants. These new immunoassays will therefore provide highly specific tools for monitoring botulinum toxin light chain endopeptidase activity and serotype identity. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Botulinum toxins are composed of a heavy and a light chain linked together by disulphide bonds. The heavy chain is responsible for binding pre-synaptically to neuronal cells and facilitates internalisation (Montecucco et al., 1994). Once inside the cell, the light chain transverses the membrane of the endocytolic vesicles and cleaves specific SNARE proteins within the cytoplasm essential for the docking and fusion of neurotransmitter containing vesicles at the nerve terminal

Abbreviations: BoNT/C1, Botulinum neurotoxin type C1; SNAP25, Synaptosomal associated protein of molecular mass 25 kDa; VAMP, Vesicle associated membrane protein, or Synaptobrevin. ⁎ Corresponding author. Fax: +44 01707 663796. E-mail address: [email protected] (R.G.A. Jones). 0022-1759/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2009.01.001

(Simpson, 2004; Schiavo et al., 2000). Subsequently, the extracellular release of neurotransmitter into the neuromuscular junction is blocked and results in a flaccid muscular paralysis. The botulinum toxin light chains are zinc dependant endopeptidases that specifically cleave one of three soluble SNARE proteins. Type A, C1 and E toxins cleave SNAP25(1–206), at positions Q197–R198, R198–A199 and R180–I181 respectively. The remaining botulinum toxin serotypes cleave VAMP at positions Q76–F77, K59–L60, Q58–K59, and A81–A82 (Type B, D, F and G toxins respectively). Type C1 toxin is unique in that it is the only serotype with two specific substrates and cleaves Syntaxin at position K253–A254 as well as SNAP25 (Schiavo et al., 1995). Botulinum toxin can cause a life-threatening, paralysing disease called botulism; however, low doses of the toxin are commonly used therapeutically to locally paralyse specific muscles for clinical or cosmetic benefit. The toxin is now

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considered the treatment of choice for disorders such as blephrospasm, strabismus and dystonia (Coffield et al., 1997). Types A and C1 induce the longest lasting muscle paralysis both in animals and man and, although BoNT/A is the most widely used therapeutic serotype, BoNT/C1 shows promise as a suitable alternative for unresponsive patients (Eleopra et al., 2006; Foran et al., 2003; Coffield et al., 1997). New retargeted botulinum toxin LHn fragments are also being developed to target pain, respiratory diseases, neuroendocrine cancer and obesity using a range of different serotypes including type C1 (Foster et al., 2006). Naturally occurring botulism in humans is most commonly associated with BoNT/A, B and E toxins. However, suspected or confirmed cases of type C1 botulism affecting humans have also occasionally been reported (Prevot et al., 1955; Oguma et al., 1990). BoNT/C1 is however, frequently associated with outbreaks of botulism in cattle, horses, foxes, mink and birds (Moeller et al., 2003; Galey et al., 2000; Wobeser et al., 1997; Frey et al., 2007; Lindstrom et al., 2004; Martinez and Wobeser, 1999; Rocke et al., 2000; Myllykoski et al., 2008). Cattle are reported to be 13 times more sensitive than mice on a weight for weight basis and current tests are often insufficiently sensitive to detect the toxin (Moeller et al., 2003). Endopeptidase immunoassays have previously been described for tetanus and botulinum type A, B, and E toxins (Hallis et al., 1996; Ekong et al., 1997; Sesardic et al., 2000; Shone et al., 2006; Kegel et al., 2007; Jones et al., 2008). However, due to the close proximity of the SNAP25 cleavage sites between type A and C1 toxins, which differ by just one amino acid, it has never previously been considered possible to distinguish them using cleavage site specific antibodies. This work describes the development of a highly specific, new endopeptidase immunoassay suitable for monitoring BoNT/ C1 or novel light chain related products. Due to the complete lack of cross reactivity with other toxin serotypes, it may also prove to be a useful, new identity or detection test in veterinary or other applications.

sheep and rabbits, respectively, following conjugation to KLH through the Cys residue. Sheep were initially immunised with Freunds complete adjuvant and then at four week intervals with Freunds incomplete adjuvant. Antibodies to SNAP25 (190–197) were raised as described previously (Jones et al., 2008). ELISA peptide binding titres were then measured. Polystyrene 96 well plates (Nunc Maxisorp) were coated with either 100 μl/well of 2 μg/ml SNAP25(191–198) or Syntaxin (254–261) in 50 mM carbonate buffer, pH 9.6, overnight at room temperature (RT), decanted and blocked with 300 μl/ well of 2.5% skimmed milk powder in phosphate buffered saline, 0.05% Tween (M-PBST) for 90 min at RT. Plates were then washed 3 times with PBST and dried. Serum samples were diluted with M-PBST and incubated for 90 min at RT, washed, and HRP labelled antibody added (rabbit anti-sheepHRP, Pierce 31480, 1in4000 dilution, or goat anti-rabbit-HRP conjugate, Sigma A0545, 1in2000 dilution in M-PBST), incubated, washed and developed. 2.4. Botulinum toxins All concentrated botulinum toxins were safely handled within a Class I safety cabinet. 2.4.1. Botulinum type C1 toxin Purified haemagglutinin complex toxin was obtained from Metabiologics (USA) at 1 mg/ml (4 × 106 LD50/ml, from Brazil strain) and diluted to 20,000 mouse LD50 /ml in Gelatine (0.2% w/v) Phosphate (50 mM di-sodium hydrogen orthophosphate) Buffer pH6.5 (GPB), aliquoted and stored frozen at −40 °C.

2. Materials and methods

2.4.2. Botulinum type A, B, D, E, F, and G toxins Purified haemagglutinin free type A, B and E toxins were prepared as described previously (Jones et al., 2008), and serotypes D, F and G complex toxins were also obtained from Metabiologics (USA) at 1 mg/ml and appropriately diluted and stored. Type G complex toxin was trypsinised as previously described for type E toxin (Jones et al., 2008).

2.1. SNAP25 peptide substrate

2.5. Toxin endopeptidase assay

A 70 amino acid SNAP25(137–206) peptide (VTNDARENEM-DENLE-QVSGI-IGNLR-HMALD-MGNEI-DTQNRQIDRI-MEKAD-SNKTR-IDEAN-QRATK-MLGSG) was synthesised as previously described (Jones et al., 2008). A 35 amino acid Syntaxin(232–266) peptide (RIEYN-VEHAVDYVER-AVSDT-KKAVK-YQSKA-RRKKI-C) of 85% purity (by HPLC) with expected mass of 4282.9 Da and found mass of 4281.6 Da (by Mass Spectrometry) was custom synthesized by Immune Systems Ltd, (Paignton, UK).

Polystyrene 96 well plates (Nunc Maxisorp) were coated with either 100 μl/well of 3 μg/ml SNAP25(137–206) or 50 μl/ well of 5 μg/ml Syntaxin(232–266) substrate in 50 mM carbonate buffer, pH9.6, overnight at RT, decanted and blocked with 300 μl/well of 5% skimmed milk powder PBST for 90 min at RT. Plates were then washed 3 times with distilled water and dried. Toxins were suitably diluted in reaction buffer and doubling dilutions performed on a substrate (SNAP25 or Syntaxin) coated plate in reaction buffer (50 mM HEPES, 20 μM ZnCl2, pH 7.0) containing 5 mM DTT and either with or without 1 mg/ml BSA (fatty acid free, globulin free, Bovine Serum Albumin, Sigma, A0281) and 0.5% Tween 20. Plates were then individually sealed and incubated at either 37 °C or RT as indicated (without stacking) for 18 h. Plates were then washed 3 times with PBST, blot dried, and 100 μl/well of primary detecting antibody, either sheep serum anti-SNAP25(191–198) at 1in800 or rabbit serum anti-Syntaxin(254–261) at 1in200 in 2.5% (w/v) skimmed milk powder PBST (antibody buffer) added, sealed individually and incubated at RT for 90 min without stacking. Plates were washed again and 100 μl/well of rabbit anti-sheep-

2.2. Peptide immunogens SNAP25(191–198) and Syntaxin(254–261) with a Cysteine tag (C-RIDEANQR and AVKYQSKA-C, respectively) were custom synthesized by Immune Systems Ltd, (Paignton, UK). 2.3. Primary detecting antibodies BoNT/C1 cleavage site-specific anti-peptide SNAP25(191– 198) and anti-Syntaxin(254–261) antibodies were raised in

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HRP (Pierce 31480, 1in4000 dilution in antibody buffer) or goat anti-rabbit-HRP conjugate (Sigma A0545, 1in2000 dilution) added and incubated as before. After washing 100 μl/well substrate solution (50 mM citric acid, pH4.0, 0.05% w/v ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and 0.05% v/v of a 30% w/v Hydrogen peroxide solution) was added and the colour allowed to develop at RT for 30–60 min. Following colour development the plates were briefly shaken and absorbance read at 405 nm using a suitable plate reader (Multiscan plate reader). Relative activities in test samples were typically calculated by parallel line analysis. 2.6. Trypsin, trypsin inhibitor and antitoxin Soyabean trypsin inhibitor (type I-S, Sigma, T9003) was added to the assay buffer to give a concentration of 1 μg/ml. Trypsin (TPCK treated, Sigma T8642) was used as indicated. A fixed C1 toxin concentration (10 LD50/ml) was mixed and incubated for 60 min at 37 °C with a variable concentration of type C antitoxin (monovalent botulinum type C, equine antitoxin, NIBSC reference number 01/508, Jones et al., 2006), and then transferred to a SNAP25 coated plate and the endopeptidase activity measured as described previously using the optimised procedure. 3. Results 3.1. Primary detecting antibodies Primary detecting antibodies were initially screened by ELISA binding assay. Two sheep were immunised with the SNAP25(191–198) conjugated peptide and their anti-peptide levels monitored (Fig. 1). A large difference in the strength of response was found between the two sheep even after a prolonged immunisation schedule that is typical when immunising with short peptides. Serum from sheep 2 at week 18 was selected for use in endopeptidase assay testing. Antibody responses from rabbits immunised with conjugated Syntaxin(254–261) were also assessed and the maximal titre sample selected (data not shown).

Fig. 2. Optimisation of Syntaxin endopeptidase assay conditions. (a) Effects of different assay buffers, with or without 0.5% v/v Tween 20 and 1 mg/ml BSA. (b) Effect of incubation temperature, at either 37 °C ■, or RT ● for 18 h in the absence of Tween and BSA (±SD, n = 8). (c) Endopeptidase assay limits of detection in the absence of Tween and BSA, BoNT/C1 (●) ± SD, n = 6 and background (n = 24) + 2SD dashed line, following incubation for 18 h at 37 °C.

3.2. Syntaxin endopeptidase assay optimisation

Fig. 1. ELISA SNAP25(191–198) peptide binding antibody levels. Sheep antiSNAP25(191–198) serum samples obtained from animal one (open symbols, broken line) and animal two (solid symbols and line) at the following time points: Week 0 (x), 6 (▲), 10 (▼), 14 (♦), 18 (●), and 22 (■).

Although no difference in sensitivity was seen either with or without Tween 20 alone, the combined presence of BSA (1 mg/ml) and Tween or BSA alone caused a decreased endopeptidase activity (~ 28 and ~ 6.5 fold respectively, Fig. 2a). Incubating at 37 °C in the absence of Tween or BSA for 18 h (overnight) increased the sensitivity of the assay ~ 30 fold compared to RT (Fig. 2b). Using these optimal conditions for the syntaxin assay, the background mean + 2SD gave a limit of detection of 4 LD50/ml (1 ng/ml) (Fig. 2c).

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3.3. SNAP25 endopeptidase assay optimisation A three fold increased BoNT/C1 enzymatic activity against SNAP25 was found with the addition of BSA (1 mg/ml, Fig. 3a). When a combination of BSA and Tween 20 were used further increases were evident (10 fold, Fig. 3a). Tween 20 alone was however, found to be the most sensitive assay system and ~ 60 fold greater than the control (without Tween or BSA). Incubating at RT for 18 h decreased the sensitivity of the assay (~ 10 fold) compared to 37 °C (Fig. 3b). Sodium chloride (0.2%) was found to inhibit the effects of BoNT/C1 (data not shown). Using these optimal conditions for the SNAP25 assay,

the background mean + 2SD gave a limit of detection of less than 0.1 LD50/ml (25 pg/ml) (Fig. 3c). 3.4. SNAP25 endopeptidase assay specificity for type A and C1 toxins Despite just one amino acid difference in the cleavage site between A and C1 toxins, no cross reactive binding was found between toxin A cleaved SNAP25 and anti-SNAP25(191–198) antibody (Fig. 4a). Conversely it was also confirmed that antiSNAP25(190–197) failed to bind type C1 cleaved SNAP25 (data not shown, see Jones et al., 2008). 3.5. Effects of type B, D, E, F, G toxins and trypsin on the SNAP25 endopeptidase assay No cross-reactivity was seen in the endopeptidase assay using type B, D, or F toxins at up to 20,000 LD50/ml (Fig. 4b). A positive signal was detected when trypsinised type E and G toxins were used (data not shown), but when 1 μg/ml soyabean trypsin inhibitor was included in the assay buffer either no endopeptidase activity (Fig. 4c) or only partial activity seen (Fig. 4d). The soyabean trypsin inhibitor however had virtually no effect on the control type C1 toxin endopeptidase activity (Fig. 4d) and the effect was confirmed by the 120 fold inhibition of trypsin activity (Fig. 4e). The type C1 assay was found to be highly sensitive for trypsin and could detect as little as 2 pg/ml in the absence of trypsin inhibitors (Fig. 4e). 3.6. Antitoxin neutralisation Specificity was also confirmed by a dose dependant neutralisation of the endopeptidase activity of type C1 toxin with a monospecific type C antitoxin (Fig. 5). 4. Discussion

Fig. 3. Optimisation of SNAP25 endopeptidase assay conditions. (a) Effects of different assay buffers, with or without 0.5% v/v Tween 20 and 1 mg/ml BSA at 37 °C. (b) Effect of incubation temperature, at either 37 °C (■), or RT (●), for 18 h in the presence of Tween (± SD, n = 8). (c) Endopeptidase assay limits of detection in the presence of Tween, BoNT/C1 (●) ± SD, n = 6 and background (n = 24) + 2SD dashed line, following incubation for 18 h at 37 °C.

New highly specific SNAP25 (191–198) and Syntaxin (254– 261) neoepitope detecting antibodies were raised and utilised in these studies to develop endopeptidase immunoassays for both BoNT/C1 neuronal substrates. The new type C1 toxin assays were found to be optimal at 37 °C for 18 h with both substrates in the absence of BSA. By contrast, the endopeptidase activity of BoNT/A, previously reported to be maximal at 37 °C over 90 min (Cai and Singh, 2001; Jones et al., 2008), when performed over 18 h in the presence of BSA and Tween was found to be optimal at RT (Jones et al., 2008). This may perhaps indicate a more temperature stable C1 toxin light chain or an interfering effect of BSA seen over longer incubation times at 37 °C. Differences between the optimal conditions with the Syntaxin and SNAP25 C1 assays were, however, found. With the Syntaxin assay no major difference was seen with or without Tween and in contrast to the SNAP25 C1 assay a combination of BSA and Tween had a strong inhibitory effect. Sodium chloride was also shown to have a strong inhibitory effect on BoNT/C1 in the SNAP25 assay. These findings may help to explain some of the contradictory C1 toxin data seen in the literature in which SNAP25 cleavage could only be shown in intact neuronal cells (Foran et al., 1996; Williamson et al., 1996).

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Fig. 4. Specificity of the BoNT/C1 SNAP25 endopeptidase assay. (a) The ability of anti-SNAP25(191–198) to differentiate between a single amino acid difference in the SNAP25 cleavage point, BoNT/C1 (●) and BoNT/A (■) after 18 h at 37 °C in the presence of Tween (±SD, n = 4). (b) BoNT/B (■), BoNT/D (▲) and BoNT/F (▼) and control BoNT/C1 (●) ± range, n = 2. (c) BoNT/E (■) and control BoNT/C1 (●) in the presence of 1 μg/ml soyabean trypsin inhibitor. (d) BoNT/G (▲) and control BoNT/ C1 (●) in the presence of 1 μg/ml soyabean trypsin inhibitor and control BoNT/C1 in the absence of inhibitor (□, dashed line). (e) Trypsin dose response curve in the presence (●) and absence (■) of trypsin inhibitor.

Currently used type C1 assays have been described as lacking the desired sensitivity for certain applications (Moeller et al., 2003). To our knowledge, this endopeptidase assay is the most sensitive type C1 assay described, with a limit of detection of just 0.1 LD50/ml compared to 1 LD50 by mass spectrometry (Boyer et al., 2005), and ~0.5 LD50/ml by the mouse local flaccid paralysis assay (Sesardic et al., 2004). The current assay takes a total of 24 h to be performed using pre-coated substrate plates, compared to 72–96 h for the mouse LD50, 48 h for the mouse local flaccid paralysis assay (Sesardic et al., 2004), and 17 h reaction incubation, prior to

spotting, drying, loading and generating spectra for mass spectrometry (Boyer et al., 2005). The highly specific nature of the detecting antibody was clearly shown by the failure of anti- SNAP25(191–198) to cross react or recognise the BoNT/A cleavage product which differs by just one amino acid residue and highlights the outstanding specificity of this C1 endopeptidase immunoassay. Likewise, type A cleavage site specific anti-SNAP25(190–197) failed to recognise the C1 cleaved peptide. With the new C1 assay no cross reactivity was found with any of the botulinum toxin serotypes (A, B, D, E, F and G) and therefore serotype identity

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Fig. 5. Antitoxin neutralisation of BoNT/C1 endopeptidase activity. A fixed toxin concentration (10 LD50/ml) was mixed and incubated for 60 min at 37 °C with a variable concentration of monovalent type C antitoxin and the SNAP25 endopeptidase activity measured as described previously (±range, n = 2).

was also obtained. This is in contrast to fluorescently labelled FRET SNAP25 assays which cannot easily distinguish between type A and C1 toxins (or contaminating trypsin-like enzymes) without an additional separation step (Hunt et al., 2008). The specificity of the BoNT/C1 assay was also confirmed by its dose dependent inhibition by type C antitoxin. Both, type C1 toxin and trypsin, share the same cleavage point. This was noted when testing trypsinised type E and G toxins which required the presence of extra soyabean trypsin inhibitor to specifically block the trypsin activity without affecting the assays ability to detect type C1 toxin endopeptidase activity. The SNAP25 C1 assay, therefore, also proved to be a highly sensitive trypsin detection system capable of measuring as little as 2 pg/ml. Although performed over a longer time course, the new assay is of equivalent sensitivity to other published trypsin assays (Koritsas and Atkinson, 1995; Lee et al., 2008). The new assay also provides a convenient way to detect trypsin like impurities in therapeutic botulinum toxin products, such as those found in serum albumin (data not shown) and as implied by Hunt et al. (2008). Identical SNAP25 substrate-coated plates can also now conveniently be applied to measure any of the three botulinum toxin serotypes (A, C1 or E) which all cleave this substrate and can be specifically detected when using the appropriate neoepitope detecting antibody. This assay system, therefore, offers the convenience of conventional immunoassays without the need for expensive specialist equipment, making it easily transferable. If used with a specific reference material, it should be possible to accurately monitor the relative enzymatic activity of any new type C1 therapeutic products. It may also provide a sensitive assay for detecting any contaminating trypsin-type activity in a wider range of therapeutic products. In summary this work describes a highly specific, new endopeptidase immunoassay suitable for monitoring BoNT/C1 or novel light chain related products. Due to the complete lack of cross reactivity with other botulinum toxin serotypes, it also provides a useful, new identity or detection test. References Boyer, A.E., Moura, H., Woolfitt, A.R., Kalb, S.R., McWilliams, L.G., Pavlopoulos, A., Schmidt, J.G., Ashley, D.L., Barr, J.R., 2005. From the mouse to the mass spectrometer: detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry. Anal. Chem. 77, 3916.

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