Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.     

A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A.

Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A). We were able to detect BoNT/A in the femtogram (10(-15)g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay. For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg. These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. The assays we developed are currently the most sensitive methods for detecting BoNT/A.     

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC₅₀s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC₅₀ of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.     

A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10⁻¹¹ M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested.     

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer.

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5–1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE’s in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.     

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.     

An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product

Tetanus neurotoxin (TeNT¹) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1 pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.     

Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ～0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.     

Determination of caderofloxacin lactate in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) method for the quantification of a newly active quinolone carboxylic acid caderofloxacin lactate in rat plasma was developed and validated after precipitation method with methanol. Chromatographic separation was achieved on a reversed-phase Shimadzu 2.0 μm C18 column (150 mm × 2.00 mm) with the mobile phase of methanol–0.02% formic acid and step gradient elution resulted in a total run time of about 10.0 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (5–2000 ng/mL) (r = 0.9998). The lowest limit of quantification (LLOQ) was 5 ng/mL and the lowest limit of detection (LLOD) was 2 ng/mL. Average recoveries ranged from 88.80 to 93.05% in plasma at the concentrations of 10, 100 and 1000 ng/mL. Intra- and inter-day relative standard deviations were 4.01–7.30 and 4.15–7.51%, respectively. This method was successfully applied in the pharmacokinetic studies in rats.     

Characterisation and physical stability of PEGylated glucagon

Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay.

Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon–PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular β-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular β-sheet was less apparent for glucagon–PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide–peptide interactions.

Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for -helix shifted to 1664 cm⁻¹, which could possibly be explained by formation of 3₁₀-helix. Neither 3₁₀-helix nor intermolecular β-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra.

In conclusion, glucagon–PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon.     

A simple high-performance liquid chromatographic method for the measurement of 2-methylsulfonylpyridine in plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method for the analysis of 2-methylsulfonylpyridine (2-MSP) in plasma has been developed. Up to 1 mL of plasma containing 2-MSP and an internal standard was extracted with 3 mL of methylene chloride, usually twice, evaporated to dryness, resuspended in mobile phase, and chromatographed on two 15-cm C8 reversed-phase LC columns in series. The mobile phase was 5% acetonitrile in water with a flow rate of 1 mL/min and ultraviolet detection was at 260 nm. Extraction of plasma produced no interfering endogenous components and the recovery of 2-MSP was 60-70%. The intraday and interday statistics of 2-MSP standard curves from 10 to 320 ng in plasma demonstrate that the assay is sensitive and precise using either human or monkey plasma and any plasma volume up to 1 mL.     

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.     

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20–135.00 ng/mL for free propranolol and 0.20–25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00–500.00 ng/mL for total propranolol and 1.00–360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (−15 °C)–thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 °C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.     

重组蝎毒抗神经兴奋肽的ELISA分析方法建立及稳定性的初步研究

目的建立重组蝎毒抗神经兴奋肽(ANEP)的ELISA分析方法并对ANEP的稳定性进行初步研究。方法以分别建立的间接ELISA和间接竞争ELISA分析方法进行ANEP含量测定,并通过方法学的优化,最终以建立的间接法ELISA方法测定了温度、pH值、反复冻融、超声对ANEP稳定性的影响。结果间接ELISA相对于间接竞争ELISA有着更好的线性关系和灵敏度,线性范围0.025~1.60μg/mL。检测限为10 ng/mL。稳定性实验表明ANEP在37,60℃的条件下放置24 h含量基本不变,pH 5~11的缓冲液中保持稳定,反复冻融、超声4 min后含量降低。结论所建立的间接ELISA方法能很好的用于ANEP的测定。ANEP的热稳定性较好,强酸性下不稳定,对反复冻融与超声等稳定性较差。     

High-performance liquid chromatographic assay for morphine in small plasma samples: application to pharmacokinetic studies in rats

In order to perform a reliable pharmacokinetic study of morphine during subchronic treatment in rats, an easy, rapid, sensitive and selective analytical method was proposed and validated. The analyte and internal standard (naloxone) were extracted from plasma samples (100 μL) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography (HPLC) along with electrochemical detection (ED). Standard calibration graphs were linear within a range of 3.5–1000 ng/mL (r = 0.999). The intra-day coefficients of variation (CV) were in the range of 5.8–8.5% at eight concentration levels (7–1000 ng/mL) and the inter-day coefficient of variation ranged from 7.4 to 8.8%. The intra-day assay accuracy was in the range of −5–10% and the inter-day assay accuracy ranged from 3.0 to 9.3% of relative error (RE). The limit of quantification was 3.5 ng/mL using a plasma sample of 100 μL (15.8% of CV and 12.8% of RE). Plasma samples were stable for 2 months at −20 °C. This method was found to be suitable for pharmacokinetic studies in rats, after subcutaneous administration of morphine (5.6 mg/kg per day) in subchronic treatment for 6 and 12 days.     

Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease*

Abstract: Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X‐ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate‐induced conformational changes for enzyme activation, we synthesized a series of transition‐state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17‐mer peptide corresponding to residues 187–203 of SNAP‐25. A more stable substrate, Nle²⁰²SNAP‐25 [187–203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time‐dependent inhibition. α‐Thiol amide analogs of Gln‐197 were incorporated via solid‐phase peptide synthesis into both 17‐mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the α‐thiol amide analogs of holotoxin A substrate are described. These substrate‐derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.     

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.     

Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.     

Specific and sensitive analysis of nefopam and its main metabolite desmethyl-nefopam in human plasma by liquid chromatography-ion trap tandem mass spectrometry

A specific and sensitive liquid chromatography–tandem mass spectrometric (LC–MS–MS) method using an ion trap spectrometer was developed for quantitation of nefopam and desmethyl-nefopam in human plasma. Nefopam, desmethyl-nefopam and the internal standard (ethyl loflazepate) were extracted in a single step with diethyl ether from 1 mL of alkalinized plasma. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v:v). It was delivered at a flow-rate of 0.3 mL/min. The effluent was monitored by MS–MS in positive-ion mode. Ionisation was performed using an electrospray ion source operating at 200 °C. Nefopam and desmethyl-nefopam were identified and quantified in full scan MS–MS mode using a homemade MS–MS library. Calibration curves were linear over the concentration range of 0.78–100 ng/mL with determination coefficients >0.996. This method was fast (total run time < 6 min), accurate (bias < 12.5%), and reproducible (intra- and inter-assay precision < 17.5%) with a quantitation limit of 0.78 ng/mL. The high specificity and sensitivity achieved by this method allowed the determination of nefopam and desmethyl-nefopam plasma levels in patients following either intermittent or continuous intravenous administration of nefopam.     

Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a zinc-metalloprotease that requires two extended exosites for optimal substrate binding and recognition of its intracellular target SNAP25. CFP and YFP connected through SNAP25 peptide (141-206) containing both exosites (CsY) has been used in a FRET-based assay for BoNT/A. To further improve the FRET efficiency in this BoNT/A substrate for in vitro high-throughput assays, we explored the feasibility of enhancing the capture of CFP emission by doubling the number of YFP acceptors. In comparison to CsY, the tandem fluorescence substrates CsYY and YsCsY enhanced the ratiometric fluorescence signal between YFP and CFP. YsCsY, containing two substrate sites, offered the greatest fluorometric change upon toxin-catalyzed cleavage. In addition to known approaches for enhancing fluorescence yield through various mutations, this alternative tandem substrate approach can boost the FRET signal and is particularly useful for substrates requiring extensive exosite recognition for specificity.