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

Jones, R. L.; Liu, Yvonne; Sesardic, Dorothea

doi:10.1016/j.jim.2009.01.001

Cited by 21 publications

(18 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resulting cleavage product on the support is subsequently detected by cleavage product specific antibodies. Cleavage of SNAP-25 derived substrates was used for the detection of BoNT/A [119,120,121], BoNT/C1 [122], and BoNT/E [120]; cleavage of synaptobrevin has also been utilized for the detection of BoNT/B and its light chain [119,123]. Detection limits of this type of assay range from 200 pg/mL (BoNT/B) [119] to as low as 40 fg/mL (BoNT/A) [120].…”

Section: Immunodetection Of Cleavage Productmentioning

confidence: 99%

“…Detection of the toxin in relevant matrices was not demonstrated, but a significant decrease in sensitivity can be expected due to suboptimal cleavage conditions. It has also been demonstrated that the antibodies involved are able to adequately discriminate among the serotype specific cleavage products, suggesting that the assay can be used as a highly specific in vitro serotyping method [122]. …”

Section: Immunodetection Of Cleavage Productmentioning

confidence: 99%

See 1 more Smart Citation

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

View full text Add to dashboard Cite

Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.

show abstract

Section: Immunodetection Of Cleavage Productmentioning

confidence: 99%

Section: Immunodetection Of Cleavage Productmentioning

confidence: 99%

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

View full text Add to dashboard Cite

show abstract

“…A few alternative approaches have been described, such as activity-based methods using synthetic substrate peptides and ELISA detection of cleavage products (Evans et al, 2004;Hallis et al, 1996;Wictome et al, 1999a, b). A peptidase activity method selective for BoNT/C1 with immunological detection has also been presented (Jones et al, 2009). Furthermore, Evans et al (2009) presented a new concept in which synaptosomes from rat brain were used for in vitro capture and activity measurement of BoNTs.…”

Section: Introductionmentioning

confidence: 99%

Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Hedeland

Moura

Båverud

et al. 2011

Journal of Medical Microbiology

View full text Add to dashboard Cite

There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

show abstract

“…Immunological detection, for example, was demonstrated for BoNTC1 [36] and BoNT-B activity [37], with the target for immunological detection being an octapeptide epitope sequence that is normally buried within the native substrates alpha-helical structure but becomes exposed following cleavage by toxin types A or E [38]. Most of these cleavage-based detection methods require specialized equipment (MS, SPR, cantilever, etc), which can limit broad utilization of the technology.…”

Section: Introductionmentioning

confidence: 99%

Multi-wavelength spatial LED illumination based detector for in vitro detection of botulinum neurotoxin A activity

Sun

Francis

Sapsford

et al. 2010

Sensors and Actuators B: Chemical

View full text Add to dashboard Cite

A portable and rapid detection system for the activity analysis of Botulinum Neurotoxins (BoNT) is needed for food safety and bio-security applications. To improve BoNT activity detection, a previously designed portable charge-coupled device (CCD) based detector was modified and equipped with a higher intensity more versatile multi-wavelength spatial light-emitting diode (LED) illumination, a faster CCD detector and the capability to simultaneously detect 30 samples. A FITC/ DABCYL Förster Resonance Energy Transfer (FRET)-labeled peptide substrate (SNAP-25), with BoNT-A target cleavage site sequence was used to measure BoNT-A light chain (LcA) activity through the FITC fluorescence increase that occurs upon peptide substrate cleavage. For fluorescence excitation, a multi-wavelength spatial LED illuminator was used and compared to our previous electroluminescent (EL) strips. The LED illuminator was equipped with blue, green, red and white LEDs, covering a spectrum of 450-680 nm (red 610-650 nm, green 492-550 nm, blue 450-495 nm, and white LED 440-680 nm). In terms of light intensity, the blue LED was found to be ~80 fold higher than the previously used blue EL strips. When measuring the activity of LcA the CCD detector limit of detection (LOD) was found to be 0.08 nM LcA for both the blue LED (2 s exposure) and the blue EL (which require ≥60 s exposure) while the limits of quantitation (LOQ) is about 1 nM. The LOD for white LED was higher at 1.4 nM while the white EL was not used for the assay due to a high variable background. Unlike the weaker intensity EL illumination the high intensity LED illumination enabled shorter exposure times and allowed multi-wavelength illumination without the need to physically change the excitation strip, thus making spectrum excitation of multiple fluorophores possible increasing the versatility of the detector platform for a variety of optical detection assays.

show abstract

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

Cited by 21 publications

References 28 publications

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Multi-wavelength spatial LED illumination based detector for in vitro detection of botulinum neurotoxin A activity

Contact Info

Product

Resources

About