In Vitro Detection and Quantification of Botulinum Neurotoxin Type E Activity in Avian Blood

Abstract

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n ‫؍‬ 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.

Botulism

“…Como desventajas, hay que utilizar animales de laboratorio y exige la utilización de pruebas de neutralización para la determinación del serotipo de toxina 62 . Existen otras pruebas alternativas que tienen similar sensibilidad y confiabilidad 104 , como las técnicas de radioinmunoensayo, inmunoprecipitación, hemaglutinación pasiva y ELISA 12,20,75,76 . También se han desarrollado varias metodologías de reacción en cadena de polimerasa (PCR) para optimizar el diagnóstico sin utilizar animales de laboratorio 25,40,48,77 .…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…As enzymatic assays, endopeptidase assays are relatively sensitive toward changes in reaction conditions, and testing of complex matrices can dramatically affect assay performance (Rasooly and Do 2008;. To reduce matrix interference, an immunoaffinity enrichment step has been introduced, where the toxin is captured from the matrix using antibody-coated magnetic microbeads prior to performing the endopeptidase reaction, resulting in assay sensitivities similar to those of the MBA (Wictome et al 1999a, b;Rasooly and Do 2008;Piazza et al 2011). Immunoaffinity enrichment is advantageous since it separates the toxin from other proteases which might cleave the SNARE peptide unspecifically, preventing false-positive results.…”

“…Some of the discrepancies can be explained by the fact that SNAP-25 is not only bound around the active site of BoNT, but also by so-called a-and b-exosites upstream and downstream of the active site (Breidenbach and Brunger 2004;Brunger et al 2008;Henkel et al 2009). With respect to assay conditions, it has been shown that certain buffer components such as NaCl can reduce or even abolish cleavage of SNAP-25 or VAMP (Ferracci et al 2011;Jones et al 2011;Piazza et al 2011).…”

“…This immunoaffinity enrichment step is not only useful for ELISAs, but is also often used in combination with other techniques (e.g., endopeptidase assays, mass spectrometry, cp. 12.4.2) (Bagramyan et al 2008;Parks et al 2011;Piazza et al 2011;Liu et al 2012).…”

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

“…In fluorescence-based methods, a pair of fluorescence donor and acceptor molecules is incorporated into the peptide substrate. The presence and activity of botulinum neurotoxin is detected by monitoring the fluorescence of the toxin cleavage products using fluorescence resonance energy transfer technology (FRET) (Schmidt and Stafford 2003;Dong, Tepp et al 2004;Rasooly and Do 2008;Gilmore, Williams et al 2011;Piazza, Blehert et al 2011). An in vitro assay (ALISSA) using a large immune-sorbent surface area for toxin capture and enrichment and a fluorogenic peptide substrate for activity measurement has been reported recently and high sensitive detection of BoNTs in complex biological matrices can be achieved by this method (Bagramyan, Barash et al 2008;Bagramyan and Kalkum 2011).…”