Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay

Abstract

† Electronic supplementary information (ESI) available: comparison of multiplex toxin detection techniques, exemplary immunisation schema for rabbit and mouse, and titer development of rabbit. AbstractProteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 mL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 mL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Fluorescent sandwich immunoassays have been also performed on beads with flow cytometry instrumentation used to detect and quantify toxin [77,78,79]. Detection of BoNT/A and BoNT/B was performed in multiplex with other toxins (e.g., cholera toxin, ricin and staphylococcal enterotoxin B (SEB)) using different color-coded beads [78,79].…”

“…Detection of BoNT/A and BoNT/B was performed in multiplex with other toxins (e.g., cholera toxin, ricin and staphylococcal enterotoxin B (SEB)) using different color-coded beads [78,79]. The use of magnetic beads in flow cytometry was shown to have several advantages including that a preconcentration step can be involved, resulting in increased sensitivity and making analysis of turbid or heterogenous samples possible as beads can be easily separated from the matrix [79]. BoNT/A and B were detected at concentrations of 21 ng/mL and 73 ng/mL, respectively, in 5-plex assay together with ricin, SEB and abrin.…”

The Case of Botulinum Toxin in Milk: Experimental Data

“…The study was performed using purified 150-kDa BoNT/A1 (Hall A) and BoNT/B1 (Okra B) and the corresponding BoNT complexes, all of which were purchased from Metabiologics, Inc. (Madison, WI). Mouse monoclonal antibody A1688 [IgG1()] was used to capture BoNT/A and BoNT/A complex, and mouse monoclonal antibody B279 [IgG2a()] to capture BoNT/B and BoNT/B complex in the sandwich ELISA (30). Antibodies were purified from hybridoma supernatants using HiTrap protein G Sepharose columns according to the manufacturer's instructions (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…The detection limits were 21 and 73 pg/mL, respectively, out of 50 lL sample volume and could be further improved by magnetic immunoaffinity enrichment. Additionally, this method worked well to detect the toxins from different food matrices (Pauly et al 2009). A similar approach was followed by Garber and colleagues for the detection of BoNT/A and five additional toxins with sensitivities of 1 ng/mL in spiked food samples (Garber et al 2010).…”

“…In bead-based immunoassays the capture antibody is immobilized onto microbeads, allowing to separate the toxin from the matrix, a step which is not possible in conventional plate-bound ELISA formats. Especially, magnetic microbeads have proved to be useful for extracting BoNT from complex and even colloidal matrices Pauly et al 2009;Garber et al 2010). 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.…”