Multiplex Detection of Microbial and Plant Toxins by Immunoaffinity Enrichment and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany.

Antibodies against a synthetic peptide designed to mimic a surface area of the H chain of botulinum neurotoxin A

“…Methods have utilized for detection the toxin's proteolytic activity of [see for example Refs. [20][21][22][23][24][25][26], molecular biology techniques [27][28][29][30][31][32], various immunological methods relying mainly on anti-toxin Abs [33][34][35][36][37][38][39][40][41][42] or on Abs in combination with other tools [43][44][45]. It should be noted that toxin serotypes exhibit different degrees of structural homology and therefore their use as immunogens produces Abs with differing levels of cross-reactivity.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…In a different approach, a multiplex immunoaffinity enrichment strategy for BoNT/A and B, their respective complexes, and other biological toxins has been used, followed by tryptic digest and MALDI-TOF MS-based detection of characteristic peptide fingerprints (Kull et al 2010). The approach turned out to be successful at detecting the toxins out of beverages with a detection limit of 300-500 fmol.…”

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

“…In this study, we used mass spectrometry to analyze ricin instead of traditional detection methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is an effective and fast technique to accurately detect ricin peptides [20,21]. It also can be used to screen biomarkers in complex systems including serum [16,22].…”