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Appl. Environ. Microbiol. doi:10.1128/AEM.01136-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Coupling Immunomagnetic Separation on Magnetic Beads with MALDI-TOF Mass Spectrometry for the Detection of Staphylococcal Enterotoxin B

Gitta Schlosser, Petr Kaer, Marek Kuzma, Zoltán Szilágyi, Alida Sorrentino, Carla Manzo, Rosa Pizzano, Livia Malorni, and Gabriella Pocsfalvi^*

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., Avellino, Italy; Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Department of Organic Chemistry, Eötvös L. University, Budapest, Hungary; Department of Organic Technology, Institute of Chemical Technology, Prague, Czech Republic; Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Research Institute for Viticulture and Enology, Klyuktet, H-3300, Eger, Hungary

^* To whom correspondence should be addressed. Email: gpocsfalvi{at}isa.cnr.it.

	Abstract

The growing importance of mass spectrometry in the identification and characterization of bacterial protein toxins is merely a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, and especially when these are combined with affinity methods. Here we present a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS) for the selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low nanogram level in complex matrices is thus an important objective. In this work, affinity molecular probe was prepared by immobilizing anti-staphylococcal enterotoxin B antibody on the surface of para-toluene-sulfonyl functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB IgG and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB can be detected directly "on-beads" by placing the molecular probe on the MALDI target plate, or alternatively, "off-beads" after its acidic elution. Application of this method to complex biological matrices is demonstrated by the selective detection of SEB present in different matrices such as cultivation media of Staphylococcus aureus strains and raw milk samples.