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Applied and Environmental Microbiology, November 2007, p. 6945-6952, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01136-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B{triangledown}

Gitta Schlosser,1,2 Petr Kacer,1,3 Marek Kuzma,1,4 Zoltán Szilágyi,1,5 Alida Sorrentino,1 Carla Manzo,1 Rosa Pizzano,1 Livia Malorni,1 and Gabriella Pocsfalvi1*

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., Avellino, Italy,1 Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Department of Organic Chemistry, Eötvös L. University, Budapest, Hungary,2 Department of Organic Technology, Institute of Chemical Technology, Prague, Czech Republic,3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic,4 Research Institute for Viticulture and Enology, Kolyukteto, H-3300 Eger, Hungary5

Received 21 May 2007/ Accepted 16 August 2007

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for 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 levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB 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 immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.

* Corresponding author. Mailing address: Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science and Technology, C.N.R., via Roma 52 a/c, 83100 Avellino, Italy. Phone: (39)-0825299208. Fax: (39)-0825781585. E-mail: gpocsfalvi{at}isa.cnr.it

{triangledown} Published ahead of print on 7 September 2007.

Applied and Environmental Microbiology, November 2007, p. 6945-6952, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01136-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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