title: Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B.
abstract: 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.
authors.affiliation: Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science and Technology, C.N.R., Avellino, Italy.
authors.forename: Gitta
authors.initials: G
authors.lastname: Schlosser
authors.forename: Petr
authors.initials: P
authors.lastname: Kacer
authors.forename: Marek
authors.initials: M
authors.lastname: Kuzma
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Szilágyi
authors.forename: Alida
authors.initials: A
authors.lastname: Sorrentino
authors.forename: Carla
authors.initials: C
authors.lastname: Manzo
authors.forename: Rosa
authors.initials: R
authors.lastname: Pizzano
authors.forename: Livia
authors.initials: L
authors.lastname: Malorni
authors.forename: Gabriella
authors.initials: G
authors.lastname: Pocsfalvi
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D004768:Enterotoxins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods*; D008892:Milk / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013211:Staphylococcus aureus / Q000737:chemistry