title: Development of an immunoaffinity solid phase microextraction method for the identification of penicillin binding protein 2a.
abstract: Methicillin resistant Staphylococcus aureus (MRSA) is a bacterium that is resistant to many antibiotics. Resistance to methicillin is related to production of penicillin binding protein 2a (PBP2a). The currently presented research involves the development of antibody-linked immunoaffinity solid phase microextraction (SPME) sorbents characterized from several aspects that can identify protein PBP2a. The on-sorbent binding constant Kd of monoclonal anti-PBP2a antibody for its antigen protein PBP2a was determined to be 4×10(-10) M. This value was obtained on the basis of the binding curve determined by selective extraction of its antigen PBP2a at different concentrations. The concentration of PBP2a captured by immunoaffinity sorbents was as low as 10ng/mL; lower concentrations could not be tested due to the sensitivity limitation of the LC-MS/MS system available. Surface density was estimated at 59 ng antibody/cm(2). To reduce non-specific binding, especially when the antigen is a protein, bovine serum albumin (BSA) was used to pretreat surfaces. The established immunoaffinity platform technology is expected to provide insights into the development of a practical, specific, sensitive and accurate assay for in vitro and in vivo diagnostics of MRSA.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Canada ON N2L 3G1.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Canada ON N2L 3G1.
authors.forename: Heather
authors.initials: H
authors.lastname: Lord
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Canada ON N2L 3G1.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Maciążek-Jurczyk
authors.affiliation: Biofont Inc., 280 Phillip Street, Suite A4-142, Waterloo, Canada ON N2L 3X1.
authors.forename: Sukhsagar
authors.initials: S
authors.lastname: Jolly
authors.affiliation: Biofont Inc., 280 Phillip Street, Suite A4-142, Waterloo, Canada ON N2L 3X1.
authors.forename: Mohamed Asim
authors.initials: MA
authors.lastname: Hussain
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Canada ON N2L 3G1. Electronic address: janusz@uwaterloo.ca.
authors.forename: Janusz
authors.initials: J
authors.lastname: Pawliszyn
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007164:Immunosorbents / Q000737:chemistry*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins / Q000032:analysis* / Q000276:immunology; D052617:Solid Phase Microextraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000900:Anti-Bacterial Agents; D007164:Immunosorbents; D046915:Penicillin-Binding Proteins
keywords: Immunoaffinity solid phase microextraction; Liquid chromatography; Penicillin binding protein (PBP2a); Staphylococcus aureus; Tandem mass spectrometry