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 4×10−10M. 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 59ng antibody/cm2. 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.