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Abstract
Different ELISA tests to detect and quantify levels of S. aureus in broth cultures were developed and compared. In all cases the assays were a modification of a “sandwich” format based on the use of common IgG as well as specific antibodies to bind protein A, an antigen localized in the cellular wall of S. aureus and partially extracted by boiling. Initially, human IgG was immobilized on the surface of microtitre plate wells in order to bind, by means of the Fc region, protein A that was present either in standard solutions or broth cultures of S. aureus treated by a boiling step. The sandwich format was completed using monoclonal (MAb) antibodies specific for protein A. The amount of bound antibody was evaluated using antiglobulins labelled with alkaline phosphatase (Ab2‐AP). Reading the absorbance at 405 nm a detection limit (LOD) of 0.6 ng/mL and 2×106 CFU/mL was found for protein A and for S. aureus, respectively. In order to improve the performance of the immunoassay, different approaches were pursued: an enzymatic amplification system (Ampli Q); the use of immunomagnetic beads employed both in a colorimetric (ELIMC=Enzyme‐Linked Immunomagnetic Colorimetric) and in an electrochemical (ELIME=Enzyme‐Linked Immunomagnetic Electrochemistry) assay. Using these systems the detection limit decreased by a factor of about 30‐fold for Ampli Q and ELIMC, and about 2000‐fold for ELIME formats. In addition, a qualitative polymerase chain reaction (PCR) method, using nuc gene primers, was set up and performed in parallel and its various parameters were optimized. This method was able to detect 102 CFU/mL. In terms of minimum detectable concentration of S. aureus and total analysis time, the performance of the PCR assay and ELIME, turned out to be comparable.
This work was supported by the Ministry of Health, special programs 1% (ISPESL Cofin 2003), and by the Sixth Framework of European Commission Project “Validation and standardization of PCR‐based methods for detection and quantitative risk assessment of foodborne pathogens (FoodPCR 2).”
