DETECTION OF STAPHYLOCOCCUS-AUREUS WITH BIOTINYLATED MONOCLONAL-ANTIBODIES DIRECTED AGAINST STAPHYLOCOCCAL TNASE COMPLEXED TO AVIDIN-PEROXIDASE IN A RAPID SANDWICH ENZYME-LINKED IMMUNOFILTRATION ASSAY (SELIFA)

For rapid identification of Staphylococcus aureus, a monoclonal antibody (MAb)-biotin-avidin-peroxidase complex, directed against the S. aureus thermostable nuclease (TNase), was formed and used in a rapid three-step sandwich enzyme-linked immunofiltration assay (sELIFA) and a three-step sandwich enzyme-linked immunosorbent assay (sELISA). The MAb-peroxidase complex was formed by incubating the biotinylated MAbs with a streptavidin-peroxidase conjugate and the complex was purified by gel permeation chromatography. When compared with a four-step MAb-based sELISA described previously, this complex permitted one reagent step to be omitted in a three-step sELISA, and the test time was significantly reduced. The test sensitivity was slightly reduced in the three-step ELISA detection limit 1.0-2.0 ng of TNase/ml) when compared to the four-step sELISA (detection limit 0.5-1.0 ng of TNase/ml). The sELIFA method was based on the filtration of bacterial culture supernates through nitrocellulose membrane disks pre-spotted with a MAb directed against the S. aureus TNase, followed by detection with the MAb-peroxidase complex (three-step sELIFA). A detection limit of 0.5-2.0 ng of TNase/ml was achieved with the three-step sELIFA, depending on the filtrate volume of culture supernates. The total test time was 10-15 min when pre-spotted and blocked membranes were used. A total of 85 bacterial strains was tested in the sELIFA. All the 28 S. aureus strains showed positive results, but none of the 57 non-S. aureus strains did so, although some of these produced thermostable nuclease activity. When 75 blood cultures were tested directly in the sELIFA, 87% of the cultures with growth of S. aureus gave a positive result whereas all of the cultures with non-S. aureus gave negative results, a diagnostic sensitivity similar to that of the routine TNase enzyme test. Thus, the three-step sELIFA has potential for the rapid confirmation of S. aureus bacteraemia and, possibly, also for detecting S. aureus by direct testing of other clinical specimens.