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 antibo dy (MAb)-biotin-avidin-peroxidase complex, directed against the S. aur eus thermostable nuclease (TNase), was formed and used in a rapid thre e-step sandwich enzyme-linked immunofiltration assay (sELIFA) and a th ree-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 g el 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 significan tly reduced. The test sensitivity was slightly reduced in the three-st ep 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 thr ough nitrocellulose membrane disks pre-spotted with a MAb directed aga inst the S. aureus TNase, followed by detection with the MAb-peroxidas e complex (three-step sELIFA). A detection limit of 0.5-2.0 ng of TNas e/ml was achieved with the three-step sELIFA, depending on the filtrat e 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, alt hough some of these produced thermostable nuclease activity. When 75 b lood cultures were tested directly in the sELIFA, 87% of the cultures with growth of S. aureus gave a positive result whereas all of the cul tures with non-S. aureus gave negative results, a diagnostic sensitivi ty similar to that of the routine TNase enzyme test. Thus, the three-s tep sELIFA has potential for the rapid confirmation of S. aureus bacte raemia and, possibly, also for detecting S. aureus by direct testing o f other clinical specimens.