For rapid identification of a monoclonal antibody (MAb)-biotin-avidin-peroxidase complex, directed against the 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 TNase, followed by detection with the MAb-peroxidase complex (three-step sELIFA). A detection limit of 0·5–l;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 strains showed positive results, but none of the 57 non- 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 gave a positive result whereas all of the cultures with non- 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 bacteraemia and, possibly, also for detecting by direct testing of other clinical specimens.


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