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.(ABSTRACT TRUNCATED AT 250 WORDS)
Brakstad
O G
OG
Applied Chemistry Division, SINTEF, Trondheim, Norway.
Maeland
J A
JA
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001360:Avidin; D016470:Bacteremia / Q000175:diagnosis / Q000382:microbiology; D001710:Biotin; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007124:Immunoenzyme Techniques; D008836:Micrococcal Nuclease / Q000276:immunology*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology; D013211:Staphylococcus aureus / Q000201:enzymology / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D001360:Avidin; D001710:Biotin; D008836:Micrococcal Nuclease