1887

Abstract

Summary

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—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.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-39-2-128
1993-08-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/39/2/medmicro-39-2-128.html?itemId=/content/journal/jmm/10.1099/00222615-39-2-128&mimeType=html&fmt=ahah

References

  1. Rappaport T, Sawyer KP, Nachamkin I. Evaluation of several commercial biochemical and immunologic methods for rapid identification of gram-positive cocci directly from blood cultures. J Clin Microbiol 1988; 26:1335–1338
    [Google Scholar]
  2. Berg K, Maeland JA. Same-day confirmation of Staphylococcus aureus bacteraemia by a thermonuclease test. APMIS 1986; 94:291–292
    [Google Scholar]
  3. Lachica RVF, Genigeorgis C, Hoeprich PD. Metachromic agar-diffusion methods for detecting staphylococcal nuclease activity. Appl Microbiol 1971; 21:585–587
    [Google Scholar]
  4. Madison BM, Baselski VS. Rapid identification of Staphy lococcus aureus in blood cultures by thermonuclease testing. J Clin Microbiol 1983; 18:722–724
    [Google Scholar]
  5. Gudding R. Differentiation of staphylococci on the basis of nuclease properties. J Clin Microbiol 1983; 18:1098–1101
    [Google Scholar]
  6. Park CE, de Melo Serrano A, Landgraf M et al. A survey of microorganisms for thermonuclease production. Can J Microbiol 1980; 26:532–535
    [Google Scholar]
  7. Shortle D. A genetic system for analysis of staphylococcal nuclease. Gene 1983; 22:181–189
    [Google Scholar]
  8. Liebl W, Rosenstein R, Gotz F et al. Use of a staphylococcal nuclease gene as DNA probe for Staphylococcus aureus. FEMS Microbiol Lett 1987; 44:179–184
    [Google Scholar]
  9. Brakstad OG, Maeland JA. Generation and characterization of monoclonal antibodies against Staphylococcus aureus thermonuclease. APMIS 1989; 97:166–174
    [Google Scholar]
  10. Green NM. Avidin 1. The use of [14C]biotin for kinetic studies and assay. Biochem J 1963; 89:585–591
    [Google Scholar]
  11. IJsselmuiden OE, Herbrink P, Meddens MJM et al. Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays. J Immunol Methods 1989; 119:35–43
    [Google Scholar]
  12. Reik LM, Maines SL, Ryan DE et al. A simple non chromatographic purification procedure for monoclonal antibodies. Isolation of monoclonal antibodies against cytochrome P450 isoenzymes. J Immunol Methods 1987; 100:123–130
    [Google Scholar]
  13. Stahli C, Miggiani V, Stocker J et al. Distinction of epitopes by monoclonal antibodies. Methods Enzymol 1983; 92:242–253
    [Google Scholar]
  14. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing principles of protein-dye binding. Anal Biochem 1976; 72:248–254
    [Google Scholar]
  15. Brakstad OG, Aasbakk K, Maeland JA. Detection of Staphy lococcus aureus by polymerase chain reaction amplification of the nucss gene. J Clin Microbiol 1992; 30:1654–1660
    [Google Scholar]
  16. Shields MJ, Siegel JN, Clark CR et al. An appraisal of polystyrene-(ELISA) and nitrocellulose-based (ELIFA) enzyme immunoassay systems using monoclonal antibodies reactive toward antigenically distinct forms of human C-reactive protein. J Immunol Methods 1991; 141:253–261
    [Google Scholar]
  17. IJsselmuiden OE, Meinardi MMHM, Van der Sluis JJ et al. Enzyme-linked immunofiltration assay for rapid sero- diagnosis of syphilis. Eur J Clin Microbiol 1987; 6:281–285
    [Google Scholar]
  18. Hoffman WL, Jump AA. Tween 20 removes antibodies and other proteins from nitrocellulose. J Immunol Methods 1986; 94:191–196
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-39-2-128
Loading
/content/journal/jmm/10.1099/00222615-39-2-128
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error