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. 1979 Jun;9(6):705–708. doi: 10.1128/jcm.9.6.705-708.1979

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

W W Schultz, T J Phipps, M Pollack
PMCID: PMC275383  PMID: 115899

Abstract

An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bjorn M. J., Iglewski B. H., Ives S. K., Sadoff J. C., Vasil M. L. Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA-103. Infect Immun. 1978 Mar;19(3):785–791. doi: 10.1128/iai.19.3.785-791.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bjorn M. J., Vasil M. L., Sadoff J. C., Iglewski B. H. Incidence of exotoxin production by Pseudomonas species. Infect Immun. 1977 Apr;16(1):362–366. doi: 10.1128/iai.16.1.362-366.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Callahan L. T., 3rd Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum. Infect Immun. 1976 Jul;14(1):55–61. doi: 10.1128/iai.14.1.55-61.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Callahan L. T., 3rd Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect Immun. 1974 Jan;9(1):113–118. doi: 10.1128/iai.9.1.113-118.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Knudsen R. C., Callahan L. T., 3rd, Ahmed A., Sell K. W. Use of microculture plates and the multiple automated sample harvester for in vitro microassay of bacterial toxins. Appl Microbiol. 1974 Aug;28(2):326–327. doi: 10.1128/am.28.2.326-327.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leppla S. H. Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect Immun. 1976 Oct;14(4):1077–1086. doi: 10.1128/iai.14.4.1077-1086.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liu P. V. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J Infect Dis. 1973 Oct;128(4):506–513. doi: 10.1093/infdis/128.4.506. [DOI] [PubMed] [Google Scholar]
  9. Liu P. V., Hsieh H. Exotoxins of Pseudomonas aeruginosa. 3. Characteristics of antitoxin A. J Infect Dis. 1973 Oct;128(4):520–526. doi: 10.1093/infdis/128.4.520. [DOI] [PubMed] [Google Scholar]
  10. Liu P. V. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J Infect Dis. 1966 Oct;116(4):481–489. doi: 10.1093/infdis/116.4.481. [DOI] [PubMed] [Google Scholar]
  11. Liu P. V., Yoshii S., Hsieh H. Exotoxins of Pseudomonas aeruginosa. II. Concentration, purification, and characterization of exotoxin A. J Infect Dis. 1973 Oct;128(4):514–519. doi: 10.1093/infdis/128.4.514. [DOI] [PubMed] [Google Scholar]
  12. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  13. Pavlovskis O. R., Pollack M., Callahan L. T., 3rd, Iglewski B. H. Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections. Infect Immun. 1977 Dec;18(3):596–602. doi: 10.1128/iai.18.3.596-602.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pollack M., Callahan L. T., 3rd, Taylor N. S. Neutralizing antibody to Pseudomonas aeruginosa exotoxin in human sera: evidence for in vivo toxin production during infection. Infect Immun. 1976 Oct;14(4):942–947. doi: 10.1128/iai.14.4.942-947.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pollack M., Taylor N. S., Callahan L. T., 3rd Exotoxin production by clinical isolates of pseudomonas aeruginosa. Infect Immun. 1977 Mar;15(3):776–780. doi: 10.1128/iai.15.3.776-780.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pollack M., Taylor N. S. Serum antibody to Pseudomonas aeruginosa exotoxin measured by a passive hemagglutination assay. J Clin Microbiol. 1977 Jul;6(1):58–61. doi: 10.1128/jcm.6.1.58-61.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Taylor N. S., Pollack M. Purification of Pseudomonas aeruginosa exotoxin by affinity chromatography. Infect Immun. 1978 Jan;19(1):66–70. doi: 10.1128/iai.19.1.66-70.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

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