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Proceedings Paper

Development of a chemiluminescent enzyme capture immunoassay for the detection of Escherichia coli
Author(s): Seref Tagi; Philip E. Pivarnik; Arthur Garth Rand
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Paper Abstract

There has been increasing demand for rapid, sensitive and specific detection of Escherichia coli as an indicator of possible pathogen contamination in foods and water. Approximately 97% of E. coli strains produce ?- glucuronidase (GUS), which could permit the use of a specific microbial enzyme as an alternative approach for detection of E. coli. A procedure was developed for chemiluminometric measurement of GUS using a 1,2-dioxetene derivative as substrate, and was compared to the fluorescent assay for GUS detection. The chemiluminescent assay was found to be 10 times more sensitive than the fluorescent assay. Induction of GUS production in E. coli was maximum when p-nitropheny 1-?-D-glucuronide was used in the growth medium at 0.3 mM concentration after 8 h. GUS was isolated from the growth medium with a 30 minute immunocapture method at 37°C. Anti E. coli GUS antibodies were covalently immobilized on magnetic beads and used for the immunocapture assay. GUS from E. coli culture was captured using the prepared magnetic-beads. Compared to the chemiluminescent assay of GUS in culture filtrate, immunomagnetic capture of GUS provided signal increases up to 81x. The method permitted the detection of 1 CPU/ml of E. coli within 8 hours incubation in growth medium. The chemiluminescent enzyme capture immunoassay developed for the detection of GUS could serve as a quantitative indicator for the presence ofviable E. coli cells. The total assay time including growth, immunocapture and enzyme assay was 9 h.

Paper Details

Date Published: 13 March 2001
PDF: 10 pages
Proc. SPIE 4206, Photonic Detection and Intervention Technologies for Safe Food, (13 March 2001); doi: 10.1117/12.418717
Show Author Affiliations
Seref Tagi, Univ. of Rhode Island (United States)
Philip E. Pivarnik, Univ. of Rhode Island (United States)
Arthur Garth Rand, Univ. of Rhode Island (United States)

Published in SPIE Proceedings Vol. 4206:
Photonic Detection and Intervention Technologies for Safe Food
Yud-Ren Chen; Shu-I Tu, Editor(s)

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