Enhanced chemiluminescent immunoassay for staphylococcal enterotoxin B in foods
Ocasio, Wilfredo
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Permalink
https://hdl.handle.net/2142/21961
Description
Title
Enhanced chemiluminescent immunoassay for staphylococcal enterotoxin B in foods
Author(s)
Ocasio, Wilfredo
Issue Date
1989
Doctoral Committee Chair(s)
Blaschek, Hans-Peter M.
Department of Study
Food Science
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Biology, Microbiology
Chemistry, Analytical
Health Sciences, Public Health
Language
eng
Abstract
An enhanced chemiluminescent immunoassay (CLIA) for the detection of staphylococcal enterotoxin B (SEB) in foods was developed. This assay utilizes the $\rho$-iodophenol enhanced oxidation of luminol by hydrogen peroxide as indicator reaction. The reaction was catalyzed by horseradish peroxidase (HRPO) conjugated to immunospecific anti-SEB immunoglobulin G (IgG). Addition of $\rho$-iodophenol to the reaction mixture resulted in $>$2,000 fold increase in light emission over the unenhanced reaction. Furthermore, light emission was not only of high intensity but also of prolonged duration. This simplified manipulation since it was not necessary to start the reaction in front of the photodetector.
Food samples were artificially contaminated and assayed by the CLIA. Food extracts were obtained and added to tubes containing a beads precoated with specific anti-SEB IgG. The samples were incubated for 1 h at room temperature (RT). Samples were removed, beads washed, and the HRPO-IgG conjugate added to the tubes. Following incubation (0.5 h at RT), the conjugate was removed, beads were washed again, and transferred to disposable cuvettes. Twenty microliters of an 18 mM $\rho$-iodophenol solution were added to the cuvette in a corner opposite to the bead. The luminescent reaction was initiated by the addition of 1 ml of luminol-hydrogen peroxide substrate solution. Light emission was recorded for 60 sec after a 30 sec delay.
The detection limit for the assay varied from 0.02 ng/ml in boiled eggs to 0.10 ng/ml in cheese, cheese food and potato salad. Statistical analyses demonstrated that the CLIA was a precise and accurate technique for the determination of SEB in the foods studied. The CLIA presented here offers the following advantages over other currently available immunoassays: (1) light emitted can be measured in seconds; (2) no radioactive or carcinogenic compounds are used; and (3) no stopping reagents are necessary.
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