Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
2001-07-11
2004-11-02
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C435S007100, C435S004000, C435S021000, C435S007900, C436S528000, C436S543000, C436S518000, C436S546000
Reexamination Certificate
active
06812036
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of mycotoxin assays. More particularly, this invention relates to a homogeneous assay that uses changes in fluorescence polarization to detect the presence of deoxynivalenol in grains.
2. Description of Related Art
Deoxynivalenol (DON), which is also known as vomitoxin, is a mycotoxin produced in various grains, such as wheat, corn, barley, oats and rye, by
Fusarium graminearum
and other Fusarium strains
1-4
. Presence of DON in food causes food refusal, vomiting and growth depression in swine, gastrointestinal illness in humans, and embryotoxicity and immunotoxicity in laboratory animals
1-4
. Because of its latent health risks, research is being carried out to explore analytical methods of detecting DON.
More generally, DON is a particularly troublesome mycotoxin within the group of mycotoxins known as trichothecenes. As shown in
FIG. 1
, trichothecenes have a common skeleton that can have different groups attached at R
1
-R
5
.
FIG. 2
identifies the R
1
-R
5
groups in
FIG. 1
for DON and for some of the other more well-known trichothecenes.
Various methods for the quantitative analysis of DON and other trichothecenes in grains are in use, such as thin-layer chromatography (TLC), gas chromatography (GC), and high-pressure liquid chromatography (HPLC). The TLC method is an official method (first action) of AOAC International. Although relatively simple, the TLC method lacks sensitivity and is only semiquantitative. In addition, most of these chromatographic methods require extensive cleanup procedures after extraction and are not suitable for field testing
4,5
.
Recently, enzyme-linked immunosorbent assay (ELISA) methods have been successfully applied to the screening of DON in grains
2
. However, ELISA methods are undesirably labor intensive, in that they typically involve several washings, liquid transfers, and incubation times. Accordingly, there is a need for a simple, yet sensitive, method for the determination of DON and other trichothecenes in grains that is rapid and field portable.
SUMMARY OF THE INVENTION
In a first principal aspect, the present invention provides a homogeneous assay for the determination of deoxynivalenol (DON) in grains. In accordance with the method, DON is extracted from a grain sample. The extract is combined with a tracer and an antibody to provide a mixture. The tracer comprises DON conjugated to a fluorophore, and the tracer is able to bind to the antibody to produce a detectable change in fluorescence polarization. The fluorescence polarization of the mixture is then measured. The measured fluorescence polarization is compared with a characterized fluorescence polarization value that corresponds to a known DON concentration.
In a second principal aspect, the present invention provides a homogeneous assay for the determination of trichothecenes in grains. In accordance with the method, trichothecene is extracted from a grain sample. The extract is combined with a tracer and an antibody to provide a mixture. The tracer comprises a predetermined trichothecene conjugated to a fluorophore, and the tracer is able to bind to the antibody to produce a detectable change in fluorescence polarization. The fluorescence polarization of the mixture is then measured. The measured fluorescence polarization is compared with a characterized fluorescence polarization value that corresponds to a known trichothecene concentration.
In a third principal aspect, the present invention provides an assay kit for the determination of DON in grains. The assay kit comprises an antibody and a tracer, each in an amount suitable for at least one assay, and suitable packaging. The tracer comprises DON conjugated to a fluorophore, and the tracer is able to bind to the antibody to produce a detectable change in fluorescence polarization.
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Jolley Michael E.
Nasir Mohammad Sarwar
Davis Deborah A
Diachemix LLC
Le Long V.
McDonnell Boehnen & Hulbert & Berghoff LLP
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