Dynamic protein signature assay

Details Dynamic protein signature assay Dynamic protein signature assay

2000-09-13

2003-11-25

Smith, Lynette R. F. (Department: 1645)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C424S009100, C424S009200, C435S007220, C435S007310, C435S007320, C435S007950, C435S029000, C435S034000, C435S035000, C435S039000, C435S070100, C435S071100, C435S243000, C435S244000, C435S245000, C435S375000, C435S390000

Reexamination Certificate

active

06653135

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and diagnostic kits for determining environmental quality, i.e., internal or external conditions that may have an effect on an organism. In particular, the invention relates to methods and diagnostic kits for determining stress induced on an organism through alterations or degradation in environmental quality. More specifically, organisms in the environment are exposed to a stressor, e.g., disease, substances to treat disease, or any other substance that may have an adverse effect on the organism, and a protein expression signature for the stressor, i.e., those proteins uniquely expressed and those proteins uniquely suppressed in response to the stressor, is determined. Protein expression signatures for one or more stressors may then be used to identify exposure to a stressor or class of stressors present in an environment.
2. Background of the Prior Art
It is desirable to provide a system that cannot only provide quantitative measurement of stressor(s), but qualitative information as well, such as the type of stressor causing the adverse effect on the organism.
Many prior art assays are confined to lethal assays. That is, laboratory animals are generally exposed to concentrations of a stressor. Presumptive evidence of no adverse effect exists if the animal or organism survives exposure.
A drawback to such systems is the fact that acute lethal affects are rarely observed. Rather, in general, adverse conditions arise slowly, which corresponds to a gradual increase in or long term exposure to a stressor. A stressor eventually, lethal responses are observed and identified. However, at this point it is generally too late to remedy. In any event, the stressor has no gross observable effect (mortality) on the organism at the time of the test. In general, such assays serve only to confirm the investigator's suspicion that there was decreased environmental quality, and that the disappearance of the organism was not due to some alternative, unexplained natural cause.
Other prior art assays, although not lethal, require extended exposure (and expense) or sophisticated procedures and/or equipment.
The present inventor had previously developed a sublethal assay that determines the presence of a stressor in an environment (see U.S. Pat. Nos. 5,149,634 and 5,250,413, the entire contents of which are incorporated herein by reference) and an assay that determines proteins synthesized by
Daphia magna
after exposure to mixtures of a cationic polyamide epichlorhydrin adduct (Kymene) and a combined assortment of water-extractable substances from chemi-thermal-mechanical pulp (see Bradley et al. (1994)). The assay of U.S. Pat. Nos. 5,149,634 and 5,250,413 involve the identification and correlation of the presence of at least three different stress proteins to a specific environmental chemical pollutant.
The present inventor has determined that although the assay disclosed in U.S. Pat. Nos. 5,149,634 and 5,250,413 is effective, there remains a need for more rapidly and accurately: (1) detecting the presence of change in environmental quality below lethal levels; (2) providing quantitative information regarding the presence of a stressor; (3) providing qualitative information, concerning the identity of the stressor; and (4) providing a simple measurement to determine the health of an organism (e.g., animal, human or other biota).
SUMMARY OF THE INVENTION
The present invention is directed to a method of determining environmental quality, i.e., internal or external conditions that may have an adverse effect on an organism, including disease, substances to treat disease or any other substance that may have an adverse effect on the organism. Specifically, a single organism or organisms are exposed to a stressor and a protein profile for the stressor, i.e., those proteins expressed and those proteins suppressed in response to exposure to the stressor, is determined. The organisms can either be exposed singly or as a population of two or more. A protein profile for an individual organism, or for a population of organisms, is thus obtained. The above steps are then repeated for a second stressor, a third stressor, etc., until a protein profile is obtained for all of the stressors that need to be monitored (or pathologies that need to be followed). Each protein profile is compared to the others to determine proteins distinct for a given stressor. These distinct proteins (present and absent) comprise the protein expression signature for each stressor. Some shock proteins, or stress proteins, are expressed and other shock proteins are suppressed in virtually all organisms in a response to stress. The term stress herein is widely used to include any internal or external condition that may have an effect on an organism. Stressors may be biological, due to diseases or parasites for example, and have an adverse effect on the organism. These ubiquitous shock proteins are expressed (or suppressed) by the organism in response to the stressor well in advance of the point at which lethal quantities are encountered and in advance of outward manifestations of disease or other adverse conditions. The stressor need not be chemical in nature. Any condition which places the organism under stress but does not necessarily kill the organism, thereby forcing it to respond to sublethal conditions, may be a stressor.
Some shock proteins are highly conserved across species and genus lines. Thus, the determination of protein expression signatures provides a ready method for sampling diverse environments and for the use of diverse organisms (from the environment or the laboratory). Investigation has shown that the expression (or suppression) of shock proteins by the organisms in question is related to the concentration or amount of stressor. Thus, the amount of stress protein expressed (or suppressed) by the organism will give a direct indication of the degree of severity of the stressor. Additionally, a variety of organisms may be sampled, to determine whether or not the stressor is toxic, both to the test animal and other organisms that may be of interest, such as domestic animals, humans and other plants and animals. Moreover, different stressors induce (or suppress) different types of shock proteins. By determining the set of shock protein(s) expressed and suppressed, it is possible to rapidly and accurately identify the stressor encountered. Accordingly, steps can be taken to eradicate or correct the stressor.


REFERENCES:
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Bradley et al., 1994. Environ. Tox. and Chem. 13(7):1043-1050.*
Blackstock et al. 1999. Trends Biotech. 17:121-127.*
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Bradley, B.P. and J.B. Ward, Detection of a major stress protein using a peptide antibody, Mar. Environ. Res. 28:471-475 (1989).
Bradley, B.P. and J.B. Ward. A molecular mechanism of adaptation in an estuarine copepod., Neth. J. Sea Res., 30:1-6 (1989).
Bradley, B.P. 1992. Are the stress proteins specific indicators of exposure or effect?, Mar. Environ. Res., 34:85-88 (1992).
Bradley, B.P., Brown, D.C., Lamonte, T.N., Boyd, S.N. and O'Neill, M.C.,

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