Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
2001-09-18
2004-01-06
Tate, Christopher R. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S007320, C435S032000
Reexamination Certificate
active
06673563
ABSTRACT:
BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates generally to the field of methods for identifying toxicants and/or isolated component substances in a sample. The types of samples which may be analyzed include either a solid sample, a liquid sample or a gaseous sample. The present invention also relates to the field of biological toxicant identification agents, as a particularly described luminescent biological reagent, for example the luminescent bacteria, are employed in the claimed isolation, identification and quantitation methods and techniques disclosed herein. The present invention also relates to the field of toxicant detecting kits, as a kit for the identification of toxicants is described employing a luminescent biological reagent.
II. Description of the Related Art
When grown in appropriate liquid culture or on semi-solid culture media, suspensions of luminescent bacteria emit a constant level of light for extended periods. Luminescent bacteria are bacteria which emit light without excitation, (i.e., they glow in the dark). The origin of the emission is biochemical, and organisms which demonstrate this characteristic are described as exhibiting the phenomenon of bioluminescence. Most known examples of luminescent bacteria are marine. Two major subclasses of the luminescent organisms are 1) free living (
Vibrio harveyi
) and 2) symbiotic (
Vibrio fischeri, Photobacterium phosphoreum, Photobacterium leiognathi
). Other major bioluminescent organisms include fire flies (
Photinus pyralis
), crustaceans (
Cyridina hilgendorfi
), dinoflagellates (
Gonyaulax polyhedra, Notiluca militaris
), fungi (
Omphalia flavida
) and the sea pansy (
Renilla reniformis
).
The luminescence of bacteria has long been known to be sensitive to a wide variety of toxic substances (e.g., heavy metals, pesticides, etc.). The exquisite sensitivity of luminescent bacteria to a variety of substances has made them a popular choice in methods for the gross detection of the presence of toxic materials. For example, the use of luminescent bacteria has been discussed for the detection of toxins on solid surfaces, such as soil
5
, and in liquid substances, such as in the analysis of waste water
3
, as well an in the detection of toxins in gaseous samples
6
.
Luminescent bacteria have also been employed in the detection of toxicants in marine environments.
2
For example, Vasseur et al. describe a Microtox luminescent bacterial assay for the detection of toxicants in water (
Photobacterium phosphoreum
)
2
.
Another variety of luminescent bacteria used in the analysis of industrial waste water is described in the Baher patent.
3
Specifically, the
Klebsiella planticola
bacteria has been used to detect the presence of substances toxic to particular microorganisms (used to purify industrial chemical plant waste waters) indicated through monitoring the luminescence of the Klebsiella.
Luminescent bacteria have also been used for detecting the presence of specific substances in a sample, including antibiotics, heavy metals, enzyme inhibitors, pesticides, microbial toxins, volatile hydrocarbons, disinfectants, and preservatives.
6
For example, the Siemens patent describes the use of a luciferase-gene-transformed microorganism for detecting the presence of a toxicant in a sample through a demonstrated reduction in the luminescent signal emitted by the luminescent bacteria in the presence of a toxic substance
6
.
Others have reported the ability to detect the presence of particular classes of chemical toxicants using luminescent bacteria, particularly phenolic compounds.
7
For example, in Strom et al., the relative toxicity of a variety of particularly defined phenolic compounds, including hydroquinone, is described using a luminescent bacterium
7
.
Thus, some species and components of luminescent bacteria have been adapted for use to simply detect the general presence of a toxic substance in a sample. In the presence of toxicants, detection of the toxins is provided by an observed diminution in luminescent emission and intensity in a variety of luminescent bacteria. However, the value of the “detection” techniques currently available is limited by an inability to identify, in an isolatable form, the substance which constitutes the “detected” toxicant or foreign substance.
No methods have been described wherein a generically “detected” toxicant may be identified in an isolatable form using a luminescent bacteria. The ability to actually identify an isolated substance as a potential “toxicant” in a sample would provide a powerful industrial and-research tool. Moreover, the ability to distinguish, by positive chemical analysis, the chemical structure of an isolated toxicant (using various chemical separation techniques known to those of skill in the art) would find great potential application in research, diagnostic medicine and industrial manufacturing processes.
Standard chemical visualization techniques for the localization of separated substances employ a variety of stains and staining procedures known to those skilled in the art (i.e., coomassie brilliant blue for gel electro-phoresis of proteins; 2-Naphthol or Resoranol for paper chromatography of sugars inhydrin for amino acid analysis with TLC). However, these techniques do not identify the potential toxicity of any visualized substance in the sample. No system has been proposed wherein a reagent may be used to provide a system wherein the potential toxicity of isolated substance in a sample may also be visualized and thereby identified.
Such a novel method for the simple, inexpensive and sensitive identification of a substance(s) in a sample or product which may be potentially lethal to an organism would also facilitate the further chemical elucidation of the chemical identity of the proposed toxicant through the subsequent use of various well known chemical analysis strategies available to those of skill in the art (such as mass spectrometry, nuclear resonance spectroscopy, infrared spectroscopy, x-ray crystallography, and chromatographic analysis). Thus, the complete chemical structure and identity of the potential toxicant could be determined if such a method, capable of identifying in an isolatable form the potential toxicant, were available. Such a system would be particularly valuable in the development of strategies to remove such identified toxicants from products intended for consumer use, and also in the development of procedures to render chemically identified toxicant(s) innocuous to animals and humans.
SUMMARY OF THE INVENTION
The present invention provides a rapid and accurate method for identifying a component substance (such as a toxin/toxicants) in a sample through the use of a luminescent biological agent employed together with chromatographic resolution techniques.
While any of a variety of luminescent bacteria may be used, those species found to be most particularly preferred for use in the practice of the present invention include
Photobacterium phosphoreum, Vibrio fischeri, Vibrio harveyi
and
Photobacterium leiognathi
. However, it is to be understood that the present inventive methods, reagents and kits may be practiced using any luminescent organism whose luminescence is specifically inhibited by an isolated component substance (for example, a potential toxicant) in a sample.
The present methods, reagents and kits may be used to isolate and identify a single toxicant, a number of individual toxicants, or a group of toxicants in or on a sample in the solid, liquid, or gaseous phase.
In part, the point of novelty of the present invention resides in the ability to identifiably isolate a component substance (for example, a toxicant) contained in a sample rapidly, and without the necessity of a separate biosensitivity assay of test sample. This is accomplished, for example, by applying a potentially toxicant-containing sample to a separation phase matrix, such as a chromatography paper sheet or a thin layer chromatography plate. The sample-exposed sheet is then exposed to a luminescent biologica
Becvar James E.
Becvar Laura E.
Clark Melody E.
Flood Michele C.
Highlander Steven L.
Slusher Stephen A.
Tate Christopher R.
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