Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-11-19
2004-11-09
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S030000
Reexamination Certificate
active
06815178
ABSTRACT:
FIELD OF THE INVENTION
The invention herein pertains to detection devices and methods, and particularly pertains to the detection of calcium containing endospores which are delivered from an airborne state to a reaction vessel.
DESCRIPTION OF THE PRIOR ART AND OBJECTIVES OF THE INVENTION
Certain light emitting photoproteins, such as those isolated from the jellyfish Aequorea aequoorea, perform a natural reaction when allowed to mix with calcium ion, Ca
++
, with the resultant production of light or chemiluminescence. Such calcium reporter photoproteins are known as aequorin.
Many gram-positive bacteria such as those of the genera
Clostridium
and
Bacillus
form specialized “resting” cells called endospores. Endospores are highly durable dehydrated cells with thick walls and additional layers. They are formed internal to the bacterial cell membrane.
When released into the environment, endospores can survive extreme heat, lack of water and exposure to many toxic chemicals and radiation. Most of the water present in the spore cytoplasm is eliminated. Such endospores do not generally carry out metabolic reactions. A strikingly large amount of an organic acid called dipicolinic acid (found in the cytoplasm), is accompanied by a large number of calcium ions. Calcium ions (Ca
++
) are combined with the dipicolinic acid as seen below:
The calcium-dipicolinic acid complex represents about ten percent of the dry weight of the endospore. As would be understood, such endospores can readily become airborne. If present in an area of human occupancy, such as an office building, home or the like, certain endospores can be life threatening when present through inadvertence, accident or deliberately introduced by bioterrorists.
Liquid scintillation spectrometers are commonly used to measure radioisotopes such as in medical research when used in an out of coincidence mode it senses both analog signals from two photomultiplier tubes to thereby act as a photon (light pulse) counter.
While various types of detection methods for certain deadly endospores such as
Bacillus anthracis
(anthrax) are known, these methods generally consist of collecting specimens from office buildings, homes or the like and thereafter delivering them to a laboratory for analysis. While such laboratory analyses may be very accurate, they are time consuming in that the collection, delivery and analytical work can take several days. Thus, those unfortunate enough to be infected with deadly endospores such as anthrax may have their medical condition diagnosed too late to save their lives.
Therefore, in view of the need for a speedy and continuous method of detecting anthrax and other calcium containing endospores which may be, for example, airborne in public buildings, the present invention was conceived and one of its objectives is to provide a device and method whereby bacillus anthracis and other calcium containing endospores can be easily and inexpensively detected.
It is an objective of the invention to provide a device for detecting certain calcium containing endospores when used as weapons or when naturally occurring such as near cattle or other animals.
It is another objective of the present invention to provide a device and method for detecting certain calcium containing endospores which is easy to operate and requires little specialized training.
It is yet another objective of the present invention to provide a method for detecting calcium containing endospores which is relatively inexpensive, to operate continuously for twenty-four hours a day.
It is still another objective of the present invention to provide a method of detecting calcium containing endospores utilizing a chelating agent and natural aequorin as derived from jellyfish.
It is still another objective of the present invention to provide a method of detection of calcium containing endospores utilizing a standard scintillation spectrometer.
Various other objectives and advantages of the present invention will become apparent to those skilled in the art as a more detailed description is set forth below.
SUMMARY OF THE INVENTION
The aforesaid and other objectives are realized by providing a scintillation spectrometer with a reaction vessel containing a chemiluminescent liquid. The reaction vessel is joined to an air pump by an intake conduit. The air pump delivers air from an office, room or the like through a conventional particulate filter capable of excluding particles greater than 20 &mgr;M, and into the chemiluminescent liquid. Calcium containing endospores such as
Bacillus anthracis
then mix with the chemiluminescent liquid in which the calcium ions contained therein are first chelated. The chelated calcium ions then react with the chemiluminescent photoprotein aequorin in the chemiluminescent liquid and produce light. This chemiluminescent reaction emits photons of light which, in one embodiment are directed through the walls of the glass reaction vessel, through a light guide and into photomultiplier tubes where they are intensified. Analog signals resulting therefrom are then delivered to a ratemeter which in turn delivers corresponding electrical signals to a chart recorder and, if desired, to a printer.
In a second embodiment of the invention the spectrometer converts the light pulses to a digital signal which are sent to a personal computer (PC) whereby the signals can be read in real time on the PC monitor.
REFERENCES:
patent: 5876960 (1999-03-01), Rosen
patent: 6232107 (2001-05-01), Bryan et al.
Rajan K. Chelation Characteristics of Calcium in Relation to Water Binding and Heat Resistance of Bacterial Endospores. Spore Research vol. 2, 527-543, 1977.*
Shimomura O. Effect of Calcium Chelators on the Ca+2 Dependent Luminescence of Aequorin. Biochem J 221(3)907-910, 1984.*
“Regeneration of the photoprotein aequorin” by Nature vol. 256 Jul. 17, 1975 pp. 236-238.
Methods in Cell Biology (R. Nuccitelli, Ed), 1994, vol. 40, pp. 305-338 Academic Press, Orlando, FL.
Article published in IEEE Engineering in Medicine and Biology, Sep./Oct. 2002 issue, Elizabeth D. Lester and Adrian Ponce,An Anthrax “Smoke” Detector,pp. 38-42.
Article on Elizabeth Lester found on www.jpl.nasa.gov web site, published Oct. 23, 2002,College Student's Curiosity Leads to Discovery,2 pages.
Article found on www.jpl.nasa.gov web site, published Oct. 23, 2002,NASA to Develop Biohazard “Smoke” Detector,2 pages.
Article found on www.nasatech.com web site, published under the NASA Tech Briefs, Jan. 2003,NASA Biohazard Monitor Mimics a Smoke Detector,1 page.
Microbiology an Introduction, 7th Edition, copyright 2001, pp. 97-98.
Biology of Microorganisms, 9th Edition, copyright 2000, pp. 91-95.
Proc. Natl. Acad. Sci. USA, vol. 75, No. 6, pp. 2611-2615, Jun. 1978.
Proc. Natl. Acad. Sci, USA, vol. 96, pp. 157-161, Jan. 1999.
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