U.S. patent application number 11/026285 was filed with the patent office on 2006-11-16 for biological particulate matter analogue.
Invention is credited to David S. Alburty, Kelly L. Brown, Robert C. Huebner, Andrew E. Page.
Application Number | 20060257855 11/026285 |
Document ID | / |
Family ID | 32986992 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257855 |
Kind Code |
A1 |
Page; Andrew E. ; et
al. |
November 16, 2006 |
Biological particulate matter analogue
Abstract
The present invention provides a biological particle analogue,
or biological analogue, that simulates a chosen biological organism
or compound. The biological analogue includes a first portion that
is not, in and of itself, recognized by the biological detection
system, and a second portion, which provides the properties
necessary for recognition by the detection system, carried by the
first portion. The biological analogue is constructed in such a way
as to include some important characteristics of the chosen
biological organism or compound, while excluding other undesirable
characteristics of the chosen biological organism or compound. The
present invention is useful in testing a variety of biological
detection systems.
Inventors: |
Page; Andrew E.; (Kansas
City, MO) ; Brown; Kelly L.; (Kansas City, MO)
; Alburty; David S.; (Drexel, MO) ; Huebner;
Robert C.; (Liberty, MO) |
Correspondence
Address: |
BLACKWELL SANDERS PEPER MARTIN LLP
720 OLIVE STREET
SUITE 2400
ST. LOUIS
MO
63101
US
|
Family ID: |
32986992 |
Appl. No.: |
11/026285 |
Filed: |
December 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10248775 |
Feb 17, 2003 |
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11026285 |
Dec 30, 2004 |
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Current U.S.
Class: |
435/5 ; 435/6.16;
435/7.32; 435/7.5 |
Current CPC
Class: |
C12Q 1/6813 20130101;
C12Q 1/6851 20130101; C12Q 1/6851 20130101; G01N 33/543 20130101;
C12Q 1/6813 20130101; C12Q 2545/113 20130101; C12Q 2563/155
20130101; C12Q 2563/155 20130101; C12Q 1/04 20130101; C12Q 2545/113
20130101 |
Class at
Publication: |
435/005 ;
435/006; 435/007.32; 435/007.5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68; G01N 33/554 20060101
G01N033/554; G01N 33/569 20060101 G01N033/569 |
Claims
1. A method for testing or calibrating a biological detection
system which detects a biological material comprising: a) providing
a biological analogue comprising: (1) a first portion; and (2) a
second portion carried by said first portion; wherein said
biological analogue acts as a surrogate for said biological
material, and further wherein said biological detection system
recognizes said biological analogue as said biological material;
and b) introducing said biological analogue into an area to be
monitored or tested by said biological detection system.
2. The method of claim 1 wherein said biological analogue has
approximately the same aerodynamic diameter as said biological
material.
3. The method of claim 1 wherein said biological analogue has
approximately the same density as said biological material.
4. The method of claim 1 wherein said biological analogue has
approximately the same physical diameter as said biological
material.
5. The method of claim 1 wherein said first portion is selected
from the group consisting of latex, polystyrene, silica,
polystyrene styrene/divinylbenzene copolymer,
polymethylmethacrylate, polyvinyltoluene styrene/butadiene
copolymer, styrene/vinyltoluene copolymer, vinyl carboxylic
acid/styrene copolymer, styrene/maleic anhydride copolymer,
amino-modified microspheres, carboxylate-modified microspheres,
oleic acid and paramagnetic beads.
6. The method of claim 1 wherein said second portion is selected
from the group consisting of DNA, RNA, PNA, protein, peptide,
carbohydrate, lipid, dipicolinic acid and antibodies.
7. The method of claim 5 wherein said second portion is selected
from the group consisting of DNA, RNA, PNA, protein, peptide,
carbohydrate, lipid, dipicolinic acid and antibodies.
8. The method of claim 1 wherein said biological material detected
by said biological detection system is selected from the group
consisting of Bacillus globigii, Bacillus anthracis, Yersinia
pestis, Orthopoxvirus, botulinum, ricin, and Salmonella.
9. The method of claim 5 wherein said biological material detected
by said biological detection system is selected from the group
consisting of Bacillus globigii, Bacillus anthracis, Yersinia
pestis, Orthopoxvirus, botulinum, ricin, and Salmonella.
10. The method of claim 6 wherein said biological material detected
by said biological detection system is selected from the group
consisting of Bacillus globigii, Bacillus anthracis, Yersinia
pestis, Orthopoxvirus, botulinum, ricin, and Salmonella.
11. The method of claim 1 wherein said second portion is a
fluorophore.
12. The method of claim 5 wherein said second portion is a
fluorophore.
13. The method of claim 6 wherein said second portion is a
fluorophore.
14. The method of claim 1 wherein said second portion is carried by
said first portion because of a linkage selected from the group
consisting of covalent linkages, ionic linkages, and
streptavidin/biotin linkages.
15. The method of claim 5 wherein said second portion is carried by
said first portion because of a linkage selected from the group
consisting of covalent linkages, ionic linkages, and
streptavidin/biotin linkages.
16. The method of claim 6 wherein said second portion is carried by
said first portion because of a linkage selected from the group
consisting of covalent linkages, ionic linkages, and
streptavidin/biotin linkages.
17. The method of claim 1 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a bacterium.
18. The method of claim 5 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a bacterium.
19. The method of claim 6 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a bacterium.
20. The method of claim 1 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a virus.
21. The method of claim 5 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a virus.
22. The method of claim 6 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a virus.
23. The method of claim 1 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a toxin.
24. The method of claim 5 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a toxin.
25. The method of claim 6 wherein said second portion is at least
one of a plurality of biologically active molecules found
associated with a toxin.
26. The method of claim 1 wherein said second portion is
microencapsulated within said first portion.
27. The method of claim 5 wherein said second portion is
microencapsulated within said first portion.
28. The method of claim 6 wherein said second portion is
microencapsulated within said first portion.
29. The method of claim 1 wherein said second portion is
synthetic.
30. The method of claim 5 wherein said second portion is
synthetic.
31. The method of claim 6 wherein said second portion is
synthetic.
32. A method for testing or calibrating a biological detection
system which detects Bacillus globigii comprising: a) providing a
biological analogue comprising: (1) a first portion; and (2) a
second portion carried by said first portion; wherein said
biological analogue acts as a surrogate for Bacillus globigii, and
further wherein said biological detection system recognizes said
biological analogue as Bacillus globigii; and b) introducing said
biological analogue into an area to be monitored or tested by said
biological detection system.
33. A method for testing or calibrating a biological detection
system that detects Bacillus globigii comprising: a) providing a
biological analogue comprising: (1) a first portion; and (2) a DNA
portion carried by said first portion; wherein said biological
analogue acts as a surrogate for said Bacillus globigii, and
further wherein said biological detection system recognizes said
biological analogue as Bacillus globigii; and b) introducing said
biological analogue into an area to be monitored or tested by said
biological detection system.
34. The method of claim 33 wherein said DNA portion is total
genomic Bacillus globigii DNA.
35. The method of claim 33 wherein said first portion is selected
from the group consisting of latex, polystyrene, silica,
polystyrene styrene/divinylbenzene copolymer,
polymethylmethacrylate, polyvinyltoluene styrene/butadiene
copolymer, styrene/vinyltoluene copolymer, vinyl carboxylic
acid/styrene copolymer, and styrene/maleic anhydride copolymer,
amino-modified microspheres, and carboxylate-modified microspheres,
oleic acid and paramagnetic beads.
36. The method of claim 33 wherein said DNA portion is carried by
said first portion because of a linkage selected from the group
consisting of covalent linkages, ionic linkages, and
streptavidin/biotin linkages.
37. A method for testing or calibrating a biological detection
system for detecting Bacillus globigii comprising: a) providing a
biological analogue comprising: (1) a silica bead; and (2) a DNA
portion carried by said silica bead because of a
streptavidin/biotin linkage; wherein said biological analogue acts
as a surrogate for said Bacillus globigii, and further wherein said
biological detection system recognizes said biological analogue as
Bacillus globigii; and b) introducing said biological analogue into
an area to be monitored or tested by said biological detection
system.
38. The method of claim 37 wherein said DNA portion is total
genomic Bacillus globigii DNA.
39. A method for testing or calibrating a biological detection
system that detects Bacillus globigii comprising: a) providing a
biological analogue comprising: (1) a polystyrene bead; and (2) a
DNA portion carried by said polystyrene bead because of an ionic
linkage; wherein said biological analogue acts as a surrogate for
said Bacillus globigii, and further wherein said biological
detection system recognizes said biological analogue as Bacillus
globigii; and b) introducing said biological analogue into an area
to be monitored or tested by said biological detection system.
40. The method of claim 39 wherein said DNA portion is total
genomic Bacillus globigii DNA.
41. A method for testing or calibrating a biological detection
system which detects a biological material comprising: a) providing
a biological analogue comprising: (1) a first portion; and (2) a
second portion comprising particles from said biological material
that have been rendered nonviable, carried by said first portion;
wherein said biological analogue acts as a surrogate for said
biological material, and further wherein said biological detection
system recognizes said biological analogue as said biological
material; and b) introducing said biological analogue into an area
to be monitored or tested by said biological detection system.
42. The method of claim 41 wherein said particles from said
biological material have been rendered nonviable by a method
selected from the group consisting of autoclaving, french pressure
cell, sonication, and proteolysis.
43. A method for testing or calibrating a biological detection
system that detects a biological material comprising: a) providing
a biological analogue comprising:a first portion which is a
polystyrene bead with an aerodynamic diameter of approximately 1
micron; and (1) a second portion which is Bacillus globigii DNA;
wherein said biological analogue tests or calibrates said
biological detection system by acting as a surrogate for said
biological material, and further wherein said biological detection
system recognizes said biological analogue as said biological
material; and b) introducing said biological analogue into an area
to be monitored or tested by said biological detection system.
44. The method of claim 45 wherein said second portion is carried
by said first portion because of an ionic linkage.
45. The method of claim 45 wherein said second portion is carried
by said first portion because of a streptavidin/biotin linkage.
46. A method for testing or calibrating a biological detection
system that detects a biological material comprising: a)
introducing a biological analogue comprising: (1) a first portion;
and (2) a second portion which is a fluorphore selected from the
group consisting of tryptophan, NADH, NADPH, flavins, tyrosine, and
pyridoxine; wherein said biological analogue tests or calibrates
said biological detection system by acting as a surrogate for said
biological material, and further wherein said biological detection
system recognizes said biological analogue as said biological
material; and b) introducing said biological analogue into an area
to be monitored or tested by said biological detection system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional application of U.S.
patent application Ser. No. 10/248,775, filed Feb. 17, 2003,
currently pending.
BACKGROUND OF INVENTION
[0002] This invention relates generally to a device and method for
emulating biological organisms, biological particles, or biological
molecules and, specifically, to an analogue for biological
organisms, biological particles, or biological molecules that is
constructed in such a way as to include some important
characteristics of that organism, particle, or molecule while
excluding other undesirable characteristics.
[0003] In recent years, as the level of sophistication in the field
of biotechnology has increased, the threat of biological weapons
use by terrorist groups and rogue nations has also increased Many
of the basic methodologies involved in the production of biological
weapons, as well as the basic starting materials, are more readily
available and less expensive than those required for nuclear or
conventional weaponry. As the threat of the use of biological
weapons grows, it is imperative that means for early detection and
identification of biological warfare agents are provided.
[0004] Some devices and methods for the detection and
identification of potential biological warfare agents are known in
the art. The U.S. Postal Service, for example, is in the process of
evaluating Northrop Grumman's Biological Detection System for use
in some of its facilities. Other detection systems are likewise
available from other manufacturers. Verifying that these systems
are operating properly and will detect the presence of biological
warfare agents, however, presents a challenge.
[0005] Devices like the Northrop Grumman Biological Detection
System are generally tested for proper operation after installation
and periodically thereafter. The best way to test such a device is
to simulate a biological event (i.e. simulate the dispersal of a
biological warfare agent in the environment of the detection
system). From a practical standpoint, however, it is not feasible
to release a potential biological warfare agent, such as anthrax,
into a facility or outdoors environment for obvious reasons. Thus,
a means of testing biological detection systems without releasing
potentially dangerous agents into the air is required.
[0006] One testing method used in the past has been to release some
sort of surrogate biological organism in place of the dangerous
biological agent to be detected by various biological detection
systems. In the Northrop Grumman device mentioned above, for
example, the organism Bacillus globigii is currently being used for
testing purposes. Use of an organism such as B. globigii is far
less dangerous than using a potentially deadly agent such as
anthrax, however any use of a living organism presents certain
problems. The risk of infection always remains when a live
biological organism is used in testing equipment in a building or
other location. Further, even if infection does not result from
such a use, there is a risk of allergic response to the organism on
the part of persons in the area both during and after the testing.
Also, immunocompromised individuals may be particularly susceptible
to the ill effects of any remaining biological organisms. During
the testing, persons in the area may wear personal protective
equipment (PPE) to minimize such risks, however some microorganisms
are notoriously persistent in the environment and may linger in the
area after the testing is concluded and regular personnel have
returned to the facility. Members of the Bacillus genus, for
example, are particularly persistent due to the formation of
endospores. Even if these immediate negative effects are not
apparent, it is undesirable to allow unchecked growth of a
microorganism within a facility after such a test has been
conducted. A full decontamination procedure after every test of the
biological detection system is time-consuming, difficult to verify,
and expensive. Such a decontamination procedure is not practical.
In addition, even when such decontamination procedures are
undertaken, the decontamination is never perfect and unwanted
biological organisms may still persist.
[0007] Finally, testing with surrogates may not be a true test of a
system since the testing program or kit may need to be altered or
replaced to detect a surrogate instead of the biological warfare
agents a system is designed to identify. Specialty kits may be
needed for various testing scenarios. A biological analogue made
with markers for an actual biological warfare agent could to used
to safely do a real test of the system and could even be employed
by inspector or oversight personnel to test the proficiency of
in-place systems and their operators.
[0008] There is, therefore, a great need for some sort of device
and/or method that will allow accurate testing of a biological
detection system without the use of living organisms. Such a device
and method must simulate a true biological organism dispersal event
as closely as possible in order to provide the accurate results
needed to ensure that a biological detection system is working
properly and will perform in the event of a true biological warfare
event.
SUMMARY OF INVENTION
[0009] In view of the foregoing, the present invention provides a
biological particle analogue that can be safely used to simulate a
predetermined biological material for purposes of testing a
biological detection system, or for other applications.
[0010] The present invention is a manufactured biological analogue
that includes more than one important characteristic from the suite
of characteristics possessed by a specific biological material that
it is meant to simulate, while excluding at least one undesirable
characteristic. In a preferred embodiment, a biological analogue of
the present invention includes a first portion and second portion,
where the second portion is carried by the first portion, that can
be recognized by a biological detection system. The second portion
may be DNA, RNA, PNA (pentose nucleic acid), protein, or other
biologically active molecules, such as dipicolinic acid,
carbohydrates, lipids or antibodies. In one alternative embodiment
of the present invention, the second portion of the biological
analogue includes at least one fluorophore, allowing the biological
analogue to simulate the fluorescing behavior of a chosen
biological organism or compound.
[0011] In another embodiment of the present invention, the
biological analogue may include DNA, or another biologically active
molecule, associated with a carrier such as oleic acid (the first
portion in this embodiment) or other semivolatile or volatile
liquid, thereby producing an aerosol.
[0012] A biological analogue constructed in accordance with the
present invention may be designed to simulate directly a biological
organism that is used in the context of biological warfare, or it
can be designed to simulate a so-called surrogate organism such as
B. globigii, commonly used to test biological detection
systems.
[0013] Biological analogues can also be used to test the effect of
a biological warfare agent release into buildings, subways,
municipal water systems, and other locations or public health
systems that may be the target of a biological warfare attack. The
time of dispersion, time course, and overall pattern of dispersion
of a biological agent can be discerned so that protective measures
can be developed. In the case of water systems, biological
analogues can be developed such that the DNA or RNA is encapsulated
in a material that will tolerate the aqueous conditions long enough
for testing to occur, then biodegrade through normal processes or
be destroyed by normal water treatment processes.
[0014] The second portion of the biological analogue can be
attached to or otherwise carried by the first portion of the
biological analogue through covalent or ionic linkages, or, in one
embodiment of the present invention, through streptavidin/biotin
linkages. The second portion could also be encapsulated within the
first portion of the biological analogue. This may present certain
advantages since it could protect the second portion from the
environment until it is released from the encapsulation material
for detection.
[0015] In another embodiment of the present invention, the first
portion of the biological analogue is paramagnetic.
[0016] The biological analogue described herein, and its method of
use, is invaluable in testing and calibration of biological
detection systems. The biological detection systems that can be
calibrated or tested using a biological analogue of the present
invention are not limited to biological warfare-related systems but
may include food screening systems or any other system wherein a
biological organism is traditionally used for testing or
calibration. The present invention provides a means of conducting
required testing without the use of live biological organisms.
Other and further advantages of the present invention are set forth
in the description and appended claims.
DETAILED DESCRIPTION
[0017] The constructs of the present invention serve as analogues
or surrogates (hereinafter termed "biological analogues") of
biological materials, and the present invention also relates to a
method of using such biological analogues to safely simulate
biological materials. As used herein, the term "biological
material" includes any biological particle, organism or
biologically active molecule. The biological analogue is preferably
of the same aerodynamic diameter as its biological counterpart and
has a portion of DNA or RNA from the biological particle attached
thereto. The term "aerodynamic diameter" is an expression of the
aerodynamic behavior of an irregularly shaped particle in terms of
the diameter of an idealized particle; that is, aerodynamic
diameter is the diameter of a sphere of unit density that has
aerodynamic behavior identical to that of the particle in question.
Thus, particles having the same aerodynamic diameter may have
different dimensions and shapes.
[0018] In addition to DNA or RNA, a portion of a protein toxin or
portions of other biological molecules may be included in the
corresponding biological analogue. Biological analogues can be
designed to simulate specific bacterial or viral organisms or any
other biological materials of interest.
[0019] The biological analogues produced in accordance with the
teachings of the present invention preferably have a first portion,
such as a particle or bead that carries a second portion, such as
DNA or RNA (or other biologically active portion) associated with
the biological material of interest. As used herein, the phrase
`associated with` means that the biological material is commonly
found as part of the biological organism or compound in question.
For example, a biological material associated with a biological
organism may be a viral protein coat, a cell surface receptor, a
portion of the genome of an organism, a compound, or any other
biological material found on, within, or linked to the biological
organism or compound.
[0020] One feature of the design of the biological analogue is that
it is the second portion used in the construction of the biological
analogue that provides the means for identifying the biological
analogue as the biological material. For example, one way of making
a Bg (Bacillus globigii) biological analogue is by constructing it
so that genomic DNA from B. globigii is attached to the bead or
other carrier. Alternatively, a fragment of DNA that is unique to
Bg (either synthetic or purified from the genome of Bg) is attached
to the bead or other carrier. The beads are disseminated and
collected, and an assay that has been developed to be specific to
Bg DNA is used to test for the presence of Bg (either the genome or
the fragment of DNA that has been attached to the beads). In this
way, the biological analogues are designed specifically to meet any
and all test requirements. The development of biological analogues
with second portions from actual biological warfare agents allows
for testing of detection systems while they are being run as they
are designed to run and not in an artificial modified testing mode.
These types of biological analogues can be safely used in the field
to do proficiency testing of in-place systems and their
operators.
[0021] A biological analogue may also be provided including wholly
synthetic DNA, RNA, protein or other material, as well as with
molecules that are partially synthetic and partially taken from the
biological organism or compound of interest. The amount of DNA, RNA
or other molecules attached to the biological analogue varies
depending on the organism being simulated and the detection system
being tested or calibrated. The amount of such molecules used, in
relation to the number of biological analogues made, will most
often be proportionately similar to the molecule-to-organism ratio
in the actual organism.
[0022] The DNA, RNA or other molecule that makes up the second
portion of the present invention may be carried by the first
portion of the present invention because of covalent, ionic or
other type of binding. The first portion of the biological analogue
may be constructed from a number of suitable materials. In a
preferred embodiment, the present invention includes a polystyrene
latex bead (the first portion) with an oligonucleotide, longer DNA
fragment, or genomic DNA covalently (the second portion) linked
thereto. Polystyrene latex beads are commercially available in a
wide range of sizes from submicron to about 100 microns.
[0023] Another embodiment of the present invention includes
microencapsulation of the second portion. This protects the second
portion from the environment until released for detection.
Microcapsules can be made in a wide range of sizes to suit the
scenario in which the biological analogues are to be used.
[0024] Biological analogues made by microencapsulation of the
second portion have certain advantages over the non-encapsulated
embodiments. First, there exists a range of materials available for
use with microencapsulation, each with different properties that
allow the practitioner to make biological analogues of various
sizes, hydrophobicity, surface charges, densities, or other
desirable characteristics. Second, microencapsulation is extremely
useful in the case of RNA biological analogues because RNA may not
be very stable in the environment unless it is protected in some
manner. Microencapsulation, or other coating methods, can protect
the RNA until the microcapsules are broken or dissolved for
detection. Finally, microencapsulation can be used to encapsulate
all types of second portions of the biological analogue, including
DNA, RNA, protein, protein fragments, and other biomolecules.
[0025] The second portions are encapsulated by mixing them with the
microencapsulation materials (which are the first portion in this
embodiment of the invention) and performing microencapsulation in
the presence of the second portions. The resulting microcapsules
contain the second portion that was present during their formation.
It is possible to re-encapsulate the microcapsules, giving them an
outer coat with desirable properties such as acid resistance, a
desired charge, water insolubility, or to simulate the cell wall or
spore coat of a microorganism.
[0026] One example of a situation in which a biological analogue
constructed in accordance with the teachings of the present
invention is useful concerns the U.S. Postal Service. As noted
above, the Postal Service is evaluating the Northrop Grumman
Biological Detection System for screening of flat mail for
biological agents. Currently, that detection system is being tested
with Bacillus globigii. The Postal Service has a need for periodic
testing and inspection of the detection system but will not allow
the use of any bacteria within its facilities. Use of live bacteria
is problematic because of the infectious, allergenic or toxic
nature of biological organisms. Further, some biological organisms
can persist for a long time after use and there may be problems
associated with unwanted growth of these organisms. For the Postal
Service, it is imperative that its Biological Detection System be
tested without using living organisms.
[0027] In accordance with the teachings of the present invention, a
B. globigii biological analogue can be constructed by linking the
required portion of B. globigii DNA (the second portion) to a
polystyrene latex bead (the first portion) of nominally the same
aerodynamic diameter as B. globigii. Once the appropriate
biological analogue is in hand, it is injected into the Biological
Detection System as an aerosol and transported through the system.
The Biological Detection System collects the B. globigii biological
analogue in the same manner in which it would collect an actual
bacterial sample from the air within and surrounding the mail.
[0028] In short, the B. globigii biological analogue acts in much
the same manner as an actual B. globigii spore. Once the biological
analogue is captured by the Biological Detection system, a sample
of the collection fluid can be analyzed. The DNA linked to the
polystyrene latex bead is removed by sonication during normal
sample processing, providing the free DNA for analysis.
[0029] In addition to scenarios in which a biological analogue is
designed to simulate one specific biological organism or compound,
biological analogues can be produced that contain target sequences
from multiple biological organisms or compounds of similar
aerodynamic diameters. In this way, a single type of biological
analogue can be used to test detection systems for multiple
biological organisms or compounds.
[0030] In addition to the target sequence for the biological
organism or compound, unique, non-naturally occurring synthetic DNA
may also be attached to biological analogues. The addition of
unique synthetic DNA has several advantages. By analyzing for the
unique synthetic DNA the biological analogues can be differentiated
from the real biological organism or compound. The addition of
unique synthetic DNA also allows for individual coding of
manufactured lots of biological analogues. These DNA codes can be
matched with the purchaser of each lot of biological analogues
sold. Through DNA analysis, inappropriate use of biological
analogues can then be traced to the purchaser of that lot.
[0031] There are a number of alternative constructions available
for the first portion of the biological analogues of the present
invention. Silica spheres, paramagnetic spheres, microencapsulated
spheres, or other solid or liquid particles can be substituted for
polystyrene latex beads, for example. Other suitable bead
compositions include, but are not limited to, polystyrene
styrene/divinylbenzene copolymer, polymethylmethacrylate,
polyvinyltoluene styrene/butadiene copolymer, styrene/vinyltoluene
copolymer, vinyl carboxylic acid/styrene copolymer, and
styrene/maleic anhydride copolymer, amino-modified microspheres,
and carboxylate-modified microspheres. A number of additional
constructions are described below. It should be noted, however,
that many other modifications and alternative constructions will be
readily apparent to one of ordinary skill in the art upon reading
the disclosure contained herein.
[0032] Attachment of DNA to beads via streptavidin/biotin linkage
provides a more robust biological analogue than does simple
ionic-based attachment of DNA to silica spheres or polystyrene
latex beads. This represents one alternative construction of a
biological analogue of the present invention. Polystyrene beads are
coated first with streptavidin and then with biotinylated B.
globigii DNA. Each bead receives an average of five genomic
equivalents of DNA. Further, the DNA attaches to the beads
according to a Poisson Distribution such that very few beads have
no genome equivalents. It is proven that sonication will disrupt
the streptavidin/biotin linkage. Therefore, these linkages may not
be suitable for use in systems wherein sonication occurs unless it
is desired that the linkages be broken. The linkages do, however,
allow the biological analogue to more closely simulate a real
biological agent in detection systems that utilize sonication to
disrupt microorganisms for subsequent identification.
[0033] Biological analogues may also be constructed based upon
dipicolinic acid as the second portion. Dipicolinic acid is a
biological molecule unique to the bacterial endospore. Using
dipicolinic acid as a biomarker on a biological analogue
construction allows that particular biological analogue to be used
as a non-biological surrogate for spore-forming bacteria such as B.
anthracis, the causative agent of anthrax. The use of dipicolinic
acid also allows the biological analogue to be used in situations
requiring non-DNA based methods of detection, such as ELISA-based
methods. Various procedures could be adapted for attaching
dipicolinic acid to polystyrene beads. A particular protocol for
attachment of dipicolinic acid to the biological analogue may be
chosen based upon the strength of the attachment, the tolerance of
the attachment to various collection means, the simplicity of the
protocol, the availability of materials, and the costs associated
with a particular protocol.
[0034] RNA-based biological analogues are important for simulation
of viral dissemination events because many viruses have RNA
genomes. Methods for creating RNA-based biological analogues are
similar to those for creating DNA-based biological analogues
described above. Microencapsulation and other methods of coating
are particularly attractive for RNA biological analogues because of
the more labile nature of RNA when exposed to the environment.
Other known techniques for protecting RNA or other biomolecules can
be used in conjunction with the biological analogues of the present
invention.
[0035] Biological analogues can also be constructed having a
fluorescent signature matching that of a certain biological agent
of interest. For example, some systems detect anthrax through
laser-induced fluorescence of the organism. A biological analogue
can be constructed that simulates the fluorescence of anthrax,
thereby allowing the biological analogue to replace anthrax or
biological surrogates used during system checks or testing.
[0036] In addition to the above constructions, paramagnetic beads
may be used to enhance the usefulness of the biological analogues.
Paramagnetic beads coated with streptavidin and biotinylated B.
globigii DNA are particularly useful. Such paramagnetic beads are
readily concentrated to verify their presence in a dilute sample or
to differentiate them from real bacteria. For example, when a 10 mL
volume of collection fluid is exposed to a magnet, the paramagnetic
beads are drawn to the magnet, thereby concentrating them into a
volume of 100-200 .mu.L prior to extraction and analysis. This
allows the presence of the biological analogue to be distinguished
from the presence of actual bacteria when such an analysis is
required. For example, shortly after testing or calibration of a
biological detection system, the system may indicate the presence
of bacterial agents in the testing area. Such an indication could
be due to a true biological event or, instead, could be due to
residual biological analogues in the environment creating a `false
positive`. If the biological analogue beads are paramagnetic, any
low-level residual amount could be easily concentrated and
detected. They also provide an easy means of concentrating
biological analogues in a reference sampler during a low-level
release scenario.
[0037] Further, such magnetized biological analogues allow for easy
cleanup after a testing scenario because they are easily removed by
magnetic means. Use of such paramagnetic beads is possible with any
of the biological analogue constructions described above, and in
other biological analogue constructions that will be readily
apparent to those of ordinary skill in the art upon reading this
disclosure.
[0038] The second portion of the biological analogue may also be
aerosolized by dissemination of the second portion in a carrier (a
first portion) such as, for example, oleic acid or other
semivolatile or volatile liquid. A biological analogue constructed
in this manner is useful for testing biological detection systems
designed to detect aerosolized biological materials.
[0039] Numerous devices for detection of biological organisms
and/or compounds are currently available. For each existing device,
the specific method of detection used is known (i.e. it is known
how the device detects and/or identifies biological organisms or
compounds). Thus, given knowledge of any specific detection system,
and the contents of the present disclosure, a biological analogue
can be constructed for use with any system. Likewise, as new
detection systems are developed over time, biological analogues can
be constructed for those systems given knowledge of how those
systems work and the contents of the present disclosure. The
following paragraphs provide information on a few exemplary
detection systems.
[0040] MesoSystems (Kennewick, Wash.) sells the BT-500 and BT-550
BioCapture.TM. detection systems. The systems use Tetracore BTA.TM.
(Bio Threat Alert) test strips. These strips utilize an
antibody-based detection system that provides a color indication
when a target organism or compound is detected. Toxins or microbes
currently detected by the system are anthrax, ricin, botulinum, and
staphylococcal enterotoxin. Depending on which of these is to be
detected, a different antibody is used in the test system. A
biological analogue for use in testing this system, and constructed
in accordance with the teachings of the present invention, would
include a biologically active portion that is capable of being
bound by the specific antibodies used in the test system.
[0041] The Portal Shield biological chemical detection system
manufactured by Sentel (Alexandria, Va.) also uses immunological
methods for detecting various biological and chemical agents. The
system uses a particle counter to trigger the device. Multiple
Portal Shield systems are positioned around military installations
and are networked together to provide quick response to attack and
to reduce the number of false alarms. Biological analogues designed
for use in testing this system would preferably be of about the
same size as the particles being emulated. Again, since the
specific immunological assays used are known, one could construct
biological analogues that react accordingly. In addition to
specifically testing the detectors response to the biological
analogue, releases at various locations around an installation
would test the systems response to attack.
[0042] Texas Instruments (Dallas, Tex.) manufactures the SPREETA
surface plasmon resonance biodetector. The biodetector is able to
measure properties such as refractive index changes, avidin-biotin
binding, antibody-antigen dissociation kinetics, thickness of
insulators, refractive index of thin dielectrics, specific
detection of small molecules, protein binding, concentrations of
analytes, attachment of DNA complements, and mixture proportions.
Depending on the specific target of the detection system, the
properties measured may varied. To test the capabilites of the
SPREETA detection system, a biological analogue can be designed to
simulate the characteristics for which the system is screening.
Since, for any given version of the SPREETA system, it will be
known what the system is looking for, a biological analogue can be
designed accordingly. For example, if the SPREETA system is looking
for specific small molecules, then those are the molecules that
make up the biologically active portion of the biological
analogue.
[0043] Advanced Analytical Technology, Inc. (AATI; Ames, Iowa) has
developed a fluorescence-tagged flow cytometer for low-level
microbe detection. Each species of microbe to be detected will give
a unique combination of side scatter and fluorescence detector
responses. In order to construct a biological analogue suitable for
testing the AATI device, a biological analogue is constructed that
provides the same set of side scatter and fluorescence detector
responses as the corresponding microbe.
[0044] The Northrop Grumman device, mentioned above, uses a
DNA-based detection system.
[0045] The following examples detail the construction of a
biological analogue for B. globigii suitable for use with the
Northrop Grumman system.
EXAMPLE
[0046] As indicated above, the U.S. Postal Service requires a means
of conducting post-installation and periodic tests of its
Biological Detection System. B. globigii is commonly used as a
biological warfare surrogate organism for such purposes, however
the U.S. Postal Service will not allow a live organism to be used
in testing on its premises. The example now described provides a B.
globigii biological analogue for use in testing such Biological
Detection Systems.
[0047] Attachment of Genomic Bg to Silica Beads by Ionic-Based
Interactions.
[0048] Because of the need to simulate the B. globigii endospore as
closely as possible, it was important to determine how many genome
equivalents of B. globigii genomic DNA to attach to each biological
analogue bead. One B. globigii endospore contains one genome of B.
globigli DNA. The DNA to be attached to the biological analogue
beads cannot, however, simply be added in an amount that
constitutes enough DNA to attach one genome equivalent to each
biological analogue bead. This is because the binding occurs in a
Poisson Distribution so that, even if the DNA binds with 100%
efficiency, a large number of biological analogue beads (around 36%
or more) will receive no DNA at all.
[0049] If sufficient DNA is added to provide five genomic
equivalents for each biological analogue bead, only 0.67% of the
beads will have no DNA attached. Around 3%, or slightly more, will
have received one genomic equivalent while around 9% receive two
genomic equivalents, 14% receive three genome equivalents, and so
forth, as shown in Table 1. TABLE-US-00001 TABLE 1 Poisson
Distribution for Five Genome Equivalents of DNA/Bead. DNA copies 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Percent 0.67 3.37 8.43 14.0
17.6 17.6 14.6 10.4 6.53 3.63 1.81 0.82 0.35 0.13 0.05 0.01
0.01
[0050] The Poisson Distribution provided in Table 1 assumes 100%
efficiency of DNA binding with the biological analogue beads.
Because 100% efficiency is not achieved in the laboratory, even
under the most ideal conditions, an experiment was conducted using
five genome equivalents per biological analogue bead, as described
below.
[0051] Total genomic DNA was prepared from B. globigii. The
concentration of DNA was determined by optical spectroscopy. The
concentration of the silica beads used was provided by the
manufacturer of the beads (Bang's Laboratories). The genomic DNA
and silica beads were combined such that five genomic equivalents
of DNA were provided for each silica bead (see Table 2, below). The
attachment of the DNA to the beads was performed by the silica bead
manufacturer's recommended protocol (adapted from TechNote 302,
"Molecular Biology", from Bang's Laboratories, "Nucleic Acid
Adsorption to Silica Microspheres" ). After attachment was
complete, the biological analogue beads were washed nine times
using 70% ethanol in order to be absolutely certain all unbound DNA
was removed. The beads were stored in the final 70% ethanol wash
solution.
[0052] The newly-created biological analogue beads were removed
from the 70% ethanol and placed in Tris/EDTA buffer (made according
to a recipe provided by Bang's Laboratories, 10 mM Tris base, 1 mM
EDTA-Na.sub.2). The biological analogue beads were then subjected
to sonication (to simulate sonication in Cepheid's GenExpert
System, part of the NG Biological Detection System). A small
aliquot of liquid containing the DNA that was removed from the
biological analogue beads during sonication was set aside for
analysis by PCR (the PCR analysis described herein was conducted by
TaqMan PCR analysis unless otherwise indicated). The biological
analogue beads were then centrifuged briefly to pellet the beads,
and another liquid aliquot containing DNA was removed for PCR
analysis.
[0053] PCR analysis indicated that 33% of the DNA was recovered
during the extraction step (in which liquid was simply removed from
the biological analogue beads after sonication). The beads were
then contrifuged and the supernatant analysed. TaqMan analysis of
this sample indicated that 29% of the DNA was recovered during the
extraction. Removing the biological analogue beads from the liquid
by centrifugation did not significantly alter the DNA yield.
[0054] PCR analysis further indicated that very little DNA was
removed during the initial washing procedure (approximately 1% or
less). The PCR reaction conducted with the first rinse from the
washing procedure described above was inhibited, preventing a
direct determination of how much DNA was washed off of the
biological analogue beads during the first rinse, however the high
DNA yields in the extraction and the very low amounts of DNA
observed in Rinses 3-9 (only the odd numbered rinses were analyzed,
see Table 2) indicate that the rinsing steps, including Rinse 1,
removed very little DNA. The final extraction, which yielded 33% of
the DNA, indicated an overall recovery of approximately 1.5 genome
equivalents per biological analogue bead, as indicated in Table 2.
This number is close to the average genome equivalents per B.
globigii endospore. TABLE-US-00002 TABLE 2 Data for Recovery of DNA
from Biological analogues. Sample Copies DNA Prep 5.10E+07 Rinse 3
5.30E+05 Rinse 5 1.10E+05 Rinse 7 4.70E+05 Rinse 9 4.60E+05
Extraction 1 1.70E+07 Extraction 2 1.50E+07
[0055] This experiment therefore demonstrated that a B. globigii
biological analogue, constructed in accordance with the teachings
of the present invention, is useful as a non-biological surrogate
for dissemination-type testing. The use of 2 .mu.m biological
analogues, with five genome equivalents per biological analogue,
provides a surrogate that quite closely resembles the actual B.
globigii endospore.
[0056] As noted above, B. globigii endospores contain one genome
copy per endospore. When these endospores are analyzed using the
GenExpert System, they are subject to sonication. This breaks open
the endospore and releases the DNA, making it available for PCR
analysis. The experiment described above demonstrates that the
biological analogue of the present invention can be subjected to
sonication, that such sonication removes DNA from the biological
analogues, and that the DNA so removed is suitable for PCR
analysis. Thus, in these respects, the behavior of the biological
analogue simulates precisely the behavior of the actual B. globigii
endospore.
[0057] Attachment of Genomic DNA to Polystyrene Beads by
Streptavidin/Biotin Interactions.
[0058] The ratio of 5 genome equivalents per bead described above
was also used in this embodiment of the present invention. "EZ-Link
Psoralin-PEO Biotin" (Pierce Laboratories) was used to biotinylate
genomic DNA from Bg. A labeling protocol was developed based on a
protocol provided by Pierce Laboratories. Reactions were set up
with differing amounts of Psoralin-PEO-Biotin and a constant amount
of DNA. The reactions were irradiated with long-wave UV light. The
excess Psoralen-PEO-Biotin was removed from the DNA by washing and
precipitation steps.
[0059] ProActive Microspheres (Bang's Laboratories) were used for
the bead component of the biological analogue. The beads were
polystyrene and were already coated with streptavidin.
[0060] Preparation of the beads and attachment of the biotinylated
genomic Bg DNA were performed using protocols provided by the
manufacturer (Bang's Laboratories). The information was collected
from the following literature: TechNotes 101, 203, and 302, which
are "ProActive Microspheres", "Washing Microspheres" and "Molecular
Biology", respectively.
[0061] In summary, the biotinylated DNA prepared above was added to
washed polystyrene beads coated with streptavidin. The reactions
were allowed to incubate and the beads (now with biotinylated DNA
attached) were washed to remove any unbound DNA and stored.
Following construction, the biological analogues were analyzed in
Cepheid's GenExpert System and were able to be detected. Additional
tests were performed to verify that the biological analogues would
remain detectable when disseminated from a nebulizer and captured
in the SpinCon collector used in Northrup Grumman's Biological
Detection System. During testing, the biological analogues were
disseminated, captured by the SpinCon, and placed in the GenExpert
system and detected.
[0062] The preceding examples illustrate potential mechanisms for
attaching biomarkers to the beads. It is to be understood that the
methods described above are only two examples of a great variety of
mechanisms for attachment of biomolecules to the beads.
Furthermore, different methods of attachment may be more
appropriate for different biomolecules (for example, dipicolinic
acid).
[0063] Additional embodiments of the present invention are also
contemplated herein. For instance, a biological analogue can be
created having more than one type of biological molecule attached
thereto (DNA and protein, for example). Alternatively, a biological
analogue could be constructed having biomolecules from different
sources attached to the same bead. For instance, a single
biological analogue could be created having DNA from both Bg and Eh
(Erwinia herbicola a plant pathogen). This biological analogue is
useful in that it provides a surrogate for both organisms without
the use of multiple versions of the product.
[0064] In addition, the biological analogues of the present
invention can be labeled with synthetic strands of DNA that can
identify a specific batch of biological analogues or otherwise
trace a biological analogue of interest to a particular sale. Thus,
a biological analogue detected with a biological detection system
could be traced back to a specific end user or purchaser.
[0065] Also, biological analogues could be produced having varying
particle sizes. For example, small particle sizes can be produced
to mimic weapon-grade biological warfare agents, while larger
particle sizes or multiple particle agglomerates may be produced to
mimic biological warfare agents that might be used by a less
sophisticated organization. It is important to be able to detect
both types of particles.
[0066] Using microencapsulation techniques, or other methods of
coating, biological analogues can be developed that have a very
short half-life in the environment in which they are being used.
This decreases the likelihood of background biological analogue
material confounding the interpretation of subsequent analyses.
[0067] Fluorescent biological analogues can be used to mimic the
fluorescent activity displayed by various bacteria. NAD/NADH.sub.2
can be incorporated into the biological analogues for this type of
testing.
[0068] Biological analogues can also be produced in dry
preparations for simulating spores found, for example, in postal
envelopes. The biological analogues can be dispensed into dry
envelopes and run through the postal machinery to evaluate the
capability of the Biological Detection System to detect "spiked"
envelopes. This is useful for an inspector performing 'spot checks'
of a postal facility. The dry biological analogue material could be
produced in different particle sizes to reflect different degrees
of sophistication on the part of those who might use actual
biological weapon agents.
[0069] Also, biological analogues can be developed to mimic a
protein of interest (a protein toxin. For example). To accomplish
this, purified protein is subjected to proteolysis rendering it
harmless. The fragments may then be attached to the beads or formed
into particles in the required size range. The biological analogues
are then disseminated, collected, and analyzed by, for instance,
ELISA-based methods or pyrolysis mass spectrometer.
[0070] Biological particles such as Bacillus anthracis, can be made
nonviable through physical techniques such as autoclaving, french
pressure cell, or sonication. Other techniques such as proteolysis
may also be employed to render the particle harmless. In the
process, under most conditions, the particles will be fragmented.
These fragments may then be attached to beads or formed into
particles, of the same aerodynamic diameter as the biological
parcle,in the laboratory or during dissemination. Through these
processes, biological analogues can be engineered to look like
actual organisms to pyrolysis mass spectrometer detections systems
and other systems that key on specific chemical constituents. A
specific example would be to take a suspension, of a known
concentration of Bacillus anthracis, autoclave it, cool it, and
adjust the concentration by adding water or evaporating liquid off
of the suspension. The concentration would be adjusted so that
dissemination of the fragments will cause agglomerations to form in
the size range of the Bacillus anthracis. The size distribution of
the formed biological analogues will be determined by the size of
the cell fragments and the number of fragments in each droplet
formed during dissemination.
[0071] In addition to the various embodiments of the present
invention that can be produced, the usefulness of the present
invention is not limited to testing or calibrating detection
systems for biological warfare agents. Additional uses are
described below, and it is contemplated that many other uses will
be readily apparent to one of skill in the art upon reading this
disclosure.
[0072] Non-military or weapons-related uses are also evident.
Airborne biological organisms, toxins and allergens, such as
Legionnaire's disease, toxic molds, and pollen, can cause serious
illness and even death. Legionnaire's disease, for instance, is
caused by the naturally occurring Legionella pneumophila bacterium
and its related serotypes. The dissemination of this organism and
its transmission to humans is not well-understood, but it is known
to involve inhalation of aerosolized water droplets containing
viable Legionella that are deposited in the lungs, causing
infection. Cooling towers, faucet nozzles, aerators, piping leaks,
and showerheads have been discovered to create such
Legionella-containing aerosols. Biological analogues can be used to
mimic Legionella in a building's water system and aerosol
production scenarios could be undertaken to realistically simulate
a contamination and dissemination event for study. Similarly,
bacterial, viral, mold and pollen biological analogues can be
customized and used to study indoor air-quality, define standards,
and test monitoring equipment.
[0073] Further uses are evident in the food industry. Food
processing plants are required to prepare Hazard Analysis Critical
Control Point (HACCP) prevention plans. These plans are specific to
a processing plant, the types of food produced, and the various
ways in which the food can become contaminated with disease-causing
organisms such as Escherichia coli, Listeria monocytogenes,
Clostridium botulinum, and Salmonella spp., for example. Testing
HACCP plans is difficult because the risk of introducing live
microorganisms is unacceptable. For testing transport and
dissemination of contaminant organisms in the food processing
environment (air, water, surfaces, and plant operators), biological
analogues can be constructed to mimic organisms of concern and then
introduced into the plant processes at risk analysis points. The
plant can then be operated and monitored for the presence of the
biological analogues and biocontrol procedures can be
evaluated.
[0074] It is understood that the invention is not limited to the
specific details described herein, which are given by way of
example only, and that various modifications and alterations,
including use of other potential and yet-to-be discovered methods
of attachment of any and all biomolecule of choice, are possible
without departing from the spirit or scope of the invention as
defined in the claims.
* * * * *