Assays for detection of Bacillus anthracis

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C435S004000, C435S005000, C435S006120, C435S007800, C435S007200, C435S007210, C435S007220, C435S007300, C435S007320, C435S810000, C435S007940, C435S007950, C435S069700, C436S501000, C436S518000, C536S023700, C424S246100

Reexamination Certificate

active

06828110

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of assays for detecting
Bacillus anthracis
, the causative agent of anthrax.
2. Background
Anthrax spores were first produced as weapons in the 1950s. Several countries including the former Soviet Union, the United States and Iraq are known to have produced anthrax weapons. Anthrax is a particularly fearsome biological warfare agent, not only because of its deadliness, but also because anthrax weapons are relatively easy to produce and deliver. Production of anthrax spores requires little more than basic laboratory equipment and growth media. Anthrax weapons are comprised of an anthrax source and an industrial sprayer that can produce aerosol particles of the appropriate size for victims to inhale. Such sprayers, for instance, can be mounted on low flying airplanes or other vehicles and used to spread anthrax over a wide area. Because of the ease and relatively small expense involved in producing and delivering anthrax weapons, such weapons are potentially highly attractive weapons of mass destruction for terrorist groups. Thus, in addition to potential organized military conflicts that may give rise to the use of such weapons, terrorist organizations are a potential threat for the use of such weapons in airports, office buildings and other centers of human activity.
Anthrax is caused by
Bacillus anthracis
, a gram-positive, sporulating bacillus.
B. anthracis
is a soil bacterium and is distributed worldwide. The organism exists in the infected host as a vegetative bacillus and in the environment as a spore. The anthrax spore is typically the infective form of the bacteria life cycle. Anthrax spores can survive adverse environmental conditions and can remain viable for decades. Animals such as cattle, sheep, goats and horses can contract the spores while grazing. Humans can contract anthrax from inoculation of minor skin lesions with spores from infected animals, their hides, wool or other products, such as infected meat (Franz et al. (1997)
J. Am. Med. Assoc
. 278(5): 399-411).
The typical mode of entry of the anthrax spore into the body, inhalation, results in an illness known as woolsorter's disease. After deposit in the lower respiratory tract, spores are phagocytized by tissue macrophages and transported to hilar and mediastinal lymph nodes. The spores germinate into vegetative bacilli, producing a necrotizing hemorrhagic mediastinitis (Franz et al., supra). Symptoms include fever, malaise and fatigue, which can easily be confused with the flu. The disease may progress to an abrupt onset of severe respiratory distress with dyspnea, stridor, diaphoresis and cyanosis. Death usually follows within 24 to 36 hours.
Because the effects of exposure to anthrax are not immediate, and because the initial symptoms are easily confused with the flu, there is a need for a fast method to detect
B. anthracis
in an environment where
B. anthracis
may have been released. This need is enhanced by the increasing number of anthrax threats that are called into governmental authorities each year. A fast method for determining whether public places have been exposed to anthrax spores in therefore essential.
Anthrax spores have S-layers, as do spores of many other archea and bacteria. Most S-layers are comprised of repeats of a single protein (Etienne-Toumelin et al.,
J. Bacteriol
. 177:614-20 (1995)). The S-layer of
B. anthracis
, however, is comprised of at least two proteins: EA1 (Mesnage et al.,
Molec. Microbiol
. 23:1147-55 (1997)) and surface array protein (SAP) (see Etienne-Toumelin, et al., supra). Fully virulent
B. anthracis
isolates are encapsulated by a capsule that encompasses the S-layer of the bacteria and prevents access of antibodies to both EA1 and SAP (Mesnage et al.,
J. Bacteriol
. 180:52-58 (1998)).
Several methods for detecting
B. anthracis
have been reported, although none are optimal for quick and reliable detection of anthrax contamination. Detection methods include those based on amplification of nucleic acids that are specific for
B. anthracis
(Lee,
J. Appl. Microbiol
. 87:218-23 (1999); Patra, G.,
FEMS Immunol. Med. Microbiol
. 15:223-31 (1996); Ramisse et al,
FEMS Microbiol. Lett
. 145(1):9-15 (1996); Bruno and Keil,
Biosens. Bioelectron
. 14:457-64 (1999); and Japanese Patent Nos. 11004693; 6261759; 6253847; and 6253846). The need to conduct time-consuming laboratory procedures to use these amplification methods limits their usefulness for quick identification of anthrax contamination. Other detection methods involve detecting spore-based epitopes of
B. anthracis
using antibodies (Yu, H.,
J. Immunol. Methods
218:1-8 (1998); Phillips et al.,
J. Appl. Bacteriol
. 64:47-55 (1988); Phillips et al.,
FEMS Microbiol. Immunol
. 1:169-78 (1988)). Other reported detection methods include an enzyme-linked lectinosorbent assay (Graham et al.,
Eur. J. Clin. Microbiol
. 3:210-2 (1984)) and a method using DNA aptamers that bind anthrax spores (Bruno et al.,
Biosens Bioelectron
. 14(5):457-64 (1999)).
Previous antibody-based detection methods for
B. anthracis
employed antibodies raised against whole anthrax spores. Such immunogens lead to the production of antibodies that cross-react with other related bacterial species. Longchamps et al., for instance, found that no antibody analyzed in their study was completely specific in recognizing anthrax spores (
J. Applied Microbiology
87:246-49 (1999)). At least one study has shown that polyclonal antibodies raised against
B. anthracis
whole spore suspensions do not react with SAP protein (Mesnage et al,
Molec. Microbial
. 23:1147-55 (1997)). Closely related bacteria that may cross react with non-specific antibodies include
B. cereus, B. thuringiensis
and
B. mycoides
(Longchamp et al., supra.; Phillips et al.,
FEMS Microbiol. Immunol
. 47:169-78 (1988)). This high degree of cross-reactivity is highly problematic for detection of anthrax because these non-toxic cross-reactive strains are widespread.
B. thuringiensis
in particular is commonly found in the soil, in part because the bacteria is sprayed on crops for its insecticidal qualities.
Therefore, a need exists for improved methods for detecting
Bacillus anthracis
in the environment. Such methods should be not only provide rapid results, but also should have little or no cross-reactivity with related species that are prevalent in nature. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
The present invention provides novel methods of detecting
Bacillus anthracis
. The methods involve contacting a test sample with a capture reagent that can bind to
B. anthracis
surface array protein (SAP), wherein the capture reagent forms a complex with SAP if SAP is present in the test sample, and detecting whether SAP is bound to the capture reagent. The capture reagent, for instance, can form a complex with the surface array protein if the surface array protein is present in the sample. Presence of the surface array protein is indicative of the presence of
B. anthracis
in the sample. In one embodiment, SAP comprises a polypeptide with the amino acid sequence shown in SEQ ID NO:1. In another embodiment, the
B. anthracis
strain is encapsulated.
The capture reagent can comprise an antibody that binds to SAP. In some embodiments, the antibody can be a recombinant antibody, such as a recombinant polyclonal or monoclonal antibody.
In a preferred embodiment, the test sample is collected from a site of suspected or threatened anthrax contamination. In another preferred embodiment, the test sample is collected using a cyclonic device. The test sample does not need to be cultured prior to contacting with the capture reagent.
In some methods of the invention, the capture reagent can be immobilized on a solid surface, such as a microtiter dish. The capture reagent can be immobilized on the solid support prior to contacting the capture reagent with the test sample.
In presently preferred embodiments, the assay methods of the invention are hig

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