Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-14
2003-03-11
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007920, C435S007940, C435S962000, C435S971000, C435S972000, C436S518000, C436S524000, C436S525000, C436S527000, C436S528000, C436S529000, C436S531000, C436S532000, C436S536000, C436S538000, C436S164000, C436S172000, C436S824000, C530S391100, C530S391300, C530S391500, C530S413000
Reexamination Certificate
active
06531278
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to capture of a target entity on a solid phase and detection of the captured target. More particularly, the invention relates to capture and detection of contaminants in biomedical, environmental, and food samples.
Considerable progress in the development of detectors of microbial contamination has been achieved in recent years. These detectors can be applied to medical, process control, and environmental fields. Such detectors must possess features such as high specificity, simplicity, sensitivity, speed, reliability, and reproducibility. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as the ligands for specific capture, numerous applications have been developed for detection of pathogenic bacteria. M. R. Blake & B. C. Weimer, Immunomagnetic Detection of
Bacillus stearothermophilus
Spores in Food and Environmental Samples, 63 J. Appl. Environ. Microbiol. 1643-1646 (1997); A. Burkowski, Rapid Detection of Bacterial Surface Proteins Using an Enzyme-linked Immunosorbent Assay System, 34 J. Biochem. Biophys. Methods 69-71 (1997); S. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods Using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L. S. Metherell et al., Rapid, Sensitive, Microbial Detection by Gene Amplification Using Restriction Endonuclease Target Sequence, 11 Mol. Cell Probes 297-308 (1997); F. Roth et al., A New Multiantigen Immunoassay for the Quantification of IgG Antibodies to Capsular Polysaccharides of
Streptococcus pneumoniae
, 176 J. Inf. Dis. 526-529 (1997).
Bacterial spores are the most heat-stable form of microorganisms, are ubiquitous in the environment, and therefore are of great concern in food products, such as milk, that receive extensive heat treatments to prolong shelf life. Spore counts in milk from around the world vary between zero and >22,000 colony forming units (cfu)/ml depending on the climate of the region. S. Chen, supra.
Bacillus stearothermophilus
spores are among the most heat-resistant spores and are found in high numbers in soil and water.
B. stearothermophilus
spores survive extreme heat and will germinate and grow at elevated product storage temperatures, which occur in foods transported in equatorial regions of the world.
While
B. stearothermophilus
is not commonly a problem, other bacilli often lead to food-borne illness or spoilage in a variety of foods.
Bacillus cereus, B. licheniformis, B. subtilis
, and
B. pumilus
, have all been implicated in outbreaks of food-borne illness and are commonly isolated from raw and heat-treated milk. M. W. Griffiths, Foodborne Illness Caused by Bacillus spp. Other than
B. cereus
and Their Importance to the Dairy Industry, 302 Int. Dairy Fed. Bulletin 3-6 (1995).
B. cereus
is also responsible for a sweet curdling defect in milk, as well as being pathogenic. W. W. Overcast & K. Atmaram, The Role of
B. cereus
, in Sweet Curdling of Fluid Milk, 37 J. Milk Food Technol. 233-236 (1973). A mesophilic heat-resistant bacillus similar to Bacillus badius, has been isolated from extreme-temperature-processed milk (D
147
=5 sec). P. Hammer et al., Pathogenicity Testing of Unknown Mesophilic Heat Resistant Bacilli from UHT-milk, 302 Int. Dairy Fed. Bulletin 56-57 (1995).
B. badius
is a mesophilic organism that grows readily at room temperature, making it a likely candidate for spoiling temperature-processed foods. There have been 52 confirmed cases of
B badius
in ultra-high-temperature treated milk in Europe and two cases outside Europe. Lack of a rapid spore assay that can be used in milk contributes to the difficulty of prediction of post-processing spoilage, thereby limiting the shelf life and product safety. H. Hofstra et al., Microbes in Food-processing Technology, 15 FEMS Microbiol. Reviews 175-183 (1994). Such an assay could be used in a hazard critical control point (HACCP) plan allowing raw materials with high spore loads to be diverted to products that do not pose a food safety risk to consumers.
The standard method for quantifying spores in milk, G. H. Richardson, Standard Methods for the Examination of Dairy Products (15
th
ed., 1985), involves heat-shock and an overnight plate count. This method is time-consuming and merely yields historical information. The food industry needs microbiological assays to yield predictive information for maximum benefit in HACCP analysis and risk assessment. An enzyme-linked immunosorbent assay (ELISA) capable of detecting >10
6
cfu/ml of
B. cereus
spores in food has been reported, but was unacceptable due to antibody cross reactivity. L. A. Metherell et al., supra.
Techniques to increase sensitivity of immunosorbent assays have focused on more efficient reporter labels, such as faster catalyzing reporter-enzymes; signal amplification, such as avidin- or streptavidin-biotin enzyme complexes; and better detectors, such as luminescence and fluorescence. L. J. Kricka, Selected Strategies for Improving Sensitivity and Reliability of Immunoassays, 40 Clin. Chem. 347-357 (1994); W. W. Overcast & K. Atmaram, supra. Immunomagnetic antigen capture is used extensively to separate and identify
Escherichia coli
and Salmonella from foods. M. R. Blake & B. C. Weimer, supra; S. Y. Rabbany et al., supra; C. Blackburn et al., Separation and Detection of Salmonellae Using Immunomagnetic Particles, 5 Biofouling 143-156 (1991); P. M. Fratamico et al., Rapid Isolation of
Escherichia coli
O157:H7 from Enrichment Cultures of Foods Using an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113 (1992); A Lund et al., Rapid Isolation of K88
+
Escherichia coli
by using Immunomagnetic Particles, 26 J. Clin. Microbiol. 2572-2575 (1988); L. P. Mansfeild & S. J. Forsythe, Immunomagnetic Separation as an Alternative to Enrichment Broths for Salmonella Detection, 16 Letters Appl. Microbiol. 122-125 (1993); A. J. G. Okrend et al., Isolation of
Escherichia coli
O157:H7 using O157 Specific Antibody Coated Magnetic Beads, 55 J. Food Prot. 214-217 (1992); Skjerve & Olsvic, Immunomagnetic Separation of Salmonella from Foods, 14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright et al., Immunomagnetic Separation as a Sensitive Method for Isolating
Escherichia coli
O157 from Food Samples, 113 Epidemiol. Infect. 31-39 (1994). These methods, however, involve either a pre-incubation or a subsequent incubation step (usually
18
-
24
hours) to increase the cell numbers for detection. Immunomagnetic capture greatly shortens
E. coli
and Salmonella testing, but long incubation times limit this method for predictive information. Immunocapture has also been used to quantitate
Bacillus anthracis
spores in soil samples using luminescence detection, A. Burkowski, supra, but these efforts have led to tests that have a detection limit of about 10
3
cfu/ml.
In view of the foregoing, it will be appreciated that providing compositions and methods for capture and detection of selected contaminants would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide compositions and methods of use thereof for capture and detection of contaminants in food, environmental samples, and other applications.
It is another object of the invention to provide compositions and methods of use thereof for capture and detection of contaminants, wherein such methods are highly specific, simple, sensitive, rapid, reliable, and reproducible.
These and other objects can be achieved by providing a composition of matter comprising a ligand component covalently bonded to a nucleic acid component. Preferably, the ligand component is a member selected from the group consisting of antibodies, antigens, lectins, saccharides, and gangliosides, and more preferably is an antibody. In a preferred embodiment, the nucleic acid component is an oligonucleotide. In another preferred embodiment, a linker is interposed between the ligand component and the
Chin Christopher L.
Clayton Howarth & Cannon, P.C.
Grun James L.
Utah State University
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