Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-15
2002-06-04
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S091500, C435S180000, C435S007500, C435S002000, C436S518000, C210S198200, C428S403000
Reexamination Certificate
active
06399317
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to detection of antigens. More particularly, the invention relates to compositions and methods for detection of selected antigens in real time. In a preferred embodiment, the invention relates to compositions and processes for sensitive detection of microbes and contaminants in food samples, environmental samples, and the like within about 30 minutes.
Considerable progress in the development of biosensors for microbial detection has been achieved in the last decade. These biosensors can be applied to medical, process control, and environmental fields. They must possess ideal features such as high specificity, simplicity, sensitivity, reliability, reproducibility, and speed. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as the recognition element 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. A. 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. A. 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 are therefore of great concern in food products (e.g., milk) that receive extensive heat treatments to prolong shelf life. Spore counts in milk from around the world vary between zero and >22,000 cfu/ml depending on the climate of the region. S. A. 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).
Bacillus stearothermophilus
spores are one of the most heat-resistant bacterial spores and are found in high numbers in soil and water. Contaminating
B. stearothermophilus
spores survive extreme heat to 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, Bacillus licheniformis, Bacillus subtilis,
and
Bacillus 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, . 1973. The Role of
Bacillus 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 and 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 UHT milk in Europe and two cases outside of Europe (P. Hammer et al., supra). 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. Rev. 175-183 (1994)). Such an assay could be used in a hazard analysis 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 (1985), involves heat-shocking and an overnight plate count. This is time-consuming and yields historical information. The food industry needs microbiological assays to yield predictive information for maximum benefit in HACCP analysis and risk assessment. An enyzme-linked immunosorbent assay (ELISA) capable of detecting >10
6
cfu/ml of
B. cereus
spores in foods has been reported, but was unacceptable due to antibody cross-reactivity (Y. H. Chang & P. M. Foegeding, Biotin-avidin Enzyme-linked Immunosorbent Assay for Bacillus Spores in Buffer and Food, 2 J. Rapid Methods and Autom. Microbiol. 219-227 (1993)).
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); P. Patel, Rapid Analysis Techniques in Food Microbiology (1994)). Immunomagnetic antigen capture is used extensively to separate and identify
Escherichia coli
and Salmonella from foods (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
O0157:H7 from Enrichment Cultures of Foods Using an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113 (1992); L. Krusell & N. Skovgaard, Evaluation of a New Semi-automated Screening Method for the Detection of Salmonella in Foods within 24 h, 20 Inter. J. Food Microbiol. 124-130 (1993); 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); E. 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)). However, these methods involve either a preincubation or a subsequent incubation step (usually 18 to 24 h) 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 luminescent detection (J. G. Bruno & H. Yu, Immunomagnetic-electrochemiluminescent Detection of
Bacillus anthracis
Spores in Soil Matrices, 62 App. Environ. Microbiol. 3474-3476 (1996)), but these efforts have led to tests that have a detection limit of 10
3
cfu/ml.
In view of the foregoing, it will be appreciated that compositions and methods for real time detection of selected antigens, such as contaminants in food and the environment, would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for rapid detection of antigens.
It is another object of the invention
Clayton Howarth & Cannon, P.C.
Portner Ginny Allen
Smith Lynette R. F.
Utah State University
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