Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-13
2004-02-10
Devi, S. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S975000, C435S007100, C435S007320, C435S007300, C436S547000, C436S533000, C436S544000
Reexamination Certificate
active
06689572
ABSTRACT:
BACKGROUND OF THE INVENTION
Although
Escherichia coli
is considered part of the normal bowel flora of healthy individuals, the microorganism is also recognized as an important pathogen in human diseases and conditions, including urinary tract infections, meningitis, and diarrhea. Therefore, a highly sensitive and specific method that can rapidly identify
E. coli
in biological samples would be useful.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a highly sensitive and specific
E. coli
agglutination assay can be performed using antibodies against total urea-extracted
E. coli
antigens. Surprisingly, the sensitivity of the assay is dramatically reduced when using antibodies against specific
E. coli
antigens, even though such a strategy proved useful in agglutination assays for other bacterium (see, e.g., U.S. Pat. No. 5,418,140). Consequently, the invention relates in part to the unexpectedly superior performance of the assay using antibodies against total urea-extracted antigens, rather than specific surface antigens.
Accordingly, the invention features a method for detecting
Escherichia coli
in a sample by providing a sample suspected of containing
Escherichia coli
; contacting the sample with beads coated with a mixture of antibodies or anti-serum specific to one or more surface antigens of
Escherichia coli
, each of the one or more surface antigens having a molecular weight of 21±2 KDa, 26±2 KDa, 31±2 KDa, 36±2 KDa, 38±2 KDa, 67±2 KDa, or 77.8±2 KDa; and observing agglutination of the beads, where the presence of the agglutination indicates the presence of
Escherichia coli
in the sample. The method can further include determining whether a microorganism in the sample produces indole or ferments lactose, both of which are characteristic of
E. coli.
The invention further features a kit for testing whether
E. coli
is present in a sample. The kit contains beads and a mixture of antibodies or antisera specific to one or more surface antigens of
Escherichic coli
, wherein each of the one or more surface antigens has a molecular weight selected from the group consisting of 21±2 KDa, 26±2 KDa, 31±2 KDa, 36±2 KDa, 38±2 KDa, 67±2 KDa, and 77.8±2 KDa. The kit can also include (1) a composition containing eosin-methylene blue for determining whether a microorganism ferments lactose, (2) a composition containing p-dimethylaminocinnamaldehyde for determining whether a microorganism produces indole, (3) a positive control reagent that is expected to agglutinate the beads coated with the mixture of antibodies, or (4) a negative control reagent that is not expected to agglutinate the beads coated with the mixture of antibodies.
As used herein, “total urea-extracted
Escherichia coli
antigens” means the antigens collected from the supernatant of a centrifuged mixture containing
E. coli
bacteria incubated in an aqueous mixture containing about 4 M urea.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.
DETAILED DESCRIPTION
The invention relates to methods of detecting
E. coli
in a sample, such as a biological sample (e.g., a serum or cerebral spinal fluid sample) using an agglutination assay. While agglutination assays have been used to detect various species of bacteria in samples, the moieties attached to the beads and useful for agglutination are different, depending on the species to be detected (see, e.g., Chang et al., J. Food Protect. 57:31-36, 1994; and Chang et al., J. Food Protect. 56:759-762, 1993). Indeed, it is thus far uncertain which moieties are needed to detect a different species of bacterium.
The present invention is based in part on the identification of the particular moiety required for a highly sensitive and specific agglutination assay for detection of
E. coli
. As shown in the example below, the
E. coli
agglutination assay is not specific enough for clinical applications unless the beads are coated (e.g., by standard covalent or non-covalent methods) with a mixture of antibodies specific for total urea-extracted
E. coli
antigens. Such a mixture of antibodies can be produced by immunizing a vertebrate with total urea-extracted
E. coli
antigens and harvesting the antibodies produced by the animal, using standard techniques such as the ones described in the example below.
Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the agglutination assays described below, utilize the present invention to its fullest extent. The following example is to be construed as merely illustrative of how one skilled in the art can isolate total urea-extracted
E. coli
antigens, immunize an animal with such antigens, and harvest antibodies produced by the animal, and are not limitative of the remainder of the disclosure in any way. Any publications cited in this disclosure are hereby incorporated by reference.
REFERENCES:
patent: 4520072 (1985-05-01), Yoshino et al.
patent: 5418140 (1995-05-01), Chang et al.
patent: 5536644 (1996-07-01), Ullman et al.
patent: 6096563 (2000-08-01), Hajizadeh et al.
patent: 6235494 (2001-05-01), Hugli
patent: 6391652 (2002-05-01), Okada et al.
patent: 98 05687 (1998-02-01), None
patent: 98 55871 (1998-12-01), None
de Graaf et al. Infect. Immun. 36: 751-758, 1982, abstract.*
Vasquez et al. Vet. Microbiol. 48: 231-241, Feb. 1996, abstract.*
Faris et al. Zentralbl. Bakteriol. Mikrobiol. Hyg (A) 259 (4): 477-484, 1985.*
Wolf et al. Infect. Immun. 57 (1): 164-173, 1989, abstract.*
McConnell et al. FEMS Microbiol. Lett. 52 (1-2): 105-108, 1989, abstract.*
Thorns et al. Vet. Record 125: 91-92, 1989.*
Rice et al. J. Clin. Microbiol. 30: 1315-1316, 1992.*
Bänffer et al., “Evaluation of a Commercial Latex . . . ,” Eur. J. Clin. Microbiol. Infect. Dis., 12:633-636, 1993.
Benge, G.R., “Detection of Salmonella . . . ,” Eur. J. Clin. Microbiol. Dis, 8(4):294-298, 1989.
Chang et al., “Development of a Latex . . . ,” Journal of Food Protection, 57(1):31-36, 1994.
Chang et al., “Evaluation of a Latex . . . ,” Journal of Food Protection, 56(9):759-762, 1993.
Chang et al., “Efficacy of a Latex . . . ,” Journal of AOAC International, 79(3):661-669, 1996.
Huang et al., “Comparison of the . . . ,” Journal of Food Protection, 60(1):6-9, 1997.
Kerr et al., “Diagnostic application of . . . ,” Journal of Applied Bacteriology, 72:302-308, 1992.
Koga et al., “Isolation and Characterization . . . ,” Journal of General Micrbiology, 129:3185-3196, 1983.
Labadie et al., “Selection of cell wall . . . ,” Journal of Applied Bacteriology, 72:220-226, 1992.
Lim et al., “Detection of Group D . . . ,” Journal of Clinical Microbiology, 25(7):1165-1168, 1987.
March et al., “Latex Agglutination Test . . . ,” Journal of Clinical Microbiology, 27(7):1675-1677, 1989.
Sowers et al., “Evaluation of Commercial Latex . . . ,” Journal of Clinical Microbiology, 34(5):1286-1289, 1996.
Zhang et al., Evaluation of a Recombinant . . . , Microbiol. Immunol., 42(6):423-428, 1998.
Boyer et al., “Lack of Clinical Usefulness of a Positive Latex . . . ,” Sep. 1993, Pediatric Infectious Disease Journal, vol. 12, No. 9; 779-780.
Huang et al., “Development of a Latex Agglutination Test for Rapid . . . ,”, Feb. 2001, Eur.J.Clin.Microbiol.Infect.Dis., vol. 20, No. 2; 97-103.
Whitfield et al., “Biosynthesis and Assembly of the Polysialic Acid . . . ”, 1984, J.Biol.Chem., vol. 259, No. 20;12776-12780.
York et al., “Multilaboratory Validation of Rapid Spotfor . . . ”, J.Clin.Microbio., vol. 38, No. 9; 3394-3398.
Chang Hsien-Chang
Chang Tsung Chain
Huang Ya Hei
Devi S.
Executive Yuan, Council of Agriculture
Fish & Richardson P.C.
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