| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 05.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 16.05.00 |
| PATENT TITLE |
Method of diagnosing periodontal disease |
| PATENT ABSTRACT |
This invention provides a method of diagnosing periodontal disease in a subject by detecting elevated concentrations of .beta.-glucuronidase in saliva from the subject. The concentration of .beta.-glucuronidase in the subject's saliva may be determined by adding to a sample of the saliva a substrate for .beta.-glucuronidase and measuring the amount of a product produced by the reaction of .beta.-glucuronidase on the substrate. Also, the concentration of .beta.-glucuronidase in the subject's saliva may be determined by adding to a sample of saliva a labeled antibody specific for .beta.-glucuronidase and measuring the amount of labeled antibody which complexes with .beta.-glucuronidase present in the saliva. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 14.11.96 |
| PATENT REFERENCES CITED |
Biol 30:235-242, 1985. Lamster, Ira Enzyme Activity in Crevicular Fluid for Detection and Prediction of Clincal Attachment Loss in Patients with Chronic Adult Periodontol, J Periodontol 59:516-523, 1988. Mandel, I., Markers of Periodontal Disease Susceptibility and Activity Derived From Saliva, Johnson NW ed. Risk Markers for Oral Disease. vol. 3 London; Cambridge University Press pp. 228-253, 1991. Lamster Ira Development of a Risk Profile for Periodontal Disease: Microbial and Host Response Factors. J. Peiodontol 65(5):511-520, 1994. Mandel, I.D. (1991) "Markers of periodontal disease susceptibility and activity derived from saliva." In: Johnson, N.W. ed. Risk markers for oral disease. vol. 3, Peridontal diseases. London: Cambridge University Press, pp. 228-253 (Exhibit J); and. Mandel, I.D. (1990) "The diagnostic uses of saliva." J Oral Pathol Med 19: 119-125 (Exhibit K). Lamster, I.B., et al. (1988) "Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients with chronic adult periodontitis." J Periodontol 59: 516-523 (Exhibit D);. Lamster, I.B., et al. (1993) "Current status of tests of tests for periodontal disease." Adv Dent Res 7: 182-190 (Exhibit E);. Lamster, I.B., et al. (1994) "The relationship of .beta.-glucuronidase activity in crevicular fluid to clinical parameters of periodontal disease: Findings from a multicenter study." J Clin Periodontal 21: 118-127(Exhibit F);. Lamster, I.B., et al. (1995) "The relationship of .beta.-glucuronidase activity in crevicular fluid to probing attachment loss in patients with adult periodontitis: Findings from a multicenter study." J Clin Periodontol 22: 36-44 (Exhibit G);. Lamster, I.B. and Grbic, J.T. (1995) "Diagnosis of periodontal disease based on analysis of the host response." Priodontol 2000 7: 83-89 (Exhibit H);. Lamster, I.B., et al. (1991) "Indicators of the acute inflammatory and humoral immune responses in gingival crevicular fluid: relationship to active periodontal disease." J. Periodontal Research 27: 261-263 (Exhibity I);. Ferguson, D.B. (1987) "Current diagnostic uses of saliva." J Dent Res 66: 420-424 (Exhibit B);. Lamster, I.B., et al. (1985) "Evaluation and modification of spectrophotometric procedures for analysis of lactate dehydrogenase, beta-glucuronidase and arylsulphatase in human gingival crevicular fluid collected with filter-paper strips." Arch. Oral Biol. 30: 235-242 (Exhibity C);. |
| PATENT CLAIMS |
What is claimed is: 1. A method of diagnosing periodontal disease in a subject which comprises: (a) determining a concentration of .beta.-glucuronidase in saliva from the subject; and (b) comparing the concentration determined in step (a) of .beta.-glucuronidase in saliva from the subject to a concentration of .beta.-glucuronidase in saliva from individuals without periodontal disease, wherein a larger concentration of .beta.-glucuronidase in saliva from the subject than in saliva from individuals without periodontal disease indicates a diagnosis of periodontal disease in the subject. 2. The method of claim 1, wherein the concentration of .beta.-glucuronidase in the subject's saliva is determined by adding to a sample of the saliva a substrate for .beta.-glucuronidase and measuring the concentration of a product produced by the reaction of .beta.-glucuronidase on the substrate. 3. The method of claim 2, wherein the substrate is 4-methylumbelliferone .beta.-D glucuronide and the product is 4-methylumbelliferone. 4. The method of claim 3, wherein the concentration of the product is measured by the fluorescence of the product. 5. The method of claim 4, wherein the concentration of the product is measured by a calorimetric device. 6. The method of claim 4, wherein the concentration of the product is measured by a fluorometer. 7. The method of claim 2, wherein the concentration of the product is measured by the fluorescence of the product. 8. The method of claim 2, wherein the substrate is phenolphthalein mono-.beta.-glucuronic acid and the product is phenolphthalein. 9. The method of claim 2, wherein the concentration of the product is measured by a calorimetric device. 10. The method of claim 2, wherein the concentration of the product is measured by a spectrometer or fluorometer. 11. The method of claim 1, wherein the concentration of .beta.-glucuronidase in the subject's saliva is determined by adding to a sample of saliva a labeled antibody specific for .beta.-glucuronidase and measuring the amount of labeled antibody which forms a complex with .beta.-glucuronidase present in the saliva. 12. The method of claim 1, wherein the concentration of .beta.-glucuronidase in the subject's saliva is determined based upon measuring the concentration of .beta.-glucuronidase in a sample derived from the saliva. 13. The method of claim 1, wherein the saliva is supernatant saliva. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately before the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein. Conventional diagnostic evaluation of periodontal disease has relied on measuring clinical parameters, such as probing depth, attachment level, and plaque accumulation, and on measuring the height of the alveolar bone using radiographs. One shortcoming of these conventional tests is that they only define the status of the periodontium at the time of examination. In the past 10 to 15 years, studies have shown that clinical parameters of periodontal disease are poor predictors of when and at which sites patients that would experience active disease (Haffajee, A. D., et al., 1993). In addition, measurement of alveolar bone loss using intraoral radiographs is of limited value because it provides only a historical record of past disease and cannot be used to determine when the loss of crestal bone occurred. Due to these limitations, researchers have investigated alternative methods for evaluating patients with periodontal disease (Lamster, I. B., et al., 1993). Previously, analysis of the host inflammatory and immune responses in periodontal disease has been performed on gingival crevicular fluid (GCF). Among the host-derived mediators shown to identify patients at risk for active periodontal disease is the lysosomal enzyme, .beta.-glucuronidase (Lamster, I. B., et al., 1988; Lamster, I. B., et al., 1995), which is a marker of primary granule release from neutrophils into the gingival crevicular fluid. Lamster and coworkers previously found that elevated levels of .beta.-glucuronidase in gingival crevicular fluid were diagnostic for existing periodontal disease, and correlated with the likelihood of future disease progression (Lamster, I. B., et al., 1994; Lamster, I. B., et al., 1995). Existing tests for .beta.-glucuronidase in gingival crevicular fluid involve collecting fluid from within the gingival crevice using such special devices as methylcellulose strips or microsyringes. The levels of .beta.-glucuronidase are measured in the fluid by adding a substrate for .beta.-glucuronidase which in the presence of the enzyme produces a product that is detectable. Unfortunately, this method is labor-intensive and requires the services of highly-trained personnel, such as a dentist or dental hygienist. Therefore, although measurements of .beta.-glucuronidase levels in gingival crevicular fluid have been shown to be a better indicator of human periodontal disease than previous conventional evaluations, the limitations of this method make its widespread use unlikely. Currently no method exists that overcomes these disadvantages and provides a simple, reliable test for diagnosing periodontal disease which could be utilized on a widespread basis. Although one might consider using samples from other body fluids such as saliva that are easy to collect, distinct drawbacks for biochemical evaluation of periodontal disease by analysis of saliva exist (Lamster, I. B. and Grbic, J. T., 1995). More specifically, based on the current state of the art as described in the scientific literature, it would have been expected to be difficult, if possible, to detect elevated levels of .beta.-glucuronidase in saliva as a means of diagnosing periodontal disease (Ding, Y., et al., 1994; Lundy, F. T. and Lamey, P -J., 1995). Thus, detection of markers in general is more difficult in saliva than in gingival crevicular fluid because constituents of saliva are derived from many sources, including the major and minor salivary glands and from gingival crevicular fluid. (Lamster, I. B. and Grbic, J. T., 1995). Further, the relatively large volumes of fluid involved when dealing with saliva would be expected to have both a masking and a diluting effect on any potentially important marker derived from gingival crevicular fluid (Lamster, I. B. and Grbic, J. T., 1995), thus making it difficult to detect the marker. Moreover, even if detection of the enzyme in saliva was possible, one would have expected that it would be difficult to distinguish between healthy and diseased subjects. Although previous studies have proposed diagnostic tests in saliva for periodontal disease using markers such as IgG, IgA and collagenase, none has ever mentioned the use of .beta.-glucuronidase. (Lamster, I. B. and Grbic, J. T., 1995). In fact, some experts have argued that elevated levels of markers in saliva would not be detectable until after disease had become widespread, thereby limiting the importance of such an approach for diagnositic purposes (Mandel, I., 1991). Furthermore, even if .beta.-glucuronidase was detectable, one could reasonably have expected that the dilution of the marker by the much larger volume of saliva would have made it impossible to distinguish between healthy and diseased subjects. Based on the foregoing, one skilled in the art would not have expected that a test for measuring the concentration of .beta.-glucuronidase in saliva would be useful for the diagnosis of periodontal disease. Unexpectedly, a saliva test based on measuring levels of .beta.-glucuronidase has not only proven useful, it has proven to be a better discriminator of periodontitis than analysis of gingival crevicular fluid or clinical parameters. SUMMARY OF THE INVENTION This invention provides a method of testing for periodontal disease in a subject which comprises detecting an elevated concentration of .beta.-glucuronidase in saliva from the subject relative to the concentration of .beta.-glucuronidase present in saliva from a healthy subject. This invention further provides that the concentration of .beta.-glucuronidase in the subject's saliva may be determined by adding to a sample of saliva or a sample derived from saliva a substrate for .beta.-glucuronidase and measuring the concentration of a product produced by the action of .beta.-glucuronidase in the substrate. In one embodiment of this invention, the product produced by the action of .beta.-glucuronidase on the substrate is characterized by the property of being fluorescent, visible or detectable by spectrophotometry. Examples of suitable subtrates include 4-methylumbelliferone .beta.-D glucuronide or phenolphthalein mono-.beta.-glucuronic acid. This invention further provides a method of testing for periodontal disease, wherein the concentration of .beta.-glucuronidase in the subject's saliva is determined by adding to a sample of saliva a labeled antibody specific for .beta.-glucuronidase and measuring the amount of labeled antibody which forms a complex with .beta.-glucuronidase present in the saliva. This invention further provides a test kit for performing the above-described method. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method of testing for periodontal disease in a subject which comprises detecting an elevated concentration of .beta.-glucuronidase in saliva from the subject relative to the concentration of .beta.-glucuronidase present in saliva from a healthy subject. As used in this application, "periodontal disease" means periodontitis which is characterized by the presence of increased probing depth (one or more sites with a depth of at least 5 mm), evidence of clinical attachment loss and demonstrable loss of alveolar bone on periapical and bitewing dental radiographs. Periodontal disease occurs as a consequence of the host's inflammatory and immune responses to subgingival bacterial pathogens. The symptoms include erythematous (red) and edematous (swollen) gingiva, pocket formation around the teeth, tooth mobility and eventually tooth loss. As used in this application, "an elevated concentration of .beta.-glucuronidase" means that the concentration of .beta.-glucuronidase in saliva from the subject is larger than the concentration of .beta.-glucuronidase in saliva from a subject who is not afflicted with periodontal disease. A saliva sample from a subject who is not afflicted with periodontal disease should have minimal or negligible amounts of .beta.-glucuronidase. These levels of .beta.-glucuronidase will be lower in comparison to the levels of this enzyme in saliva from periodontitis patients. Further, as used in this application, "a healthy subject" is defined as one who is not afflicted with human periodontal disease. This invention also provides a method of testing for periodontal disease in a subject by detecting an elevated concentration of .beta.-glucuronidase, wherein the concentration of .beta.-glucuronidase in the subject's saliva is determined by adding to a sample of the saliva a substrate for .beta.-glucuronidase and measuring the amount of a product produced by the action of .beta.-glucuronidase in the substrate. In a preferred embodiment, the sample of saliva is unstimulated. As used in this application, "unstimulated" saliva means that the subject will expectorate in a collection vessel without stimulation of salivary flow, for example, by having the subject chew on a piece of paraffin film or a tart candy. As used in this application, a "substrate" means any molecule upon which the enzyme .beta.-glucuronidase acts to produce a product that is neither .beta.-glucuronidase nor the substrate. Substrates for the enzyme .beta.-glucuronidase are well-known in the art, and include 4-methylumbelliferone .beta.-D glucuronide which yields the product 4-methylumbelliferone, and phenolphthalein mono-.beta.-glucuronic acid which yields the product phenolphthalein. Methods of measuring the concentration of product produced by the action of .beta.-glucuronidase are also well-known in the art. In one preferred embodiment of the invention, the amount of the product is measured based upon the product being characterized by the property of being fluorescent. In another embodiment of the invention, the amount of the product is measured based upon the product being detectable by colorimetry. Due to negligible levels of .beta.-glucuronidase in healthy individuals, the elevated concentrations of .beta.-glucuronidase in periodontitis patients will act upon certain substrates so that one can either visualize and measure by calorimetric devices the concentration of .beta.-glucuronidase in a saliva sample. In another embodiment of the invention, the amount of the product is measured based upon the product being detected by spectrophotometry. For example, when .beta.-glucuronidase acts upon phenolphthalein mono-.beta.-glucuronic acid in an alkaline solution, the product produced is phenolphthalein, the concentration of which may be measured using a spectrophotometer. This invention further provides a method of testing for periodontal disease in a subject by detecting an elevated concentration of .beta.-glucuronidase by adding to a sample of saliva a labeled antibody specific for .beta.-glucuronidase and measuring the amount of labeled antibody which forms a complex with .beta.-glucuronidase present in the saliva. The labeled antibody may be a polyclonal or monoclonal antibody. In one embodiment, the labeled antibody is a purified, labeled antibody. In another embodiment, the labeled antibody may consist of two antibodies. For instance, a first antibody may specifically bind to .beta.-glucuronidase and not be labeled. A second antibody is directed against the first antibody and is labeled with a detectable marker or an enzyme which gives rise to a detectable marker. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof. The detectable marker may be, for example, radioactive or fluorescent. In another embodiment of this invention, the concentration of .beta.-glucuronidase in the subject's saliva may be determined based upon measuring the concentration of .beta.-glucuronidase in a sample derived from saliva. As used in this application, "derived from saliva" means a sample initially collected as whole saliva, but due to manipulation of the sample, only specific components remain in the sample. Methods of manipulating samples in such manner are well-known in the field. In one such embodiment, the sample derived from the saliva is supernatant saliva. As used herein, "supernatant saliva" is saliva that has had the cellular components removed. Methods of removing cellular components from saliva are well known in art, and include filtration, ion exchange chromatography and precipitation. In one such embodiment, the whole saliva is centrifuged and the supernatant is collected. In an embodiment of the above-described method, the sample saliva or the sample derived from saliva is mixed and incubated with the appropriate substrate at room temperature (20-24.degree. C.). In another embodiment, the mixing and incubation step occur over a period of about fifteen minutes. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |