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  Assays for trichomonal and other hydrolasesUSPTO Application #: 20080026417Title: Assays for trichomonal and other hydrolases Abstract: The release by trichomonads of a hydrolase that hydrolyzes a narrowly defined class of substrates at a low pH without interference from hydrolases that are unrelated to trichomoniasis is the basis for a selective diagnostic assay for trichomoniasis that measures hydrolysis of any of these substrates by vaginal fluid at a low pH. Selective assays for trichomoniasis are also obtained by removing particulate matter from a sample of vaginal fluid to extract a fraction devoid of particles greater than a selected size, and where desired, combining the extracted fraction with any of certain specified hydrolase inhibitors, then testing the fraction for enzymatic hydrolase activity. These qualities of trichomoniasis are the basis for a series of diagnostic tests and test devices that produce results that are detectable by visual and other means with a high degree of accuracy. (end of abstract) Agent: Townsend And Townsend And Crew, LLP - San Francisco, CA, US Inventors: Paul J. Lawrence, Mark A. Hughes, Aulena Chaudhuri, Terrence J. Andreasen USPTO Applicaton #: 20080026417 - Class: 435018000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Hydrolase The Patent Description & Claims data below is from USPTO Patent Application 20080026417. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a divisional application of co-pending U.S. patent application Ser. No. 11/353,497 filed Feb. 13, 2006, which is a continuation application of U.S. patent application Ser. No. 10/269,917, filed Oct. 10, 2002, and issued as U.S. Pat. No. 7,041,469 on May 9, 2006, the contents of which are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention resides in the field of hydrolases (i.e., hydrolytic enzymes), and in particular, methods and devices for the detection of hydrolase activity in a sample or specimen and the diagnosis of disease based on the detection of hydrolase activity. A specific area of interest of this invention is the detection of trichomoniasis in female subjects by assaying for the presence of enzymatically active trichomonal hydrolases in a vaginal fluid specimen. [0004] All literature cited in this specification, including patents, technical articles and books, are incorporated by reference in their entirety. [0005] 2. Description of the Prior Art [0006] Trichomoniasis is a clinically important Sexually Transmitted Disease (STD) caused by the protozoan Trichomonas vaginalis. In 1955, the World Heath Organization estimated approximately 170 million new cases of trichomoniasis would arise annually among adults worldwide, with higher prevalence and incidence rates in both developing and industrialized countries than for any other sexually transmitted disease. Trichomoniasis is frequently undetected because most infected men and approximately 50% of infected women are asymptomatic. Trichomoniasis in women may cause discomfort or a foul smelling vaginal discharge, and can be associated with adverse clinical sequelae. Trichomoniasis can increase the risk the human immunodeficiency virus (HIV) transmission and infection (Laga et al., AIDS 7(1):95-102 (1993) and WHO Press Release WHO/64 (1995)). [0007] Trichomonads produce specific hydrolases called proteinases that hydrolyze proteins including, but not limited to, IgG, IgM, and IgA antibodies (Provenzano and Alderete, Infect. Immun. 63(9):3388-3395 (1995)). These proteinases also damage secretory leukocyte hydrolase inhibitor (SLPI), a protective factor normally present in vaginal fluid that inhibits HIV viral entry into human monocytic cells (Draper et al., J. Infect. Dis. 178(3):815-819 (1998)). Trichomonas vaginalis (T. vaginalis) is also thought to play a role in promoting cervical cancer (Yap et al., Genitourin. Med. 71(6):402-404 (1995)). Pregnant women infected with T. vaginalis at mid-gestation are more likely to have a low birth weight infant or to deliver preterm (Cotch et al., Sex. Transm. Dis. 24:353-360 (1997)). [0008] The most common method for diagnosing trichomoniasis is wet mount microscopy, a microscopic examination of vaginal fluid specimens for T. vaginalis. This is a labor-intensive method which requires a microscope and a skilled technician, and fails to detect approximately half of the infected women (Baron et al., Laboratory Diagnosis of Female Genital Tract Infections, Cumitech 17A, American Society for Microbiology (1993)). Trichomoniasis can be diagnosed with high sensitivity by culturing vaginal fluid specimens, but this method requires culture media and long-term growth in a controlled-temperature incubator, in addition to the use of a microscope and a skilled technician. Due to the labor involved, the expense of culture media and supplies, the training required, and the delay of up to a week to detect trichomonal growth, few clinics or laboratories routinely use culture to diagnose trichomoniasis. [0009] A diagnostic test based on DNA amplification and detection has recently been developed by Lawing et al., J. Clin. Microbiol. 38(10):3585-3588 (2000). This test is sensitive but, like culture, not rapid enough to produce the results during the patient's visit to the clinic. Due to the cost of the DNA-based test kits and the training and laboratory equipment required to perform these tests, this methodology is not widely used in clinics or in medical practice in general, particularly in the parts of the world where the need for a trichomoniasis test is greatest. [0010] Thus, to date there is no rapid, accurate, cost-effective and simple method or test device for point-of-care diagnosis of trichomoniasis. Numerous studies have demonstrated that T. vaginalis produces a variety of hydrolases; see for example Garber et al., Can. J. Microbiol. 35:903-909 (1989). Two studies have demonstrated that vaginal fluid specimens from women with trichomoniasis contain detectable levels of hydrolases that disappear after infected women are treated and cured of the infection (Alderete et al., Genitourin. Med. 67(6):469 (1991), and Garber and Lemchuck-Favel, Parasitol. Res. 80(5):361-365 (1994)). Unfortunately, the laboratory-based hydrolase detection methods used in the studies reported by these authors required equipment, skill and training and were labor-intensive and slow, so that the results were obtained only after several hours. For example, the method used by Aldrete et al. for detecting trichomonal hydrolases involved a four-step process to produce gelatin-acrylamide zymograms: first, trichomonal hydrolases were electrophoretically concentrated into discrete bands in a sheet of polyacrylamide gel; second, the hydrolases were allowed to digest gelatin which had been immobilized within the polyacrylamide gel; third, the gel was stained with a general protein stain; and fourth, the gel was destained in a lengthy washing process which eventually revealed the hydrolase-digested gelatin which appeared as clear bands on the darkly stained background gel (the zymogram). Trichomonal hydrolases were detected in the Garber et al. study by polyacrylamide gel electrophoresis followed by immunoblotting, a similarly complex procedure that involved the use of rabbit antibodies to visualize a specific hydrolase band. Both methods require a high level of expertise and costly equipment and take many hours to complete, and are therefore unsuitable for point-of-care testing. [0011] The zymogram procedure described above can also be performed by using synthetic fluorogenic substrates to detect the trichomonal hydrolases once the hydrolases have been separated by gel electrophoresis. These substrates typically contain one or more amino acids linked to a fluorogenic reporter group that becomes fluorescent only after it is enzymatically cleaved from the peptide group by a hydrolase. Unfortunately, this procedure still entails the unwieldy polyacrylamide gel electrophoresis step prior to testing for hydrolase activity with the fluorogenic substrates. Moreover, hydrolysis of the substrate can be observed only by examining the electrophoretic gels for bands that fluoresce under ultraviolet light. Both the gelatin-digestion method and the fluorogenic substrate method were utilized in a study of intracellular and secreted T. vaginalis hydrolases reported by North et al., Mol. Biochem. Parasitol. 39:183 (1990). Several fluorogenic substrates were identified which could be used to detect trichomonal hydrolases. Unfortunately, the methods used in this study are impractical for a point-of-care clinical diagnostic test, since gel electrophoresis is slow and cumbersome and observation of fluorescence requires instrumentation or a darkroom and ultraviolet illuminator. The difficulty is that vaginal fluid contains many different hydrolases secreted by a variety of sources, including bacteria, which are present at extremely high levels, white blood cells, vaginal epithelial cells, and others. Each method relies on electrophoretic separation to achieve selective detection of the trichomonal hydrolases. Without electrophoretic separation, it could not be determined if the hydrolytically active bands were derived from trichomonads or from some other source of hydrolytic activity in a vaginal fluid specimen. [0012] A trichomoniasis test is therefore needed that can be performed by attending clinicians quickly, simply, inexpensively and accurately while the patient is still present. It would be particularly beneficial to be able to perform the test with a disposable device that is inexpensive and easy to use and one that rapidly produces accurate results. SUMMARY OF THE INVENTION [0013] A series of discoveries has now been made that permit a sample of vaginal fluid to be tested for the presence of T. vaginalis in a fast, accurate, and efficient manner. These discoveries also lead to methods and test devices for detecting the presence of enzymes in general, including various types of hydrolases, that are indicative of a variety of physiological conditions. The specimens in which the detections are performed may be any bodily fluids, vaginal fluid being but one example. [0014] One discovery is that trichomonads release a particular hydrolase into vaginal fluid that actively hydrolyzes a narrowly defined class of substrates at a low pH without interference from other hydrolases that are unrelated to trichomoniasis and that are also present, or frequently present, in vaginal fluid. A diagnosis of trichomoniasis can thus be made by assaying vaginal fluid for hydrolytic activity against one or more members of this class of substrates at low pH. Hydrolysis of the substrate is readily converted to a signal that is either machine-readable or visually detectable. [0015] This discovery can be implemented in various diagnostic methods and test materials. One example is the use of an implement that is capable of retaining liquid together with a solid support on whose surface are deposited the assay components, in distinct regions if necessary, depending on the particular assay methodology. The implement can be applied to the surface and the assay result can be read directly on or in the implement. The implement can for example be a swab, dropper, pipette, or other liquid transfer device, and the deposited assay components can include a substrate and an indicator. Swabs are particularly convenient implements since a swab can easily be wetted with the sample, then rubbed on the operative surface of the solid support, causing the assay components deposited on the surface to adhere to the swab. This allows the user to perform the assay determination by simply noting whether a detectable change, preferably a color change, has occurred on the swab. [0016] A further discovery is that trichomonal hydrolase activity, without being restricted to low pH, can be separated from other sources of hydrolase activity in a specimen of bodily fluid by size exclusion. Thus, by extracting from a specimen of bodily fluid, particularly vaginal fluid, a fraction that is devoid of particulate matter above a certain size threshold, one can detect the presence or absence of trichomoniasis in the specimen by determining whether hydrolytic cleavage of the substrate has occurred. Selectivity toward trichomonal hydrolase activity can be improved further by combining size exclusion with the use of a narrowly defined class of hydrolase inhibitors. Thus, in preferred embodiments of this discovery, a sample of vaginal fluid is processed to extract a fraction that is devoid of particulate matter above a certain size threshold and, in the presence of one or more of these inhibitors, the extracted fraction is brought into contact with an appropriate substrate to detect hydrolase activity. Restricting the substrate to a narrowly defined class provides even greater selectivity for trichomonal hydrolase activity. [0017] Related to the discovery addressed in the preceding paragraph is the discovery of a test device that includes a migration path through which a sample of bodily fluid that is applied to the device travels by capillary force. The device contains porous material along the migration path to filter out particulate matter above a selected size threshold. The device is useful for the detection of any soluble enzyme that is not adhered to cells or other particulate matter in the bodily fluid, and bodily fluids on which the device can be used include vaginal fluid, urine, blood, saliva, and various others. The device also contains a substrate that is acted upon by the enzyme of interest and an indicator that produces a signal as a result of action of the enzyme upon of the substrate. In assays for trichomoniasis, for example, the substrate will be one that is hydrolyzed by trichomonal hydrolase activity. An alternative to the substrate-indicator combination is a substrate that includes a chromogen that undergoes a color change as a direct result of the action of the enzyme of interest. For enzymes that cause cleavage of the substrate, the chromogen may undergo a color change upon release of the chromogen from the remainder of the substrate. Hydrolases, including trichomonal hydrolases, are examples of such enzymes. Regardless of whether a substrate-indicator combination is used or a substrate is used that includes a chromogen whose color changes upon action of the enzyme, the substrate, the indicator if one is present, or both substrate and indicator are positioned far enough along the migration path that the visual signal is attributable only to hydrolytic activity in the filtered liquid. In preferred such devices, one of the class of inhibitors referred to above is also included to further improve the selectivity of the assay. [0018] Among the advantages that are offered by these discoveries, methods, compositions and devices are accuracy and selectivity in the diagnosis of T. vaginalis, tests that can be performed quickly with minimal training or instruction, and the ability to perform the tests in test devices that are self-contained with all reagents included and requiring only the application of the liquid test specimen. These and other objects, features, aspects, and advantages of the invention, as well as various embodiments of the principles forming the basis for the various discoveries, will be apparent from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS [0019] FIG. 1 is a vertical cross section of a test device in accordance with this invention, in which the test result is read on the test device itself. [0020] FIG. 2 is a vertical cross section of a second test device in accordance with this invention, representing a variation of the test device of FIG. 1. Continue reading... Full patent description for Assays for trichomonal and other hydrolases Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Assays for trichomonal and other hydrolases patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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