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United States Patent im
Thieme et al.
US005714341A [ii] Patent Number:  Date of Patent:
 SALIVA ASSAY METHOD AND DEVICE
 Inventors: Thomas R. Thieme, Independence; B.
Melina Cimler. Portland; Nanette M.
Klimkow, Beaverton. all of Qreg.
 Assignee: Epitope, Inc., Beaverton, Qreg.
 Appl. No.: 219,977
 Filed: Mar. 30, 1994
 Int CL6 C12Q 1/40; G01N 21/00;
 U.S. CI 435/22; 435/4; 435/29;
435/805; 435/810; 436763; 422/56; 422/58; 422/61; 422/102; 128/760; 128/762; 128/769;
 Field of Search 435/22. 4, 29,
435/805, 810; 436/63; 422/56, 58, 61. 102;
128/760, 762. 769, 771
 References Cited
U.S. PATENT DOCUMENTS
2.850,903 9/1958 Schwerin 435/22
4,582,795 4/1986 Shibuya et al 435/34
4,853,325 8/1989 Vodian et al 435/5
5,036,000 7/1991 Palmer et al 435/26
5,056,521 10/1991 Parsons et al 128/635
5,103,836 4/1992 Goldstein et al 128/760
5,149,623 9/1992 Carison et al 435/5
5,334,502 8/1994 Sangha 435/7.21
5,339,829 8/1994 Thieme et al 128/760
5,479,937 1/1996 Thieme et al 128/760
FOREIGN PATENT DOCUMENTS
0 083 941 7/1983 European Pat. Off. .
52O408A2 12/1992 European Pat. Off. .
WO 91/14944 10/1991 WIPO .
W093/11434 6/1993 WIPO .
Thieme et al, J. Clin. Microbiol., vol. 30. No. 5, pp. 1076-1079, May 1992.
Winn-Deen et al, Clin. Chem., vol. 34. No. 10, pp. 2005-2008. 1988.
Wallenfels et al. Chemical Abstracts, vol. 100. p. 206. Ref. #81626s, 1984 (Aerzl. Lab. 1983, 29(11). 339^8).
Primary Examiner—Cecilia J. Tsang
Assistant Examiner—Abdel A. Mohamed
Attorney, Agent, or Firm—Townsend and Townsend and
An improved method for determining the presence of an analyte in an oral fluid sample. A portion of the sample is mixed or contacted with a chromogenic substrate effective to produce a colored product upon reaction with a-amylase present in the sample. After a selected reaction time, the reaction mixture is inspected to determine the level of such colored product produced. Production of a level of colored product above a selected threshold within the reaction time indicates that an effective volume of undiluted oral fluid sample has been collected which is sufficient to allow detection of the analyte. A device for use in the method is also disclosed.
20 Claims, 2 Drawing Sheets
U.S. Patent Feb. 3, 1998 Sheet 2 of 2
SALIVA ASSAY METHOD AND DEVICE
FIELD OF THE INVENTION
The present invention relates to methods of analyzing saliva for the presence of selected analytes.
Parry, J. V., et al.. Lancet 2:72-75 (1987). Thieme, T.. et al., J. Clin. Microbiol. 20:1076-1079 (1992a).
Thieme, T., et al., "Oral Fluid Sampling for Determination of HIV-I Antibody Serostatus," Abstract from Vm Int'l. Conf. on AIDS. pg. C328 (1992b).
BACKGROUND OF THE INVENTION
A number of analytical procedures and devices are commonly used to test body fluids for the presence of substances of diagnostic value. Most procedures today are based on analysis of blood or urine. An advantage of blood as a test fluid is that analytes are often present at relatively high concentrations, and measurements of these concentrations can often provide information about a patient's health. Urine is useful for diagnostic testing when the blood component of interest (e.g., a low molecular weight drug or hormone) is concentrated during urine formation. However, the urine concentration of an analyte does not usually reflect the physiologically active amount of the analyte in blood.
Although saliva has not been commonly used as a body fluid in medical diagnosis, numerous studies (Parry et al., 1987; Thieme et al., 1992 a. b) have demonstrated that oral fluids may be used for diagnostic testing for antibodies or antigens specific for various human or animal pathogens. Oral fluids have also been shown to be useful in measuring the body levels of therapeutic drugs, drugs of abuse, and naturally occurring hormones.
Devices for collection of oral fluid have been described. U.S. Pat. Nos. 4,418.702 and 4,580,577 show an absorbent pad for collecting oral fluid, and a barrel-piston arrangement for extracting the fluid from the pad.
U.S. Pat. No. 4.774,962 describes an absorbent pad for collecting saliva, and a centrifuge tube and tube-insert for removing oral fluid from the pad by centrifugation.
U.S. Pat. No. 5,056,521 describes an apparatus for includes a barrel-piston arrangement having a nonreactive absorbent swab secured to the piston. The barrel and piston are used to squeeze the fluid sample from the swab into a glucose monitoring instrument.
PCT Publication No. WO 91/13355 and U.S. Pat. No. 5,103.836 disclose a method and device for collecting analytes from the oral cavity for testing. The method employs an absorbent pad impregnated with the salts of a hypertonic solution effective to recover a high concentration of the analyte.
EPO Publication No. 520408 A2 and U.S. Pat. No. 5,260,031 describe sampling devices for collecting biological fluids such as saliva. The '031 patent describes a device that includes a piece of filter paper fixed to a holder for collecting a saliva sample, and an indicator for determining that a selected volume of sample has been collected.
One difficulty in the testing of saliva is that the saliva sample may be relatively dilute, because of a recent intake of liquids, for example. Where the sample is to be tested for an analyte whose presence indicates a specific disease state, a dilute saliva sample can give rise to a negative test result because the analyte is too dilute to be detected.
A further problem with saliva is that unlike blood, which can be easily recognized by virtue of its color, saliva is colorless and may therefore be indistinguishable in appearance from water. In situations where an insurance represen
5 tative must collect a saliva sample from a test subject, the subject may be able to evade the test by providing a water sample rather than a saliva sample.
Since false negatives can seriously undermine the reliability of analyte testing in saliva, it would be desirable to
to have a method that provides rapid assessment of the dilution state of a collected saliva sample. Such a method could significantly enhance the reliability of saliva-based diagnostic tests, by establishing whether a collected sample is a saliva sample that is sufficiently concentrated to allow
15 reliable detection of the analyte of interest.
SUMMARY OF THE INVENTION
The present invention relates to an improvement for methods of determining the presence of an analyte in a saliva
20 sample, where a variable volume of sample is collected from the mouth of a human subject for testing in an analyte assay.
In the method of the invention, a portion of the sample is contacted with a chromogenic substrate effective to produce a colored product upon reaction with a-amylase present in
25 the sample. After a selected reaction time, the reaction mixture is inspected to determine the level of colored product produced within the selected time. Production of a level of colored product above a selected threshold level indicates that an effective volume of undiluted saliva has
30 been collected that is sufficient to allow detection of the analyte in the analyte assay.
In one embodiment, the chromogenic substrate is localized on a collection device which includes (i) an absorbent
35 pad for collecting oral fluid when the pad is placed in a subject's mouth, and (ii) a reaction region containing the substrate, which is located downstream from the pad on the device for absorbing oral fluid from the pad after oral fluid has been absorbed by the pad. The device may additionally include a detection region downstream from the reaction region for timing purposes, such that the reaction region is inspected after the fluid reaches the detection region.
In another embodiment, the sample is collected in an absorbent pad which is then submerged in a vessel having a
45 known volume of reaction medium containing the amylase substrate.
The method is useful in assays for detecting a variety of analytes including immunoglobulins, viral and other microbial antigens, hormones and hormone derivatives, and drugs 50 and drug metabolites. In one embodiment, the level of colored product produced in the reaction is used as an indicator of the level of total immunoglobulins in the sample.
In another aspect the invention includes a device which 55 can be used in accordance with the method described above. The device includes (i) an absorbent pad for collecting oral fluid when placed in a subject's mouth, and (ii) reagent means for producing a threshold level of a colored reaction product when oral fluid from the absorbent pad is contacted 60 with the reagent means for a selected time period. An above-threshold level of colored reaction produced within the selected time period indicates that an effective volume of undiluted saliva sample sufficient to allow detection of the analyte in such assay has been collected. 65 In one embodiment, the reagent means includes a selected volume of a solution containing the chromogenic amylase substrate. Submersion of the adsorbent pad, after collection