Threshold glucose detection in urine

Details Threshold glucose detection in urine Threshold glucose detection in urine

2003-04-16

2004-01-27

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Optical result

Including reagent preparation

C436S095000, C436S169000, C422S051000, C422S067000, C422S068100, C435S010000, C435S015000

Reexamination Certificate

active

06682937

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a dry chemistry test device for glucose detection in liquid samples. More particularly, the invention is directed to a test device for threshold detection and quantification of glucose in urine and to methods of making and using the same.
BACKGROUND AND SUMMARY OF THE INVENTION
The detection of glucose levels is important in the treatment of diabetes. Test devices for glucose testing of the blood and urine of human diabetics, including many dry chemistry test devices or test strips, are described in numerous papers and patents. Glucose tests have been available in a dry test strip format for more than three decades. Analysis of blood and urine using test strips plays an important role in the diagnosis and treatment of human diabetes.
The history of dry chemistry disposable test-devices for glucose detection is summarized in U.S. Pat. Nos. 3,964,871 and 5,563,042. Quick and simple glucose determination in body fluids using dry chemistry test devices under field conditions is important for the early detection, diagnosis, and control of both human and animal diabetes. Key requirements for dry chemistry colorimetric tests are to detect glucose in a wide range of concentrations and to achieve easily discernable color distinctions at variously defined glucose levels.
For detection of relatively low glucose levels with test strips, several reagents have been proposed. U.S. Pat. No. 3,886,045 describes a non-toxic indicator comprising 4-aminoantipyrene and phenolic compounds that can form colored quinones in the presence of glucose. In U.S. Pat. No. 4,427,770, there is described a similar formulation using 4-aminoantipyrene, in a two pad test format to detect a wide range of glucose levels. U.S. Pat. No. 5,824,491 reports that 4-aminoantipyrene produces a gradual concentration/reflectance response from 0-400 mg/dL, with a linear concentration/reflectance response in the 150-400 mg/dL glucose range.
A sensitive and stable glucose test device was also described in U.S. Pat. No. 5,183,742. The test-device comprises a support having printed or coated detection reagent region on a surface. However, with only one peroxidatively active indicator, it proved difficult to obtain well discernable color changes over the broad range of 20-500 mg/dL.
Other indicator types have been used for detection and quantification of relatively high glucose levels and of a relatively wide range of glucose levels. In U.S. Pat. Nos. 4,303,753 and 4,340,669, an indicator in combination with a polymer (iodide +poly(vinylpyrrolidone)) is described for high range (500-1000 and 1000-10,000 mg/dL) glucose detection. Iodide salts have also been used in conjunction with a blue dye that fades in the presence of hydrogen peroxide. See, e.g., U.S. Pat. No. 3,814,668. The two components act in concert to provide a continuum of indicator colors.
A threshold color control system was described in U.S. Pat. No. 5,036,000. The system was designed for measurement of NAD(P)H. Analytes react to form NAD(P)H which is, in turn, oxidized by a chromogen. Non-chromogen competing compounds, such as Fe(III) compounds, are added to prevent a visible color change until a predetermined amount of NAD(P)H is exceeded. When the predetermined amount of NAD(P)H is exceeded, a single, easy to discern, color change is produced.
The above-mentioned reagents and test devices were generally designed for human urine testing. However, diabetes also afflicts animals. For example, feline diabetes is quite common, affecting about 0.2% to 1.0% of the cat population. In this affliction, glucose levels in the blood must be controlled. As with humans afflicted with diabetes, hyperglycemic cats are treated with insulin injections. While insulin treats the hyperglycemia, proper treatment of diabetes also requires periodic monitoring of glucose levels. Since elevated glucose levels in blood usually lead to elevated glucose levels in urine, periodic monitoring of glucose levels in the urine would provide needed screening for proper animal treatment.
While both the patent and non-patent literature is replete with reference to glucose testing systems and devices, few dry chemistry glucose tests have been developed specifically for testing the body fluids of animals. European Pat. No. 0 060 133 describes a method and device for nonenzymatic glucose measurement in animal body fluids. Recently, a test strip product (Petstix™ 7, Bayer Agricultural Division (Etobicoke, Ontario, Canada)) for animal urine testing (including glucose test) was introduced in the market. Petstix 7 uses the same color blocks as N-Multistix™ SG (Bayer Diagnostics Division (Elkhart, Ind.)), which is marked with U.S. Pat. No. 3,814,668.
There are several limitations and specific requirements for animal urine testing with test strips. For example, there are salient compositional differences between cat urine and human urine. One such difference is that cat urine has a significantly higher specific gravity than does human urine. Because of this difference, false readings may be obtained if test strips designed for human use are used for glucose detection in cats. Another limitation involves the problems in collecting a urine sample from the animal. In use with animal body fluids, it is also preferable to have a test device wherein the results are not dependent upon the amount of sample applied and the time of exposure to ambient conditions prior to activation, and wherein the results are stable for a prolonged period of time subsequent to activation.
SUMMARY OF THE INVENTION
The present invention is directed to a method, reagent composition, and dry chemistry test device for threshold glucose detection, particularly in cats. The device is in the form of an indicator impregnated substrate that is preferably cut into pieces suitable for being distributed onto cat litter. The indicator/substrate combination is selected so that after being wetted by animal urine, the indicator result remains discernable for a sufficient period of time to allow it to be observed by the pet owner or caretaker. Thus, the preferred indicator composition should not only be able to withstand the ambient conditions of a litter box and prolonged exposure to these conditions until activation by animal urine, but it should also have good stability even after activation with urine. The indicator reagent preferably should also be selected to indicate whether the reagent device has been contacted by animal urine, regardless of whether abnormal glucose levels are present.
The reagent composition used in the present invention is based on a standard glucose enzymatic system including glucose oxidase and peroxidase, and a combination of peroxidatively active indicators (chromogens). The combination of chromogens of the present invention provides for good color differentiation corresponding to a variety of urine glucose concentrations. Furthermore, glucose detection in cat urine is preferably based on an established sensitivity threshold or concentration threshold. At glucose concentrations less than the threshold, there would be no discernible color change on the test device. To achieve the desired sensitivity threshold, in addition to the combination of chromogens, the reagent composition includes a scavenger of peroxide/oxidized chromogens. The reagent composition undergoes a color change only when exposed to urine having a glucose concentration at or above the threshold level, and, when such occurs, easily visually discernible color changes indicate higher glucose concentrations.
In one preferred embodiment the test device is prepared using a cellulose paper substrate impregnated with a solution of the reagent composition. The paper is then dried and cut into small but visually detectable pieces to provide a device that may be distributed on cat litter. The inactivated device remains glucose sensitive for several days even at high humidity. After contact with cat urine, a color indicative of glucose concentration develops and persists for a period of time sufficient to all

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