Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
1999-10-15
2003-05-27
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S021000
Reexamination Certificate
active
06569637
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a reagent for diagnosing renal dysfunction, which contains luciferin and luciferase. The present invention also relates to a method for analyzing a urine sample, which is useful to diagnose renal dysfunction and to determine an optimal dose of a nephrotoxic drug, the method including determining free adenosine 5′-triphosphate (hereinafter, referred to as “ATP”) in the urine sample by a bioluminescence technique using the above-mentioned diagnostic reagent.
BACKGROUND ART
As a conventional method for diagnosing renal dysfunction, for example, the following methods are known: a method in which urea in a blood sample is determined (JP-B-61-47382); a method in which creatinine in a blood sample is determined (JP-B-60-38666); and a method in which albumin and an albumin degradation substance in a urine sample are determined (JP-A-5-302922).
Although numbers of methods are known for diagnosing renal dysfunction by determining components in a urine or a blood sample, such methods are disadvantageous in that they take time to obtain the results of the determination.
In the field of clinical medicine, many drugs are known to be toxic to kidney. In particular, cisplatin, which is frequently used in chemotherapy (a major therapy for patients with urinary carcinoma) has an excellent carcinostatic action but yet it has strong toxicity to kidney. Therefore, excessive administration of cisplatin arises a risk of causing serious effects on the treatment of a patient.
Accordingly, it is known to avoid the side-effects as much as possible by detecting hypersensitivity of a patient caused by such drug or by an excessive administration of such drug, determining an optimal dose (a maximum acceptable dose) of such drug, and analyzing increase and decrease of metabolite &bgr;
2
-microglobulin (hereinafter, referred to as “&bgr;
2
-MG”) that usually appears in urine or &bgr;-N-acetyl-D-glucosaminidase (hereinafter, referred to as “NAG”) contained in a urine sample.
However, the above-mentioned methods are disadvantageous in that they take time to obtain the results of the determination and in that they have some other drawbacks. Thus, they have not yet met the needs.
Specifically, the method of measuring the metabolite &bgr;
2
-MG in a urine sample requires an expensive reagent and a special device. The method of measuring an NAG activity in a urine sample by using &rgr;-nitrophenyl-&bgr;-D-glucosaminide as a substrate or by using cresolsulfonphthaleinyl-N-acetyl-&bgr;-D-glucosaminide as a substrate employs a calorimetric analysis that has drawbacks such as low sensitivity and a wide reference value that does not allow diagnosis unless the progress of the renal dysfunction is severe enough.
DISCLOSURE OF THE INVENTION
Thus, the present invention aims at providing a method that can be used for diagnosing renal dysfunction and for determining an optimal dose of a drug that is toxic to kidney, and aims at providing a reagent used in the method.
In order to solve the above problem, the present inventors have undergone intensive studies and directed their attention on free ATP contained in a urine sample of a patient suffering from renal dysfunction. They determined the free ATP based on bioluminescence using luciferin/luciferase luminescent reagent, and noticed that the ATP levels in the urine of the patient suffering from common renal dysfunction is likely to be high, finding significant difference between the ATP level of the patient and that of a healthy individual. Accordingly, they found out that ATP can be used as an index for diagnosing renal dysfunction, and that renal dysfuction can be diagnosed by accurately determining free ATP in a urine sample in a short time by a very simple operation. Furthermore, they collected daily the urine from patients who have been administered with carcinostatic such as cisplatin or methotrexate that is toxic to kidney. The urine samples were added with ATP to react therewith for a prescribed period of time and the ATP levels in the urine samples were measured using luciferin and luciferase. The obtain ed ATP levels and a control ATP level obtained before the reaction were compared to determine the ATP degradation activities in the urine samples. These activities were found to correlate well with that of the conventional diagnoses for renal dysfunction based on changes in NAG activities or &bgr;
2
-MG in urine samples. Similarly, urine samples were added with adenosine 5′-diphosphate (hereinafter, referred to as “ADP”) to react therewith for a prescribed period of time, and the ATP levels in the urine samples were measured using luciferin and luciferase. The obtained ATP levels and a control ATP level obtained before the reaction were compared to determine the ATP production activities of the urine samples. These activities were also found to correlate well with that of the conventional diagnoses for renal dysfunction.
The present invention was completed based on the above-described findings.
The first aspect of the present invention provides a diagnostic reagent for renal dysfunction, comprising luciferin and luciferase.
The second aspect of the present invention provides a method for analyzing a urine sample, comprising the steps of reacting luciferin and luciferase with a urine sample and determining the amount of free ATP in the urine sample; a method for analyzing a urine sample, comprising the steps of adding ATP to a urine sample for reaction for a certain period of time, determining the amount of ATP in the urine sample using luciferin and luciferase, and comparing the determined ATP amount to an ATP amount of a control obtained before the reaction; and a method for analyzing a urine sample, comprising the steps of adding ADP to a urine sample for reaction for a certain period of time, determining the amount of ATP in the urine sample using luciferin and luciferase, and comparing the determined ATP amount to an ATP amount of a control obtained before the reaction.
The third aspect of the present invention provides a diagnostic kit for renal dysfunction, comprising (i) a diagnostic reagent for renal dysfunction containing luciferin and luciferase, (ii) a buffer solution for diluting a urine sample, and (iii) ATP standard reagent; and a diagnostic kit for renal dysfunction, comprising (i) a diagnostic reagent for renal dysfunction containing luciferin and luciferase, (ii) a buffer solution for diluting a urine sample, and (iii) ADP standard reagent.
The fourth aspect of the present invention provides a diagnostic method for renal dysfunction, comprising the steps of reacting luciferin and luciferase with a urine sample and determining the amount of free ATP in the urine sample; a diagnostic method for renal dysfunction comprising the steps of adding ATP to a urine sample for reaction for a certain period of time, determining the amount of ATP in the urine sample using luciferin and luciferase, and comparing the determined ATP amount to an ATP amount of a control obtained before the reaction; and a diagnostic method for renal dysfunction comprising the steps of adding ADP to a urine sample for reaction for a certain period of time, determining the amount of ATP in the urine sample using luciferin and luciferase, and comparing the determined ATP amount to an ATP amount of a control obtained before the reaction.
The fifth aspect of the present invention provides a use of a composition for diagnosing renal dysfunction, comprising luciferin and luciferase.
Hereinafter, the present invention will be described in more detail.
A diagnostic reagent for renal dysfunction according to the invention is prepared by dissolving luciferin, luciferase and magnesium ion (or other metal ions) in a suitable buffer. Preferably, the diagnostic reagent of the invention is a commercially available product such as “CheckLite™”, an ATP measurement kit available from Kikkoman Corp.
Preferable concentration ranges of components in the diagnostic reagent of the invention are shown below. Although the diagnostic reagent may be
Aoyagi Kazumasa
Koiso Kenkiti
Nakajima Moto-o
Gitomer Ralph
Kikkoman Corporation
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