Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-12-22
2002-11-26
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S018000
Reexamination Certificate
active
06485926
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for measuring protease. More specifically, the invention relates to a method for measuring protease, which enables accurate diagnosis of malignancy of cancer cells such as infiltrative and metastatic activity, degree of progress of periodontal diseases such as alveolar pyorrhea, destructive pathological conditions in rheumatoid arthritis and the like.
2. Related Art
It is known that various proteases such as matrix metalloproteinases and plasminogen activator are involved in infiltration and metastasis of cancer cells, progress of periodontal diseases such as alveolar pyorrhea, progress of tissue destruction in rheumatoid arthritis and the like, wound healing process, ontogenesis process and the like. As methods for detecting and quantifying such proteases, there are known immunoassay methods which utilize antibodies, immunoblotting methods and electrophoresis zymography methods and the like. Further, as a method for measuring protease activity in tissues, there is known the so-called in situ zymography method, which is disclosed in FASEB Journal, Vol. 9, July, pp.974-980, 1995 or International Patent Publication WO97/32035.
In the method for measuring protease activity disclosed in International Patent Publication WO97/32035, colorants such as Amido black, Coomassie Blue and Ponceau can be used for staining thin membranes such as those of gelatin. However, when the thin membranes are stained with such colorants, tissue slices adhered to the thin membranes are also simultaneously stained. Therefore, it may become difficult to detect traces of digestion, i.e., accurately measure protease activity.
SUMMARY OF THE INVENTION
Therefore, an object of the present invention is to provide means for solving the aforementioned problem. More specifically, the object of the present invention is to provide an improvement that enables more precise analysis of expression of protease activity in the method for measuring protease activity disclosed in International Patent Publication WO97/32035.
The inventors of the present invention conducted various studies to achieve the aforementioned object. As a result, they found that, when a thin membrane containing a protease substrate is colored with a colorant beforehand, the staining step of the thin membrane becomes unnecessary, and expression of protease activity in a tissue or cells on the thin membrane can be analyzed more precisely. Furthermore, they also found that, when cell nuclei are stained with a dye of a color different from that of the colorant contained in the thin membrane, it becomes possible to simultaneously observe information of nuclear morphology and traces of digestion, and thus it becomes markedly easier to detect localization of protease activity. The present invention was achieved on the basis of these findings.
The present invention thus provides a method for measuring protease activity, which comprises the steps of:
(1) bringing a biosample containing a protease into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant and a protease substrate; and
(2) washing the thin membrane with an aqueous medium and detecting traces of digestion formed on the thin membrane by the action of the protease.
According to another aspect of the present invention, there is provided a method for measuring protease activity, which comprises the steps of:
(1) bringing a biosample containing a protease into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant and a protease substrate;
(2) washing the thin membrane with an aqueous medium and detecting traces of digestion formed on the thin membrane by the action of the protease; and
(3) staining the biosample, preferably a tissue slice or cell nuclei, on the thin membrane.
According to a further aspect of the present invention, there is provided a method for measuring protease activity, which comprises the steps of:
(1) bringing one of two or more substantially continuous slices of a biosample into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant and a protease substrate;
(2) bringing another slice of said slices into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant, a protease substrate, and a protease inhibitor;
(3) washing the thin membranes with an aqueous medium and detecting traces of digestion formed on the thin membranes by the action of the protease; and
(4) comparing the traces of digestion on the thin membrane used in the step (1) with the traces of digestion on the thin membrane used in the step (2).
The present invention further provides a method for measuring protease activity, which comprises the steps of:
(1) bringing one of two or more substantially continuous slices of a biosample into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant and a protease substrate;
(2) bringing another slice of said slices into contact with a crosslinked and/or substantially water-insoluble thin membrane that is formed on a support surface and contains at least one colorant selected from the group consisting of an emulsion-dispersed colorant and a solid-dispersed colorant, a protease substrate, and a protease inhibitor;
(3) washing the thin membranes with an aqueous medium and detecting traces of digestion formed on the thin membranes by the action of the protease;
(4) staining the biosample, preferably tissue slices or cell nuclei, on the thin membranes,; and
(5) comparing the traces of digestion on the thin membrane used in the step (1) with the traces of digestion on the thin membrane used in the step (2).
According to preferred embodiments of these methods of the present invention, there are provided the aforementioned methods wherein cell nucleus staining is performed as the staining of the biosample; the aforementioned methods wherein hematoxylin or Methyl Green is used for the staining of cell nuclei; and the aforementioned methods wherein the biosample is a tissue, a cell, or a body fluid obtained from a mammal including a human. The thin membrane may be crosslinked or contain a hardening agent. The thin membrane may have a monolayer structure or multilayer structure. The thin membrane may contain two or more kinds of colorants, and in a thin membrane having a multiplayer structure, each layer may contain a different colorant.
According to a preferred embodiment of the present invention, there are provided the aforementioned methods wherein the biosample is those isolated and collected from a mammal including a human, preferably a patient, mammal suspected to have a disease, experimental animal or the like. As the biosample, there can be used, solid samples such as tissue pieces, as well as non-solid samples such as samples containing cells or tissue fragment collected from tissues by auction, blood, lymph and saliva. For example, a preferred embodiment of the present invention includes the aforementioned method wherein the biosample is a cancer tissue, lymph node, tissue of periodontal disease, gingival crevicular fluid, tissue or fluid contained in destructive morbid tissues (e.g., synovial fluid of rheumatic morbidity, exud
Nakamura Koki
Naruse Hideaki
Nemori Ryoichi
Fuji Photo Film Co. , Ltd.
Gitomer Ralph
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