Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-12
2001-04-03
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007900, C435S007940, C435S023000, C436S116000
Reexamination Certificate
active
06210912
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for detecting the onset of chronic rejection after organ transplantation by examining body fluids drawn out of body, and more specifically, the present invention relates to methods for detecting rejection (chronic rejection) emerging at the chronic stage after organ transplantation by assaying nitrogen trioxide (NO
3
), matrix metalloproteinase-2 (referred to as “MMP-2”) or the precursor thereof in body fluids, and a method for assaying MMP-2 or the precursor thereof in urine.
BACKGROUND OF THE INVENTION
In patients with kidney transplantation, generally, chronic rejection is diagnosed by renal biopsy. However, the patients are suffered from periodic renal biopsy, which may deteriorate the renal function concomitantly. Furthermore, such chronic rejection proceeds slowly, with the resultant poor subjective symptoms, and therefore, most patients reject painful renal biopsy.
Meanwhile, renal function tests giving the indicators of the progress of chronic rejection have been practiced because renal function is damaged due to chronic rejection. At the tests, urine protein, urine albumin, and urine transferrin etc. have been assayed as the indicators of the acceleration of protein permeability, reflecting glomerular abnormality, and blood creatine, blood urea nitrogen (BUN), blood &bgr;
2
-microglobulin and blood &agr;
1
-microglobulin have been assayed as the indicators of the decrease of glomerular filtration rate (GFR). However, the change of the test values in the progress of chronic rejection is mild, and when abnormal test values are detected, the chronic rejection has already exerted symptoms at the end stage. So far, not any test method has been known, which can enable early detection of chronic rejection by assaying endogenous substances in body fluids such as urine and blood. Since no such simple test method is currently present, patients with poor subjective symptoms of chronic rejection fall into the end stage, with no appropriate treatment, so that the patients frequently lose their renal function.
It is an object of the present invention to provide simple methods capable of detecting chronic rejection at its early stage after organ transplantation.
DISCLOSURE OF THE INVENTION
NO
3
and NO
2
are final metabolites of NO radical in biological organisms. No radical is generated from granulocytes and macrophages and the like during inflammation and organ damage Therefore, these metabolites are clinically detected during inflammation, ischemic cardiac diseases due to organ damage, shock, acute rejection after transplantation, and bacterial infection. However, no report about NO radical during chronic rejection after transplantation has been issued. Thus, whether or not NO, is generated in body due to rejection which progresses chronically, and whether or not NO, in body fluids possibly serves as a marker at tests of chronic rejection have been examined, so that it has been found that NO
3
in body fluids can serve as a marker at the detection of chronic rejection.
Furthermore, MMP-2, an enzyme degrading collagen composing the basal membrane, works during restoration and regeneration of tissues. For cancers, still furthermore, the enzyme is considered to work to decompose collagen during the infiltration of cancer cells into the basal membrane. After intensive investigations, the inventors have found that MMP-2 or the precursor thereof never leaks, or only a trace amount of them leaks even if it does leak, into body fluids such as urine in normal subjects or patients with no onset of chronic rejection after kidney transplantation, while the amount of MMP-2 or the precursor in the body fluids such as urine in patients with chronic rejection after organ transplantation markedly increases. In accordance with the present invention, it is provided methods for detecting rejection after organ transplantation by assaying endogenous substances such as NO
3
and MMP-2 or the precursor in body fluids such as blood and urine, based on the finding that the concentrations of these endogenous substances secreted into body fluids such as blood and urine are significantly increased, following the onset of chronic rejection after organ transplantation; a testing kit for use according to the method; and a method for assaying MMP-2 or the precursor in urine.
More specifically, the present invention relates to
(1) a method for detecting the onset of chronic rejection at an early stage after organ transplantation, by examining body fluids drawn out of body;
(2) a method for detecting chronic rejection after organ transplantation, characterized by detecting NO
3
, MMP-2 or the precursor thereof in body fluids;
(3) the method described above in (2), characterized by detecting NO
3
in body fluids;
(4) the method described above in (2), characterized by detecting MMP-2 or the precursor thereof in body fluids;
(5) the method described above in (2), (3) or (4), wherein the body fluids comprise human urine or blood.
(6) the method described above in (2), characterized by detecting MMP-2 or the precursor thereof in an urine sample;
(7) the method described above in (2), (3), (4), (5) or (6), wherein the organ transplantation is kidney transplantation;
(8) the method described above in (6), characterized by presetting a cut-off value for determining the presence or absence of chronic rejection after kidney transplantation within a range of 0.1 to 10.0 ng/ml as the concentration of the MMP-2 precursor in an urine sample;
(9) the method described above in (8), characterized by presetting the cut-off value within a range of 0.5 to 5.0 ng/ml;
(10) the method described above in (6), characterized by assaying the MMP-2 precursor concentration and creatine concentration in an urine sample, and presetting a cut-off value of an index after correction on a creatine concentration basis for determining the presence or absence of chronic rejection after kidney transplantation within a range of 0.1 to 20.0 &mgr;g/g·creatine;
(11) the method described above in (10), characterized by presetting the cut-off value within a range of 0.5 to 10.0 &mgr;g/g·creatine;
(12) the method described in any of (2) and (4) through (11), wherein the detection is carried out by immunoassay;
(13) the method described above in (12), wherein the immunoassay is enzyme immunoassay;
(14) the method described above in (12) or (13), wherein the immunoassay is sandwich assay;
(15) a method for assaying urine MMP-2 or the precursor thereof by immunoassay;
(16) a kit for detecting the onset of chronic rejection at an early stage after organ transplantation, by examining body fluids drawn out of body;
(17) a kit for detecting the onset of chronic rejection after organ transplantation, y by detecting nitrogen trioxide (NO
3
), or matrix metalloproteinase (MMP)-2 or the precursor thereof in body fluids;
(18) a kit for detecting the onset of chronic rejection after organ transplantation, by detecting MMP-2 or the precursor thereof in urine or blood;
(19) the kit described above in (18), wherein the detection is carried out by immunoassay;
(20) a method for diagnosing chronic rejection at an early stage after organ transplantation, by examining body fluids;
(21) a diagnostic method of chronic rejection after organ transplantation, characterized by detecting nitrogen trioxide (NO
3
) or matrix metalloproteinase (MMP)-2 or the precursor thereof in body fluids; and
(22) the diagnostic method described above in (21), wherein the body fluids comprise urine or blood.
REFERENCES:
patent: 4-183397 (1992-06-01), None
patent: 7-159402 (1995-06-01), None
Lewis et al. Circulation 93 (4):720-729, Feb. 1996.*
Langrehr et al. Nitrate and Nitrate Production following Orthotopic Liver Transplantation, Portland Press Proc. 8 (Biology of Nitric Oxide, 3) 527-530, 1994.*
Langhrehr et al. Journal of Clinical Investigation, 90 (2): 679-83, 1992.*
Langhrehr et al. Surgery, 112:395-402, 1992.*
J.M. Langrehr, ‘Detection of Nitric Oxide by Electron Paramagnetic Resonance Spectroscopy during Rejection and Graft-Versus-Ho
Isoda Atsuko
Koyama Isamu
Mae Takako
Nemoto Kyuichi
Saiga Kan
DeCloux Amy
Nields & Lemack
Nippon Kayaku Kabushiki Kaisha
Saunders David
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