WO2006016687A1

WO2006016687A1 - Method of judging ruptured aneurysm and judgment reagent

Info

Publication number: WO2006016687A1
Application number: PCT/JP2005/014872
Authority: WO
Inventors: Hiroshi Hazui; Masayoshi Nishimoto; Hitoshi Takeshita; Yasuhiko Ohkaru
Original assignee: Dainippon Sumitomo Pharma Co., Ltd.
Priority date: 2004-08-10
Filing date: 2005-08-09
Publication date: 2006-02-16
Also published as: JPWO2006016687A1

Abstract

It is intended to provide a method of judging ruptured aneurysm which comprises detecting the D-dimer concentration in the blood separated from a human subject, comparing the thus detected concentration with the concentration(s) of a ruptured aneurysm patient and/or a patient suffering from analogous syndrome, or comparing with a cut-off value having been determined between ruptured aneurysm and analogous syndrome, and thereby judging the possibility of the onset of ruptured aneurysm; a judgment reagent to be used in performing the above method; and so on.

Description

Specification

TECHNICAL FIELD The present invention relates to a ruptured abdominal aortic aneurysm (RAAA) by detecting the concentration of D-dimer in blood. ), A determination reagent used for carrying out the method, and a commercial package containing the determination reagent and the like. Background art

D-dimer is an in vivo protein that is used as a marker of hyperfibrinolytic system enhancement in order to understand in detail the pathogenesis of blood coagulation / fibrinolysis system enhancement. Specifically, it is used as a marker for diagnosing disseminated intravascular coagulation syndrome (D I C) and various thrombotic diseases, understanding the pathophysiology, and determining treatment effects.

In addition, the blood D-dimer concentration in patients with acute myocardial infarction is increased compared to that of healthy individuals. The power is lost. (For example, “The American Journal of Medicine”, (US), 1 9 9 2 years 1 February, 93rd, p. 6 5 1— 6 5 7).

However, the use of D-dimer as a biochemical marker to determine ruptured abdominal aortic aneurysms has never been reported. For example, "Journal of Vascular Surgery" (United States), 1 99 9 10th, 30th, No. 4,; 6 4 1-6 5 0, and "Journal of Vascular Surgery", (United States ), April 2000, No. 35, No. 4, p. 6 6 1-6 65, before surgery for patients with ruptured and non-ruptured abdominal aortic aneurysms in the lower kidney The blood D-dimer concentration is measured and the measured value in each patient is listed.

However, in these documents, D-dimer was only measured as one of the markers for judging blood coagulation and fibrinolytic system abnormalities. It does not describe a method for determining a ruptured abdominal aortic aneurysm using D-dimer concentration as an index. In addition, these documents compare the detected D-dimer concentration in blood with the D-dimer concentration in blood of patients with asthma of ruptured abdominal arterial aneurysms such as acute myocardial infarction described later. There is no description on how to determine a ruptured abdominal aneurysm. Disclosure of the invention

Ruptured abdominal aortic aneurysm accompanied by severe abdominal pain is one of the diseases belonging to acute abdomen, a high fatality rate, and an urgent disease. Therefore, it is very important in the field of lifesaving medicine to diagnose and appropriately treat this disease quickly and accurately in the early stages of onset.

Acute abdomen is a general term for diseases that have a rapid complaint of abdominal pain and require early treatment. In the acute abdomen, there are many diseases having abdominal pain that is indistinguishable from symptoms of ruptured abdominal aortic aneurysm, that is, ruptured abdominal aortic aneurysm. Therefore, in life-saving medical settings, it may be difficult to distinguish between a ruptured abdominal aortic aneurysm and its analogy when determining whether or not a ruptured abdominal aortic aneurysm has occurred.

For example, acute myocardial infarction, which is one of the symptom of ruptured abdominal aortic aneurysm, has a high fatality rate like ruptured abdominal aortic aneurysm, and is a disease that requires prompt diagnosis and treatment at an early stage of onset. Pain in the vicinity of the upper abdomen (epigastric pain), which is indistinguishable from symptoms of abdominal aortic aneurysm, may occur. For this reason, it may be difficult for doctors, especially non-specialists, to distinguish clearly between ruptured abdominal aortic aneurysms and acute myocardial infarction.

If a patient with ruptured abdominal aortic aneurysm, which is a bleeding disorder, is mistakenly treated with thrombolysis, which is a treatment for acute myocardial infarction with a hemorrhagic disease, the patient may be killed by massive bleeding. There is. Therefore, it is very important in the field of lifesaving medicine to distinguish ruptured abdominal aortic aneurysms from those of ruptured abdominal aortic aneurysms such as acute myocardial infarction and to provide appropriate treatment for ruptured abdominal aortic aneurysms. It is.

In the current medical setting, ruptured abdominal aortic aneurysms can be diagnosed using common diagnostic methods for patients with abdominal diseases such as abdominal X-ray, ultrasonic tomography, computed tomography (CT) and Imaging techniques such as magnetic resonance imaging (MRI) have been used. Using these methods, doctors directly and visually observe the lesions of the organs and blood vessels to grasp detailed information such as the shape and size of the lesions.

However, many of these diagnostic methods are [1] long measurement times, [2] patients need to be held in a constant position for a long time, [3] fasting may be necessary, [4] The device itself is large and special, so it is difficult to carry, and it is necessary to move the patient himself to the facility where the device is located. [5] Angiographic contrast agents may be required, [6] ] There is a lack of quickness and simplicity in terms of the need for specialists who require special technology.

Therefore, in the field of lifesaving medicine, in addition to the conventional methods for diagnosing the general abdominal disease described above, a ruptured abdominal aorta that uses a biochemical marker in the blood to reduce the burden on the patient quickly. The provision of an aneurysm determination method is desired.

In view of such circumstances, the present invention is used to implement a method for determining a ruptured abdominal aortic aneurysm by detecting D-dimer concentration in blood as a biochemical marker, and the determination method. It is an object of the present invention to provide a commercial package containing a reagent for determination and the like, as well as the reagent for determination. The present inventors analyzed the blood of 14 patients with ruptured abdominal aortic aneurysm, and 3 cases of acute myocardial infarction, 3 patients with ureteral stone, 1 of them has hydronephrosis), perforated gastric ulcer and peritonitis 3 patients with peritonitis), 1 patient with perforated duodenal ulcer and peritonitis (general peritonitis), 1 patient with colonic diverticulitis and peritonitis, and 1 patient with adhesion ileus The D-dimer concentration contained in the sample was measured. As a result, it was found that the D-dimer concentration in blood of a patient who developed ruptured abdominal aortic aneurysm was significantly higher than that of a patient who developed ruptured abdominal aortic aneurysm. Was completed.

That is, the present invention is as follows.

(1) A method for determining a ruptured abdominal aortic aneurysm by determining the possibility of developing a ruptured abdominal aortic aneurysm by detecting the concentration of D-dimer in blood separated from humans.

(2) Rupture to determine the possibility of developing a ruptured abdominal aortic aneurysm by detecting the concentration of D-dimer in blood isolated from humans with a symptomatic ruptured abdominal aortic aneurysm A method for determining an abdominal aortic aneurysm.

(3) The detected D-dimer concentration in the blood is determined based on the pre-measured D-dimer concentration in the blood of the patient with ruptured abdominal aortic aneurysm and / or the pre-measured ruptured abdominal aortic aneurysm. The determination method according to the above (1) or (2), wherein the possibility of developing a ruptured abdominal aortic aneurysm is compared with the D-dimer concentration in blood.

(4) Compared the detected D-dimer concentration in the blood with the cut-off value of D-dimer set between ruptured abdominal aortic aneurysm and ruptured abdominal aortic aneurysm, If the concentration is equal to or higher than the force-off value, it is judged that a ruptured abdominal aortic aneurysm is likely to develop. If the concentration is less than the force-off value, a ruptured abdominal aortic aneurysm is developed. The determination method described in (1) or (2) above, which is determined to be unlikely.

(5) The determination according to any one of (1) to (4) above, wherein the D-dimer concentration is detected by an immunochemical method using an antibody that recognizes D-dimer. Method.

(6) The determination method according to the above (5), wherein the immunochemical method is any one of an enzyme immunochemical method, a latex agglutination method, and an immunochromatography method.

(7) Reagent or determination kit for determination of ruptured abdominal aortic aneurysm used for carrying out the determination method described in (5) or (6) above and comprising an antibody that recognizes D-dimer .

(8) The determination reagent or determination kit according to (7) above, wherein the antibody is a monoclonal antibody.

(9) A commercial package containing the determination reagent according to (7) above and a description related to the reagent, wherein the reagent is used to determine a ruptured abdominal aortic aneurysm. A commercial package that states that it can or should be used for an application.

(10) A commercial package containing the determination kit described in (7) above and a description relating to the kit, wherein the kit is attached to the description or the package, the kit being a rupturable abdominal aortic aneurysm A commercial package that can be used for or should be used in the determination of

By using the determination method and determination reagent of the present invention, the possibility of developing a ruptured abdominal aortic aneurysm can be determined using the D-dimer concentration in blood as an indicator, and a doctor. In consideration of this judgment result, the patient can be treated appropriately. Brief Description of Drawings

FIG. 1 is a distribution diagram of D-dimer concentration in the blood of each patient measured in Example 1.

FIG. 2 is an ROC curve of D-dimer concentration prepared in Example 2. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention develops a ruptured abdominal aortic aneurysm by detecting the concentration of D-dimer in blood isolated from a human, for example, a human having a suspicion of a ruptured abdominal aortic aneurysm. The present invention relates to a method for determining a ruptured abdominal aortic aneurysm that determines the possibility of being present.

Ruptured abdominal aortic aneurysm is a disease having a pathological condition in which an enlarged abdominal aortic aneurysm is ruptured in the retroperitoneal cavity or the abdominal cavity. Since the aortic aneurysm often increases without the patient's subjective symptoms, it is difficult to predict the onset of this disease early. An aortic aneurysm is a condition in which the aortic wall weakened by factors such as arteriosclerosis and inflammation cannot withstand blood pressure, and the arterial lumen is locally expanded.

A typical clinical symptom of this disease is severe abdominal pain. Depending on the location and size of the lesion, the pain may extend to the lumbar region or back region without being limited to the abdomen.

Therefore, in the present invention, “a symptom of suspected ruptured abdominal aortic aneurysm” means a clinical symptom of ruptured abdominal aortic aneurysm as described above.

In the present invention, `` determination of ruptured abdominal aortic aneurysm '' refers to the development of a ruptured abdominal aortic aneurysm, not only to determine the possibility, but also to the possibility of developing this disease. If judged, it also means estimating the severity of the disease state based on the detected D-dimer concentration.

In the present invention, detection of D-dimer concentration in blood separated from human is not particularly limited and can be performed by any method. In this specification, “detection of D-dimer concentration” includes quantitatively measuring D-dimer concentration, semi-quantitatively measuring D-dimer concentration within a certain range, and above a certain concentration. D—Qualitatively determine the presence or absence of dimer Anything to do is included.

Examples of methods for detecting the concentration of D-dimer in blood include various chromatographic methods such as immunochemical methods and HPLC methods. Among them, in particular, antibodies that recognize D-dimer (hereinafter referred to as “anti-D”). It is preferable to detect it by an immunochemical method using “-dimer antibody”. In addition, when it is desired to determine the severity and course of the pathological condition of a ruptured abdominal aortic aneurysm, a method that can quantitatively measure the D-dimer concentration is preferred.

There are no particular limitations on the immunochemical method used to detect the D-dimer concentration in the blood. For example, enzyme immunoassay (EIA method), latex agglutination, immunochromatography, radioimmunoassay Method (RIA method), Fluorescence immunoassay method (FIA method), Luminescence immunoassay method, Spin immunoassay method, Nephelometry method for measuring turbidity associated with antigen-antibody complex formation, Use of antibody solid phase membrane electrode Examples thereof include an enzyme sensor electrode method, an immunoelectrophoresis method, a Western plot method, etc. for measuring a potential change due to binding to an antigen. Among these, it is preferable to detect the D-dimer concentration by EIA method, latex agglutination method or immunochromatography method.

The EIA method includes a competing method in which the enzyme-labeled antigen and the antigen in the sample are competing, and a non-competing method in which no competing is performed. Among these methods, two types of antibodies, particularly monoclonal antibodies, are used. The Sandwich enzyme-linked immunosorbent solid phase assay (sandwich ELISA method), which is a kind of non-competitive method, is particularly preferable in terms of specificity to the antigen (D-dimer) and ease of measurement.

According to the sandwich ELISA method, D-dimer is detected between two types of antibodies that recognize different epitopes present in D-dimer, namely, immobilized anti-D-dimer antibody and enzyme-labeled anti-D-dimer antibody. Sandwich (Sandwich) can detect D-dimer concentration. That is, the concentration of D-dimer can be detected by measuring the amount of labeled antibody bound to D-dimer captured by the immobilized antibody.

There is also a method using avidin-piotin reaction as a kind of sandwich ELISA method. According to this method, D-dimer in blood is captured with an immobilized anti-D-dimer antibody, and an antigen-antibody reaction occurs between the captured D-dimer and anti-D-dimer antibody labeled with piotin. Then, by adding enzyme-labeled streptavidin, the D-dimer concentration can be detected in the same manner as described above.

The latex agglutination method is an immunochemical method using an agglutination reaction between antibody-sensitized latex particles and an antigen. The detection of D-dimer concentration by this method involves the immunoreaction of anti-D_dimer antibody-sensitized latex particles with D-dimer in the sample blood and measuring the degree of aggregation of the resulting latex particles. Can be implemented.

The immunochromatography method is a method in which all immunochemical reaction systems are held on a sheet-like carrier, and the operation is completed only by adding blood. The procedure for detecting D-dimer concentration by this method is as follows. First, when the blood sample is dripped into the carrier, D-dimer in the blood and the anti-D-dimer antibody on the carrier labeled with a labeled substance (gold colloid, etc.) undergo an immunoreaction, resulting in an immune response. A complex is generated. This complex develops on the carrier and recognizes another epitope immobilized on the specific site. When captured by the anti-D-dimer antibody, the label accumulates and the degree of accumulation is observed with the naked eye. The D-dimer concentration can be detected.

There is no need for special measuring equipment to perform immunochromatography. This is an advantageous method when a quick judgment such as judgment or emergency is required. This method is suitable when semi-quantitatively measuring the concentration of D-dimer within a certain range, or when qualitatively determining the presence or absence of D-dimer above a certain concentration. In this case, it can be easily set so that a positive result will be obtained if a D-dimer concentration above the cut-off value is detected, and a negative result will be obtained if it is less than the cut-off value.

In addition, immunochemical methods such as the above-mentioned sandwich ELISA method and latex agglutination method for the purpose of detecting the concentration of D-dimer in blood are already known, and the measurement reagent and measurement of D-dimer based on these methods are known. Kits are commercially available as described below. By using these reagents and kits, it is possible to easily detect the D-dimer concentration in blood separated from humans.

In the present invention, the blood of human blood as a sample may be whole blood, serum, or plasma, and these may be appropriately treated by processing blood collected from humans according to a conventional method. , Obtainable.

The method for determining a ruptured abdominal aortic aneurysm is based on the D-dimer concentration in the blood thus detected, for example, the average value of the D-dimer concentration in the blood of a patient with a ruptured abdominal aortic aneurysm measured in advance. By comparing with the distribution, the possibility of developing a ruptured abdominal aortic aneurysm can be determined.

Specifically, if the detected D-dimer concentration in the blood is close to the average value of D-dimer concentrations measured in patients with ruptured abdominal aortic aneurysms measured in advance, a ruptured abdominal aortic aneurysm developed. It can be judged that there is a high possibility that

According to the present inventors, the average value of the D-dimer concentration in the blood of 14 patients with ruptured abdominal aortic aneurysm was 16.5 μg / L, as shown in Examples below. Met.

In addition, by comparing the detected D-dimer concentration in the blood with the average value and distribution of the blood D-dimer concentration in patients with ruptured abdominal aortic aneurysms, the ruptured abdominal aortic aneurysm It is possible to determine the possibility of developing.

Specifically, if the detected D-dimer concentration in the blood is close to the average value of D-dimer concentrations in patients with pre-measured ruptured abdominal aortic aneurysms, ruptured abdominal aortic aneurysm It can be judged that the possibility of developing the disease is low.

According to the present inventors, as shown in Example 1 below, the average D-dimer concentration in the blood of 43 patients with ruptured abdominal aortic aneurysm was 0.5 7 μg / mL. Yes, it is very different from the D-dimer concentration in the blood of patients with ruptured abdominal aortic aneurysms (see Figure 1).

In the present invention, “ruptured abdominal aortic aneurysm” means a disease that belongs to acute abdomen and has abdominal pain that is indistinguishable from symptoms of ruptured abdominal aortic aneurysm. For example, acute myocardial infarction, ureteral calculus, perforated gastric ulcer, perforated duodenal ulcer, colonic diverticulitis, peritonitis, and adhesive ileus are examples of ruptured abdominal aortic aneurysms. The outline of the pathology of these syndromes is as follows.

“Acute myocardial infarction (AMI)” is a disease in which coronary arteries are occluded and myocardial cells are killed because nutrients and oxygen do not reach the heart muscle. The World Health Organization (WHO) has identified two or more of the following: A. Chest pain, B. ECG abnormalities, and C. Creatine kinase 1 MB Increased myocardial divergence enzymes in the blood, such as N-aspartate aminotransferase. It shows the diagnostic criteria that those who meet the requirements develop this disease. .

This disease has increased rapidly in Japan due to recent westernization of eating habits. ing. In addition, this disease suddenly develops with the main symptoms of severe chest pain and dyspnea, and depending on the size of the myocardial infarction, it may lead to cardiac arrest at the same time as the onset. It is. For this reason, patients often die within 1 to 2 hours after the onset of disease. Prompt and accurate diagnosis of this disease and appropriate measures corresponding to it are important for lifesaving. As described above, the typical symptom of acute myocardial infarction is severe chest pain. However, there are cases in which upper abdominal pain that is indistinguishable from that of ruptured abdominal aortic aneurysm, that is, epigastric pain, develops as a chief complaint.

“Ureter olithiasis” is a disease that develops when renal stones descend into the ureter and block the ureter. When the ureter is obstructed, urine flow disturbance occurs, urine flows back into the kidney, and the internal pressure of the kidney increases. Therefore, severe pain similar to ruptured abdominal aortic aneurysms is often associated with the abdomen, lumbar region, or back.

“Perforated gastric ulcer” and “perforated duodenal ulcer” are caused by deep ulcers on the surface of the mucous membrane of the stomach and duodenum, resulting in holes in the stomach and duodenal walls. It is a disease that develops. The disease begins with sudden upper abdominal pain, particularly severe epigastric pain, which persists.

“Diverticulitis of the colon” means that the colonic diverticulum (the intestinal tract's internal pressure increases and the colonic mucosa protrudes from the wall) is inflamed due to bacterial infection, etc., resulting in localized tenderness in the lower abdomen. It is a disease accompanied.

"Peritonitis" is a disease that develops due to bacterial contents leaking into the abdominal cavity due to perforation of the digestive tract into the abdominal cavity and causing inflammation due to bacterial infection of the peritoneum. . Symptoms of the disease are persistent pain that spreads throughout the abdomen. The case where inflammation spreads throughout the peritoneum is called generalized peritonitis.

"Adhesive intestinal obstruction J is an open hand Of the intestinal tract contents caused by adhesion of the intestines or between the intestine and the mesentery, the abdominal wall, and other organs due to surgery or intestinal inflammation, etc. It is a passage obstacle. Increased intestinal peristaltic movements often result in intermittent colic attacks in the abdomen.

As described above, the ruptured abdominal aortic aneurysm and the ruptured abdominal aortic aneurysm are similar in that they have severe abdominal pain. It may be difficult to differentiate from the disease.

In another embodiment of the present invention, a D-dimer force-off value is set in advance between a ruptured abdominal aortic aneurysm and a ruptured abdominal aortic aneurysm and detected. If the D-dimer concentration is greater than or equal to the cutoff value (positive), it is likely that a ruptured abdominal aortic aneurysm has developed, and the detected concentration is less than the cutoff value (negative) ), It can be determined that the possibility of developing a ruptured abdominal aortic aneurysm is low.

The “cut-off value” is a value that can satisfy both high diagnostic sensitivity (prevalence of correct diagnosis) and high diagnostic specificity (prevalence of non-diagnostic diagnosis) when determining the disease based on this value. is there. For example, in the case of the present invention, a patient who develops a ruptured abdominal aortic aneurysm has a high positive (force-off value or higher) rate, and a patient who develops an anatomy of a ruptured abdominal aortic aneurysm The D-dimer concentration, which shows a high negative (less than cut-off value) rate, can be set as the cut-off value.

The method for calculating the cut-off value is well known in this field. For example, in many patients with ruptured abdominal aortic aneurysms and patients with ruptured abdominal aortic aneurysms, the concentration of D-dimer in the blood is measured, and ruptured abdominal aortic aneurysms and ruptured abdominals at any concentration Obtain the diagnostic sensitivity and specificity of the diagnosis of aortic aneurysm. Characteri sti c) Create a curve. ROC curves can also be created using commercially available analysis software (see Figure 2). Then, the D-dimer concentration when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can be obtained, and that value can be used as the cutoff value. In addition, for example, the diagnosis efficiency at any concentration (the ratio of the total number of correctly diagnosed cases and non-diagnosed cases to the total number of cases) is calculated, and the concentration at which the highest diagnosis efficiency is calculated is calculated as the force-off value. You can also

For example, if the ruptured abdominal aortic aneurysm is a group consisting of acute myocardial infarction, ureteral stone, perforated gastric ulcer, perforated duodenal ulcer, colonic diverticulitis, peritonitis, and adhesive ileus The cut-off value of D-dimer in the blood that was set between the abdominal aortic aneurysm and the above-mentioned asymptomatic group was 1.1 μg Zm (Example 2). Such cut-off values are not specific and will vary depending on the patient population used to set the cut-off values.

Even when the same sample is used, the measured D-dimer concentration may vary depending on the measurement method and reagent used, so the cut-off value must be set for each measurement method and reagent used. .

The severity of the pathological condition of ruptured abdominal aortic aneurysm can also be estimated based on the detected D-dimer concentration. For example, the concentration of D-dimer can be positively correlated with the severity of ruptured abdominal aortic aneurysm pathology.

When carrying out the determination method of the present invention, other methods that are known general examination methods, such as abdominal simple radiography, ultrasonic tomography, computer tomography (CT), and magnetic Combined with resonance imaging (MRI), etc., the accuracy of determination of ruptured abdominal aortic aneurysms is higher.

In addition, the present invention is used for detecting the D-dimer concentration in blood by an immunochemical method and carrying out the above-described determination method of the present invention. The present invention relates to a determination reagent or determination kit for a ruptured abdominal aortic aneurysm comprising a mer antibody.

The anti-D-dimer antibody contained in the determination reagent or determination kit recognizes D-dimer present in blood separated from human and exhibits specific immunoreactivity with D-dimer. Any of polyclonal antibodies and monoclonal antibodies may be used as long as they have them. Of these, a monoclonal antibody is preferable in terms of stable supply of the antibody and in terms of high specificity and homogeneity with respect to D-dimer.

Such an anti-D-dimer antibody can be produced by a known means, and is contained in the reagent or kit in a free state, a labeled state, or a solid phase.

Polyclonal antibodies are obtained by immunizing animals such as mice, rats, and rabbits with D-dimer that has been separated and purified from human blood by a conventional method, and collecting the blood for known treatment. Can be manufactured. As an immunizing antigen, a recombinant D-dimer obtained by genetic engineering techniques or an equivalent (fragment) thereof can be used instead of the above D-dimer.

Monoclonal antibodies are obtained by collecting the spleen cells of the immunized animal and conducting cell fusion with myeloma cells, screening for antibody-producing cells, cloning, etc. by the method of Milstein et al. It can be produced by establishing a cell line to produce and culturing it.

When the determination reagent of the present invention is based on sandwich ELISA, the anti-D-dimer antibody thus obtained is in the form of a solid-phased anti-D-dimer antibody and an enzyme-labeled anti-D-dimer antibody. include.

Solid-phased anti-D-dimer antibody is obtained by solidifying the antibody obtained as described above. (For example, it can be produced by binding to a microplate well or plastic beads). The binding to the solid phase is usually performed by dissolving the antibody in an appropriate buffer such as citrate buffer and bringing the surface of the solid phase into contact with the antibody solution for an appropriate time (1-2 days). it can.

Furthermore, in order to suppress non-specific adsorption and non-specific reactions, a phosphate buffer solution in which bovine serum albumin (BSA), bovine milk protein, etc. are dissolved is brought into contact with the solid phase, and the solid phase that has not been coated with the antibody. In general, the phase surface portion is blocked with the BSA or bovine milk protein.

The enzyme-labeled anti-D-dimer antibody can be produced by binding (labeling) an enzyme with an anti-D-dimer antibody that recognizes an epitope different from the above-mentioned immobilized antibody. Examples of the enzyme for labeling the antibody include alkaline phosphatase, gnorecosoxidase, Λ-oxidase, β-galactosidase, and the like. The binding between these enzymes and the anti-D-dimer antibody can be performed by a method known per se, for example, the dartaldehyde method, the maleimide method, or the like.

In addition, when an avidin-piotin reaction is used in the ELI IS method, the piotin-labeled anti-D-dimer antibody contained in the determination reagent of the present invention can be produced, for example, by using a commercially available biotin labeling kit. When performing Sandwich ELISA, in addition to the anti-D-dimer antibody, as required, standard substances, washing solutions, reagents for enzyme activity measurement (substrate agent, substrate solution, reaction stop solution, etc.), enzyme-labeled streptogram Avidin (when using avidin-piotin reaction) etc. is used. Therefore, the present invention may be in the form of a determination kit containing these as constituent reagents in addition to the anti-D-dimer antibody.

As the substrate agent, an appropriate one is selected according to the selected labeling enzyme. For example, if you selected peroxidase as the enzyme, Direndiamine (OPD), tetramethylbenzidine (TMB), etc. are used, and p-nitrotrophic phosphate (PNPP), etc., is used when al-force phosphatase is selected. In addition, as the reaction stop solution and the substrate solution, conventionally known ones can be used as appropriate depending on the selected enzyme.

In addition, many D-dimer measurement reagents and measurement kits by sandwich ELISA method are sold (for example, “Acerachrome D-dimer” (trade name) manufactured by Boehringer Mannheim Co., Ltd., “ These reagents and the like can also be used as the determination reagent or determination kit of the present invention.

When the determination reagent of the present invention is based on the latex agglutination method, the anti-D-dimer antibody is contained in the reagent or the kit in the form of a latex-sensitized anti-D-dimer antibody. The production of Latustus-sensitized anti-D-dimer antibody, ie, sensitization (binding) of latex particles with anti-D-dimer antibody, can be carried out by methods known in the art, for example, chemical bonding methods (carposimidyadal as a cross-linking agent). Or a physical adsorption method).

In carrying out the latitudus agglutination method, a dilution stabilizing buffer, a standard antigen or the like is used as necessary in addition to the above-described latex-sensitized antibody. Therefore, the present invention may be in the form of a determination kit containing these as a constituent reagent in addition to the latex-sensitized antibody.

Numerous reagents and kits for measuring D-dimer using the latex agglutination method are on the market (for example, Elpiaace D-D dimer (trade name) manufactured by Diatron and Coagsol manufactured by Nippon Roche). These reagents and the like can also be used as the determination reagent or determination kit of the present invention. When the determination reagent of the present invention is based on immunochromatography, the anti-D-dimer antibody is contained in the reagent in the form of a solid-phased anti-D-dimer antibody and a labeled anti-D-dimer antibody. As the labeled product in the labeled anti-D-dimer antibody, those known in this field can be used as appropriate, and among them, it is preferable to use a gold colloid. The determination reagent of the present invention by such an immunochromatography method is produced according to the method described in “Cl inical Biochemistry”, 2010, Vol. 34, p. 2 5 7-2 63. be able to. The determination reagent of the present invention based on the immunochromatography method gives a positive result when a D-dimer concentration equal to or higher than a predetermined force-off value is detected, and gives a negative result when it is less than the force-off value. It is preferably used when setting to exit. For example, if a positive result is obtained using the determination reagent, it is determined that a ruptured abdominal aortic aneurysm is likely to develop, and if a negative result is obtained, It can be judged that the possibility of developing a ruptured abdominal aortic aneurysm is low.

In addition to the above anti-D-dimer antibody, standard substances, buffers, etc. are used for the immunochromatography as necessary. Therefore, the present invention may be in the form of a determination kit containing these as constituent reagents in addition to the anti-D-dimer antibody.

The determination reagent of the present invention is provided in the form of a commercial package in an actual medical field. Such a commercial package contains the determination reagent of the present invention and a description relating to the reagent (so-called “package insert”). And in the said description and Z or the said commercial package, it is described that the determination reagent of this invention can be used for determination of a ruptured abdominal aortic aneurysm, or should be used.

Further, the determination kit of the present invention is also provided in the form of a commercial package similar to the above-described determination reagent in an actual medical field. Example

Hereinafter, the present invention will be described more specifically based on examples.

Example 1; Measurement of D-dimer concentration in blood of patients with ruptured abdominal aortic aneurysm and patients with ruptured abdominal aortic aneurysm

Among emergency outpatients, patients diagnosed with ruptured abdominal aortic aneurysm (14 patients), patients diagnosed with acute myocardial infarction (34 patients), patients diagnosed with ureteral stones (3 patients) Patients, 1 patient with hydronephrosis), 3 patients with perforated gastric ulcer and peritonitis (generic peritonitis), 3 patients with perforated duodenal ulcer and peritonitis (generic) Peritonitis) (1 patient), confirmed diagnosis of colonic diverticulitis and peritonitis (1 patient), patients with definitive diagnosis of adhesion ileus (1 patient) The concentration of D-dimer in the blood collected at 1 visit was measured as follows, and the results were obtained.

Informed consent was obtained from these patients.

(1) D-dimer measurement

D-dimer measurement reagent “STA Liatest D-dimer” (trade name) (trade name) (standard value: 0.4 / g / ml) manufactured by Roche Diagnostics, which uses the latex agglutination method as the measurement principle. And measured as follows. 1. 8 ml of sodium citrate blood collection tube from patient's vein (includes 0.2% ml of Insepak® C coagulation test (Sekisui Chemical Co., Ltd., 3.1% 3% sodium citrate)) Whole blood was collected. Thereafter, the blood collection tube was gently tumbled and mixed, and centrifuged at 300 rpm for 5 minutes at room temperature. The collected blood tube after centrifugation was directly set on a STA manufactured by Roche Diagnostix, which is an automatic D-dimer measurement device, and analyzed. Analytical conditions were: Sample blood volume 50 μL, buffer (reagent 1 attached to the D-dimer measurement reagent above; Tris (Hydroxymethyl) aminomethane 1 1.5 mg / mL, sodium azide 1 mg / mL, Na C 1 2 3.5 mg / mL, methylbaraoxybenzoic acid [preservative] 0 SS mg ZmL) 1 00 L, STA equipment dilution for D-dimer measurement reagent 50 L, latex-sensitized anti-D-dimer antibody solution (reagent 2 attached to D-dimer measurement reagent above; human D- da one more mouse monoclonal antibodies 5 5 μ g L, latex 0. 6 5 mg / m L _s © shea serum albumin 3 mg Zm L, methyl rose oxy repose Kosan [preservatives] 0. 9 5 mg / mL, sodium azide l mgZmL) 1 50 μL was automatically added and measured at a minute-digit time of 240 seconds and a reaction temperature of 37 ° C. The calibration curve range of this measurement reagent was D-dimer concentration 0.2-4.0 μgZmL, and high-value samples exceeding this concentration range were subject to retesting. The high-value sample was diluted in advance with the above-mentioned diluent for STA equipment at a predetermined dilution factor, and applied again to the STA apparatus to measure the D_dimer concentration in the sample. D-Dima concentration measurements were computer processed and managed online.

(2) Measurement results

The measurement results are shown in [Table 1] below. Figure 1 shows the distribution of measured values for each patient. The concentration of D-dimer in the blood of patients with such ruptured abdominal aortic aneurysms is acute myocardial infarction, ureteral stone, perforated gastric ulcer, perforated duodenal ulcer, colonic diverticulitis, peritonitis, and It was much higher than in patients with adhesive ileus. [table 1 ]

D-dimer concentration in blood of patients with ruptured abdominal aortic aneurysm and patients with ruptured abdominal aortic aneurysm

Example 2: Determination of ruptured abdominal aortic aneurysm by force-off value

(1) Setting the cut-off value

ROC curve analysis of the measured values obtained in Example 1 above was performed using “M ed C a 1 c” (trade name) manufactured by ROC analysis software Med C alc S oftware (Benoregie). Between a ruptured abdominal aortic aneurysm and a group of ruptured abdominal aortic aneurysms consisting of acute myocardial infarction, ureteral stone, perforated gastric ulcer, perforated duodenal ulcer, colonic diverticulitis, peritonitis, and adhesive ileus D—Dimer cut-off value was calculated. The calculated value was 1.7 μg / m. Figure 2 shows the ROC curve at that time. The 2 product (AUC; area under curve) was 0.99 2 which was very close to 1. This means that D-dimer in the blood is a useful marker to distinguish ruptured abdominal aortic aneurysms from the group of ruptured abdominal aortic aneurysms.

(2) Applying cut-off value

Using the force-off value (1.7 μg / mL) set in (1) above, the ruptured abdominal aortic aneurysm case in Example 1 was treated as a group of ruptured abdominal aortic aneurysms (rapid myocardial infarction). Ureteral stone, perforated gastric ulcer, perforated duodenal ulcer, colonic diverticulitis, peritonitis, and adhesive ileus). The diagnostic accuracy (diagnostic sensitivity, diagnostic specificity and diagnostic efficiency) at that time is shown in [Table 2] below. Here, diagnostic sensitivity means the percentage of positive cases (prevalence of correct diagnosis) in patients with ruptured abdominal aortic aneurysm, and diagnosis specificity is negative cases in patients with an asymptomatic group of ruptured abdominal aortic aneurysm (no-correct diagnosis) Means the percentage of The diagnostic efficiency is the ratio of the total number of positive cases in patients with ruptured abdominal aortic aneurysm and negative cases in the group of ruptured abdominal aortic aneurysms to the total number of cases. The diagnostic criteria established for diagnosing a disease indicate that the disease can be diagnosed more accurately.

[Table 2]

Determination of ruptured abdominal aortic aneurysm

As shown in Table 2 above, the cut-off value obtained in the previous section (1) The diagnostic accuracy when using the criteria for cervical aortic aneurysm was very high. This means that a ruptured abdominal aortic aneurysm can be accurately determined by using the cut-off value. Industrial applicability

According to the method for determining a ruptured abdominal aortic aneurysm of the present invention, it is possible to determine the possibility of developing a ruptured abdominal aortic aneurysm using the D-dimer concentration in the blood as an index. Appropriate treatment can be applied to the patient after considering the results. In addition, the determination reagent of the present invention is useful for carrying out the determination method by detecting the D one-dimer concentration. This application is based on Japanese Patent Application No. 2 0 0 4 _ 2 3 2 8 6 1 filed in Japan (filing date: August 10, 2000) It is included.

Claims

The scope of the claims

1. A method for determining a ruptured abdominal aortic aneurysm that determines the possibility of developing a ruptured abdominal aortic aneurysm by detecting the concentration of D-dimer in blood separated from humans.

2. Ruptureability to determine the possibility of developing a ruptured abdominal aortic aneurysm by detecting the concentration of D-dimer in blood isolated from humans with a suspicious ruptured abdominal aortic aneurysm A method for determining an abdominal aortic aneurysm.

3. The detected D-dimer concentration in the blood is determined based on the pre-measured D-dimer concentration in the blood of the patient with ruptured abdominal aortic aneurysm and Z or the pre-measured ruptured abdominal aortic aneurysm. The determination method according to claim 1 or 2, wherein the possibility of developing a ruptured abdominal aortic aneurysm is compared with the D-dimer concentration in blood.

4. Compare the detected D-dimer concentration in the blood with the D-dimer force toff value set between ruptured abdominal aortic aneurysm and ruptured abdominal aortic aneurysm. If the concentration is greater than or equal to the cut-off value, it is determined that there is a high possibility that a ruptured abdominal aortic aneurysm has occurred. If the concentration is less than the cut-off value, a ruptured abdominal aortic aneurysm is developed. 3. The determination method according to claim 1 or 2, wherein it is determined that there is a low possibility that the information is present.

5. The determination method according to any one of claims 1 to 4, wherein the D-dimer concentration is detected by an immunochemical method using an antibody that recognizes D-dimer.

6. The determination method according to claim 5, wherein the immunochemical method is any one of an enzyme immunochemical method, a latex agglutination method, and an immunochromatography method.

7. A reagent or a determination kit for determining a ruptured abdominal aortic aneurysm, which is used for carrying out the determination method according to claim 5 or 6 and contains an antibody that recognizes D-dimer.

8. The determination reagent or determination kit according to claim 7, wherein the antibody is a monoclonal antibody.

9. A commercial package comprising the determination reagent according to claim 7 and a description related to the reagent, wherein the reagent is used for the determination of a ruptured abdominal aortic aneurysm A commercial package that states that it can or should be used.

10. A commercial package including the determination kit according to claim 7 and a description related to the kit, wherein the kit is used for determining a ruptured abdominal aortic aneurysm. A commercial package that states that it can or should be used for an application.