JP2006250862A - Method for and kit for detecting pregnancy toxemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a kit, capable of sharply and accurately detecting pregnancy toxemia, its severity and the like. <P>SOLUTION: The method for detecting pregnancy toxemia includes at least a step of measuring bikunin and/or a complex of serum origin hyaluronan binding protein and hyaluronic acid in a specimen, and correlating measurement result with pregnancy toxemia. The kit for sensing the pregnancy toxemia includes at least following constituents (A) and (B): hyaluronic acid binding protein (A) and anti-inter-α-trypsin inhibitor antibody (B). Alternatively, the kit for detecting the pregnancy toxemia includes at least following constituents (A) and (B), bikunin (A) and antibikunin antibody (B). In the case a large quantity of the bikunin or the complex of serum origin hyaluronan binding protein and hyaluronic acid is detected in the specimen, the detection result can be associated with a state of being pregnancy toxemia or having a high probability of pregnancy toxemia. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel detection method and detection kit for pregnancy toxemia.

The abbreviations used in the application documents are as follows.
BSA: bovine serum albumin
HA: hyaluronic acid (hyaluronan)
HABP: hyaluronan binding protein
HRP: horseradish peroxidase
ITI: inter-alpha-trypsin inhibitor
PBS: phosphate buffered saline
PBS-T: PBS containing 0.1% Tween 20
ROC curve: receiver operating characteristic curve
SHAP: serum-derived hyaluronan associated protein
SHAP-HA complex: complex of SHAP and HA TMB: 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine)
UTI: bikunin
Preeclampsia is one in which one or more symptoms of hypertension, proteinuria, and edema are observed in pregnancy, and these symptoms are not simply caused by accidental pregnancy complications and develop after 20 weeks of pregnancy and delivery. Those that return to normal by 12 weeks later. Since pregnancy toxemia may be accompanied by intrauterine growth retardation, it is necessary to detect it early and to apply diet and drug therapy. In pregnancy, both mother and child are strongly required to be safe. Therefore, when pregnancy toxemia is diagnosed, it is important to appropriately evaluate whether the pregnancy toxemia is mild or severe.

  Hereinafter, the prior art considered closest to the present invention will be described.

  Non-Patent Document 1 describes that HA concentration in serum increases in pregnant women with preeclampsia.

  However, there is no description or suggestion of detecting the presence or severity of pregnancy toxemia by measuring SHAP-HA complex or UTI.

  Patent Document 1 is characterized in that a SHAP-HA conjugate in a specimen is reacted with an anti-ITI antibody to form an anti-ITI antibody- (SHAP-HA conjugate) complex, and this complex is measured. A method for measuring a SHAP-HA conjugate in a sample and a kit for use in the method are described. In addition, this document describes that a SHAP-HA conjugate in a sample is measured, thereby diagnosing arthritis and liver disease.

  However, there is no description or suggestion of detecting the presence or severity of pregnancy toxemia by measuring SHAP-HA complex or UTI.

Osmels, R.A. G. W. (Osmers, R.G.W.) et al., 1998, American Journal of Obstetrics and Gynecology, 178, No. 2, p. 341-345 Japanese Patent Laid-Open No. 10-82784

  An object of the present invention is to provide a method and kit capable of sensitively and accurately detecting pregnancy toxemia. Furthermore, this invention makes it a subject to provide the method and kit which can detect the severity of pregnancy toxemia sensitively and correctly.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have found that pregnancy toxemia can be detected sensitively and accurately by measuring the SHAP-HA complex and UTI in a specimen. It came to complete. Furthermore, it has also been found that the severity of pregnancy toxemia can be detected sensitively and accurately by measuring the SHAP-HA complex in the specimen, and the present invention has been completed.

That is, the present invention includes a method for detecting pregnancy toxemia (hereinafter, “method of the present invention”) comprising at least a step of measuring SHAP-HA complex and / or UTI in a specimen and associating the measurement result with pregnancy toxemia. ).
The present invention also includes a method for detecting pregnancy toxemia (hereinafter referred to as “method 1 of the present invention”) comprising at least a step of measuring a SHAP-HA complex in a specimen and associating the measurement result with pregnancy toxemia. I will provide a. This sample is preferably a blood-derived sample.
The measurement of the SHAP-HA complex is preferably performed by an immunoassay. This immunoassay is preferably performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen into contact with a solid phase to which HABP is fixed.
(2) A step of detecting a SHAP-HA complex in the specimen bound to the solid phase.

The present invention also provides a pregnancy toxia detection kit (hereinafter referred to as “the present invention kit 1”), which comprises at least the following components (A) and (B).
(A) HABP
(B) An anti-ITI antibody or the present invention is a method for detecting pregnancy toxemia (hereinafter referred to as “method 2 of the present invention”) comprising at least a step of measuring UTI in a sample and associating the measurement result with pregnancy toxemia. .)I will provide a. This specimen is preferably urine.

The UTI measurement is preferably performed by an immunoassay. This immunoassay is preferably performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen and “polypeptide that binds to UTI” into contact with a solid phase to which UTI is fixed.
(2) A step of detecting “polypeptide that binds to UTI” bound to the solid phase.
The present invention also provides a pregnancy toxia detection kit (hereinafter referred to as “the present invention kit 2”), which comprises at least the following components (A) and (B).
(A) UTI
(B) Anti-UTI antibody

  The method of the present invention is extremely useful because pregnancy toxemia can be detected and discriminated easily, quickly, sensitively and accurately. Among them, the method 1 of the present invention is extremely useful because it can easily and quickly detect, and discriminate not only the presence or absence of pregnancy toxemia but also the severity of pregnancy toxemia. In addition, the kits 1 and 2 of the present invention are extremely useful because the detection and discrimination of such pregnancy toxemia can be carried out more easily and quickly.

Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention.
<1> Method of the Present Invention The method of the present invention is a method for detecting pregnancy toxemia, comprising at least a step of measuring a SHAP-HA complex and / or UTI in a sample and associating the measurement result with pregnancy toxemia. . In the method of the present invention, both the SHAP-HA complex and UTI in the sample may be measured, or only one of them may be measured. Hereinafter, the method 1 and 2 of the present invention will be described separately.
<1> -1 Method 1 of the present invention
The method 1 of the present invention is a method for detecting pregnancy toxemia, comprising at least a step of measuring a SHAP-HA complex in a specimen and associating the measurement result with pregnancy toxemia.
The method 1 of the present invention was applied as a method for detecting preeclampsia because it was revealed by the present invention that the SHAP-HA complex is significantly increased in specimens of animals with preeclampsia. is there.
The “sample” here is not particularly limited as long as the amount of the SHAP-HA complex contained in the sample varies due to pregnancy toxemia. Examples of such specimens include those derived from biological tissues in which the amount of the SHAP-HA complex varies due to pregnancy toxemia, those derived from body fluids, and the like.
Examples of those derived from body fluids include blood-derived specimens. The term “blood-derived specimen” here includes blood itself, specimens obtained by subjecting blood to some kind of treatment, and the like. Examples of the “specimen obtained by subjecting blood to some kind of treatment” include those obtained by separating from blood cell components of blood (for example, serum and plasma), and the SHAP-HA complex fraction purified from blood. Etc. are exemplified. Of these, serum or plasma is preferable, and serum is particularly preferable.
The animal from which the specimen is derived is not particularly limited as long as it is a mammal that can cause pregnancy toxemia, but it is preferably a human.
The method for measuring the SHAP-HA complex in the sample is not particularly limited as long as it is a method capable of detecting and measuring the SHAP-HA complex present in the sample. The term “measurement” in the present invention is a concept including not only quantitatively measuring a certain substance but also qualitatively measuring (detecting the presence or absence of a certain substance).
Such measurement of the SHAP-HA complex in the specimen can be performed by, for example, a physicochemical method (for example, a chromatography method) or a biological method (for example, an immunoassay method). Especially, it is preferable to carry out by an immunoassay from viewpoints of simplicity and rapidity. Examples of the immunoassay include enzyme immunoassay (ELISA) and radioimmunoassay (RIA), but any method can be employed.

The term “immunoassay” in the present application document includes not only a measurement method using an antigen-antibody reaction but also a measurement using affinity between biomolecules (for example, affinity between HABP and HA, etc.). Law is also encompassed.
When the SHAP-HA complex in the sample is measured by an immunoassay, it is preferably performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen into contact with a solid phase to which HABP is fixed.
(2) A step of detecting a SHAP-HA complex in the specimen bound to the solid phase.
Hereinafter, each step will be described.
Step (1):
Step (1) is a step in which the specimen is brought into contact with the solid phase to which HABP is fixed.

  The solid phase to which HABP is fixed is not particularly limited as long as it can fix HABP and is insoluble in water, a sample, and a reaction solution. Examples of the shape of the solid phase include a plate (for example, a well of a microplate), a tube, a bead, a membrane, a gel, a fine solid phase carrier (a synthetic polymer particle such as gelatin particles, kaolin particles, latex, etc.), etc. Can do. Of these, a microplate is desirable from the viewpoint of accurate quantitativeness and ease of use.

  Examples of the solid phase material include polystyrene, polypropylene, polyvinyl chloride, nitrocellulose, nylon, polyacrylamide, polyallomer, polyethylene, glass, and agarose. Among these, a plate made of polystyrene is preferable.

  As a method for fixing HABP on the surface of such a solid phase, a general method such as a physical adsorption method, a covalent bond method, a comprehensive method (for example, immobilized enzyme, 1975, Kodansha, 9-75). (See page).

  Among these, the physical adsorption method is preferable because the operation is simple and frequently used.

  As an example of a specific method of physical adsorption of HABP, for example, a buffer solution of HABP is brought into contact with a solid phase, and it is about 0 ° C. to 10 ° C., preferably about 0 ° C. to 5 ° C., about 6 hours to 24 hours. The method of leaving still preferably for 10 hours-20 hours is illustrated.

  In addition, a surface portion to which HABP is not fixed may remain on the surface of the solid phase to which HABP is fixed, and the SHAP-HA complex present in the sample is fixed nonspecifically there. There is a risk that accurate measurement results cannot be obtained. Therefore, it is preferable that the blocking substance is brought into contact with the solid phase before the sample is brought into contact with the solid phase, and the portion where HABP is not fixed is coated. Examples of such blocking substances include serum albumin (such as BSA), casein, skim milk, gelatin, and pluronic. Moreover, what is marketed as a blocking substance can also be used.

  As an example of a specific method of blocking, for example, a buffer solution of BSA is brought into contact with a solid phase, and it is about 0 ° C. to 37 ° C., preferably about 10 ° C. to 30 ° C., about 15 minutes to 2 hours, preferably 30 The method of leaving still for about minutes-about 2 hours is illustrated.

  The contact method between the solid phase and the specimen is not particularly limited as long as the HABP molecule fixed to the solid phase and the SHAP-HA complex molecule present in the specimen come into contact with each other. For example, the sample may be added to the solid phase for contact, the solid phase may be added to the sample for contact, or both may be added simultaneously to separate containers. The contact method is not limited to these, and can be appropriately determined by those skilled in the art according to the shape and material of the solid phase.

  After contacting both of these, in order to sufficiently bind HABP fixed to the solid phase and the SHAP-HA complex in the specimen, for example, incubation is performed at about 0 ° C. to 37 ° C. for about 30 minutes to about 2 hours. It is preferable.

  After this reaction, the solid phase and the liquid phase are separated. If necessary, it is preferable to wash the surface of the solid phase with a washing solution to remove non-specifically adsorbed substances and components in the specimen that have not reacted.

As the cleaning solution, for example, a buffer solution (for example, PBS, phosphate buffer solution, Tris-HCl buffer solution, etc.) to which a nonionic surfactant such as a Tween surfactant is added can be used.
SHAP present in a sample by contacting the sample with a solid phase to which HABP is fixed
-The HA part in the HA complex binds to HABP immobilized on the solid phase, and the SHAP-HA complex binds to the solid phase via HABP. And if there is much quantity of SHAP-HA complex which exists in a test substance, corresponding to this, many SHAP-HA complexes will couple | bond with a solid phase via HABP.
Step (2):
Step (2) is a step of detecting the SHAP-HA complex in the specimen bound to the solid phase. The method for detecting the SHAP-HA complex bound to the solid phase is not particularly limited, and the SHAP-HA complex bound to the solid phase may be directly detected physicochemically and bound to the SHAP-HA complex. You may detect using polypeptide etc. This polypeptide preferably binds specifically to the SHAP-HA complex.

An example of “polypeptide that binds to a SHAP-HA complex” is, for example, an anti-ITI antibody. The anti-ITI antibody binds to the SHAP portion of the SHAP-HA complex.
As used herein, “polypeptide” includes not only such an antibody itself but also its antigen binding site (
Polypeptides or the like that retain Fab) are also included. Examples of the “polypeptide retaining an antibody Fab” include a fragment containing an antibody Fab.

  The “antibody” herein may be either polyclonal or monoclonal, but is preferably a monoclonal antibody from the viewpoint of specificity, homogeneity, reproducibility, large-scale and permanent productivity, and the like. .

  For example, the production of an anti-ITI antibody can be carried out by a general antibody production method using ITI or SHAP-HA complex as an antigen, or a commercially available product can be used.

A “polypeptide that binds to a SHAP-HA complex” typified by an anti-ITI antibody may be labeled with a labeling substance or labeled so as to facilitate detection. The labeling substance that can be used for labeling is not particularly limited as long as it can be used for labeling ordinary proteins. For example, enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, glucose oxidase, etc.), radioisotope ^{(125 I, 131 I, 3} H , etc.), a fluorescent dye (fluorescein isothiocyanate (FITC), 7- amino-4-methylcoumarin-3-acetate (AMCA), dichlorotriazinyl aminofluorescein (DTAF) Tetramethylrhodamine isothiocyanate (TRITC), Lisamine Rhodamine B (Lissamine Rhodamine B), Texas Red, Phycoerythrin (PE), Umbelliferone, Europium, Phycocyanin, Tricolor, Cyanine, etc.) , Chemiluminescent substances (such as luminol) , Haptens (dinitrofluorobenzene, adenosine monophosphate (AMP), 2,4-dinitroaniline, etc.), specific binding pairs (biotin and avidin (streptavidin, etc.), lectin and sugar chain, agonist and agonist receptor Examples include one of heparin and antithrombin III (ATIII).

In addition, a “polypeptide that binds to a SHAP-HA complex” typified by an anti-ITI antibody is bound to a SHAP-HA complex bound to a solid phase, and then a second polypeptide that binds to the polypeptide. You may detect using a peptide (for example, secondary antibody etc.). This second polypeptide is preferably pre-labeled with a labeling substance as described above.
By detecting such a “polypeptide that binds to the SHAP-HA complex” or a labeling substance that binds to the second polypeptide that binds to this, the SHAP-HA in the sample bound to the solid phase Complexes can be detected.

  As a method for detecting the labeling substance, a known detection means corresponding to the type of the labeling substance can be appropriately selected. For example, when one substance (for example, biotin) of a specific binding pair is used as a labeling substance, an enzyme (for example, peroxidase) to which the other substance (for example, streptavidin) that specifically binds to this is bound. Add to form a specific binding pair. Next, a substrate for the enzyme (for example, hydrogen peroxide (when the enzyme is peroxidase)) and a coloring substance (for example, TMB, diaminobenzidine, etc.) are added thereto, and the degree of color development of the product by the enzyme reaction is measured by absorbance. The labeling substance can be detected by measuring with the above.

  For example, when a radioisotope, a fluorescent dye, or a chemiluminescent substance is used as a labeling substance, a method of measuring radioactivity count, fluorescence intensity, fluorescence polarization, emission intensity, or the like is exemplified.

  Through such detection of the labeling substance, the SHAP-HA complex in the sample bound to the solid phase can be detected, and thereby the SHAP-HA complex in the sample can be measured. Since this method is a so-called sandwich method, if a large amount of labeling substance is detected, it means that the amount of the sandwich complex is large, that is, the SHAP-HA complex in the sample is large.

When qualitative measurement of the SHAP-HA complex (detection of the presence or absence of the SHAP-HA complex) is desired, the presence or absence of detection of the labeling substance can be directly used as the measurement result. In addition, when a quantitative measurement of the SHAP-HA complex (such as measurement of the concentration of the SHAP-HA complex) is desired, the absorbance, radioactivity count, fluorescence intensity, emission intensity, etc. are directly used in the SHAP-HA complex. Can be an indicator of the amount of. It is also possible to prepare a calibration curve or a relational expression in advance using a standard solution of a known concentration of SHAP-HA complex, and use this to determine the concentration of the SHAP-HA complex in the sample.
The measurement result of the SHAP-HA complex in the specimen can be associated with pregnancy toxemia as follows.

  According to the present invention, it was revealed that the SHAP-HA complex is significantly increased in the specimens of animals with preeclampsia. Therefore, when the measurement result of the SHAP-HA complex in the sample (the amount of the SHAP-HA complex in the sample) is higher than the measurement result in a healthy animal (an animal that is not gestational toxemia), Can be associated with “is a symptom” or “highly likely to have pregnancy toxemia”. Further, if this measurement result is equal to or lower than the measurement result in a healthy animal, it can be related to “not pregnancies” or “not likely to have preeclampsia”.

In addition, the correlation between the measurement result of the SHAP-HA complex in the specimen and pregnancy toxemia is not only related to “presence / absence of pregnancy toxemia” but also the severity of pregnancy toxemia and the degree of progression of the disease state. Detection is also included. According to the present invention, it has been clarified that the SHAP-HA complex in the specimen is significantly increased in severe pregnancy toxemia. For example, the amount of the SHAP-HA complex in a specimen of an individual is measured, When it is higher than a certain level, it can be related to “severe pregnancy toxemia” or “highly likely to have severe pregnancy toxemia”, and conversely, there is an amount of SHAP-HA complex in the sample When it is lower than a certain level, it can be associated with “a mild preeclampsia” or “highly likely a mild preeclampsia”. The “certain level” mentioned here can be appropriately determined by those skilled in the art based on the results of “Table 2” shown in the examples described later and others.
In addition, when the SHAP-HA complex in the sample is regularly measured and the amount tends to increase, “pregnancy toxemia is progressing” or “pregnancy toxemia may be progressing” High ”can be associated. On the other hand, if the tendency is to decrease, it can be related to “pregnancy toxemia is in an improvement direction” or “possibility that pregnancy toxemia is in an improvement direction”. Moreover, when there is no change in quantity, it can be related to "there is no change in the progression (improvement) of pregnancy toxemia" or "there is a high possibility that there is no change in the progression (improvement) of pregnancy toxemia". Thereby, it can also be applied to monitoring of pregnancy toxemia.

  The measurement result (amount of the SHAP-HA complex in the sample) that serves as a reference for association with pregnancy toxemia may be the concentration obtained using the calibration curve or the relational expression described above. It may be a ratio to the amount in the specimen.

Although the present invention is a “detection method” for pregnancy toxemia, it goes without saying that it also includes the idea of “screening method” and “diagnosis method”.
<1> -2 Method 2 of the present invention
The method 2 of the present invention is a method for detecting pregnancy toxemia, comprising at least a step of measuring UTI in a specimen and associating the measurement result with pregnancy toxemia.
In the method 2 of the present invention, it has been clarified by the present invention that not only the SHAP-HA complex but also UTI is significantly increased in the specimen of an animal with pregnancy toxemia. Applied.
The “sample” in the method 2 of the present invention can be the same as that described in the method 1 of the present invention, but urine is particularly preferable. Other explanations are the same as those obtained by replacing the SHAP-HA complex in the method 1 of the present invention with “UTI” and ITI with “UTI”, respectively.

However, when UTI is measured by the immunoassay method in the method 2 of the present invention, it is preferably performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen and “polypeptide that binds to UTI” into contact with a solid phase to which UTI is fixed.
(2) A step of detecting “polypeptide that binds to UTI” bound to the solid phase.
Hereinafter, each step will be described.
Step (1):
Step (1) is a step in which the specimen and “polypeptide that binds to UTI” are brought into contact with the solid phase to which UTI is fixed.

As described above, the “solid phase to which UTI is fixed”, “polypeptide that binds to UTI” and the like are described as follows. Is the same as the replacement of
Note that the contact in step (1) may be simultaneous, or the UTI-immobilized solid phase and the specimen may be contacted and then the “polypeptide that binds to UTI” may be contacted. Alternatively, after contacting the specimen with “polypeptide that binds to UTI”, it may be brought into contact with “solid phase to which UTI is fixed”. The treatment after the contact is also the same as the method 1 of the present invention.
By bringing these three members into contact, “UTI present in the specimen” and “UTI fixed to the solid phase” compete with each other for “polypeptide that binds to UTI”. As a result, if the amount of UTI present in the specimen is large, the “polypeptide that binds to UTI” bound to the solid phase decreases. Conversely, if the amount of UTI present in the specimen is small, the “polypeptide that binds to UTI” that binds to the solid phase will increase.

Step (2):
(2) A step of detecting “polypeptide that binds to UTI” bound to the solid phase.
Detection of “polypeptide that binds to UTI” bound to the solid phase can also be carried out in the same manner as “detection of SHAP-HA complex in specimen bound to solid phase” in the above-described Method 1 of the present invention. Therefore, the method for detecting this polypeptide is not particularly limited, and may be directly detected physicochemically, such as a second polypeptide that binds to this “polypeptide that binds to UTI” (for example, a secondary antibody). You may detect using. Examples of the “polypeptide that binds to UTI” include an anti-UTI antibody (as described above, the anti-ITI antibody in the method 1 of the present invention may be read as an anti-UTI antibody).
The UTI in the sample can be measured by detecting a “polypeptide that binds to UTI” bound to the solid phase and a labeling substance that is bound to the second polypeptide that binds to it. As understood from the above description, this method is a so-called inhibition method. Therefore, if a large amount of labeling substance is detected from the solid phase, it means that the amount of UTI in the sample is small.

It should be noted that the accuracy of detection of pregnancy toxemia can be further increased by performing the method 1 of the present invention and the method 2 of the present invention together.
<2> Invention kit 1
The kit 1 of the present invention is a detection kit for pregnancy toxemia, comprising at least the following components (A) and (B).
(A) HABP
(B) Anti-ITI Antibody The kit 1 of the present invention can be used for the detection of the presence or absence of pregnancy toxemia, the detection of the severity, etc. according to the method 1 of the present invention.
As described above, HABP, which is a component of the kit 1 of the present invention, is used for binding to a solid phase. This HABP may be a constituent component of the kit 1 of the present invention in a state of being fixed to a solid phase in advance.

  Further, as described above, the anti-ITI antibody that is a component of the kit 1 of the present invention is used for detecting the SHAP-HA complex in the specimen bound to the solid phase.

  The other description regarding the kit 1 of the present invention is the same as that of the method 1 of the present invention. The kit 1 of the present invention is not particularly limited as long as it contains at least the above-described constituents. Further, a standard product of a known concentration of a SHAP-HA complex that is a standard for preparing a calibration curve or a relational expression, or detection of a labeling substance Reagents and the like can be added as components. In addition to these components, the blocking substance, the cleaning solution, the sample diluent, the enzyme reaction stop solution, and the like may be included. Furthermore, the kit of the present invention may contain a positive control (QC control) for keeping the execution level between measurement batches at a constant level.

  These components can be stored in separate containers and stored as a kit that can be used according to the method 1 of the present invention at the time of use.

The kit of the present invention is a “detection kit” for pregnancy toxemia, but needless to say includes the concept of “screening kit” and “diagnostic kit”.
<3> Invention kit 2
The kit 2 of the present invention is a detection kit for pregnancy toxemia, comprising at least the following components (A) and (B).
(A) UTI
(B) Anti-UTI antibody The kit 2 of the present invention can be used for detection of pregnancy toxemia according to the method 2 of the present invention. UTI, which is a component of the kit 2 of the present invention, is used for binding to a solid phase as described above. This UTI may be a constituent component of the kit 2 of the present invention in a state of being fixed to a solid phase in advance.

  The anti-UTI antibody that is a component of the kit 2 of the present invention corresponds to the “polypeptide that binds to UTI” in the method 2 of the present invention. Other explanations are the same as those of the kit 1 of the present invention.

  The kit 1 of the present invention and the kit 2 of the present invention may be combined into one kit. By using this kit, the accuracy of detection of pregnancy toxemia can be further increased.

Hereinafter, the present invention will be described in detail by way of examples.
<1> Materials The materials used in this example will be described below.
1. Specimen samples (serum and urine) were collected from the mothers of 60 normal pregnant women with single pregnancy and 20 pregnant women with preeclampsia (of which 7 were severe and 13 were mild). did. All were collected after obtaining written informed consent.

A case where hypertension (systolic blood pressure is 140 mmHg or more or diastolic blood pressure is 90 mmHg or more) and proteinuria (300 mg / day) occurs after the 20th week of pregnancy was determined as “pregnancy toxemia”. Among them, when systolic blood pressure is 140 mmHg or more and less than 160 mmHg or diastolic blood pressure is 90 mmHg or more and less than 110 mmHg, and proteinuria (300 mg / day or more and less than 2 g / day) occurs, It was determined as “pregnancy toxemia”. In addition, when the systolic blood pressure was 160 mmHg or more or the diastolic blood pressure was 110 mmHg or more and proteinuria (2 g / day or more) was occurring, it was determined as “severe pregnancy toxemia”.
2. Other reagents The following reagents were used.
・ HA measurement kit (Hyaluronan Assay Kit) (Seikagaku Corporation)
・ TMB solution (TMB microwell peroxidase substrate; KPL)
・ HABP (Seikagaku Corporation)
・ Rabbit anti-human ITI antibody (DAKO)
・ HRP labeled goat anti-rabbit immunoglobulin antibody (Jackson)
・ Human UTI (Mochida Pharmaceutical Co., Ltd.)
・ Rabbit anti-human UTI antibody (distributed from Institute of Molecular Medical Science, Aichi Medical University)
<2> Method 1. Measurement of HA The concentration of HA in the sample was measured using an HA measurement kit. First, the HABP solid-phased microplate included in the kit was washed 3 times with PBS-T, and 50 μl of a sample diluent (serum diluted 5 to 10 times with 1% BSA / PBS-T), and 50 μl of biotin-labeled HABP solution included in the kit was added to each well and incubated at 37 ° C. for 1 hour. Thereafter, the wells were washed with PBS-T, and 100 μl of HRP-labeled streptavidin solution contained in the kit was added to each well, and further incubated at 37 ° C. for 1 hour.

After washing the wells with PBS-T, 100 μl of TMB solution was added to each well, and color was developed by incubating at 37 ° C. for 30 minutes. The color reaction was stopped by adding 100 μl of 1M HCl to each well. Thereafter, the absorbance at 450/630 nm (absorbance at 450 nm−absorbance at 630 nm) was measured for each well. This measurement was performed three times for each specimen.
2. Measurement of SHAP-HA complex The concentration of the SHAP-HA complex in the specimen was measured according to the method described in J. Biol. Chem., 278, p32710-32718 (2003). HABP (prepared to 4 μg / ml with 0.1 M sodium carbonate buffer (pH 9.5)) is added to the wells of the microtiter plate and allowed to stand at 4 ° C. for 15 hours, so that HABP is deposited on the surface of the wells. Coated. Thereafter, the wells were washed twice with 200 μl of PBS, and then blocked by adding 200 μl of 3% BSA (dissolved in PBS-T) to the wells and allowing to stand at room temperature for 1 hour. Thereafter, after washing 3 times with 200 μl of PBS-T, 50 μl of sample diluent (serum diluted 5 to 10 times with 1% BSA / PBS-T) was added to each well, and the mixture was added at 37 ° C. Incubated for hours.
Thereafter, the wells were washed, and rabbit anti-human ITI antibody (diluted 3000 times with 1% BSA / PBS-T) and HRP-labeled goat anti-rabbit immunoglobulin antibody (diluted 3000 times with 1% BSA / PBS-T) 25 μl was added to each well and incubated at 37 ° C. for 1 hour.
Thereafter, the wells were washed three times, and 50 μl of TMB solution was added to each well, and color was developed by incubating at 37 ° C. for 10 minutes. The color reaction was stopped by adding 50 μl of 1M HCl to each well. Thereafter, the absorbance at 450/650 nm (absorbance at 450 nm−absorbance at 650 nm) was measured for each well. This measurement was performed three times for each specimen.
3. Measurement of UTI The concentration of UTI in the specimen was measured as follows.
Human UTI (prepared to 2 μg / ml with 0.1 M sodium carbonate buffer (pH 9.5)) was added to the wells of a microtiter plate and allowed to stand overnight at 4 ° C. Coated. Next, 2% BSA was added to the wells and left standing at 4 ° C. for blocking. Then, after washing 3 times with PBS-T, 50 μl of the sample (urine diluted 5 to 10 times with PBS) and rabbit anti-human UTI antibody were added to each well, and the mixture was incubated at 37 ° C. for 1 hour. Incubated.
Thereafter, the wells were washed, 100 μl of HRP-labeled goat anti-rabbit immunoglobulin antibody (diluted 3000 times with 1% BSA / PBS-T) was added to each well, and incubated at 37 ° C. for 1 hour.
Thereafter, the wells were washed three times with PBS-T, and 50 μl of TMB solution was added to each well, and color was developed by incubating at 37 ° C. for 10 minutes. The color reaction was stopped by adding 50 μl of 1M HCl to each well. Thereafter, the absorbance at 450/650 nm (absorbance at 450 nm−absorbance at 650 nm) was measured for each well. This measurement was performed three times for each specimen.
4). Statistical analysis Analysis of significant differences was performed by unpaired t-test, χ ² test, and two-way analysis of variance (ANOVA). A P <0.05 is considered statistically significant. The accuracy of detection of pregnancy toxemia by measurement of serum HA, serum SHAP-HA complex and urine UTI was analyzed by ROC curve.
<3> Results Table 1 shows the results of measuring serum HA, serum SHAP-HA complex, and urine UTI concentrations in normal pregnant women and pregnant women with preeclampsia.

From Table 1, it was shown that the HA concentration in serum, the SHAP-HA complex concentration in serum, and the UTI concentration in urine were significantly higher than those in normal pregnant women. The finding that the serum concentration increases due to pregnancy toxemia is consistent with the finding in Non-Patent Document 1. In addition, the measurement value of SHAP-HA complex in serum and UTI in urine had a lower P value than the measurement result of HA in serum. Therefore, it was shown that the SHAP-HA complex in blood and UTI in urine are superior parameters that easily reflect pregnancy toxemia as compared to HA in blood.
Table 2 shows the results of measuring serum HA, serum SHAP-HA complex, and urine UTI concentrations in pregnant women with severe and mild preeclampsia.

  Table 2 shows that the concentration of the SHAP-HA complex in the serum of pregnant women with severe pregnancy toxemia is significantly higher than that of mild pregnant women. In contrast, serum HA and urine UTI concentrations were not significantly different between severe and mild. Therefore, it was shown that the SHAP-HA complex in blood is an excellent parameter that can discriminate between severe and mild preeclampsia and reflects the severity and pathological progression.

  Furthermore, the accuracy of detection of pregnancy toxemia by measurement of serum HA, serum SHAP-HA complex and urine UTI in pregnant women with severe and mild preeclampsia was analyzed by ROC curve. The results are shown in FIG. In FIG. 1, the horizontal axis indicates specificity, and the vertical axis indicates sensitivity.

  FIG. 1 also shows that the SHAP-HA complex in the serum is a parameter that can detect the severity of pregnancy toxemia particularly accurately.

From the above results, it was shown that by measuring the SHAP-HA complex and UTI in the specimen, it is possible to more sensitively detect whether or not it is pregnancy toxemia than when HA is measured. Furthermore, it was shown that by measuring the SHAP-HA complex, the severity of pregnancy toxemia can be detected sensitively and accurately.
<4> Production of Invention Kit 1 Invention kit 1 having the following constitution was produced. This kit can be used for detection of pregnancy toxemia, detection of severity, etc. through measurement of the SHAP-HA complex by the sandwich method.
1. One 96-well immunoplate with HABP attached 2. 2. Rabbit anti-human ITI antibody 3. HRP-labeled goat anti-rabbit immunoglobulin antibody One TMB solution5. One stop solution (1M HCl) 6. Cleaning solution (PBS-T)
7). SHAP-HA complex standard solution 1 set <5> Production of kit 2 of the present invention Kit 2 of the present invention having the following constitution was produced. This kit can be used for detection of pregnancy toxemia or the like through measurement of UTI by an inhibition method.
1. One 96-well immunoplate to which human UTI is adhered 2. 2. Rabbit anti-human UTI antibody 3. HRP-labeled goat anti-rabbit immunoglobulin antibody One TMB solution5. One stop solution (1M HCl) 6. Cleaning solution (PBS-T)
7). 1 set of UTI complex standard solution

  The method of the present invention and the kit of the present invention can be used for detection of the presence or absence of pregnancy toxemia, detection of severity, and the like.

FIG. 2 shows ROC curves for HA in serum, SHAP-HA complex in serum and UTI measurement in urine of pregnant women with severe and mild preeclampsia.

Claims (11)

  A method for detecting pregnancy toxemia, comprising at least a step of measuring a “complex of a serum-derived hyaluronan-binding protein and hyaluronic acid” and / or bikunin in a sample and associating the measurement result with pregnancy toxemia.   A method for detecting pregnancy toxemia, comprising at least a step of measuring a “complex of a serum-derived hyaluronan-binding protein and hyaluronic acid” in a specimen and associating the measurement result with pregnancy toxemia.   The detection method according to claim 2, wherein the sample is a blood-derived sample.   4. The detection method according to claim 2, wherein the measurement of “complex of serum-derived hyaluronan-binding protein and hyaluronic acid” is performed by an immunoassay. The detection method according to claim 4, wherein the immunoassay is performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen into contact with a solid phase to which a hyaluronic acid binding protein is fixed.
(2) A step of detecting “complex of serum-derived hyaluronan-binding protein and hyaluronic acid” in the sample bound to the solid phase. A detection kit for pregnancy toxemia, comprising at least the following components (A) and (B):
(A) Hyaluronic acid binding protein (B) Anti-inter-α-trypsin inhibitor antibody   A method for detecting pregnancy toxemia, comprising at least a step of measuring bikunin in a sample and associating the measurement result with pregnancy toxemia.     The detection method according to claim 7, wherein the specimen is urine.   The detection method according to claim 7 or 8, wherein bikunin is measured by an immunoassay. The detection method according to claim 9, wherein the immunoassay is performed by a method including at least the following steps (1) and (2).
(1) A step of bringing a specimen and a “polypeptide that binds to bikunin” into contact with a solid phase to which bikunin is fixed.
(2) A step of detecting “polypeptide binding to bikunin” bound to the solid phase. A detection kit for pregnancy toxemia, comprising at least the following components (A) and (B):
(A) Bikunin (B) Anti-bikunin antibody

Images