WO1999067638A1

WO1999067638A1 - Method and reagents for detecting dystocia

Info

Publication number: WO1999067638A1
Application number: PCT/JP1998/002821
Authority: WO
Inventors: Shuichi Hiyamuta; Akihiko Kadota
Original assignee: Idemitsu Kosan Co., Ltd.
Priority date: 1998-06-24
Filing date: 1998-06-24
Publication date: 1999-12-29
Also published as: WO1999067638A9

Abstract

A method for detecting dystocia by quantitating human ceruloplasmin or activated human ceruloplasmin in a test sample, in particular, by an immunoassay with the use of a specific reaction between human ceruloplasmin or activated human ceruloplasmin and an antibody reacting specifically therewith; and detection reagents containing this antibody. The above detection method and reagents make it possible to conveniently detect the possibility of the onset of dystocia, in particular, premature rupture and fetal asphyxia at the early stage.

Description

Satsuki Itoda »

Abnormal labor detection method and detection reagent

Technical field

The present invention relates to a detection method and a detection reagent that can easily and easily detect the possibility of abnormal labor.

Background art

Cell mouth plasmin is a type of acute-phase protein synthesized in the liver, and its amount is known to increase or decrease due to the presence of various diseases. Then, methods for measuring and detecting diseases by immunoassay utilizing this phenomenon have been developed. For example, the applicant of the present application discloses a reagent and a method for detecting Wilson's disease in Japanese Patent Application Laid-Open No. 6-265545.

However, there is no known detection method or detection reagent that can easily detect in advance the possibility of abnormal labor such as premature rupture or fetal distress. For example, in the case of premature rupture that occurs before the expected date of parturition or fetal asphyxia in which the fetus is in a state of asphyxia at the time of parturition, each high-risk group is selected to check for bacterial infection and to monitor the fetus by echo At present, it is possible to detect the possibility of the occurrence by performing ringing, and it is difficult to target all pregnant women due to the complexity of the testing method. Since such abnormal delivery may cause fetal pulmonary blood disorder, intellectual impairment, maternal postpartum fever, etc. I have.

In addition, hypertensive preeclampsia causes swelling, etc. in pregnancy toxemia with hypertension. There is a growing demand for the development of detection methods and detection reagents that can easily detect the possibility of fetal asphyxia. o

A second object of the present invention is to provide a detection method capable of easily and early detecting the possibility of abnormal labor, particularly, the onset of premature rupture and fetal asphyxia. Another object of the present invention is to provide a detection method and a detection reagent which can more easily and quickly detect the possibility of the onset while achieving the object of the present invention. A third object of the present invention is to provide a detection method capable of detecting the possibility of the onset of fetal asphyxia in a pregnant woman exhibiting hypertensive pregnancy toxemia early, easily, quickly and accurately.

Disclosure of the invention

As a result of intensive studies, the present inventors have found that the above problems can be solved by grasping the amount of human celluloplasmin or the amount of activated human celluloplasmin in a test sample. The present invention has been completed based on such findings.

That is, the gist of the present invention is as follows.

(1) A method for detecting the possibility of abnormal delivery consisting of measuring the amount of human celluloplasmin or the amount of active human cell plasmin in the test sample o

Tested by immunoassay using the specific reaction between an antibody that specifically reacts with human cell plasmin or activated human celluloplasmin and human cell plasmin or activated cell plasmin. A method for detecting the possibility of abnormal labor that involves measuring the amount of human celluloplasmin or activated human celluloplasmin in a sample.

(3). The method according to (1) or (2), wherein the abnormal delivery is selected from premature rupture or fetal distress.

(4). Mid-pregnancy and pregnancy collected from pregnant women with hypertensive pregnancy toxemia. For detecting the possibility of fetal asphyxia by measuring the amount of human celluloplasmin or the amount of activated cell-mouth plasmin in a test sample during the second trimester of pregnancy

(5) Test sample by immunoassay using specific reaction between an antibody that specifically reacts with human celluloplasmin or activated human celluloplasmin and human celluloplasmin or activated cell plasmin. The detection method according to the above (4), wherein the amount of human celluloplasmin or activated human celluloplasmin is measured.

(6) A detection reagent consisting of an antibody that specifically reacts with human cell plasmin or activated human celluloplasmin to detect the possibility of abnormal labor

(7). The detection reagent according to (6), wherein the abnormal delivery is selected from premature rupture or fetal distress.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail.

Human celluloplasmin is a type of acute-phase protein synthesized in the liver, and the existence of active and inactive human celluloplasmin is known. The term "human celluloplasmin" as used in the present invention means a general term for activated human celluloplasmin and inactive human celluloplasmin.

In the detection method that achieves the first object of the present invention, the possibility of abnormal delivery is detected by quantifying the amount of human celluloplasmin or the amount of activated human celluloplasmin. Then, a known method can be employed. Examples of such a method include a paraffin diammine activity measurement method, a laser-nephelometry method, a single radial immunodiffusion method, and an immunoassay method. The test sample is not limited as long as it is derived from human, but human body fluid or human effluent can be used.

Human fluid is a general term for all fluids that fill the space between the blood vessels, tissues, and cells in the human body, specifically, blood, lymph fluid, cerebrospinal fluid, etc.

. The buffer solution in the case where a test sample using a serum 1 0 ⁴ to 1 0

It is preferable to use a ^five- fold dilution.

Human excretion refers to all liquids excreted from the human body.Specifically, urine, sweat, tears, or secretions are secreted. Indicates the liquid to be used. As a test sample urine, in the case of using a sweat and tears rather to preferred and Mochiiruko to measured without dilution, 1 0 ¹ to 1 0 ³ times with buffer or the like in the case of using the fluid secreted It is preferable to use a diluted one.

On the other hand, the timing of the measurement is not particularly limited, but is preferably in the second trimester (from the 21st to the 32nd week) or in the third trimester (after the 33rd week). It is preferable to use and measure.

In this measurement, if a higher amount of human celluloplasmin or active human celluloplasmin than that of a healthy pregnant woman is detected, abnormal labor such as premature rupture of water or fetal asphyxia during labor may occur. is expected.

The reason why a higher amount of human celluloplasmin or active human cell plasmin is detected than in healthy pregnant women may be an increase in the detection value as an acute response to bacterial infection. Here, a healthy pregnant woman is a pregnant woman without physical abnormalities such as preeclampsia and aplastic anemia.

Mean pregnant women did not cause abnormal delivery.

In the above-mentioned measuring method, an immunoassay method is employed to achieve the second object of the present invention. Regarding the test sample and the measurement timing, the same embodiment as described above can be used. This method uses a human cell mouth plus Is an immunoassay using a specific reaction between an antibody that specifically reacts with activated human celluloplasmin and human celluloplasmin or activated human celluloplasmin, to determine whether human celluloplasmin or activated type is present in the test sample. This is a detection method that measures cell opening plasmin. In addition, as a detection reagent which achieves this object, there is a detection reagent comprising an antibody which specifically reacts with plasmin of human cell type or activated human plasmin.

Antibodies that specifically react with human cell plasmin or active human celluloplasmin, which are used as reagents to achieve the second object of the present invention, are human celluloplasmin or active human celluloplasmin. A monoclonal antibody or a polyclonal antibody may be used as long as it can react specifically with the monoclonal antibody, but a monoclonal antibody is preferred. Human celluloplasmin monoclonal antibody, which is preferably used as a detection reagent, reacts specifically with human cell plasmin. In addition, the active cell-mouth plasmin monoclonal antibody specifically reacts with the active human cell plasmin, that is, it reacts with the active human cell-mouth plasmin, but reacts with the inactive human cell plasmin. Does not react with

As such a human celluloplasmin antibody, human celluloplasmin monoclonal antibody ID-1 (Hybridoma (1994) vo1.13, No.2, pp.139-1-1 4 1) The hybridoma producing ID-1 has been deposited with the National Institute of Bioscience and Biotechnology (formerly called the Institute for Microorganisms and Industrial Technology) of the National Institute of Advanced Industrial Science and Technology, and its accession number is F ERMBP-413.

In addition, the activated cell mouth plasmin monoclonal antibody is an activated cell mouth plasmin monoclonal antibody ID-2 (Hybridom a (1994) vol.13, No.2, pp.139-141. The hybridoma producing this ID-2 has been deposited with the National Institute of Bioscience and Human-Technology (formerly the same name, the Institute of Microbiology and Industrial Technology) of the National Institute of Advanced Industrial Science and Technology, and its accession number is FE RM BP-4-145 (fine (Transferred from No. 1242, No. 1).

The detection of human celluloplasmin in the test sample is based on the specific reaction between an antibody that specifically reacts with human celluloplasmin or active human celluloplasmin and the cell opening plasmin or type of active human celluloplasmin. It can be performed by immunoassay utilizing the reaction.

Methods for immunoassays are well known and include the Sandwich method for monoclonal and monoclonal antibodies, the sandwich method for monoclonal and monoclonal antibodies, and gold colloid. Any of the currently used antibody-based detection methods such as staining method, agglutination method, latex method, and chemiluminescence method can be used.

For example, detection of human celluloplasmin or activated human celluloplasmin in a sample can be carried out based on a conventional sandwichizer. That is, for example, a primary antibody is immobilized on a gel, and then a test sample is added to react the primary antibody. After washing, a secondary antibody is reacted. After washing, the secondary antibody is measured. This can be done o

Specifically, an antibody that reacts with human cell plasmin can be used as the primary antibody, and the above-described human cell plasma monoclonal antibody or active antibody can be used as the secondary antibody. A human celluloplasmin monoclonal antibody can be used. The primary antibody and the secondary antibody may be reversed.

The measurement of the secondary antibody is based on the immunoglobulin of the animal from which the secondary antibody is derived. The reaction can be performed by reacting a labeled antibody (for example, an enzyme-labeled antibody) and measuring the labeled product (for example, an enzyme). Alternatively, it can also be carried out by using a labeled secondary antibody and measuring the labeled product.

For the measurement of a labeled substance, the labeled substance may be, for example, an enzyme, an isotope, or an anti-cell opening plasmid such as a monoclonal antibody or a polyclonal antibody labeled with a fluorescent substance. By measuring the amount of the labeled substance after reacting the antibody, the amounts of the human cell plasmin and the activated human celluloplasmin in the test sample can be measured.

In the detection method which achieves the third object of the present invention, the amount of human celluloplasmin or active human celluloplasmin in a test sample collected from a pregnant woman exhibiting hypertensive pregnancy toxin symptom is determined during the second trimester of pregnancy. Measure in the latter period. As a quantification method at this time, a detection method that achieves the first object of the present invention can be employed, but a detection method that achieves the second object of the present invention is preferably employed.

In this case, if the amount of human plasmin or active human plasmin in the middle of pregnancy is higher than that of healthy pregnant women, and in the latter half of pregnancy, the amount of plasmin or active human plasmin decreases to the same level or lower than that of healthy pregnant women. In addition, the possibility of fetal asphyxia is accurately detected.

This is because during the second trimester, the intracellular defense system (antioxidant system) increases the amount of human celluloplasmin or active human cell plasmin, and in late pregnancy, the system becomes more severe. Seems to have fallen and this amount has fallen.

Therefore, it is not necessary to strictly understand the meanings of the second and third trimesters.In test samples collected from pregnant women with hypertensive pregnancy toxin symptoms, it was found that human cell plasmin or active human celluloplasmin was not detected. Abnormal on quantity If an increase and subsequent decline is observed, then the possibility of fetal asphyxia is predicted ^

By adopting the detection method that achieves the third object of the present invention, the possibility of fetal asphyxia in a pregnant woman exhibiting hypertensive pregnancy toxin symptoms can be detected quickly, easily, quickly and accurately. Echo monitoring can be continued to address fetal asphyxia.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1

Serum plasmin in the serum collected from pregnant women during the third trimester (week 35 to week 37) was measured by the following method.

That is, the human cell plasmin monoclonal antibody ID-1 as a primary antibody was immobilized on 96 μl permeate plate. Fixation was carried out by adding 3 ig Zml of the antibody solution to each gel at 100a1 and incubating at 30 ° C for 1.5 hours. Next, the gel was washed three times with a phosphate buffer (PBS) containing 0.5% by volume of a surfactant Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.).

Then, the casein solution (made by Snow Brand Co., Ltd.) was diluted 4-fold, and the diluted solution was added to each well at a rate of 3001 and incubated at 30 ° C for 2 hours to perform blocking. After that, washing was performed in the same manner as above. Then, serum collected from a pregnant woman in the test sample (late pregnancy (week 35 to week 37)) was washed with PBS without Tween 20 for 50,000. The solution was diluted 100-fold into each pellet, and incubated at 30 ° C for 1.5 hours. To prepare a calibration curve, a standard human cellulos plasmin solution (0, 10, 20, 30, 40, 50, 50 ng / ml) was used. 0 1 Each of the cells was put in each cell and incubated similarly.

After washing in the same manner as above, as a secondary antibody, a PBS solution containing 100 ng / ml of a peroxidase-labeled human celluloplasmic clonal monoclonal antibody Tween 20 (human celluloplasmin) was used. (The monoclonal antibody was manufactured by Chemicon Co., Ltd.) Each 100 μl of the gel was added, and the mixture was incubated at 30 ° C. for 1.5 hours.

After washing in the same manner as above, ABTS (2,2-azinobis (3-ethylpentiziazolin-6-sulfonate diammonium salt); Sumitomo Bee Cry Aqueous solution was added to each pellet in 1001 and allowed to react for 10 minutes at 30 ° C. Then, 100 μl of a reaction stop solution was added to each pellet, and the wavelength was 4 μm. The absorbance at 15 nm was measured to quantify the amount of plasmin in the human cell mouth.

Separately, the amount of human celluloplasmin in serum of 34 normal pregnant women in the third trimester (standard) was measured by the laser nephelometry method, and the distribution of human celluloplasmin obtained from the measurement results was measured. 9 Upper limit of 5% confidence limit 91.4 8 mg Pregnant women different from the above 34 who showed a value of more than 8 mg Z deciliters The occurrence of water or fetal distress was observed.

The results are shown in Table 1. One in the table indicates that no measurement was made. Example 2

Active human celluloplasmin in serum collected from pregnant women during the third trimester (weeks 35 to 37) was measured by the following method.

That is, the measurement was performed in exactly the same manner as in Example 1, except that the active human cell plasmin monoclonal antibody ID-2 was used as the primary antibody, and the amount of the active human celluloplasmin was determined. did.

As in Example 1, separately from 34 normal pregnant women in the third trimester (standard) The amount of active human cell plasmin in the serum was measured, and the upper limit of the 95% confidence limit in the distribution of the amount of active human celluloplasmin determined from the measurement results was 91.488 mg / dec. Observation after measurement was continued in 48 pregnant women who showed a value greater than the liter, and the occurrence of premature rupture or fetal asphyxia at delivery was observed.

The results are shown in Table 1. One in the table indicates that no measurement was made.

Example 1 Example 2 Healthy pregnant woman

Number of samples (example) 48 48 34 Human cell mouth plasmin concentration Average 116. 69

(mg / τsil) Upper limit 0000 Active human cell Rasmin concentration Average 115.69 83.28

(mg / deciliter) Upper limit 89.83 Number of cases of early-stage rupture (example) 15 15 Number of cases of fetal asphyxia (example) 15 15 In Table 1, 15 out of 8 pregnant women with fetal asphyxia showed a value greater than or equal to the 95% confidence limit for the amount of human plasmin or activated human plasmin. Similarly, fifteen cases of early rupture were observed. From the above results, it can be seen that the possibility of fetal asphyxia or early rupture of water can be easily and quickly detected by using the measuring method or the measuring reagent of the present invention.

Examples 3 to 5

A test sample was prepared using the serum of the second trimester (week 21 to week 29) collected from a pregnant woman with hypertensive preeclampsia, except that the test sample was prepared in the same manner as in Example 1 or 2. The amount of human celluloplasmin or active human cell plasmin was determined. In addition, observation after measurement was continued, and the occurrence of fetal asphyxia during delivery was observed.

Separately, the serum levels of human celluloplasmin and active human celluloplasmin were measured in 65 healthy pregnant women during the second trimester, and the values of human celluloplasmin and active human celluloplasma were determined from the measurement results. The upper limit of each 95% confidence limit in the distribution of skin levels is 91.4 8 mg Z deciliter and pregnant women who show 89.8 3 mg Z deciliter or higher are pregnant. Serum was also collected at the later stage (week 35 to week 37), and the amounts of human celluloplasmin and active human celluloplasmin were measured in the same manner as described above. After that, further observation was continued to observe the onset of fetal asphyxia during delivery.

The results are shown in Table 2. One in the table indicates that no measurement was made. Table 2 Examples Comparative examples Comparative examples Healthy pregnant women

3 1 2 Number of measurements (example) 1 1 1 34 65

Outbreak of hypertensive toxemia Yes Yes Yes No

Pregnancy Measured human ceruloplasmin concentration 109.53 105.92 100.65 ― ― Pregnancy (mg / liter)

Medium Upper limit ― 1 ― ― 85.41 Activated value 128.78 77.76 98.68 ― 1 Human cerulobrasmin concentration

(mg / liter "liter") Upper limit-111.02

Pregnancy Human ceruloplasmin concentration 73. 17 90. 86 96. 27 pregnancy (mg / liter)

After Upper limit 11-91.48 Activated value 65.54 100.73 91.84 Human ceruloplasmin concentration

(mg / liter "liter") Upper limit 89.83

Occurrence of fetal asphyxia Yes No No No Table 2 shows that in the second trimester, the amount of human celluloplasmin or active human celluloplasmin was higher than the upper limit of healthy pregnant women, and in the second trimester, the amount was lower than or equal to the upper limit of healthy pregnant women. Pregnant women with hypertensive pregnancy toxin who have a positive amount of plasmin or activated human plasmin may have a higher likelihood of developing fetal asphyxia than in Example 4 or Example 5. I understand.

Example 6

Vaginal secretions of 102 pregnant women in late pregnancy (week 35 to week 37) adsorbed on a cotton swab were immersed in 1 ml of phosphate buffered saline, extracted, and PBS was extracted. In addition, the dilution of the secretion fluid diluted 100-fold was used as the test sample, human celluloplasmin monoclonal antibody was used as the primary antibody, and 200 ng per ml of peroxydamine was used as the secondary antibody. The procedure was performed in the same manner as in Example 1 except that ze-labeled human cellulosin monoclonal antibody ID-2 was used, and the amount of active human cell plasmin in vaginal secretions was measured.

After that, observation was continued and the occurrence of rupture in the first half was observed.

Of the 102 women, the group of 5 pregnant women with premature rupture was observed and the group of 7 pregnant women without rupture of premature rupture. The upper and lower limits of the 95% confidence limit in the mean value of the amount of toseruloplasmin and its distribution were determined.

The results are shown in Table 3.

Clothing 3

Table 3 shows that the amount of active human celluloplasmin in the secretion fluid in which the premature rupture occurred was higher than the amount of active human celluloplasmin in the secretion fluid of healthy pregnant women who delivered normally. At the same time, their measurements were clearly distributed in different areas.

This indicates that pregnant women with high levels of activated human celluloplasmin in secretions during the second trimester (weeks 35 to 37) are more likely to develop premature rupture.

Industrial applicability

Quantification of human celluloplasmin or active human celluloplasmin collected from pregnant women enables early and easy detection of abnormal labor, especially the possibility of early premature rupture and fetal asphyxia. Can be.

Abnormal parturition, especially by quantifying human cell plasmin or active human celluloplasmin collected from a pregnant woman by a specific immunoassay or a method using a specific immunoassay reagent, etc. Early, simple, and rapid detection of the possibility of fetal asphyxia Wear.

By measuring human celluloplasmin or active human celluloplasmin collected from hypertensive preeclampsia in the second and third trimesters, the possibility of fetal asphyxia can be detected early and accurately.

Measurement of human celluloplasmin or active human celluloplasmin collected from hypertensive preeclamptics in the second and third trimesters using a specific immunoassay may reduce the possibility of fetal distress. It can be detected easily, quickly and accurately at an early stage.

Claims

The scope of the requiem

1. A method for detecting the possibility of abnormal delivery, comprising measuring the amount of human celluloplasmin or active human celluloplasmin in a test sample.

2. A test sample obtained by immunoassay utilizing the specific reaction of an antibody that specifically reacts with human cell plasmin or active human celluloplasmin with human celluloplasmin or active human celluloplasmin A method for detecting the possibility of abnormal delivery, comprising measuring the amount of plasmin in the human cell mouth or the amount of activated human celluloplasmin.

3. The detection method according to claim 1 or 2, wherein the abnormal delivery is selected from premature rupture or fetal distress.

4. Fetal asphyxia is possible by measuring the amount of human celluloplasmin or activated celluloplasmin in test samples collected during the second and third trimesters of pregnant women with hypertensive toxemia. Sex detection method.

5. Test sample by immunoassay using the specific reaction between the antibody that specifically reacts with human cell plasmin or activated human celluloplasmin and human celluloplasmin or activated human celluloplasmin The detection method according to claim 4, wherein the amount of plasmin in the human cell mouth or the amount of plasmin in the activated human cell mouth is measured.

6. A detection reagent consisting of an antibody that reacts specifically with human cell plasmin or activated human celluloplasmin to detect the possibility of abnormal labor.

7. The detection reagent according to claim 6, wherein the abnormal delivery is selected from premature rupture or fetal distress.