Application of SP70 in preparation of health monitoring reagent for pregnant women and fetuses in gestation period

Abstract

The invention discloses application of SP70 in preparation of a health monitoring reagent for pregnant women and fetuses in the gestation period. The reagent for detecting SP70 in serum is applied to preparation of a reagent for monitoring abnormal pregnancy of pregnant women and intrauterine growth and development conditions of fetuses. The serum SP70 concentration of a normal pregnant woman is obviously higher than that of a healthy physical examinee, the serum SP70 concentration of threatened abortion and ectopic pregnancy patients is obviously lower than that of the normal pregnant woman, and the diagnosis efficiency of threatened abortion and ectopic pregnancy can be obviously improved by combining SP70 with hCG detection. In addition, the serum SP70 concentration of the pregnant women is related to the intrauterine growth condition of the fetuses, the serum SP70 concentration of the pregnant women with small fetuses is remarkably lower than that of the pregnant women with normal fetuses, and the serum SP70 concentration of the pregnant women with large fetuses is remarkably higher than that of the pregnant women with normal fetuses. Therefore, the reagent for detecting the SP70 in the serum can be applied to preparation of reagents or auxiliary reagents for monitoring abnormal pregnancy of pregnant women in the gestation period and intrauterine growth and development conditions of fetuses.

Description

Technical Field

[0001] The invention belongs to the field of biological diagnosis and relates to the application of tumor specific protein 70 (SP70) in the preparation of abnormal pregnancy and fetal intrauterine growth and development monitoring reagents (abnormal pregnancy includes threatened abortion and ectopic pregnancy). Background Art

[0002] Pregnancy is a complex natural process involving a series of stages from the development of the embryo and fetus in the mother's body from the fertilized egg to the final delivery. This process is affected by many factors, such as the hormone level in the pregnant woman, the nutritional status during pregnancy, and the function of the placenta. Pregnancy does not always go smoothly. Abnormalities in any link of pregnancy may cause diseases in the pregnant woman or the fetus. When the fertilized egg implants outside the uterus, the fetus develops abnormally in the uterus, or the mother suffers specific organ damage due to pregnancy, the pregnant woman will suffer pregnancy complications. These diseases include threatened abortion, ectopic pregnancy, and gestational hypertension. Fetal abnormalities include abnormal fetal growth and development, birth defects, and fetal distress. In recent years, due to various reasons such as abnormal maternal endocrine and immune function, abnormal placental function and development, the widespread use of assisted reproductive technology, and increased pollution in the living environment, the risk of diseases in pregnant women and fetuses during pregnancy is increasing. Relevant studies at home and abroad have shown that diseases related to pregnant women and fetuses during pregnancy not only threaten the health of the mother and fetus, but can also cause death in severe cases. Threatened abortion is one of the most common complications of early pregnancy, with an incidence rate of 20-25%. The current global prevalence of ectopic pregnancy is about 5-8%, which is one of the causes of maternal and perinatal mortality. Therefore, early prevention and intervention of these conditions and strengthening of pregnancy care can reduce the occurrence of adverse maternal and fetal events.

[0003] Standardized prenatal examinations are an important measure for early detection of pregnancy diseases. Appropriate early intervention and treatment can significantly reduce the economic burden and mortality of pregnancy diseases. Prenatal examinations include obstetric ultrasound, fetal heart rate monitoring, and laboratory index examinations during pregnancy. Ultrasound is the most commonly used prenatal screening method, which can observe fetal growth parameters, blood flow index, placenta and amniotic fluid volume, but its results are highly subjective and related to the operation level of clinicians. Moreover, imaging examinations such as ultrasound can only reflect changes in anatomical structures, but cannot reflect the metabolic level and placental function of pregnant women. Monitoring of fetal heart rate during pregnancy has been widely used in clinical practice, but there are still differences in the reliability and consistency of the results, and there is a certain rate of missed detection. In terms of monitoring the health of pregnant women and evaluating the intrauterine growth and development of the fetus, blood indicators play an irreplaceable and important role. Abnormal fluctuations in laboratory indicators during pregnancy can indicate abnormal conditions of pregnant women or fetuses, but the sensitivity and specificity are limited. Moreover, many pathophysiological mechanisms such as the pathogenesis of pregnancy diseases and causes of death are still unclear or have not been clearly elucidated, which makes it challenging to determine the appropriate timing and frequency of prenatal monitoring. Under the current circumstances, it is indeed impossible to avoid missed diagnosis of fetal or pregnant women's abnormalities. Therefore, finding reliable markers for monitoring abnormal pregnancy and intrauterine growth and development of the fetus during pregnancy has important theoretical and clinical application value. Since clinical blood samples are easy to obtain, the results are more objective, and do not rely on the operator's experience, more and more researchers are trying to find blood markers for monitoring abnormal pregnancy and intrauterine growth and development of the fetus during pregnancy.

[0004] SP70 is a biologically active protein discovered by the inventors through monoclonal antibody library technology in the early stage, which can be targeted, recognized and bound by the monoclonal antibody NJ001. In the serum of patients with various abnormal proliferative tumors such as lung cancer, pancreatic cancer, and breast cancer, the SP70 positive rate is significantly higher than that of benign diseases and healthy control groups, and is highly correlated with the proliferation of tumor cells in patients. Further studies have shown that this protein can promote tumor cell proliferation, invasion and metastasis of tumor cells. Because it is highly expressed in highly proliferative tumor cells, lowly expressed in low-proliferation normal cells, and can promote the proliferation of tumor cells, it is also called proliferation stimulating protein (PSP). However, there is currently no literature reporting on SP70 as an indicator for monitoring abnormal pregnancy and fetal growth and development in pregnant women. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide the use of SP70 as a detection target in the preparation of monitoring reagents or auxiliary reagents for abnormal pregnancy and fetal intrauterine growth and development during pregnancy (abnormal pregnancy includes threatened abortion and ectopic pregnancy).

[0006] Another object of the present invention is to provide a reagent for detecting SP70 in serum for use in preparing a reagent or auxiliary reagent for monitoring abnormal pregnancy and fetal intrauterine growth and development during pregnancy (abnormal pregnancy includes threatened abortion and ectopic pregnancy).

[0007] Pregnancy is a process of high cell proliferation in a non-disease state. Preliminary tests have found that SP70 is highly expressed in the peripheral blood of pregnant women. By directly detecting amniotic fluid precipitation by immunofluorescence, cells expressing SP70 were also found. According to our latest clinical research, the concentration of serum SP70 in pregnant women with normal pregnancy is significantly higher than that in non-pregnant women, and the concentration of SP70 increases with the number of days of pregnancy, and decreases rapidly after delivery. The changes in SP70 levels are basically consistent with the normal pregnancy process, which suggests that SP70 in the serum of pregnant women may come from the fetus and its appendages. When a pregnant woman has threatened abortion or ectopic pregnancy, the serum SP70 level decreases significantly, indicating that SP70 can indicate adverse pregnancy events in early pregnancy and has clinical application value in pregnancy care. In addition, we found that the serum SP70 concentration of pregnant women with smaller fetal growth and development is significantly lower than that of those with normal fetal growth and development, and the serum SP70 concentration of pregnant women with larger fetal growth and development is significantly higher than that of those with normal fetuses. Therefore, SP70 can be used as an indicator for monitoring fetal intrauterine growth and development, and has potential predictive value for fetal growth and development. Based on the above scientific research results, the technical solution of the present invention is formed:

[0008] The application of SP70 as a detection target in the preparation of monitoring reagents or auxiliary reagents for abnormal pregnancy and fetal intrauterine growth and development during pregnancy.

[0009] As a preferred embodiment of the present invention, the abnormal pregnancy is any one of threatened abortion or ectopic pregnancy.

[0010] The use of reagents for detecting SP70 in serum in the preparation of reagents or auxiliary reagents for monitoring abnormal pregnancy and intrauterine growth and development of fetuses during pregnancy.

[0011] As a preferred embodiment of the present invention, the abnormal pregnancy is any one of threatened abortion or ectopic pregnancy.

[0012] As a preferred embodiment of the present invention, the reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum.

[0013] As a preferred embodiment of the present invention, the reagent for detecting SP70 in serum comprises the monoclonal antibody NJ001-1 against SP70, and the monoclonal antibody NJ001-1 is secreted by the hybridoma cell line NM001-1 with a preservation number of CCTCC NO: C201172.

[0014] The use of a reagent for detecting SP70 in serum combined with a reagent for detecting HCG in the preparation of an auxiliary diagnostic reagent for abnormal pregnancy in pregnant women and / or a reagent for monitoring the intrauterine growth and development of the fetus or an auxiliary detection reagent.

[0015] As a preferred embodiment of the present invention, the reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum, and the reagent for detecting HCG is a reagent for quantitatively detecting HCG.

[0016] As a preferred embodiment of the present invention, the reagent for detecting HCG is a reagent for detecting HCG by chemiluminescence method.

[0017] An auxiliary diagnosis kit for abnormal pregnancy in pregnant women and / or a reagent or auxiliary detection kit for monitoring the intrauterine growth and development of the fetus, comprising a reagent for quantitatively detecting SP70 in serum and a reagent for quantitatively detecting HCG.

[0018] As a preferred embodiment of the present invention, the reagent for quantitatively detecting SP70 in serum comprises the monoclonal antibody NJ001-1 against SP70, and the monoclonal antibody NJ001-1 is secreted by the hybridoma cell line NM001-1 with a preservation number of CCTCC NO: C201172.

[0019] Beneficial Effects

[0020] The present invention first found that the concentration of SP70 in the serum of pregnant women was significantly higher than that of non-pregnant women (P < 0.01), and there was a significant positive correlation between the concentration and the number of days of amenorrhea. With the increase of pregnancy time, the concentration of SP70 in the serum of pregnant women gradually increased, continued until before delivery, and rapidly decreased after delivery (P < 0.01). The changes in the concentration of SP70 in the serum of pregnant women during pregnancy and delivery showed a certain regularity, and this change trend was basically consistent with the progress of pregnancy. The present invention also found that the concentration of SP70 in the serum of pregnant women with normal pregnancy was significantly higher than that of patients with threatened abortion and ectopic pregnancy (P < 0.01). Human chorionic gonadotropin (hCG) will decrease to varying degrees when threatened abortion and ectopic pregnancy occur, so it is clinically used to distinguish between pregnant women with normal pregnancy and pregnant women with threatened abortion or ectopic pregnancy. Receiver operating characteristic curve (ROC) curve analysis was performed based on the serum SP70 concentration, hCG concentration and the corresponding diagnostic outcomes of pregnant women. The results showed that the areas under the ROC curve (AUC) of SP70 alone for diagnosing threatened abortion and ectopic pregnancy were 0.792 and 0.840, respectively, the areas under the ROC curve of hCG alone for diagnosing threatened abortion and ectopic pregnancy were 0.714 and 0.816, respectively, and the areas under the ROC curve of SP70 combined with hCG for diagnosing threatened abortion and ectopic pregnancy were 0.896 and 0.935, respectively. The diagnostic efficacy of SP70 alone for diagnosing threatened abortion and ectopic pregnancy and SP70 combined with hCG for diagnosing threatened abortion and ectopic pregnancy were higher than that of hCG alone. This suggests that SP70, as a new laboratory indicator, has the potential value of clinical diagnosis of diseases such as threatened abortion and ectopic pregnancy during pregnancy. In addition, the serum SP70 concentration of pregnant women is related to the intrauterine growth of the fetus. The serum SP70 concentration of pregnant women with smaller fetuses is significantly lower than that of those with normal fetal growth, and the serum SP70 concentration of pregnant women with larger fetuses is significantly higher than that of those with normal fetal growth (P < 0.01). SP70 can be used as a laboratory indicator to monitor the intrauterine growth of the fetus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Serum SP70 concentrations in normal pregnant women and normal non-pregnant women. A: Serum SP70 concentrations in normal pregnant women (n=298); B: Serum SP70 concentrations in normal non-pregnant women (n=52) and normal pregnant women at different stages of pregnancy (early pregnancy n=49, mid-pregnancy n=127, late pregnancy n=181); C: Serum SP70 concentrations in parturients before and after delivery (n=8)

[0022] Figure 2Serum SP70 concentration in normal pregnant women and patients with threatened abortion. A: Serum SP70 concentration in normal pregnant women (n=298) and patients with threatened abortion (n=17); B: Serum hCG concentration in normal pregnant women (n=298) and patients with threatened abortion (n=17); C: ROC curve analysis of the diagnostic value of SP70 concentration, hCG concentration and the combination of SP70 and hCG for threatened abortion

[0023] Figure 3 Serum SP70 concentration in normal pregnant women and patients with ectopic pregnancy. A: Serum SP70 concentration in normal pregnant women (n=298) and patients with ectopic pregnancy (n=25); B: Serum hCG concentration in normal pregnant women (n=298) and patients with ectopic pregnancy (n=25); C: ROC curve analysis of the diagnostic value of SP70 concentration, hCG concentration and the combination of the two for ectopic pregnancy

[0024] Figure 4 Serum SP70 concentrations in pregnant women with normal fetal growth (n=308), small fetal growth (n=78), and large fetal growth (n=42) DETAILED DESCRIPTION

[0025] The sample sources of each of the following embodiments are different.

[0026] Example 1 Changes and analysis of maternal serum SP70 concentration during normal pregnancy

[0027] SP70 detection kit (ELISA method) was used to detect the serum SP70 concentration of 298 normal pregnant women, the serum SP70 concentration of 8 parturients before and after delivery, and the serum SP70 concentration of 52 healthy physical examination subjects. The serum SP70 concentration of the 52 healthy physical examination subjects was used as the normal control.

[0028] 1) Take out the tumor-specific protein 70 detection kit (ELISA method) from the 4°C refrigerator and equilibrate at room temperature for about 30 minutes;

[0029] 2) Prepare 1× working concentration washing buffer by mixing 20× concentrated washing solution, and then put the washing solution into the washing solution bucket in the plate washer after mixing thoroughly.

[0030] 3) Take out the planned amount of anti-SP70 enzyme-labeled reaction plate, seal the remaining microwell strips and place them in a refrigerator at 4°C

[0031] save;

[0032] 4) Take out 5 tubes of standard products from the kit, with concentrations of A = 0.00 ng / mL, B = 3.75 ng / mL, C = 7.50 ng / mL, D = 15.00 ng / mL, and E = 30.00 ng / mL. Add 120 μL of deionized water to each tube, mix thoroughly with a micro vortex mixer, and collect the residual liquid on the tube wall and tube cover with a micro instant centrifuge to prepare the SP70 standard product;

[0033] 5) Take out the quality control products L1 and L2 provided in the kit, add 1200 μL of deionized water to each tube, and mix thoroughly with a micro vortex mixer to prepare the quality control products;

[0034] 6) Add 100 μL of SP70 standard, quality control and patient serum sample to the corresponding detection wells of the ELISA reaction plate in sequence and complete the operation within 10 minutes. After sealing the plate with plastic film, place the reaction plate at 37°C.

[0035] Incubate on the water bath shelf for 60 min;

[0036] 7) After incubation, discard the supernatant and set the parameters of the plate washer: add 250 μL of washing solution to each well and wash 4 times; after the 4th wash, remove the reaction plate and pat dry the remaining liquid;

[0037] 8) Take out the antibody diluent and dilute the monoclonal antibody NJ001 100 times (e.g., add 10 μL of monoclonal antibody to 990 μL of antibody diluent and mix), to prepare the monoclonal antibody working solution. Add 100 μL of monoclonal antibody working solution to each well, seal the plate with plastic film, and incubate the reaction plate on a 37°C water bath shelf for 30 minutes;

[0038] 9) Repeat step 7;

[0039] 10) Take the antibody diluent and dilute the enzyme-labeled secondary antibody 100 times (e.g., add 10 μL of secondary antibody to 990 μL of antibody diluent and mix), to prepare the enzyme-labeled secondary antibody working solution. Add 100 μL of enzyme-labeled secondary antibody working solution to each well, seal the plate with plastic film, and incubate the reaction plate on a 37°C water bath shelf for 30 minutes;

[0040] 11) Repeat step 7 and set the plate washing times to 5 times; after the 5th plate washing, remove the reaction plate and pat dry the remaining liquid;

[0041] 12) Add 100 μL TMB to each well and color develop for 15-20 min at room temperature in the dark;

[0042] 13) Add 50 μL of stop solution to each well and tap the edge of the reaction plate to mix for about 30 seconds;

[0043] 14) Within 15 minutes after adding the stop solution, measure the OD value of each well at 450 nm using a microplate reader;

[0044] 15) Check that the quality control is under control, record the OD value of the standard and draw a standard curve, and calculate the SP70 concentration of the sample to be tested.

[0045] According to the clinical classification standard for pregnancy, pregnancy is divided into three periods: early pregnancy before 14 weeks of pregnancy, early pregnancy from 14th to 27th weeks of pregnancy, and late pregnancy from 14th to 27th weeks of pregnancy. +6 The period from the 28th week to delivery is called mid-pregnancy, and the period from the 28th week to delivery is called late pregnancy.

[0046] The results are as follows Figure 1 As shown in A, the serum SP70 concentration of pregnant women was significantly positively correlated with the number of days of amenorrhea (correlation coefficient r = 0.512, P < 0.01). The serum SP70 concentration of pregnant women was significantly higher than that of healthy people, and there were statistical differences in the serum SP70 levels of pregnant women at different gestational periods, with the highest level in late pregnancy, followed by mid-pregnancy, and the lowest level in early pregnancy ( Figure 1 B, P<0.01). The serum SP70 level of parturients on the first day after delivery was significantly lower than that on the day before delivery ( Figure 1 C, P<0.001).

[0047] Example 2 Diagnostic value of pregnant women's serum SP70 concentration for threatened abortion

[0048] SP70 detection kit (ELISA method) was used to detect the serum SP70 concentration of 49 normal pregnant women and 17 patients with threatened abortion. The diagnostic criteria for threatened abortion refer to "Obstetrics and Gynecology (9th Edition)" (People's Medical Publishing House): a small amount of vaginal bleeding and lower abdominal pain before 28 weeks of pregnancy, no discharge of pregnancy products. Serum hCG is positive, ultrasound examination shows a gestational sac in the uterine cavity, gynecological examination shows that the cervix is ​​not open, the fetal membrane is not ruptured, and the size of the uterus is consistent with the number of weeks of amenorrhea.

[0049] The results are as follows Figure 2 As shown in A, the serum SP70 concentration of normal pregnant women is significantly higher than that of patients with threatened abortion. One of the clinical applications of hCG is to distinguish pregnant women with normal pregnancy from pregnant women with threatened abortion. The hCG test uses Beckman Coulter AU5800 automatic biochemical analyzer and supporting reagents (chemiluminescence method). Figure 2 As shown in B, the serum hCG concentration of normal pregnant women is significantly higher than that of patients with threatened abortion. ROC curve analysis was performed based on the serum SP70 concentration, hCG concentration and the corresponding diagnostic outcomes. The results are shown in Figure 2 As shown in C: In terms of the incidence of threatened abortion diagnosed by a single indicator, the AUC of SP70 reached 0.792, which was greater than that of hCG. When the two indicators were combined, the AUC could reach 0.896, and the diagnostic efficiency was significantly better than that of a single indicator.

[0050] Example 3 Diagnostic value of maternal serum SP70 concentration for ectopic pregnancy

[0051] SP70 detection kit (ELISA method) was used to detect the serum SP70 concentration of 49 normal pregnant women and 25 ectopic pregnancy patients. The diagnostic criteria for ectopic pregnancy refer to "Obstetrics and Gynecology (9th Edition)" (People's Medical Publishing House): no gestational sac in the uterine cavity during ultrasound examination, positive serum hCG, history of amenorrhea, adnexal mass found by abdominal palpation, and visible embryo and primitive heart tube pulsation.

[0052] The results are as follows Figure 3 As shown in A, the serum SP70 concentration of normal pregnant women is significantly higher than that of ectopic pregnancy patients. When ectopic pregnancy occurs, the serum hCG growth rate of abnormal embryo development is slower than that of normal embryos. Figure 3 As shown in B, the serum hCG concentration of normal pregnant women is significantly higher than that of patients with ectopic pregnancy. ROC curve analysis was performed based on the serum SP70 concentration, hCG concentration and the corresponding diagnostic outcomes. The results are shown in Figure 3 As shown in C: In the diagnosis of ectopic pregnancy with a single indicator, the AUC of SP70 is 0.840, which is greater than hCG. When the two indicators are combined, the AUC can reach 0.935, which is significantly better than a single indicator ( Figure 3 C).

[0053] Example 4 The value of maternal serum SP70 concentration in monitoring fetal intrauterine growth

[0054] The SP70 detection kit (ELISA method) was used to detect the serum SP70 concentration of 428 pregnant women. According to the uterine ultrasound report of the pregnant women, the intrauterine growth and development of the fetus were divided into normal fetal growth, small fetal growth and large fetal growth. The obstetric ultrasound judgment standard for fetal growth: the fetus's biparietal diameter or head circumference is 2 standard deviations or more lower than the average level of fetuses of the same gestational age, which means the fetus is small; the fetus's biparietal diameter or head circumference is 2 standard deviations or more higher than the average level of fetuses of the same gestational age, which means the fetus is large; and the fetus is in between the two. There were 308 normal fetuses, 78 small fetuses, and 42 large fetuses. The results are as follows Figure 4 As shown, the serum SP70 concentration of pregnant women with smaller fetuses was significantly lower than that of pregnant women with normal fetal growth, and the serum SP70 concentration of pregnant women with larger fetuses was significantly higher than that of pregnant women with normal fetal growth (P < 0.01).

Claims

1. Application of SP70 as a detection target in the preparation of auxiliary diagnostic reagents for abnormal pregnancy in pregnant women and / or monitoring reagents or auxiliary monitoring reagents for fetal intrauterine growth and development.

2. The use according to claim 1, characterized in that: The abnormal pregnancy is any one of threatened abortion or ectopic pregnancy.

3. Use of reagents for detecting SP70 in serum in the preparation of auxiliary diagnostic reagents for abnormal pregnancy in pregnant women and / or reagents for monitoring intrauterine growth and development of the fetus or auxiliary monitoring reagents.

4. The use according to claim 3, characterized in that: The abnormal pregnancy is any one of threatened abortion or ectopic pregnancy.

5. The use according to claim 4, characterized in that: The reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum.

6. The use according to claim 5, characterized in that: The reagent for detecting SP70 in serum contains the monoclonal antibody NJ001-1 of SP70, and the monoclonal antibody NJ001-1 is secreted by the hybridoma cell line NM001-1 with the deposit number of CCTCCNO: C201172.

7. Use of a reagent for detecting SP70 in serum in combination with a reagent for detecting hCG in the preparation of an auxiliary diagnostic reagent for abnormal pregnancy in pregnant women and / or a reagent for monitoring the intrauterine growth and development of the fetus or an auxiliary detection reagent.

8. The use according to claim 7, characterized in that: The reagent for detecting SP70 in serum is a reagent for quantitatively detecting SP70 in serum, and the reagent for detecting hCG is a reagent for quantitatively detecting hCG.

9. A kit for assisting in diagnosing abnormal pregnancy in pregnant women and / or a reagent or an auxiliary detection kit for monitoring the intrauterine growth and development of the fetus, characterized in that Contains reagents for quantitatively detecting SP70 in serum and reagents for quantitatively detecting hCG.

10. The kit according to claim 9, characterized in that The reagent for quantitatively detecting SP70 in serum comprises the monoclonal antibody NJ001-1 of SP70, and the monoclonal antibody NJ001-1 is secreted by the hybridoma cell line NM001-1 with the deposit number of CCTCC NO: C201172.

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