Multi-project composite prenatal screening quality control product and preparation method thereof

Abstract

The invention discloses a multi-project composite prenatal screening quality control product and a preparation method thereof, and belongs to the technical field of prenatal screening quality control products. The quality control product comprises the following components: a human serum matrix; the natural or human protein comprises 5 to 200 IU / mL of AFP, 5 to 200000 mIU / mL of HCG (human chorionic gonadotropin), 5 to 150 mIU / mL of Free beta HCG, 10 to 1200 pg / mL of statin A, 10 to 8000 mIU / L of PAPP-A, 1 to 24 ng / mL of uE3, 20 to 5000 pg / mL of PlGF and 10 to 20000 pg / mL of sFlt-1. An anti-Free [beta] HCG protein antibody; a protein protective agent; a preservative; and a freeze-drying protective agent. The quality control product covers many items, solves the technical problem of mutual interference of HCG and Free beta HCG, and can be used as a third-party quality control product.

Description

Technical Field

[0001] The invention belongs to the technical field of prenatal screening quality control products and relates to a multi-item composite prenatal screening quality control product and a preparation method thereof. Background Art

[0002] Prenatal screening is a means of preventing the birth of most congenital defects. It screens out high-risk groups by testing certain specific indicators in the blood of pregnant women. Prenatal screening includes three tests: 21-trisomy syndrome (Down syndrome), 18-trisomy syndrome and neural tube defect serum biochemical immunological screening.

[0003] 1. Prenatal screening for Down syndrome Down's syndrome (DS), also known as trisomy 21, is a condition in which the patient has one more chromosome in the 21st pair than normal people. It is the most common chromosomal aneuploidy disease. Down's syndrome screening is performed by testing the levels of PAPP-A (pregnancy-associated plasma protein A), AFP (alpha-fetoprotein), HCG (human chorionic gonadotropin), uE3 (estriol) and Inhibin A (inhibin A) in the serum of pregnant women at a specific gestational week, and combining the clinical information of the pregnant woman such as age, gestational week, weight, smoking, and insulin-dependent diabetes, and calculating the risk value through risk assessment software.

[0004] 2. Prenatal screening for trisomy 18 Trisomy syndrome is also known as Edwards syndrome. The incidence of live births is about 1 / 6000. It is caused by non-disjunction of chromosomes during cell division and is divided into three types: standard type, translocation type and mosaic type. Its prenatal serological screening and Down syndrome screening together show "three lows" in the three biochemical markers of maternal serum AFP, HCG and uE3 during the second trimester.

[0005] 3. Prenatal screening for neural tube defects Neural tube defect (NTD) refers to congenital structural abnormalities of the brain and spine, which are caused by the failure of the neural tube to close due to certain factors during embryonic development. The incidence rate is about (1.4-2) / 1000, including anencephaly, encephalocele, spina bifida and other malformations. Maternal serum AFP testing is an effective means of screening NTD. When the positive cut-off value is set at 2.5MOM, approximately 85% of singletons and 80% of twins can be screened. It should be emphasized here that serum AFP can only screen for open NTDs, and occult spina bifida cannot be screened because their serum AFP is not increased.

[0006] 4. Prediction of preeclampsia The background of pregnancy-induced hypertension in pregnant women is complex, especially the cause of preeclampsia is still unclear, and an effective and highly specific method for predicting preeclampsia has not yet been established. A large number of studies have verified that angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and soluble endoglin (sEng), can play a certain role in predicting early preeclampsia in mid-pregnancy. The sFlt-1 / PlGF ratio has clinical value for short-term prediction of preeclampsia. When the sFlt-1 / PlGF ratio is ≤38, the negative predictive value (excluding preeclampsia within 1 week) is 99.3%; when the sFlt-1 / PlGF ratio is >38, the positive predictive value (predicting preeclampsia within 4 weeks) is 36.7%.

[0007] Alpha-fetoprotein (AFP) is the most common protein in fetal serum. It can transport substances such as bilirubin and hormones, avoid rejection reactions between the liver and the mother, and play an important role in fetal growth and development. Under normal pregnancy conditions, there is a large concentration difference between fetal and maternal serum AFP levels. The reason is that the three-layer barrier effect of the fetus, placenta and amnion keeps the maternal serum AFP level at a low level. Monitoring maternal AFP levels can help understand changes in placental blood flow and the permeability of fetal membranes during pregnancy. The pathophysiological changes of PE are characterized by damage to vascular endothelial cells and spasm of small arteries throughout the body. The direct harm to pregnancy is mainly to reduce placental blood flow, which reduces the amount of AFP secreted by the fetus and transported to the maternal blood through the placenta.

[0008] Human chorionic gonadotropin (HCG), free β subunit of human chorionic gonadotropin (Free β HCG): HCG is a glycoprotein hormone secreted by placental syncytiotrophoblasts. It is composed of two subunits, α and β. The structure of the β subunit is quite different from that of other hormones. It is not easy to cross-react when used for detection, and can accurately indicate the actual secretion of HCG. HCG and Free β HCG increase rapidly in early pregnancy, reaching a peak at 8 to 10 weeks, and then decline after about two weeks. Both HCG and Free β HCG in the maternal blood of Down syndrome fetuses show a continuous increase, so they can be used as screening indicators.

[0009] Free estriol (uE3) is a metabolite of estradiol. uE3 is mainly synthesized by the fetal-placental unit after 10 weeks of pregnancy and enters the maternal circulation. In pregnancies with Down syndrome, the level of uE3 in maternal serum is lower than that in normal pregnancies. It is used as an indicator for Down syndrome screening during the second trimester.

[0010] Pregnancy-associated plasma protein A (PAPP-A) is also secreted by placental syncytiotrophoblast cells. In unaffected pregnancies, the level of PAPP-A in maternal serum increases rapidly in the first trimester and more slowly in the second trimester. In pregnancies affected by Down syndrome, serum PAPP-A generally decreases; in terms of the rate of decrease, the first trimester is much faster than the second trimester. Therefore, PAPP-A is used as an indicator for early pregnancy screening for Down syndrome.

[0011] Inhibin A is a glycoprotein composed of two subunits, α and β. The level of inhibin in maternal serum increases in early pregnancy, gradually decreases after the 10th week, and stabilizes at 15 to 25 weeks. The level of inhibin A in the serum of pregnant women with Down syndrome fetuses is twice that of ordinary pregnant women.

[0012] Soluble fms-like tyrosine kinase-1 (sFlt-1) is a soluble receptor for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), which is released into the maternal circulation and participates in endothelial dysfunction. Soluble fms-like tyrosine kinase-1 is an endogenous anti-angiogenic protein formed by the placenta, which works by binding and neutralizing the pro-angiogenic proteins VEGF and PlGF. The results of some scholars have confirmed that when VEGF and PLGF bind to sFlt, PLGF and VEGF signals can be transmitted to cells, thereby effectively blocking angiogenesis.

[0013] CN110106247A discloses a quality control product and a preparation method thereof, a kit and a method for detecting fetal trisomy 21 and 18 syndrome. The quality control product is a quality control product of a kit for detecting fetal trisomy 21 and 18 syndrome, and the quality control product contains a positive reference product, a negative reference product and a detection limit reference product.

[0014] CN116358954B discloses a composite quality control product and its preparation method and a prenatal screening kit, wherein the matrix liquid is a buffer matrix, and contains at most six prenatal screening quality control products, namely, AFP, HCG, Free β HCG, uE3, inhibin A, and PAPP-A. Including: alpha-fetoprotein 8ng / mL~12ng / mL, free β subunit of human chorionic gonadotropin 5mIU / mL~20mIU / mL, human chorionic gonadotropin 800mIU / mL~1200mIU / mL, inhibin A 64pg / mL~96pg / mL, pregnancy-related protein A 1200mIU / L~1800mIU / L and unconjugated estriol 0.8ng / mL~1.2ng / mL; or, the composite quality control product includes: alpha-fetoprotein 56ng / mL~96ng / mL, human chorionic gonadotropin free β subunit 40mIU / mL~100mIU / mL, human chorionic gonadotropin 4000mIU / mL~6000mIU / mL, the inhibin A2 40pg / mL~360pg / mL, pregnancy-related protein A4 800mIU / L~7200mIU / L and unconjugated estriol 3.2ng / mL~4.8ng / mL.

[0015] Alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), free β subunit of human chorionic gonadotropin (Free β HCG), inhibin A, pregnancy-associated plasma protein A (PAPP-A), estriol (uE3), placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1), these serological biomarkers for prenatal screening each have their own clinical significance, and accurate detection of the concentration of these markers is very important for prenatal screening. However, a technical difficulty faced in the preparation of prenatal screening composite quality control products is that the two raw materials HCG and Free β HCG interfere with each other. High concentrations of HCG cause the detection value of Free β HCG to increase abnormally and exceed the upper limit of the target value. Therefore, the concentration of HCG in CN116358954B is only 4000mIU / mL to 6000mIU / mL, which cannot meet actual needs. The normal HCG value in non-pregnant people is <5mIU / mL, while the value during pregnancy can reach more than 100,000mIU / mL.

[0016] There is an urgent need to develop new composite quality control products for prenatal screening, including markers such as AFP, HCG, Free β HCG, inhibin A, PAPP-A, uE), PlGF and sFlt-1, to achieve prenatal screening and eclampsia prediction, solve the technical problem of mutual interference between the two raw materials HCG and Free β HCG, so that HCG can reach the prenatal screening concentration and the concentration of Free β HCG can meet the measurement needs during pregnancy. Summary of the invention

[0017] The purpose of the present invention is to provide a multi-item composite prenatal screening quality control product and a preparation method thereof, covering the relevant serological test items involved in the entire pregnancy, solving the technical problem of mutual interference between HCG and Free β HCG, so that HCG reaches the prenatal screening concentration, and the concentration of Free β HCG can meet the measurement requirements during pregnancy, and can be used as a third-party quality control product for product quality inspection of different manufacturers. The purpose of the present invention is achieved by the following specific technical solutions.

[0018] A multi-item composite prenatal screening quality control product, characterized in that it includes the following components: human serum matrix; natural or human proteins, including alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), free β subunit of human chorionic gonadotropin (Free β HCG), inhibin A (Inhibin A), pregnancy-associated plasma protein A (PAPP-A), estriol (uE3), placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1); anti-Free β HCG protein antibody; protein protective agent; preservative; freeze-drying protective agent; wherein the concentration range of each protein is: AFP: 5-200 IU / mL; HCG: 5-200000mIU / mL; Free β HCG: 5-150 mIU / mL; uE3: 1-24 ng / mL; PAPP-A: 10-8000 mIU / L; Inhibin A: 10-1200 pg / mL; PlGF: 20-5000 pg / mL; sFlt-1: 10-20000 pg / mL.

[0019] The multi-item composite prenatal screening quality control product provided by the present invention is aimed at the serological detection involved in the prenatal screening stage to prevent most fetal congenital defects. The items included are relatively comprehensive and can cover the relevant serological test items involved in the entire pregnancy. The quality control product of the present invention solves the technical problem of mutual interference between HCG and Free β HCG by adding anti-Free β HCG protein antibodies, so that HCG reaches the prenatal screening concentration, and the concentration of Free β HCG can meet the measurement requirements during pregnancy without affecting other markers. The quality control product of the present invention uses raw serum or commercial human serum as the matrix liquid to minimize the matrix effect, which is close to clinical samples; it is suitable for most models on the market and can be used as a third-party quality control product for product quality inspection of different manufacturers.

[0020] Furthermore, the human serum matrix is ​​selected from one of normal human serum and hormone-free human serum, and the serological test results of HBsAg, HCV-Ab, HIV-Ab, and TP are negative. The matrix effect can be minimized, consistent with the clinical sample matrix, avoiding the detection deviation caused by the buffer matrix; at the same time, it ensures that the quality control product has no biological contamination risk.

[0021] Furthermore, the protein protective agent is selected from one or both of DBP and HPD, and the concentration is 0.5-3 g / L. The stability can be enhanced, the stability deviation is <5% in the 24-month validity period, and the validity period of the quality control product is extended; the protein denaturation during the freeze-drying process is reduced, and the detection value after reconstitution is ensured to be stable.

[0022] Furthermore, the lyoprotectant includes: lyoprotectant A: one or more selected from sucrose, trehalose, lactose, and dextran, with a concentration of 5-100 g / L; lyoprotectant B: one or more selected from glycine, histidine, and arginine, with a concentration of 5-50 g / L; lyoprotectant C: one or two selected from mannitol and sorbitol, with a concentration of 2-50 g / L. The combination of these lyoprotectants can reduce ice crystal damage, increase the reconstitution speed of lyophilized powder, and improve long-term storage stability. After the lyophilized powder is stored at 2-8°C for 24 months, the relative deviation of each indicator is <5%.

[0023] Furthermore, the preservative is selected from one or more of sodium azide, Proclin 300, Krovin 500, and gentamicin sulfate, with a concentration of 0.1%-0.5%. These preservatives can inhibit microorganisms and prevent the quality control product from deteriorating, and low concentrations do not affect the reaction of the detection reagent.

[0024] Furthermore, the quality control product is in the form of a lyophilized powder, with a stability of ≥14 days after reconstitution and a shelf life of ≥24 months at 2-8°C. It is convenient for transportation and long-term storage, flexible in use, and supports multiple testing needs.

[0025] The preparation method of the above-mentioned quality control product comprises the following steps: (1) Filtering human serum matrix; (2) adding a preservative, a lyophilization protective agent, and a protein protective agent to the human serum matrix filtered in step (1) and mixing well; (3) Add PlGF and sFlt-1 protein in sequence and adjust to the target concentration; (4) Add AFP, PAPP-A, and inhibin A protein and adjust to the target concentration; (5) Add HCG, Free β HCG protein and anti-Free β HCG protein antibody and adjust to the target concentration; (6) Equilibrate at 25-37°C for 24-48 hours; (7) Store at 2-8°C after aliquoting and freeze-drying.

[0026] Furthermore, in step (5), the amount of anti-Free β HCG protein antibody added is controlled so that the detection value of Free β HCG is stabilized in the range of 5-150 mIU / mL.

[0027] A kit, a quality control product provided according to the present invention.

[0028] The application of the quality control product provided by the present invention in prenatal screening includes at least one of the following: risk assessment of Down syndrome, trisomy 18, neural tube defects or preeclampsia; laboratory internal quality control or inter-laboratory quality evaluation of prenatal screening serological detection systems. It can support the entire process of prenatal screening (early, middle and late stages) and risk assessment of multiple diseases, and improve the consistency and standardization of the detection system.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects. Multi-project coverage: Eight serological indicators fully cover the needs during pregnancy, filling the market gap. Matrix optimization: The human serum matrix reduces detection deviation, and the freeze-drying process improves stability. The technical problem of mutual interference between the two raw materials HCG and Free β HCG is solved, so that HCG reaches the prenatal screening concentration, and the concentration of Free β HCG can meet the measurement requirements during pregnancy, and can be used as a third-party quality control product for product quality inspection of different manufacturers. The preparation method is simple and controllable, suitable for large-scale production. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is described clearly and completely below. Obviously, the described implementation is only a part of the implementation of the present invention, not all of the implementation. Based on the implementation of the present invention, all other implementations obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention. Example 1

[0031] The steps for preparing multi-item composite prenatal screening quality control products are as follows.

[0032] (1) Take 2000 ml of human serum and filter it for later use.

[0033] (2) Add preservative Krovin 500 0.1%, sucrose 20 g / L, glycine 10 g / L, mannitol 10 g / L, and protein protectant DBP 1.0 g / L to (1) and mix thoroughly.

[0034] (3) Add proteins PLGF, sFlt-1, AFP, PAPP-A, inhibin A, HCG, and Free β HCG to keep their concentrations within the target range.

[0035]

[0036] The concentration before adding anti-Free β HCG protein antibody can only be adjusted to the concentration in the table below.

[0037]

[0038] Different concentrations of anti-Free β HCG protein antibodies were added to Level 1 and Level 2, and the values ​​of 8 items were measured as shown in the following table.

[0039]

[0040] From the above, it can be seen that after adding anti-Free β HCG protein antibodies, the measured value of Free β HCG can be within the set target concentration range without affecting other items.

[0041] After the test is completed, it is packaged into 7 mL brown vials, dried in low-temperature vacuum until it is freeze-dried, and then stored at 2-8°C. Example 2

[0042] Uniformity test of the quality control product prepared in Example 1.

[0043] When selecting samples for uniformity testing, the production volume of the quality control materials is not considered. Ten minimum packaging units of quality control materials are randomly selected and randomly numbered 1-10. Each packaging unit is measured three times.

[0044] Measurement order: Considering the random variation of the measurement system caused by factors such as time, the three measurements are performed in different orders, such as 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9.

[0045] The data processing and judgment standards of the uniformity test results refer to the pharmaceutical industry standard of the People's Republic of China YY / T1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents".

[0046] The experimental results of quality control product level 1 and level 2 are shown in the table below.

[0047]

[0048] The results showed that the CV of the quality control product test results between bottles was less than 15%, and the uniformity between bottles met the requirements. Example 3

[0049] The quality control product prepared in Example 1 was tested for stability during its shelf life.

[0050] The quality control products were stored at 2℃~8℃ in the dark. Two bottles were taken out after 0, 12 and 24 months respectively. Each bottle was tested 3 times. The average value of the test results at each time point was calculated, and the relative deviation between the test results at the end of the stable period and 0 month was calculated. The results are shown in the table below.

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] It can be seen from the above test results that the quality control product prepared in Example 1 has good shelf life stability.

[0068] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. The protection scope of the present invention is defined by the claims and their equivalent technical solutions.

Claims

1. A multi-item composite prenatal screening quality control product, characterized in that: Includes the following components: Human serum matrix; Natural or human proteins, including AFP, HCG, Free β HCG, Inhibin A, PAPP-A, uE3, PlGF, and sFlt-1; Anti-Free β HCG protein antibody; Protein protectant; preservative; Lyoprotectants; The concentration range of each protein is: AFP: 5-200 IU / mL; HCG: 5-200,000 mIU / mL; Free β HCG: 5-150 mIU / mL; uE3: 1-24 ng / mL; PAPP-A: 10-8000 mIU / L; Inhibin A: 10-1200 pg / mL; PlGF: 20-5000 pg / mL; sFlt-1: 10-20000 pg / mL.

2. The quality control product according to claim 1, characterized in that: The human serum matrix is ​​selected from normal human serum and hormone-free human serum, and the serological test results of HBsAg, HCV-Ab, HIV-Ab and TP are negative.

3. The quality control product according to claim 1, characterized in that: The protein protective agent is selected from one or both of DBP and HPD, and the concentration is 0.5-3 g / L.

4. The quality control product according to claim 1, characterized in that: The lyophilization protective agent includes: Lyoprotectant A: one or more selected from sucrose, trehalose, lactose, dextran, with a concentration of 5-100 g / L; Lyoprotectant B: one or more selected from glycine, histidine, and arginine, with a concentration of 5-50 g / L; Lyoprotectant C: selected from one or both of mannitol and sorbitol, with a concentration of 2-50 g / L.

5. The quality control product according to claim 1, characterized in that: The preservative is selected from one or more of sodium azide, Proclin 300, Krovin 500, and gentamicin sulfate, and the concentration is 0.1%-0.5%.

6. The quality control product according to claim 1, characterized in that: The quality control product is in the form of a lyophilized powder with a stability of ≥14 days after reconstitution and a shelf life of ≥24 months when stored at 2-8°C.

7. The method for preparing the quality control product according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Filtering human serum matrix; (2) adding a preservative, a lyophilization protective agent, and a protein protective agent to the human serum matrix filtered in step (1) and mixing well; (3) Add PlGF and sFlt-1 protein in sequence and adjust to the target concentration; (4) Add AFP, PAPP-A, and inhibin A protein and adjust to the target concentration; (5) Add HCG, Free β HCG protein and anti-Free β HCG protein antibody and adjust to the target concentration; (6) Equilibrate at 25-37°C for 24-48 hours; (7) Store at 2-8°C after aliquoting and freeze-drying.

8. The preparation method according to claim 7, characterized in that: In the step (5), the amount of anti-Free β HCG protein antibody added is controlled so that the detection value of Free β HCG is stabilized in the range of 5-150 mIU / mL.

9. A kit, characterized in that: Comprising a quality control product according to any one of claims 1 to 6.

10. Use of the quality control product according to any one of claims 1 to 6 in prenatal screening, characterized in that: The application includes at least one of the following: Risk assessment for Down syndrome, trisomy 18, neural tube defects, or preeclampsia; Intra-laboratory quality control or inter-laboratory quality assessment of serological testing systems for prenatal screening.

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