A multi-item composite prenatal screening quality control product and its preparation method

Abstract

A multi-item composite prenatal screening quality control product and its preparation method belong to the technical field of prenatal screening quality control products. The quality control product includes the following components: human serum matrix; natural or human proteins, including AFP 5-200 IU / mL, HCG 5-200000 mIU / mL, Free β HCG 5-150 mIU / mL, inhibin A 10-1200 pg / mL, PAPP-A 10-8000 mIU / L, uE3 1-24 ng / mL, PlGF20-5000 pg / mL and sFlt-1 10-20000 pg / mL; anti-Free β HCG protein antibody; protein protective agent; preservative; lyophilization protective agent. The quality control product of the present invention covers many items, solves the technical problem of mutual interference between HCG and Free β HCG, and can be used as a third-party quality control product.

Description

Technical Field

[0001] The present 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 for high-risk individuals by testing certain specific indicators in the pregnant woman's blood. Prenatal screening includes three tests: serum biochemical and immunological screening for trisomy 21 (Down syndrome), trisomy 18, and neural tube defects.

[0003] 1. Prenatal screening for Down syndrome

[0004] Down syndrome (DS), also known as trisomy 21, is a condition characterized by an extra chromosome in the 21st pair of chromosomes, making it the most common chromosomal aneuploidy. Down syndrome screening involves measuring the levels of PAPP-A (pregnancy-associated plasma protein A), AFP (alpha-fetoprotein), human chorionic gonadotropin (HCG), estriol (uE3), and inhibin A in maternal serum at a specific gestational age. Risk assessment software then calculates the risk of pregnancy based on the mother's age, gestational age, weight, smoking status, and insulin-dependent diabetes mellitus.

[0005] 2. Prenatal screening for trisomy 18

[0006] Trisomy syndrome, also known as Edwards syndrome, occurs in approximately 1 in 6,000 live births. It is caused by nondisjunction of chromosomes during cell division and is categorized into standard, translocation, and mosaic forms. Prenatal serological screening, along with Down syndrome screening, reveals a "triple low" in maternal serum biochemical markers: AFP, HCG, and uE3.

[0007] 3. Prenatal screening for neural tube defects

[0008] Neural tube defects (NTDs) are congenital structural abnormalities of the brain and spine caused by failure of the neural tube to close during embryonic development. They occur in approximately 1.4 to 2 cases per 1,000 births and include anencephaly, encephalocele, and spina bifida. Maternal serum AFP testing is an effective method for screening NTDs. With a positive cutoff value of 2.5 MOM, it can screen approximately 85% of singleton pregnancies and 80% of twin pregnancies. It is important to emphasize that serum AFP can only screen for open NTDs; conditions such as occult spina bifida cannot be screened because these conditions do not show elevated serum AFP levels.

[0009] (IV) Prediction of preeclampsia

[0010] Hypertensive disorders complicating pregnancy present in a complex context, particularly in the context of preeclampsia, whose etiology remains unclear. To date, effective and specific methods for predicting preeclampsia remain elusive. Numerous studies have validated the role of angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and soluble endoglin (sEng), in predicting early-onset preeclampsia during the second trimester. The sFlt-1 / PlGF ratio has clinical value for short-term prediction of preeclampsia. A sFlt-1 / PlGF ratio ≤ 38 has a negative predictive value (excluding preeclampsia within 1 week) of 99.3%, while a ratio > 38 has a positive predictive value (predicting preeclampsia within 4 weeks) of 36.7%.

[0011] Alpha-fetoprotein (AFP) is the most common protein in fetal serum. It transports substances such as bilirubin and hormones, preventing rejection between the liver and the mother, and plays a vital role in fetal growth and development. During normal pregnancy, there is a significant difference in fetal and maternal serum AFP levels. This is due to the three-layer barrier function of the fetus, placenta, and amniotic membrane, which keeps maternal serum AFP levels low. Monitoring maternal AFP levels helps understand changes in placental blood flow and fetal membrane permeability 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 due to reduced placental blood flow, which reduces the amount of AFP secreted by the fetus that is transported across the placenta into the maternal blood.

[0012] Human chorionic gonadotropin (HCG) and free β-subunit of human chorionic gonadotropin (Free β-HCG): HCG is a glycoprotein hormone secreted by the syncytiotrophoblast cells of the placenta. It is composed of two subunits, α and β. The β-subunit differs significantly from other hormones in structure, making it less likely to cross-react and accurately indicating the actual level of HCG secretion. HCG and free β-HCG levels increase rapidly during early pregnancy, reaching a peak at 8-10 weeks, then decline after approximately two weeks. Both HCG and free β-HCG levels in maternal blood of fetuses with Down syndrome are consistently elevated, making them suitable screening indicators.

[0013] Free estriol (uE3) is a metabolite of estradiol. After 10 weeks of gestation, uE3 is primarily synthesized by the fetoplacental unit and enters the maternal circulation. In pregnancies with Down syndrome, maternal serum uE3 levels are lower than in normal pregnancies. It is used as a marker for Down syndrome screening during the second trimester.

[0014] Pregnancy-associated plasma protein A (PAPP-A) is also secreted by placental syncytiotrophoblast cells. In unaffected pregnancies, maternal serum PAPP-A levels increase rapidly in the first trimester and more slowly in the second trimester. In pregnancies affected by Down syndrome, serum PAPP-A levels typically decrease, with the rate of decrease being much greater in the first trimester than in the second. Therefore, PAPP-A is used as a marker for early pregnancy screening for Down syndrome.

[0015] Inhibin A is a glycoprotein composed of two subunits, α and β. Maternal serum inhibin levels rise in early pregnancy, gradually decline after the 10th week, and stabilize between 15 and 25 weeks. Inhibin A levels in maternal serum of fetuses with Down syndrome are twice as high as those in normal pregnant women.

[0016] Soluble fms-like tyrosine kinase-1 (sFlt-1) is a soluble receptor for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). It is released into the maternal circulation and is implicated in endothelial dysfunction. Soluble fms-like tyrosine kinase-1 is an endogenous anti-angiogenic protein produced by the placenta that acts by binding and neutralizing the pro-angiogenic proteins VEGF and PlGF. Research has demonstrated that when VEGF and PlGF bind to sFlt, PLGF and VEGF signals are transmitted into cells, effectively blocking angiogenesis.

[0017] CN110106247A discloses a quality control product and its preparation method, 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.

[0018] CN116358954B discloses a composite quality control product and its preparation method and prenatal screening kit, wherein the matrix liquid is a buffer matrix and contains at most six prenatal screening quality control products: AFP, HCG, Free β HCG, uE3, inhibin A, and PAPP-A. The products include: alpha-fetoprotein 8ng / mL to 12ng / mL, free β subunit of human chorionic gonadotropin 5mIU / mL to 20mIU / mL, human chorionic gonadotropin 800mIU / mL to 1200mIU / mL, inhibin A 64pg / mL to 96pg / mL, pregnancy-associated 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 A 4800mIU / L~7200mIU / L and unconjugated estriol 3.2ng / mL~4.8ng / mL.

[0019] 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 serum biomarkers for prenatal screening each have their own clinical significance, and accurate measurement of their concentrations is crucial for prenatal screening. However, a technical challenge in preparing composite quality control products for prenatal screening is the mutual interference between HCG and Free β-HCG. High HCG concentrations can lead to abnormally elevated Free β-HCG test values, exceeding the upper limit of the target value. Consequently, the HCG concentration in CN116358954B is only 4000mIU / mL to 6000mIU / mL, which does not meet practical needs. The normal HCG value in non-pregnant people is <5mIU / mL, while the value during pregnancy can reach more than 100,000mIU / mL.

[0020] 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

[0021] The present invention aims to provide a multi-item composite prenatal screening quality control product and its preparation method, covering relevant serological test items involved in the entire pregnancy period. This product addresses the technical problem of mutual interference between HCG and Free β-HCG raw materials, ensuring that HCG concentrations reach prenatal screening levels while maintaining Free β-HCG concentrations that meet pregnancy measurement requirements. This product can be used as a third-party quality control product for product quality inspections by different manufacturers. This objective of the present invention is achieved through the following specific technical solutions.

[0022] A multi-item composite prenatal screening quality control product, characterized by comprising 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; lyophilization 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.

[0023] The multi-item composite prenatal screening quality control product provided by the present invention targets serological tests 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, which minimizes the matrix effect and 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.

[0024] Furthermore, the human serum matrix is ​​selected from normal human serum and hormone-depleted human serum, and the results of serological tests for HBsAg, HCV-Ab, HIV-Ab, and TP are negative. This minimizes matrix effects, is consistent with clinical sample matrices, avoids detection bias caused by buffer matrices, and ensures that quality control products are free of biological contamination risks.

[0025] Furthermore, the protein protectant is selected from one or both of DBP and HPD, with a concentration of 0.5-3 g / L. This can enhance stability, with a stability deviation of <5% over a 24-month shelf life, extending the shelf life of the quality control product; reduce protein denaturation during the freeze-drying process, and ensure stable test values ​​after reconstitution.

[0026] Furthermore, the lyoprotectants include: 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; and lyoprotectant C: one or both selected from mannitol and sorbitol, with a concentration of 2-50 g / L. This combination of lyoprotectants can reduce ice crystal damage, increase the reconstitution rate of the lyophilized powder, and enhance long-term storage stability, with the relative deviation of each parameter of the lyophilized powder being <5% after 24 months of storage at 2-8°C.

[0027] 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% to 0.5%. These preservatives can inhibit microorganisms and prevent the quality control product from deteriorating, while the low concentration does not affect the reaction of the detection reagent.

[0028] 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 when stored at 2-8°C. This facilitates transportation and long-term storage, is flexible to use, and supports multiple testing needs.

[0029] The preparation method of the above-mentioned quality control product comprises the following steps:

[0030] (1) Filtering human serum matrix;

[0031] (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;

[0032] (3) Add PlGF and sFlt-1 protein in sequence and adjust to the target concentration;

[0033] (4) Add AFP, PAPP-A, and inhibin A protein and adjust to the target concentration;

[0034] (5) Add HCG, Free β HCG protein, and anti-Free β HCG protein antibody and adjust to the target concentration;

[0035] (6) Equilibrate at 25-37°C for 24-48 hours;

[0036] (7) Store at 2-8°C after aliquoting and freeze-drying.

[0037] 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.

[0038] A kit, comprising a quality control product according to the present invention.

[0039] The quality control products provided in this invention are used in prenatal screening, including at least one of the following: risk assessment for Down syndrome, trisomy 18, neural tube defects, or preeclampsia; and intra-laboratory quality control or inter-laboratory quality assessment of prenatal screening serological testing systems. These products can support the entire prenatal screening process (early, mid-, and late) and risk assessment for multiple diseases, improving the consistency and standardization of testing systems.

[0040] Compared with the existing technology, the present invention has the following beneficial technical effects. Multi-item coverage: Eight serological indicators comprehensively cover pregnancy needs, filling a market gap. Matrix optimization: The human serum matrix reduces detection bias, and the freeze-drying process improves stability. It solves the technical problem of mutual interference between HCG and Free β HCG, allowing HCG to reach a prenatal screening concentration. Furthermore, 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 by different manufacturers. The preparation method is simple and controllable, making it suitable for large-scale production. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention. Example 1

[0042] The steps for preparing multi-item composite prenatal screening quality control products are as follows.

[0043] (1) Take 2000 ml of human serum and filter it for later use.

[0044] (2) Add the 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.

[0045] (3) Add proteins PLGF, sFlt-1, AFP, PAPP-A, inhibin A, HCG, and Free β HCG to keep their concentrations within the target range.

[0046]

[0047] Before adding anti-Free β HCG protein antibody, the concentration can only be adjusted to the concentrations in the table below.

[0048]

[0049] Different concentrations of anti-Free β HCG protein antibodies were added to Levels 1 and 2, and the values ​​of the eight items were measured as shown in the following table.

[0050]

[0051] From the above, it can be seen that the addition of anti-Free β HCG protein antibodies can keep the measured value of Free β HCG within the set target concentration range without affecting other parameters.

[0052] After the test is completed, the sample is divided into 7 mL brown vials, vacuum-dried at low temperature to a freeze-dried state, and then stored at 2-8°C. Example 2

[0053] Uniformity testing of the quality control product prepared in Example 1.

[0054] 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.

[0055] Measurement sequence: Considering the random variation of the measurement system caused by factors such as time, the three measurements are performed in different sequences, 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.

[0056] The data processing and judgment standards of the uniformity test results refer to the pharmaceutical industry standard YY / T1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents" of the People's Republic of China.

[0057] The experimental results of quality control product levels 1 and 2 are shown in the table below.

[0058]

[0059] 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

[0060] Stability test of the quality control product prepared in Example 1.

[0061] The quality control product was stored at 2°C to 8°C in the dark. Two bottles were removed at 0, 12, and 24 months, and each bottle was tested three times. The average of the test results at each time point was calculated, and the relative deviation between the test result at the end of the stable period and the test result at 0 months was calculated. The results are shown in the table below.

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] It can be seen from the above test results that the quality control product prepared in Example 1 has good shelf life stability.

[0079] Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention. The scope of protection of the present invention is defined by the claims and their equivalents.

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 antibodies; 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 HBsAg, HCV-Ab, HIV-Ab, and TP serological test results 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, with a concentration of 0.5-3 g / L.

4. The quality control product according to claim 1, characterized in that: The lyoprotectant comprises: 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 both selected from 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, with a concentration of 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 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 the quality control product according to any one of claims 1 to 6.

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