Serum matrix alpha fetoprotein heteroplasmal quality control product and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to an alpha fetoprotein heteroplasmal quality control product of a serum matrix and a preparation method of the alpha fetoprotein heteroplasmal quality control product. According to the quality control product disclosed by the invention, sodium periodate is introduced for treating a human serum matrix for the first time, so that the efficiency of identifying AFP-L3 by LCA is greatly improved, the feeding amount of antigens is reduced, the cost is saved, and stable mass production of the AFP-L3 quality control product becomes possible.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a serum-based alpha-fetoprotein heterogeneous quality control product and a preparation method thereof. Background Art

[0002] Primary hepatic carcinoma (PHC) is a common malignant tumor, of which 75%-85% is hepatocellular carcinoma (HCC). AFP is currently a commonly used tumor marker for the diagnosis of PHC, and AFP-L3% (AFP-L3 / AFP) is an important supplement to AFP. It can be used to identify the nature of elevated AFP (benign / malignant). Combined with different cutoff values, it facilitates early screening for HCC, supplements screening for small HCCs, and can also assist in the diagnosis, disease progression monitoring, and prognosis of HCC.

[0003] Due to methodological limitations, AFP-L3% detection is a challenging aspect of the three liver cancer tests. Testing is primarily performed using affinity adsorption centrifugation, magnetic microparticle chemiluminescence immunoassay, and microfluidic immunofluorescence. Different brands of testing systems and associated reagents utilize antibodies targeting different tumor marker antigens. These antibodies vary in their source, binding mode, calibrator traceability, and instrument detection principles, leading to varying test results and reference ranges across different testing systems.

[0004] In vitro diagnostic reagents require extensive testing with quality control materials throughout their development, production, supervision, and use to monitor, evaluate, confirm, and validate methods, products, operations, environments, and facilities. Therefore, the importance of quality control materials can be imagined. However, there are currently no AFP-L3 quality control materials on the market, let alone serum-based AFP-L3 quality control materials. This makes it difficult to measure and regulate the differences in AFP-L3 caused by different detection methods.

[0005] Quality control products are composed of main biological raw materials and biological excipients. Biological raw materials include various biologically active antigens, antibodies, sera, etc. Biological excipients refer to a type of biological raw materials used as protein protective agents in the production process, mainly including serum, serum albumin, etc.

[0006] Affinity adsorption centrifugation and magnetic microparticle chemiluminescence immunoassays are also used to detect AFP variants. After the sample is passed through a lentil lectin (LCA)-coupled affinity medium (magnetic beads), AFP-L3 in the sample is specifically adsorbed. After elution with an eluent, AFP detection is performed. Neither method has established AFP-L3 calibrators or quality controls. AFP detection using affinity adsorption centrifugation allows the use of reagents from different manufacturers. Different manufacturers use their own AFP quality controls to regulate the detection system. Differences in antibody sites, detection methods, and measurement values between manufacturers can lead to variations in the final test results. The magnetic microparticle chemiluminescence method also cannot avoid matrix effects caused by samples processed with different batches of AFP-L3 separation reagents, resulting in unstable test results.

[0007] Microfluidic immunofluorescence is the earliest method to achieve automated detection of AFP-L3. Its detection principle is to identify AFP in the sample with a double antibody sandwich, separate the AFP into heterogeneous AFP-L1 / L2 / L3 through LCA electrophoresis, and quantify AFP-L3 by fluorescence grayscale. This method is based on an immunofluorescence platform that is inconsistent with the mainstream magnetic microparticle chemiluminescence platform on the market. Its accompanying calibrants are not compatible with the magnetic microparticle chemiluminescence platform detection system. In addition, this method is limited by detection throughput and is expensive, so it has not been widely accepted by the market.

[0008] The serum-based AFP-L3 calibrator prepared by the present invention can fill the market gap and is used for quality control of AFP-L3 detection kits (magnetic particle chemiluminescence method).

[0009] According to WS / T 641-2018, "Guidelines for Internal Quality Control of Quantitative Assays in Clinical Laboratories," quality control products should have a similar or identical matrix to the patient sample being tested. They should be homogeneous and stable, and, if conditions permit, can be stored for a year or more. Inter-bottle variability should be less than the variability of the analytical system. Therefore, developing a stable serum-based quality control product for AFP isoforms is an urgent need in clinical laboratories. Summary of the Invention

[0010] In view of this, the present invention provides a serum-based AFP heterogeneous quality control product and a preparation method thereof, providing clinical laboratories with a serum-based AFP-L3 quality control product that is easy to use and has good stability, so as to meet the clinical laboratory's needs for AFP-L3 quality control.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a method for preparing an alpha-fetoprotein heterogeneous substance quality control matrix liquid, which is characterized by comprising: mixing raw materials and sodium periodate, stirring at a low speed at 4°C in the dark for 30 to 60 minutes, adding glycine and stirring at a low speed at room temperature for 15 to 30 minutes, inactivating for 30 to 120 minutes to obtain a reaction product, returning the reaction product to room temperature, mixing the product with a preservative, and filtering the product to obtain the alpha-fetoprotein heterogeneous substance quality control matrix liquid;

[0013] The low speed is 5 to 35 rpm, which can be 5 rpm, 10 rpm, 15 rpm, 20 rpm, 25 rpm, 30 rpm or 35 rpm;

[0014] The raw material includes at least one of serum and plasma.

[0015] The present invention also provides an alpha-fetoprotein heteromorphic quality control matrix liquid prepared by the above preparation method.

[0016] The present invention also provides an alpha-fetoprotein heterogeneous quality control product, comprising the above-mentioned alpha-fetoprotein heterogeneous quality control product matrix liquid.

[0017] In some specific embodiments of the present invention, the above-mentioned alpha-fetoprotein heterogeneous quality control product also contains AFP-L3 antigen, which can be natural AFP-L3 antigen derived from placenta, natural AFP-L3 antigen derived from cells, natural AFP-L3 antigen derived from ascites, recombinant AFP-L3 antigen expressed by eukaryotes, or synthetic AFP-L3 antigen.

[0018] The present invention also provides a method for preparing an alpha-fetoprotein heterogeneous quality control product, comprising: mixing AFP-L3 antigen with the above-mentioned alpha-fetoprotein heterogeneous quality control product matrix liquid according to a required concentration, stirring evenly, and freeze-drying to obtain the alpha-fetoprotein heterogeneous quality control product.

[0019] In some specific embodiments of the present invention, the concentration of the sodium periodate in the above preparation method is any value of 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, or a range between any two values.

[0020] In some specific embodiments of the present invention, the concentration of glycine in the above preparation method is any value among 0.5 M, 1 M, 1.5 M, 2 M, or a range between any two values.

[0021] In some specific embodiments of the present invention, the volume ratio of the sodium periodate to the raw material in the above preparation method is 1:98.

[0022] In some specific embodiments of the present invention, the volume ratio of the glycine to the raw material in the above preparation method is 1:98.

[0023] In some specific embodiments of the present invention, the volume ratio of the preservative to the raw material in the above preparation method is any value among 0.1%, 0.15%, 0.2%, or a range between any two values.

[0024] In some specific embodiments of the present invention, the serum in the above preparation method is human serum, and the plasma is human plasma.

[0025] In some specific embodiments of the present invention, the preservative in the above preparation method includes Proclin300.

[0026] In some specific embodiments of the present invention, the inactivation temperature in the above preparation method is 56° C. for 30 to 120 min.

[0027] In some specific embodiments of the present invention, the pore size of the filtration in the above preparation method is 0.22 μm.

[0028] In some specific embodiments of the present invention, the AFP-L3 antigen in the above preparation method can be a natural AFP-L3 antigen derived from placenta, a natural AFP-L3 antigen derived from cells, a natural AFP-L3 antigen derived from ascites, a recombinant AFP-L3 antigen expressed by a eukaryote, or a synthetic AFP-L3 antigen.

[0029] The serum-based AFP-L3 quality control provided herein can be prepared in a lyophilized form or in a liquid form. However, liquid forms cannot be stored at 2-8°C and must be stored at -20°C or below, increasing transportation and storage costs. Furthermore, the shelf life may be shorter than that of the lyophilized form.

[0030] The natural AFP-L3 extracted from placental tissue used in the serum-based AFP-L3 quality control product of the present invention can be used for quality control of an AFP-L3 detection kit (magnetic particle chemiluminescence method).

[0031] The present invention also provides a reagent comprising the above-mentioned alpha-fetoprotein heterogeneous substance quality control product matrix solution or the above-mentioned alpha-fetoprotein heterogeneous substance quality control product, and acceptable excipients or auxiliary agents.

[0032] The present invention also provides a kit, characterized in that it includes the above-mentioned alpha-fetoprotein heterogeneous quality control product matrix liquid or the above-mentioned alpha-fetoprotein heterogeneous quality control product.

[0033] The present invention also provides a device, characterized in that it includes the above-mentioned alpha-fetoprotein heterogeneous substance quality control product matrix liquid or the above-mentioned alpha-fetoprotein heterogeneous substance quality control product, and acceptable components.

[0034] The serum-based AFP-L3 quality control provided by the present invention is characterized in that the serum matrix is treated with sodium periodate, which improves the separation efficiency of LCA magnetic beads, reduces the amount of antigen administered, and saves costs. It is also characterized in that after desugaring, the matrix is inactivated at 56°C and filtered to obtain a quality control matrix liquid. The matrix of this quality control is similar to the matrix of clinical samples, thus effectively avoiding the occurrence of matrix effects. The selected AFP-L3 antigen is a natural placental extract with a stable source and low preparation cost. Referring to YY / T 1652-2019 General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents, the AFP-L3 quality control provided by the present invention, which meets the requirements for uniformity and stability, can provide quality control for clinical AFP-L3 detection.

[0035] Existing sources of AFP-L3 antigens fall into two main categories: native and recombinant. The structure of native antigens is closer to the antigen's natural state in the human body. A comparison of AFP-L3 antigens extracted from three different sources shows that placental sources are more stable and have higher AFP-L3 content. Cell culture-derived AFP-L3 content is also relatively high, but there is significant batch-to-batch variability. AFP-L3 from ascites fluid is affected by individual differences among liver cancer patients and has significant batch-to-batch variability. Recombinant antigens, however, are limited by the expression system itself. Prokaryotic expression systems produce insufficient glycosylation, resulting in lower AFP-L3 content. Eukaryotic expression systems express proteins with some glycosylation, resulting in higher AFP-L3 content compared to prokaryotic expression. Both native AFP-L3 antigens extracted from the three different sources and eukaryotic-expressed AFP-L3 antigens can be used as AFP-L3 quality control materials. Placental AFP extraction is less expensive because it is a relatively readily available biological material that does not require customized modification, and the extraction process may not require overly complex techniques or equipment. Cell culture is relatively expensive, involving complex processes and high material costs, and requiring specialized laboratory and operating equipment. The cost of extracting AFP from ascites fluid falls somewhere in between, primarily depending on the cost of testing and processing.

[0036] Based on the comparison of AFP-L3 antigen content, batch-to-batch differences in antigens, and antigen acquisition costs, natural AFP-L3 antigen from placenta was selected to prepare serum-based AFP-L3 quality control products, which are stable and economical and can be used for quality control of AFP-L3 detection. DETAILED DESCRIPTION

[0037] The present invention discloses a serum-based alpha-fetoprotein heterogeneous quality control product and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The present invention provides a serum-based alpha-fetoprotein isoform quality control product and its preparation method, which are used for quality control of alpha-fetoprotein isoform detection kits. The serum matrix used in this quality control product needs to be added with 0.5-5 mM sodium periodate and stirred at a low speed (5-35 rpm) at 4°C in the dark for 30-60 minutes, preferably 1 mM sodium periodate; after adding 5-20 mM glycine or lysine, stir at a low speed (5-35 rpm) at room temperature for 15-30 minutes, preferably 10 mM glycine; and heated at 56°C for 30 minutes for inactivation, followed by terminal filtration. Specifically, the preparation steps include the following:

[0039] S1: Prepare 80 mL of 0.1 M sodium acetate buffer (pH = 4.5), add 2.14 g of sodium periodate, dissolve thoroughly, and dilute to 100 mL to prepare a 100 mM sodium periodate stock solution.

[0040] S2: Take 980 mL of normal human pooled serum, add 10 mL of sodium periodate stock solution to a final concentration of 1 mM, and stir at a low speed (5-35 rpm) for 30-60 min at 4°C in the dark to remove sugars.

[0041] S3: Add 7.5 g of glycine to 80 mL of purified water and dilute to 100 mL to prepare a 1 M glycine stock solution.

[0042] S4: Add 10 mL of glycine stock solution to S2 to a final glycine concentration of 10 mM. Stir at room temperature at low speed (5-35 rpm) for 15-30 min to terminate the reaction.

[0043] S5: Inactivate the serum prepared in S4 at 56°C for 30-120 min and cool to room temperature for later use;

[0044] S6: Add 0.2% (v / v) preservative Proclin 300 to the inactivated desugared human serum prepared in S5, stir well, and filter with a 0.22 μm sterile filter membrane to obtain the quality control matrix solution;

[0045] S7: Add appropriate amount of AFP-L3 antigen to the quality control matrix liquid prepared in S6, stir evenly, and prepare the liquid quality control product of required concentration;

[0046] S8: The liquid quality control product obtained in S7 is packaged according to the specified specifications and freeze-dried using a vacuum freeze dryer to obtain the serum-based AFP-L3 quality control product, which is stored at 2-8°C.

[0047] The normal human mixed serum can be replaced with hormone-free serum or plasma, and the subsequent steps remain unchanged.

[0048] Antigens added to the treated serum matrix can be natural AFP-L3 extracted from placental tissue, natural AFP-L3 extracted from ascites, natural AFP-L3 derived from cells, or recombinant AFP-L3 expressed in eukaryotes, with natural AFP-L3 extracted from placental tissue being preferred. This quality control product, for the first time, uses sodium periodate to treat the human serum matrix, significantly improving the efficiency of AFP-L3 recognition by LCA, reducing the amount of antigen administered, and saving costs. It also fills a gap in the market for AFP-L3 quality control products and enables stable mass production of AFP-L3 quality control products.

[0049] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0050] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0051] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0052] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0053] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0054] The room temperature mentioned in this application is not particularly limited and can be 18°C to 25°C, specifically 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C.

[0055] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0056] The present invention will be further described below with reference to the embodiments.

[0057] Example 1: Preparation method of sugar-removing quality control matrix solution of the present invention

[0058] Take 980 mL of commercial human serum matrix solution, add 10 mL of sodium periodate stock solution to a final concentration of 1 mM, stir at low speed (5-35 rpm) at 4°C in the dark for 30-60 min to remove sugar, add 10 mL of glycine stock solution to a final concentration of 10 mM, stir at low speed (5-35 rpm) at room temperature for 15-30 min to terminate the reaction, inactivate at 56°C for 30-120 min, and cool to room temperature; add 0.2% (v / v) preservative Proclin 300, stir evenly, and filter with a 0.22 μm sterile filter membrane to obtain the desugared quality control matrix solution.

[0059] Comparative Example 1: Preparation of Quality Control Matrix Solution without Sugar Removal

[0060] Take 1000 mL of serum matrix solution, inactivate it at 56°C for 30-120 min, and cool it to room temperature; add 0.2% (v / v) preservative Proclin 300, stir evenly, and filter it with a 0.22 μm sterile filter membrane to obtain the unsaccharide-removed quality control matrix solution.

[0061] Example 2: Preparation of AFP-L3 Quality Control in Serum Matrix

[0062] Take the sugar-free quality control matrix liquid prepared in Example 1, add an appropriate amount of natural AFP-L3 antigen extracted from placenta, stir evenly, and prepare a liquid quality control product of the required concentration; package according to the specified specifications, and freeze-dry using a vacuum freeze dryer to obtain the serum-based AFP-L3 quality control product, which is stored at 2~8°C.

[0063] Test Example 1: Assessment of AFP-L3 Antigen Content in the Preparation of Quality Control Products Using Sugar-Removed and Non-Sweetened Matrix Solutions

[0064] AFP-L3 quality control products were prepared using the sugar-removed control matrix solution of Example 1 and the unsugar-removed control matrix solution of Comparative Example 1, respectively. Equal amounts of placenta-extracted natural AFP-L3 antigen were added to each solution according to the method described in Example 2. Two lyophilized control products, Control 1 (sugar-removed serum matrix + placenta-derived natural AFP-L3 antigen) and Control 2 (unsugar-removed serum matrix + placenta-derived natural AFP-L3 antigen), were reconstituted and stored at 2-8°C. AFP-L3% (AFP-L3 / AFP) was measured for Control 1 and Control 2 using the AutoLumo A2000 plus detection system and an AFP-L3 and AFP kit. The average value of duplicate wells for each quality control point was calculated. The results are shown in Table 1.

[0065] Table 1: Comparison of AFP-L3% content in quality control products prepared with different matrix solutions

[0066]

[0067] The results showed that the AFP-L3% of the quality control product 1 provided by the present invention was 10 percentage points higher than that of the quality control product 2; the AFP-L3 detection efficiency of the quality control product prepared by adding an equal amount of natural AFP-L3 antigen extracted from the placenta to the sugar-removed quality control matrix liquid was greatly improved compared with the quality control product prepared by adding an equal amount of natural AFP-L3 antigen extracted from the placenta to the sugar-removed quality control matrix liquid.

[0068] Comparative Example 2: Preparation of AFP-L3 antigen from ascites (natural)

[0069] Ascites samples from patients with liver cancer were collected and centrifuged at 3000 g for 10 min. The supernatant was collected and then heated in a 56°C water bath for 30-45 min. After natural cooling, it was purified using a Sepharose 4B affinity chromatography column. After impurities were removed, it was eluted with a high-salt eluent and dialyzed before ultrafiltration and concentration. The storage concentration was 5 mg / mL and the samples were aliquoted and frozen at -20°C for later use.

[0070] Comparative Example 3: Preparation Method of Cell-derived AFP-L3 Antigen (Natural)

[0071] HepG2 hepatoma cell line was cultured in DMEM medium containing 10% serum until the confluence reached 80%-90%. The culture supernatant was collected and centrifuged at 3000 g for 10 min. The supernatant was purified using a Sepharose 4B affinity chromatography column. After impurities were removed, the supernatant was eluted with a high-salt eluent, dialyzed, and concentrated by ultrafiltration. The supernatant was then aliquoted and frozen at -20°C for later use.

[0072] Comparative Example 4: Preparation Method of Recombinantly Expressed AFP-L3 Antigen (Prokaryotic)

[0073] The AFP-L3 gene was cloned into the pET28a vector and transformed into Escherichia coli Rosetta (DE3) for expression. After IPTG induction and ultrasonic disruption, AFP-L3 was almost entirely present in the precipitate as inclusion bodies. After impurities were removed with 2 M urea solution, the precipitate was dissolved with 8 M urea and renatured using urea gradient dialysis until the solution was free of urea. The solution was then aliquoted and frozen at -20°C for later use.

[0074] Comparative Example 5: Preparation Method of Recombinantly Expressed AFP-L3 Antigen (Eukaryotic)

[0075] The AFP-L3 gene was cloned into the pSecTag2A vector and transfected into a CHO cell line for expression. After hygromycin B selection and induction of expression, the cell supernatant was collected and purified using a Sepharose 4B affinity chromatography column. After impurities were removed, the supernatant was eluted with a high-salt eluent, dialyzed, and concentrated by ultrafiltration. The supernatant was then aliquoted and frozen at -20°C for later use.

[0076] Test Example 2: Assessment of AFP-L3 Antigen Content from Different Sources

[0077] Placenta-extracted natural AFP-L3 antigen and AFP-L3 antigen prepared in different batches according to different methods in Comparative Examples 2 to 5 were added to the control matrix solution of the present invention in Example 1, and 5 AFP-L3 control products were prepared according to the method described in Example 2, including control product 1 (serum matrix + placenta-derived natural AFP-L3 antigen), control product 3 (serum matrix + ascites-derived natural AFP-L3 antigen), control product 4 (serum matrix + cell-derived natural AFP-L3 antigen), control product 5 (serum matrix + prokaryotic expression recombinant AFP-L3 antigen), and control product 6 (serum matrix + eukaryotic expression recombinant AFP-L3 antigen). After reconstitution, the 5 lyophilized control products were stored at 2 to 8 ° C. The AFP-L3% (AFP-L3 / AFP) of control products 1 to 5 was determined using the AutoLumo A2000 plus detection system and the AFP-L3 and AFP kit. Each quality control point was tested in duplicate, and the mean value was calculated to compare the differences between the five quality control products, AFP-L3%, prepared from different batches of antigen. The results are shown in Table 2.

[0078] Table 2: AFP-L3% content and deviation of different antigens and batches

[0079]

[0080] It can be seen from the experimental data in Table 2 that the AFP-L3% in the quality control products 1 and quality control products 4 (Comparative Example 3) provided by the present invention is the highest, with an average of 30.7% and 30.6%, and the inter-batch difference CV of different batches of antigens for quality control product 1 is 10.9%, which is better than 19.6% of quality control product 4; the AFP-L3 proportion in quality control product 4 (Comparative Example 3) is 26.4%, but the inter-batch difference CV of the antigen reaches 39.2%, and the AFP-L3% in the ascites of different liver cancer patients has significant differences; the AFP-L3 proportion in quality control product 5 (Comparative Example 4) is only 8.7%, which is related to the inherent defects of the prokaryotic expression system, insufficient glycosylation level, and the inter-batch difference CV of the antigen reaches 35.3%; the inter-batch difference CV of the antigen for quality control product 6 (Comparative Example 5) is the best at 9.9%, but the AFP-L3% is only 17.4%, which is much lower than that of quality control product 1. Compared with the recombinant AFP-L3 antigen derived from genetic engineering, the natural antigen is structurally closer to the antigen structure in the human body and is more rigorous in the preparation of quality control products. Among natural antigens from different sources, the cost of extracting AFP-L3 antigen from the placenta is the lowest. The placenta is a relatively easy-to-obtain biological material and does not require modification. The extraction process does not require overly complex technology or equipment. The source is stable and the difference between antigen batches is small. The cost of cell culture is relatively high, and the process is complex and the cost of materials such as cell culture is high. The cost of extracting AFP-L3 from ascites may be between the two, but it involves extracting ascites from liver cancer patients, and there are large individual differences between different patients.

[0081] In summary, the AFP-L3% in Controls 1 (placenta-derived natural AFP-L3 antigen), 3 (ascites-derived natural AFP-L3 antigen), 4 (cell-derived natural AFP-L3 antigen), and 6 (eukaryotically expressed recombinant AFP-L3 antigen) all reached above 10% and are derived from stable and reliable sources, making them effective quality controls for AFP-L3. Furthermore, based on the above comparison results, Control 1 (placenta-derived natural AFP-L3 antigen) was selected for further validation analysis.

[0082] Test Example 3: Freeze-thaw, reconstitution, and frozen storage stability assessment of the quality control product of the present invention

[0083] The freeze-dried AFP-L3 quality control product (prepared according to the method described in Example 2) was reconstituted and tested. The test time points were set at 0 days, 1 day, 3 days, 1 month of freezing at -20°C, 2 months of freezing at -20°C, and 3 freeze-thaw cycles. The average value of each quality control point was calculated by testing the replicates. The differences in freeze-thaw, reconstitution, and frozen storage stability were compared. The results are shown in Table 3.

[0084] Table 3: Freeze-thaw, reconstitution, and frozen storage conditions for reconstitution stability assessment of AFP-L3 quality control products

[0085]

[0086] The requirement of detection value deviation being less than 10% is satisfied. As can be seen from the experimental data in Table 3, the AFP-L3 quality control product provided by the present invention was frozen and thawed three times, frozen for two months, and reconstituted for three days, and the detection value deviation of the three quality control points was within ±10.0%.

[0087] Test Example 4: Thermal Accelerated Stability Assessment of the Quality Control Product of the Present Invention

[0088] The thermal accelerated destruction stability of the quality control product of the present invention (prepared according to the method described in Example 2) was evaluated. The quality control products were placed in a 37°C incubator in reverse order for 14 days, 10 days, and 7 days, respectively. After the time was up, they were taken out at the same time and returned to room temperature. They were then evaluated together with the control stored at 2-8°C. The stability changes of the quality control products after thermal accelerated destruction were compared. The results are shown in Table 4.

[0089] Table 4: Thermal Accelerated Stability Assessment of the Quality Control Products of the Present Invention

[0090]

[0091] As can be seen from the data in Table 4, after the quality control product of the present invention is placed at 37 degrees for 7 days, 10 days, and 14 days, the stability variation is less than 10% compared with the quality control product stored at 2-8 degrees, which meets the requirements.

[0092] Test Example 5: Real-time stability assessment of the quality control product of the present invention

[0093] The accompanying test kit (including the quality control product prepared according to the method described in Example 2) was frozen and stored below -20°C, with one set removed for each use. The stability of the quality control product of the present invention was evaluated at 2-8°C for 0, 3, 6, 9, and 12 months. The results were compared with the 0-month test results. The range of the quality control product's real-time storage stability at 2-8°C was observed to determine the shelf life of the quality control product. The results are shown in Table 5.

[0094] Table 5: Real-time stability assessment of the quality control products of the present invention

[0095]

[0096] As shown in Table 5, after 13 months of storage at 2-8°C, the variation in the quality control product of the present invention compared to the value at 0 months was less than 10%, which meets the requirements. Therefore, the shelf life of the quality control product of the present invention can be set at 12 months.

[0097] Test Example 6: Uniformity Assessment of the Quality Control Product of the Present Invention

[0098] Perform the homogeneity test according to the following method and calculate the coefficient of variation within the bottle ( ) and the inter-bottle coefficient of variation ( ), the result should not be greater than 10%.

[0099] Ten sets of quality control products (prepared according to the method shown in Example 2) were randomly selected and numbered 1 to 10 for each concentration, and were measured three times in the following order.

[0100] Measurement order: 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.

[0101] Record the measurement results and calculate according to formula 1 to formula 10 、 、 、 and :

[0102] Formula 1:

[0103] Formula 2:

[0104] Formula 3:

[0105] Formula 4:

[0106] Formula 5:

[0107] Formula 6:

[0108] Formula 7:

[0109] Formula 8:

[0110] Formula 9:

[0111] Formula 10:

[0112] Where:

[0113] -variance;

[0114] — degrees of freedom;

[0115] — mean square;

[0116] — Test value;

[0117] -sample Number of repeated measurements;

[0118] —Number of valid measurements;

[0119] —Inter-bottle standard deviation;

[0120] —Repeatability standard deviation;

[0121] — measurement or calculation results;

[0122] —Grand average.

[0123] When the statistical results When the value is less than or equal to 10, it is considered that the uniformity between the quality control bottles is good and can be used as a calibrator. The average value of all test results is calculated and then the and Calculation; when the statistical results When the value is greater than 10, it is considered that the uniformity between the quality control bottles is poor and it is not suitable to be used as a quality control product, and it is no longer calculated. and .

[0124] When the statistical results When the standard deviation between bottles is replaced by the standard deviation within the bottle, .

[0125] The results are shown in Table 6.

[0126] Table 6: Uniformity assessment results of the quality control products of the present invention

[0127]

[0128] As shown in Table 6, the intra-bottle and inter-bottle coefficients of variation of the AFP-L3 control product of the present invention are both less than 10%, which meets the requirements.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a quality control matrix solution of an alpha-fetoprotein heterogeneous substance, characterized in that: include: The raw materials and sodium periodate are mixed, stirred at a low speed of 5-35 rpm for 30-60 min at 4°C in the dark, glycine is added, stirred at a low speed of 5-35 rpm for 15-30 min at room temperature, and inactivated for 30-120 min to obtain a reaction product, which is then returned to room temperature and mixed with a preservative, filtered, and the alpha-fetoprotein heterogeneous quality control matrix solution is obtained; The raw material includes at least one of serum and plasma.

2. The preparation method according to claim 1, wherein: The concentration of the sodium periodate is 50-500 mM; The concentration of glycine is 0.5~2 M; The volume ratio of the sodium periodate to the raw material is 1:98; The volume ratio of the glycine to the raw material is 1:

98.

3. The preparation method according to claim 1 or 2, wherein The serum is human serum, and the plasma is human plasma.

4. The preparation method according to any one of claims 1 to 3, characterized in that The preservatives include Proclin 300.

5. The preparation method according to any one of claims 1 to 4, characterized in that The inactivation temperature is 56°C.

6. A quality control matrix solution of alpha-fetoprotein heterogeneities obtained by the preparation method according to any one of claims 1 to 5.

7. A quality control product for alpha-fetoprotein heterogeneity, characterized in that: Contains the alpha-fetoprotein heterogeneous quality control matrix liquid according to claim 6.

8. The alpha-fetoprotein heterogeneous quality control product according to claim 7, characterized in that: Also contains native AFP-L3 antigen of placental origin.

9. A method for preparing a quality control product of an alpha-fetoprotein heterogeneous substance, characterized in that: include: The AFP-L3 antigen is mixed with the alpha-fetoprotein heterogeneous quality control product matrix solution according to claim 6 according to the required concentration, stirred evenly, and freeze-dried to obtain the alpha-fetoprotein heterogeneous quality control product.

10. The preparation method according to claim 9, characterized in that The AFP-L3 antigen is a natural AFP-L3 antigen derived from placenta.

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