Compound quality control product for Alzheimer disease as well as preparation method and application thereof

Abstract

The invention discloses a compound quality control product for Alzheimer's disease as well as a preparation method and application of the compound quality control product, every 100 mL of the quality control product comprises a matrix, a compound biomarker, 10-50 mg of a blocking agent, a protective agent, 0.5-5 g of chloride, 1-5 g of buffer salt and 0.01-0.1 g of a preservative, the pH value is controlled to be 6.0-8.0, and purified water is used for fixing the volume; the composite biomarker is any one or combination of Alzheimer's disease biomarker recombinant protein or beta amyloid polypeptide pure product solution standard substances; the blocking agent is HHB-H1 (Hepatitis B Receptor The protective agent in each 100 mL of the quality control product is prepared from 1 g to 10 g of polyethylene glycol, 0.5 g to 5 g of a surfactant, 0.5 g to 5 g of saccharides, 0.5 mL to 5 mL of a 0.5 M EDTA (Ethylene Diamine Tetraacetic Acid) solution and 0.1 g to 1.0 g of EGTA (Ethylene Glycol Terephthalate); the matrix in every 100 mL of the quality control product is any one of human plasma, artificial plasma, bovine serum albumin and calf serum; according to the invention, 11 detection items or indexes can be monitored at the same time, comprehensive quality control data can be provided, and the diagnosis accuracy is improved; the performance of the whole detection system can also be evaluated.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and relates to an Alzheimer's disease composite quality control product, its preparation method and application. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease. At present, the research on diagnostic kits for Alzheimer's disease has become one of the hot issues in the field of immunoassay. With the continuous introduction of various Alzheimer's disease diagnostic kits on the market, it is particularly important to ensure the accuracy, authenticity and effectiveness of the detection results of these kits. Quality control is of great significance.

[0003] Quality control products play a crucial role in laboratory analysis and clinical testing. The lack of effective quality control products will lead to serious problems such as the unreliability of detection results, the concealment of instrument and reagent problems that cannot be detected in time, the reduced controllability of experimental conditions, non-compliance with regulations and standards, and the decline of customer trust.

[0004] There are a wide variety of Alzheimer's disease diagnostic kits on the market, and the quality control products used are mostly internal single-item quality control products provided by kit manufacturers. These internal single-item quality control products often lack fairness, independence and objectivity, and it is difficult to meet the standardized and normalized requirements of laboratory quality control. Moreover, single-item quality control products cannot meet the need to simultaneously monitor multiple detection items or indicators. If multiple detections or indicators need to be controlled, different single-item quality control products need to be replaced, thus wasting inspection time and laboratory resources.

[0005] Therefore, the research and development of Alzheimer's disease composite quality control products is of great significance. Different from composite quality control products for different diseases, Alzheimer's disease composite quality control products need to include various types of Alzheimer's disease biomarkers. These biomarkers not only have a large variety, but also a high degree of structural similarity. How to make Alzheimer's disease biomarkers stably coexist in the same system for a long time and not interfere with each other has become an urgent technical problem to be solved. Summary of the Invention

[0006] The purpose of the present invention is to research and develop an Alzheimer's disease composite quality control product. By studying the components and preparation method of the composite quality control product, Alzheimer's disease biomarkers can stably coexist in the same system for a long time and not interfere with each other, so as to ensure that the composite quality control product of the present invention can accurately reflect the performance of different target detection items.

[0007] The technical solution adopted by the present invention provides an Alzheimer's disease composite quality control product. The key lies in that every 100 mL of the quality control product includes a matrix, composite biomarkers, 10 mg to 50 mg of a blocker, a protective agent, 0.5 g to 5 g of chloride, 1 g to 5 g of buffer salts, and 0.01 g to 0.1 g of a preservative. The pH value is controlled within the range of 6.0 to 8.0, and it is made up to volume with purified water; the above-mentioned composite biomarkers are any one or combination of Alzheimer's disease biomarker recombinant proteins or β-amyloid polypeptide pure product solution standards; the above-mentioned blocker is HBR-H1; the protective agent in every 100 mL of the quality control product consists of 1 g to 10 g of polyethylene glycol, 0.5 g to 5 g of a surfactant, 0.5 g to 5 g of saccharides, 0.5 mL to 5 mL of a 0.5 M EDTA solution, and 0.1 g to 1.0 g of EGTA; the matrix in every 100 mL of the quality control product is any one of 60 g to 90 g of human plasma, 60 g to 90 g of artificial plasma, 0.1 g to 2 g of bovine serum albumin, and 0.1 g to 2 g of calf serum.

[0008] Specifically, the above-mentioned Alzheimer's disease biomarker recombinant proteins are NfL, GFAP, BD-Tau, pTau-181, pTau-231, pTau-212, pTau-205, pTau-217, and Tau; the above-mentioned β-amyloid polypeptide pure product solution standards are Aβ40 and Aβ42.

[0009] Further, the above quality control products are divided into high-level concentration, medium-level concentration, and low-level concentration quality control products; in the above high-level concentration quality control products, when the quality control product contains 9 Alzheimer's disease biomarker recombinant proteins or 2 β-amyloid polypeptide pure product solution standards, it is not necessary that the quality control product contains all 9 Alzheimer's disease biomarker recombinant proteins or 2 β-amyloid polypeptide pure product solution standards pointed out by the present invention. The concentration ranges of the 9 Alzheimer's disease biomarker recombinant proteins contained should meet the following requirements: the concentration range of NfL is 22 pg / mL to 40 pg / mL, the concentration range of GFAP is 1000 pg / mL to 3500 pg / mL, the concentration range of BD-Tau is 12 pg / mL to 20 pg / mL, the concentration range of pTau-181 is 4 pg / mL to 8 pg / mL, the concentration range of pTau-231 is 4.0 pg / mL to 10 pg / mL, the concentration range of pTau-212 is 4 pg / mL to 8 pg / mL, the concentration range of pTau-205 is 6 pg / mL to 12 pg / mL, the concentration range of pTau-217 is 0.6 pg / mL to 1.2 pg / mL, the concentration range of Tau is 18 pg / mL to 40 pg / mL. The concentration ranges of the 2 β-amyloid polypeptide pure product solution standards contained should meet the following requirements: the concentration range of Aβ40 is 80 pg / mL to 150 pg / mL, and the concentration range of Aβ42 is 20 pg / mL to 50 pg / mL;

[0010] In the above medium-level concentration quality control product, when the quality control product contains 9 Alzheimer's disease biomarker recombinant proteins or 2 pure β-amyloid polypeptide solution standards, it is not necessary that the quality control product contains all 9 Alzheimer's disease biomarker recombinant proteins or 2 pure β-amyloid polypeptide solution standards pointed out by the present invention. The concentration ranges of the 9 Alzheimer's disease biomarker recombinant proteins contained should meet the following requirements: the concentration range of NfL is 15 pg / mL to 20 ng / mL, the concentration range of GFAP is 150 pg / mL to 300 pg / mL, the concentration range of BD-Tau is 6 pg / mL to 10 pg / mL, the concentration range of pTau-181 is 1 pg / mL to 3 pg / mL, the concentration range of pTau-231 is 1.5 pg / mL to 3.0 pg / mL, the concentration range of pTau-212 is 1 pg / mL to 3 pg / mL, the concentration range of pTau-205 is 2 pg / mL to 5 pg / mL, the concentration range of pTau-217 is 0.3 pg / mL to 0.5 pg / mL, the concentration range of Tau is 5 pg / mL to 15 pg / mL. The concentration ranges of the 2 pure β-amyloid polypeptide solution standards contained should meet the following requirements: the concentration range of Aβ40 is 15 pg / mL to 25 pg / mL, and the concentration range of Aβ42 is 5 pg / mL to 10 pg / mL;

[0011] In the above low-level concentration quality control product, when the quality control product contains 9 Alzheimer's disease biomarker recombinant proteins or 2 pure β-amyloid polypeptide solution standards, it is not necessary that the quality control product contains all 9 Alzheimer's disease biomarker recombinant proteins or 2 pure β-amyloid polypeptide solution standards pointed out by the present invention. The concentration ranges of the 9 Alzheimer's disease biomarker recombinant proteins contained should meet the following requirements: the concentration range of NfL is 8 pg / mL to 14 ng / mL, the concentration range of GFAP is 60 pg / mL to 120 pg / mL, the concentration range of BD-Tau is 1 pg / mL to 5 pg / mL, the concentration range of pTau-181 is 0.5 pg / mL to 0.9 pg / mL, the concentration range of pTau-231 is 0.5 pg / mL to 1.4 pg / mL, the concentration range of pTau-212 is 0.4 pg / mL to 0.8 pg / mL, the concentration range of pTau-205 is 0.5 pg / mL to 1.8 pg / mL, the concentration range of pTau-217 is 0.05 pg / mL to 0.2 pg / mL, the concentration range of Tau is 1 pg / mL to 3 pg / mL. The concentration ranges of the 2 pure β-amyloid polypeptide solution standards contained should meet the following requirements: the concentration range of Aβ40 is 5 pg / mL to 10 pg / mL, and the concentration range of Aβ42 is 1 pg / mL to 4 pg / mL.

[0012] Preferably, the surfactant is any one of Triton X-100, Triton X-200 or Tween-20; the preservative is any one of sodium azide, thimerosal or Proclin 300; the chloride is sodium chloride or potassium chloride; the buffer salt is any one of HEPES, PBS or Tris; the saccharide is any one of sucrose, trehalose or inulin; the polyethylene glycol is any one of PEG1000, PEG2000, PEG3000 or PEG4000.

[0013] Furthermore, in the above preparation method, a buffer solution is first prepared with the buffer salt, then EDTA solution, EGTA and chloride are added, and stirred until dissolved. The pH value range is measured. If the pH value is not within the range of 6.0 - 8.0, it is adjusted with a pH regulator. Then saccharide, polyethylene glycol, surfactant and preservative are added and stirred evenly. Finally, the matrix or the matrix solution prepared with the matrix, the blocker and the composite biomarker are added, stirred evenly, made up to the target volume, filtered through a membrane and then sub-packed, and assigned values to obtain the composite quality control product in liquid form; the solvent for preparing the buffer solution is purified water. Since human plasma and artificial plasma are already in liquid form, they can be directly added; bovine serum albumin and calf serum are solids, so they need to be first dissolved and prepared into the corresponding matrix solution before use.

[0014] Even further, in the above preparation method, the composite quality control product in liquid form is also freeze-dried to obtain the composite quality control product in solid form; the conditions for the freeze-drying are: the cold trap temperature is -50°C to -30°C, the vacuum degree of the drying chamber is 1 Pa to 3 Pa, and the drying time is 10 h to 20 h; the rotation speed of the stirring is 800 r / min to 1500 r / min, and the stirring time is 2 min to 15 min.

[0015] Specifically, in the above freeze-drying process, a freeze-drying excipient of 1% - 8% of the mass of the composite quality control product in liquid form can also be added.

[0016] It should be noted that the above quality control product is used for preparing a diagnostic kit for Alzheimer's disease.

[0017] It should be noted that the above quality control product is used for the inspection of an inspection system including instruments, reagents and calibration products.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention studies a composite quality control product for Alzheimer's disease, which contains 11 biomarkers of Alzheimer's disease. Among them, it not only includes 2 pure β-amyloid polypeptide solution standards, namely national standard substances, but also includes 9 recombinant proteins, basically covering common Alzheimer's disease diagnostic kits on the market.

[0020] The composite quality control product of the present invention can monitor up to 11 test items or indicators simultaneously, can provide more comprehensive quality control data, and helps to more accurately evaluate the performance of the entire detection system; the present invention can comprehensively evaluate the overall operation of the laboratory and discover potential systematic problems, rather than accidental errors of a single item. Therefore, the present invention can improve the stability and reliability of the detection system, and through more comprehensive monitoring, significantly reduce the incidence of false positives and false negatives, and improve the accuracy of diagnosis.

[0021] Using the composite quality control product of the present invention can improve efficiency. Using a multi-item composite quality control product can reduce the number of quality control products to be processed in each experiment, thereby saving time and laboratory resources, reducing operation steps and complexity, reducing the probability of errors, and improving work efficiency.

[0022] The present invention can also enhance the consistency and comparability of results. Multi-item composite quality control products usually undergo strict standardization to ensure consistency between different batches, making the results more comparable.

[0023] The present invention has higher cost-effectiveness. Although the unit price of multi-item composite quality control products may be relatively high, due to their high efficiency and reduced usage, the overall cost is often lower, avoiding unnecessary waste and reducing the overall operating cost.

[0024] In summary, multi-item composite quality control products have obvious advantages in terms of comprehensiveness, efficiency, consistency, cost-effectiveness, and reliability, and are indispensable tools in modern laboratories and clinical tests. Detailed implementation mode

[0025] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] For those not specified in the embodiments, they can be carried out according to conventional conditions; for reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] Examples 1 to 11

[0028] Examples 1 to 11 of the present invention are Alzheimer's disease composite quality control products with different components. The types and dosages of components in each 100 mL of the composite quality control product are shown in Table 1 or Table 2; if the pH value of the composite quality control product is not within the range of 6.0 to 8.0, use NaOH solution or hydrochloric acid solution for adjustment to make the pH value of the composite quality control product meet the requirements, which are respectively denoted as quality control product sample 1 to quality control product sample 11.

[0029] Table 1: Types and Dosages of Each Component in 100 mL of the Composite Quality Control Product Sample (Examples 1 to 5) Table 2: Types and Dosages of Each Component in 100 mL of the Composite Quality Control Product Sample (Examples 6 to 11) Note: Bovine serum albumin in Table 1 and Table 2 is represented by BSA, and calf serum is represented by CS; "——" indicates that the corresponding substance is not added during the preparation.

[0035] Example 12

[0036] Taking the components of Example 3 as an example, the preparation process of the composite quality control product of the present invention is described as follows:

[0037] Before preparing the composite quality control product, 0.15 g of BSA needs to be dissolved in 5 mL of purified water, and vortexed until the BSA is completely dissolved to obtain a BSA solution for standby; prepare 10 M NaOH solution and 10 M hydrochloric acid solution as pH regulators for standby; prepare 0.5 M EDTA solution for standby.

[0038] The specific steps for preparing the composite quality control product are as follows:

[0039] Step S1, take a 150 mL beaker, add 80 mL of purified water, weigh 1.2 g of HEPES, 1.7 g of sodium chloride, and 0.2 g of EGTA, add them to the beaker, and stir with a magnetic stirrer at 1000 r / min for 10 min until the solid reagents are completely dissolved;

[0040] Step S2, add 1.0 mL of 0.5 M EDTA solution and 300 μL of 10 M NaOH solution, stir with a magnetic stirrer at 1000 r / min for 3 min, measure the pH value with a pH meter, which should be within 6.0 to 8.0. If it is not within the range, adjust the pH value with 10 M NaOH solution or 10 M hydrochloric acid solution;

[0041] Step S3: Add 0.05 g of Proclin 300, 2.5 g of sucrose, 2.0 g of PEG 2000, and 1 g of Triton X-100 into a beaker, and stir with a magnetic stirrer at 1000 r / min for 5 min;

[0042] Step S4: Add 5 mL of the prepared BSA solution, 20 mg of blocker HBR-H1, 850 pg of Aβ40 standard substance, 200 pg of Aβ42 standard substance, 1000 pg of NfL recombinant antigen, 8000 pg of GFAP recombinant antigen, 300 pg of BD-Tau recombinant antigen, 70 pg of pTau-181 recombinant antigen, 80 pg of pTau-231 recombinant antigen, 60 pg of pTau-212 recombinant antigen, 10 pg of pTau-217 recombinant antigen, 100 pg of pTau-205 recombinant antigen, and 200 pg of Tau recombinant antigen into the beaker, and stir with a magnetic stirrer at 1000 r / min for 3 min; Transfer to a volumetric flask and make up to 100 mL with purified water;

[0043] It should be noted that since the product specifications and forms of the purchased standard substances and recombinant antigens are different, and it is difficult to accurately directly weigh the pg level of biomarkers, it is necessary to perform gradient dilution on each biomarker to obtain a biomarker stock solution with a suitable concentration. When preparing the composite quality control product, use a pipette to take the stock solution containing the corresponding mass of the biomarker;

[0044] Step S5: After filtering with a 0.22 μm filter membrane, dispense into vials, assign values, and obtain the composite quality control product in liquid form, denoted as quality control sample 3. The composite quality control product in liquid form needs to be stored frozen at -20 °C or below.

[0045] Example Thirteen

[0046] Taking the components of Example 3 as an example, the preparation process of the composite quality control product of the present invention is described. The preparation process is the same as that of Example Twelve, except that the stirring speed and stirring time in each step are adjusted. Specifically:

[0047] In Step S1, the speed of the magnetic stirrer is 800 r / min and the stirring time is 15 min;

[0048] In Step S2, the speed of the magnetic stirrer is 1500 r / min and the stirring time is 2 min;

[0049] In Step S3, the speed of the magnetic stirrer is 1200 r / min and the stirring time is 5 min;

[0050] In Step S4, the speed of the magnetic stirrer is 1500 r / min and the stirring time is 2 min.

[0051] Example 14

[0052] Prepare the quality control product sample according to the steps of Example 12. Steps S1 to S3 are the same as those in Example 12. When performing Step S4, add 2 g of mannitol as the excipient for freeze-drying. After stirring, add purified water to make the volume up to 100 mL. After filtering with a 0.22 μm filter membrane, aliquot into vials and perform freeze-drying.

[0053] The specific parameters of freeze-drying are as follows: the cold trap temperature is -45°C, the vacuum degree in the drying chamber is 2 Pa, and the drying time is 16 h. The freeze-dried powder sample 1 is prepared. The freeze-dried powder sample needs to be stored refrigerated at 2°C to 8°C. The refrigeration temperature in this example is selected as 4°C.

[0054] Example 15

[0055] Prepare the quality control product sample according to the steps of Example 12. Steps S1 to S3 are the same as those in Example 12. When performing Step S4, add 1 g of mannitol as the excipient for freeze-drying. After stirring, add purified water to make the volume up to 100 mL. After filtering with a 0.22 μm filter membrane, aliquot into vials and perform freeze-drying.

[0056] The specific parameters of freeze-drying are that the cold trap temperature is -30°C, the vacuum degree in the drying chamber is 1 Pa, and the drying time is 20 h. The freeze-dried powder sample 2 is prepared.

[0057] Example 16

[0058] Prepare the quality control product sample according to the steps of Example 12. Steps S1 to S3 are the same as those in Example 12. When performing Step S4, add 8 g of mannitol as the excipient for freeze-drying. After stirring, add purified water to make the volume up to 100 mL. After filtering with a 0.22 μm filter membrane, aliquot into vials and perform freeze-drying.

[0059] The specific parameters of freeze-drying are that the cold trap temperature is -50°C, the vacuum degree in the drying chamber is 3 Pa, and the drying time is 10 h. The freeze-dried powder sample 3 is prepared.

[0060] Comparative Example 1

[0061] The implementation method of this comparative example is the same as that of Example 12, except that in Step S3, no blocking agent of any kind is added, and a liquid-form composite quality control reference product is prepared, denoted as reference product 1. The specific components are shown in Table 3.

[0062] The components of this comparative example are the same as those of Example 3 except that no blocking agent is added.

[0063] Comparative Example 2

[0064] The implementation method of this comparative example is the same as that of Example 12, except that when performing Step S3, the blocking agent added is casein, and a composite quality control product reference in liquid form is prepared, denoted as Reference 2. For the specific components, see Table 3.

[0065] Except for the type and dosage of the blocking agent, the components of this comparative example are the same as those of Example 3.

[0066] Comparative Example 3

[0067] The implementation method of this comparative example is the same as that of Example 12, except that when performing Step S3, the blocking agent added is mouse IgG, and a composite quality control product reference in liquid form is prepared, denoted as Reference 3. For the specific components, see Table 3.

[0068] Except for the type and dosage of the blocking agent, the components of this comparative example are the same as those of Example 3.

[0069] Comparative Example 4

[0070] The implementation method of this comparative example is the same as that of Example 12, except that when performing Step S3, the blocking agent added is MAK-33-IgG Poly, and a composite quality control product reference in liquid form is prepared, denoted as Reference 4. For the specific components, see Table 3.

[0071] Except for the type and dosage of the blocking agent, the components of this comparative example are the same as those of Example 3.

[0072] Comparative Example 5

[0073] The implementation method of this comparative example is the same as that of Example 12, except that when performing Step S1, EGTA is not added, and a composite quality control product reference in liquid form is prepared, denoted as Reference 5. For the specific components, see Table 3.

[0074] Except for not adding EGTA and increasing the dosage of EDTA, the components of this comparative example are the same as those of Example 3.

[0075] Comparative Example 6

[0076] The implementation method of this comparative example is the same as that of Example 12, except that when performing Step S2, the 0.5M EDTA solution is not added, and a composite quality control product reference in liquid form is prepared, denoted as Reference 6. For the specific components, see Table 3.

[0077] Except for not adding the 0.5M EDTA solution and increasing the dosage of EGTA, the components of this comparative example are the same as those of Example 3.

[0078] Table 3: Types and dosages of each component in every 100 mL of the composite quality control product reference

[0079]

[0080]

[0081] Note: In Table 3, "——" indicates that the corresponding substance was not added during the preparation.

[0082] Tests and Analyses

[0083] In accordance with the requirements of "YY / T 1662-2019 Quality Control Substances for Biochemical Analysis" and "YY 1549-2024 Calibrators for Biochemical Analyzers", the UD-X type automatic single molecule fluorescence immunoassay analyzer produced by Youdi Biotechnology (Shijiazhuang) Co., Ltd. and its company's supporting detection kits were used to verify the quality control samples 1-11, freeze-dried powder samples 1-3 and reference samples 1-6.

[0084] I. Acceptable Range

[0085] The quality control samples 1-11, freeze-dried powder samples 1-3 and reference samples 1-6 were respectively tested using the kits, and the results are shown in Tables 4-13.

[0086] The target values in Tables 4-13 are based on the "Guidelines for the Registration Review of Quality Control Substances - Research on the Assignment of Quality Control Substances" and

[0087] "YY / T 1662-2019 Quality Control Substances for Biochemical Analysis". The assignment of each quality control substance adopts the 4×5 model for assignment, that is, each concentration of each biomarker is detected 2 times in the morning and afternoon every day, that is, detected 4 times a day, and continuously detected for 5 days, and the mean value is calculated, that is, the target value and the standard deviation value, represented by SD. The quality control range for this concentration level of this biomarker is the target value ± 3SD.

[0088] At the same time, the quality control range was confirmed, that is, other batches of kits were used for detection, and the detection results were still within the quality control range.

[0089] Table 4: Test Results of Acceptable Range (Quality Control Sample 1 and Quality Control Sample 2)

[0090]

[0091] Table 5: Test Results of Acceptable Range (Quality Control Sample 3 and Quality Control Sample 4)

[0092]

[0093]

[0094] Table 6: Test Results of Acceptable Range (Quality Control Sample 5 and Quality Control Sample 6)

[0095]

[0096] Table 7: Acceptable Interval Test Results (Quality Control Sample 7 and Quality Control Sample 8)

[0097]

[0098]

[0099] Table 8: Acceptable Interval Test Results (Quality Control Sample 9 and Quality Control Sample 10)

[0100]

[0101] Table 9: Acceptable Interval Test Results (Quality Control Sample 11 and Lyophilized Powder Sample 1)

[0102]

[0103]

[0104] Table 10: Acceptable Interval Test Results (Lyophilized Powder Sample 2 and Lyophilized Powder Sample 3)

[0105]

[0106] Table 11: Acceptable Interval Test Results (Reference Standard 1 and Reference Standard 2)

[0107]

[0108]

[0109] Table 12: Acceptable Interval Test Results (Reference Standard 3 and Reference Standard 4)

[0110]

[0111] Table 13: Acceptable Interval Test Results (Reference Standard 5 and Reference Standard 6)

[0112]

[0113]

[0114] As can be seen from the results in Tables 4 to 13, in the liquid-form composite quality control product samples and the freeze-dried powder samples in solid form of the present invention, the detection results of each biomarker are closer to the theoretical values. In Control 1, no blocker is used, and the target value is significantly higher than the theoretical value, indicating that the detection results of individual biomarkers are on the high side due to non-specific binding between different biomarkers; in Controls 2 to 4, other blockers are selected. Although the detection results of individual biomarkers are closer to the theoretical value than in Control 1, they are still on the high side, indicating that non-specific binding between different biomarkers still affects the accuracy of the detection results; in Controls 5 and 6, only one metal chelator is added, and the test results show a downward trend, indicating that some metal ions in the system hinder the binding of antigens and antibodies.

[0115] It is found that the selection of blockers is particularly important. Blockers play an important role in biochemical and immunological experiments, but the effects of different blockers vary. The blocker HBR-H1 selected in the present invention belongs to an active blocker, which is a heterophilic antibody blocker and has a certain protective effect in addition to blocking in the present invention. It can also prevent the sample from being degraded by enzymes or damaged by other factors during the experiment, thereby maintaining the integrity and activity of the sample and making the target value closer to the theoretical value.

[0116] The present invention also finds that better results can be obtained and interference can be reduced to the greatest extent only by using EDTA and EGTA in combination. This is because EDTA can form stable chelates with metal ions through a total of six coordination sites of two N atoms and four O atoms, and is particularly suitable for binding magnesium ions; while EGTA is more inclined to selectively bind calcium ions. The two can complement each other in function. The combined use may not only help improve the detection accuracy or efficiency, but also can be used as an auxiliary component to help maintain the stable state of some protein molecules.

[0117] II. Homogeneity test

[0118] Perform tests on quality control product sample 3, freeze-dried powder sample 1, Control 4 and Control 5. Randomly select 10 minimum package units of quality control products for each sample, and randomly number them 1 to 10, and test them 3 times in the following order respectively.

[0119] Measure in the 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), record the measurement results, and calculate according to Formulas 1 to 11.

[0120]

[0121] SS 瓶内 = SS 总和 - SS瓶间 ··············Formula 3

[0122]

[0123]

[0124] In formula 1 to formula 11, SS-variance; v-degree of freedom; MS-mean square; FF test value; n i - the number of measurements of the i-th bottle; n0- the number of effective measurements; S bb -Standard deviation between bottles; S r - Repeatability standard deviation; CV- coefficient of variation; S 总 -Standard deviation of all measurement data; x-measurement or calculation result; - Overall average.

[0125] When F≤F 0.05(v1,v2) When , there is no need to calculate CV 总 , it is believed that there is no significant difference in the assignment results between the calibration bottles and between bottles, and the uniformity between bottles is good: if F≤1, CV 瓶内 =CV 瓶间 , if 1<F≤F 0.05(v1,v2) , CV should be calculated separately 瓶内 and CV 瓶间 ;

[0126] When F>F 0.05(v1,v2) When the value of the calibration sample is considered to be different within the bottle and between bottles, the coefficient of variation CV of all measurement data should be calculated according to formula 9. 总 :If CV 总 If the CV is less than or equal to one third of the allowable error of the item, the product is considered to be suitable for use as a calibrator and has good uniformity between bottles. 总 If it is greater than one-third of the allowable error of this item, it is considered that the product has poor uniformity between bottles and is not suitable as a calibrator.

[0127] Calculate the standard deviation S between bottles according to the formula bb , standard deviation in bottle S r , coefficient of variation CV between bottles and CV within bottles. When the statistical result F value is ≤1, the standard deviation within the bottle is used instead of the standard deviation between bottles, that is, S bb =S r .

[0128] The allowable error of this item is 25%. In this test, one third of the allowable error of this item is 8.33%. 0.05(v1,v2) =2.392814, in this test, 2.393 is taken. The results are shown in Tables 14 and 15.

[0129] Table 14: Homogeneity test (QC sample 3 and lyophilized powder sample 1)

[0130]

[0131]

[0132] Table 15: Homogeneity test (Reference standard 4 and Reference standard 5)

[0133] As can be seen from Table 14 and Table 15, the F values of each biomarker in QC sample 3 and lyophilized powder sample 1 are all less than 1 and F 0.05(v1,v2) , it can be considered that there is no obvious difference in the assigned values of each biomarker within and between vials, and the CV between vials is less than 10%. Further, it shows that the homogeneity of each biomarker in the liquid-form QC product and solid-form prepared by the present invention is good, and the composite QC product can meet the homogeneity requirements.

[0134] However, in Reference standard 4, the F value of some biomarkers > F 0.05(v1,v2) , indicating that there is a difference in the assigned values of this biomarker within and between vials. Among them, the CV of Aβ40, GFAP and pTau-231 总 exceeds 8.33%, indicating that the between-vial homogeneity of this part of biomarkers is poor. Although the CV of BD-Tau 总 does not exceed 8.33%, its CV 瓶间 is greater than 10%, indicating that the between-vial homogeneity of this biomarker is also poor; the F value of some biomarkers is 1 < F ≤ 2.393, indicating that there is a difference in the assigned values of this biomarker within and between vials. CV 瓶内 and CV 瓶间 should be calculated separately. The CVs of NfL, pTau-212 and pTau-205 瓶间 are all greater than 10%, and the between-vial homogeneity of the corresponding biomarkers is still poor. Generally speaking, the between-vial homogeneity of each biomarker in Reference standard 4 cannot fully meet the requirements. Therefore, Reference standard 4 is not suitable as a composite QC product. Similarly, analyzing the results of Reference standard 5, it is found that the between-vial homogeneity of each biomarker in Reference standard 5 cannot fully meet the requirements. Similarly, Reference standard 5 is not suitable as a composite QC product,

[0135] III. Stability

[0136] The variance was used to analyze the results of the stability study data of QC sample 3, lyophilized powder sample 1, Reference standard 4 and Reference standard 5. The F value and probability (p) value were calculated according to the variance analysis table recorded in Table 16. For a 95% inclusion probability, p ≥ 0.05 indicates that the trend is not significant, otherwise the trend is significant.

[0137] Table 16: Analysis of Variance Table

[0138]

[0139]

[0140] 1. Expiry Stability

[0141] Quality control sample 3, reference standard 4 and reference standard 5, and all composite quality control products in liquid form shall be stored below -20°C and can be stably stored for 12 months. Detection shall be carried out at 0, 3, 6, 9, 12, and 13 months of storage, with each detection repeated 3 times. Trend analysis shall be carried out according to Table 16, and the change trend shall not be significant. The results are shown in Tables 17 to 19;

[0142] Lyophilized powder sample 1 and all composite quality control products in solid form shall be stored refrigerated at 2°C to 8°C. In the present invention, the storage temperature of 4°C is used as the verification temperature and can be stable for 18 months. Detection shall be carried out at 0, 3, 6, 9, 12, 15, 18, and 20 months of storage, with each detection repeated 3 times. Trend analysis shall be carried out according to Table 16, and the change trend shall not be significant. The results are shown in Table 20.

[0143] Table 17: Results of Expiry Stability Test of Quality Control Sample 3

[0144]

[0145] Table 18: Results of Expiry Stability Test of Reference Standard 4

[0146]

[0147]

[0148] Table 19: Results of Expiry Stability Test of Reference Standard 5

[0149]

[0150] Table 20: Results of Expiry Stability Test of Lyophilized Powder Sample 1

[0151]

[0152]

[0153] As can be seen from the results in Tables 17 to 20, quality control sample 3 and lyophilized powder sample 1 are stable within the expiry period under the corresponding storage conditions; while for reference standard 4 and reference standard 5, when stored for 12 months, some biomarkers showed an obvious decreasing trend, and when stored for 13 months, almost all biomarkers decreased significantly, indicating insufficient stability of reference standard 4 and reference standard 5.

[0154] 2. Freeze-thaw Stability

[0155] The freeze-thaw stability study was conducted on quality control product sample 3, freeze-dried powder sample 1, reference standard 4, and reference standard 5. Quality control products in this field can be repeatedly frozen and thawed at least 3 times. Detection was carried out respectively after 0, 1, 2, and 3 times of freeze-thawing, with each detection repeated 3 times. Trend analysis was performed according to Table 16, and the change trend should not be significant. The results are shown in Tables 21 and 22.

[0156] Table 21: Results of Freeze-thaw Stability Test (Quality Control Product Sample 3 and Freeze-dried Powder Sample 1)

[0157]

[0158] Table 22: Results of Freeze-thaw Stability Test (Reference Standard 4 and Reference Standard 5)

[0159]

[0160]

[0161] As can be seen from the results of Tables 21 and 22, the quality of quality control product sample 3 and freeze-dried powder sample 1 remains stable after being repeatedly frozen and thawed 3 times; while for reference standard 4 and reference standard 5, after being repeatedly frozen and thawed 2 times, some biomarkers showed an obvious decreasing trend, and after being repeatedly frozen and thawed 3 times, all biomarkers decreased significantly, indicating insufficient freeze-thaw stability.

[0162] 3. Refrigerated Stability

[0163] The refrigerated stability study was conducted on quality control product sample 3, freeze-dried powder sample 1, reference standard 4, and reference standard 5. They were stored stably at 2℃ - 8℃ for 9 days respectively. Detection was carried out respectively on days 0, 1, 3, 5, 7, and 9 after refrigeration, with each detection repeated 3 times. Trend analysis was performed according to Table 16, and the change trend should not be significant. The results are shown in Tables 23 - 26.

[0164] Table 23: Results of Refrigerated Stability Test (Quality Control Product Sample 3)

[0165] Marker Refrigerated for 0 days Refrigerated for 1 day Refrigerated for 3 days Refrigerated for 5 days Refrigerated for 7 days Refrigerated for 9 days Aβ40 —— 0.895 0.569 0.668 0.452 0.325 Aβ42 —— 0.789 0.852 0.623 0.589 0.458 NfL —— 0.698 0.781 0.589 0.458 0.521 GFAP —— 0.745 0.775 0.625 0.568 0.485 BD-Tau —— 0.852 0.685 0.745 0.624 0.589 pTau-181 —— 0.568 0.753 0.662 0.452 0.512 pTau-231 —— 0.741 0.723 0.641 0.523 0.325 pTau-212 —— 0.456 0.787 0.598 0.369 0.338 pTau-217 —— 0.885 0.621 0.598 0.412 0.458 pTau-205 —— 0.695 0.552 0.478 0.538 0.514 Tau —— 0.745 0.623 0.432 0.352 0.443

[0166] Table 24: Results of Refrigerated Stability Test (Freeze-dried Powder Sample 1)

[0167]

[0168]

[0169] Table 25: Results of Refrigerated Stability Test (Reference Standard 4)

[0170] Marker Refrigerated for 0 days Refrigerated for 1 day Refrigerated for 3 days Refrigerated for 5 days Refrigerated for 7 days Refrigerated for 9 days Aβ40 —— 0.263 0.158 0.056 0.023 0.036 Aβ42 —— 0.569 0.235 0.125 0.045 0.012 NfL —— 0.623 0.338 0.036 0.038 0.025 GFAP —— 0.751 0.521 0.025 0.045 0.033 BD-Tau —— 0.523 0.263 0.015 0.033 0.014 pTau-181 —— 0.395 0.156 0.095 0.038 0.036 pTau-231 —— 0.485 0.235 0.087 0.047 0.025 pTau-212 —— 0.585 0.336 0.036 0.045 0.005 pTau-217 —— 0.652 0.125 0.205 0.044 0.012 pTau-205 —— 0.369 0.085 0.036 0.025 0.005 Tau —— 0.448 0.097 0.025 0.008 0.022

[0171] Table 26: Results of Cold Storage Stability Test (Reference Standard 5)

[0172]

[0173]

[0174] As can be seen from the results in Tables 23 to 26, the quality of Quality Control Sample 3 and Lyophilized Powder Sample 1 remains stable after 9 days of cold storage; while for Reference Standard 4 and Reference Standard 5, after 5 days of cold storage, some biomarkers showed an obvious decreasing trend, and after 7 days of cold storage, all biomarkers decreased significantly, indicating insufficient cold storage stability.

[0175] Application Effect

[0176] The composite quality control product prepared by the present invention can be used to prepare a diagnostic kit for Alzheimer's disease, and can also be used for the inspection of an inspection system including instruments, reagents and calibrators.

[0177] Quality Control Sample 3 contains 11 biomarkers and has stable quality. The following takes Quality Control Sample 3 as an example for illustration:

[0178] Case 1: If the quality control of pTau-217 shows out-of-control at multiple levels, whether it is an increase or a decrease, while the levels of other biomarkers are within the quality control range, it indicates that there may be a malfunction in the pTau-217 detection system including calibrators, reagents and analyzers.

[0179] It can be checked and processed according to the following steps:

[0180] Step 1: Preliminary verification: Repeatedly detect the pTau-217 quality control product to verify whether the out-of-control phenomenon is consistent;

[0181] Step 2: Detailed troubleshooting: If the repeated detection results are still out of control, the following aspects can be checked:

[0182] Confirm whether the last calibration is within the validity period;

[0183] Check the expiration dates of the calibrator and the quality control product to ensure they are not expired;

[0184] Check whether all the reagents and consumables used are within the validity period and whether the storage conditions meet the requirements;

[0185] Evaluate the operating status of the analyzer, including maintenance records and recent performance test results;

[0186] Step 3: Problem solving, recalibration and re-inspection:

[0187] Once specific problems are identified, corresponding corrective measures should be taken. After the problems are solved, a comprehensive recalibration of the pTau-217 detection system should be carried out to ensure that the system performance is restored to the best state;

[0188] Use quality control samples for re-detection to confirm that the system has returned to normal and is within the quality control range.

[0189] Situation 2: If there are out-of-control phenomena at multiple levels for Aβ42, NfL, GFAP, BD-Tau, pTau-181, and pTau-231, whether it is an increase or a decrease, that is, multiple items have problems at multiple levels. This situation indicates that there may be serious problems with the entire detection system. A comprehensive inspection and adjustment of the detection system, including instruments, reagents, operating procedures, etc., are required to eliminate all potential failure points and ensure the system resumes normal operation.

[0190] Situation 3: If only the quality control sample of pTau-217 shows an out-of-control phenomenon at a low level, this problem is more likely to be caused by accidental errors. It can be observed continuously to see if a similar bias occurs again, or a re-inspection can be carried out to confirm whether there are systematic problems.

Claims

1. Alzheimer's disease combined quality control product, characterized in that, Each 100 mL of the quality control product contains matrix, composite biomarker, 10 mg - 50 mg of blocker, protective agent, 0.5 g - 5 g of chloride, 1 g - 5 g of buffer salt, and 0.01 g - 0.1 g of preservative, with the pH value controlled within the range of 6.0 - 8.0, and made up to volume with purified water; the composite biomarker is any one or combination of Alzheimer's disease biomarker recombinant protein or β-amyloid polypeptide pure product solution standard; the blocker is HBR-H1; the protective agent in each 100 mL of the quality control product consists of 1 g - 10 g of polyethylene glycol, 0.5 g - 5 g of surfactant, 0.5 g - 5 g of saccharide, 0.5 mL - 5 mL of 0.5 M EDTA solution, and 0.1 g - 1.0 g of EGTA; the matrix in each 100 mL of the quality control product is any one of 60 g - 90 g of human plasma, 60 g - 90 g of artificial plasma, 0.1 g - 2 g of bovine serum albumin, and 0.1 g - 2 g of calf serum.

2. The Alzheimer's disease composite quality control product according to claim 1, wherein The Alzheimer's disease biomarker recombinant protein is NfL, GFAP, BD-Tau, pTau-181, pTau-231, pTau-212, pTau-205, pTau-217, and Tau; the β-amyloid polypeptide pure product solution standard is Aβ40 and Aβ42.

3. The Alzheimer's disease composite quality control product according to claim 2, wherein The quality control product is divided into high-level concentration, medium-level concentration, and low-level concentration quality control products; in the high-level concentration quality control product, when the quality control product contains Alzheimer's disease biomarker recombinant protein or β-amyloid polypeptide pure product solution standard, the concentration range of NfL is 22 pg / mL - 40 pg / mL, the concentration range of GFAP is 1000 pg / mL - 3500 pg / mL, the concentration range of BD-Tau is 12 pg / mL - 20 pg / mL, the concentration range of pTau-181 is 4 pg / mL - 8 pg / mL, the concentration range of pTau-231 is 4.0 pg / mL - 10 pg / mL, the concentration range of pTau-212 is 4 pg / mL - 8 pg / mL, the concentration range of pTau-205 is 6 pg / mL - 12 pg / mL, the concentration range of pTau-217 is 0.6 pg / mL - 1.2 pg / mL, the concentration range of Tau is 18 pg / mL - 40 pg / mL, the concentration range of Aβ40 is 80 pg / mL - 150 pg / mL, and the concentration range of Aβ42 is 20 pg / mL - 50 pg / mL; In the medium-level concentration quality control product, when the quality control product contains the Alzheimer's disease biomarker recombinant protein or the β-amyloid polypeptide pure product solution standard, the concentration range of NfL is 15 pg / mL to 20 pg / mL, the concentration range of GFAP is 150 pg / mL to 300 pg / mL, the concentration range of BD-Tau is 6 pg / mL to 10 pg / mL, the concentration range of pTau-181 is 1 pg / mL to 3 pg / mL, and the concentration range of pTau-231 is 1.5 pg / mL to 3.0 pg / mL. The concentration range of pTau-212 is 1 pg / mL to 3 pg / mL, the concentration range of pTau-205 is 2 pg / mL to 5 pg / mL, the concentration range of pTau-217 is 0.3 pg / mL to 0.5 pg / mL, and the concentration range of Tau is 5 pg / mL to 15 pg / mL. The concentration range of Aβ40 is 15 pg / mL to 25 pg / mL, and the concentration range of Aβ42 is 5 pg / mL to 10 pg / mL. In the low-level concentration quality control product, when the quality control product contains the Alzheimer's disease biomarker recombinant protein or the β-amyloid polypeptide pure product solution standard, the concentration range of NfL is 8 pg / mL to 14 pg / mL, the concentration range of GFAP is 60 pg / mL to 120 pg / mL, the concentration range of BD-Tau is 1 pg / mL to 5 pg / mL, the concentration range of pTau-181 is 0.5 pg / mL to 0.9 pg / mL, the concentration range of pTau-231 is 0.5 pg / mL to 1.4 pg / mL, the concentration range of pTau-212 is 0.4 pg / mL to 0.8 pg / mL, the concentration range of pTau-205 is 0.5 pg / mL to 1.8 pg / mL, the concentration range of pTau-217 is 0.05 pg / mL to 0.2 pg / mL, the concentration range of Tau is 1 pg / mL to 3 pg / mL, the concentration range of Aβ40 is 5 pg / mL to 10 pg / mL, and the concentration range of Aβ42 is 1 pg / mL to 4 pg / mL.

4. The Alzheimer's disease composite quality control product according to claim 1, wherein The surfactant is any one of Triton X-100, Triton X-200, or Tween-20; the preservative is any one of sodium azide, sodium thimerosal, or Proclin 300; the chloride is sodium chloride or potassium chloride; the buffer salt is any one of HEPES, PBS, or Tris; the sugar is any one of sucrose, trehalose, or inulin; the polyethylene glycol is any one of PEG1000, PEG2000, PEG3000, or PEG4000.

5. The preparation method of the Alzheimer's disease composite quality control product according to any one of claims 1-4, characterized in that, The described preparation method is to first prepare a buffer solution using a buffer salt, then add an EDTA solution, EGTA, and a chloride, stir until dissolved, measure the pH value range. If the pH value is not within the range of 6.0 - 8.0, adjust it using a pH regulator, add sugars, polyethylene glycol, a surfactant, and a preservative, stir evenly, and finally add a matrix or a matrix solution prepared with a matrix, a blocker, and a composite biomarker. After stirring evenly, make up the volume to the target volume, filter through a membrane and then aliquot, and assign values to obtain a composite quality control product in liquid form; the solvent for preparing the buffer solution is purified water.

6. The preparation method of the Alzheimer's disease composite quality control product according to claim 5, wherein, The described preparation method further includes freeze-drying the composite quality control product in liquid form to obtain a composite quality control product in solid form; the conditions for the freeze-drying are: the cold trap temperature is -50°C to -30°C, the vacuum degree of the drying chamber is 1 Pa to 3 Pa, and the drying time is 10 h to 20 h; the rotation speed of the stirring is 800 r / min to 1500 r / min, and the stirring time is 2 min to 15 min.

7. The preparation method of the Alzheimer's disease composite quality control product according to claim 6, wherein, During the freeze-drying process, a freeze-drying excipient of 1% - 8% of the mass of the composite quality control product in liquid form can also be added.

8. Use of the Alzheimer's disease composite quality control product according to any one of claims 1-4, characterized in that, The described quality control product is used for preparing a diagnostic kit for Alzheimer's disease.

9. Use of the Alzheimer's disease composite quality control product according to any one of claims 1-4, characterized in that, The described quality control product is used for the inspection of an inspection system including an instrument, reagents, and calibrators.

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