Antinuclear antibody profile quality control product and preparation method and application thereof

Abstract

This application belongs to the field of in vitro diagnostic technology, specifically relating to an antinuclear antibody (ANA) profile quality control product, its preparation method, and its application. The preparation method of the ANA profile quality control product provided in this application includes: providing a plasma sample containing ANA antibodies as raw material, screening co-positive raw materials as materials to be concentrated; ultrafiltration concentration of the raw materials to be concentrated to prepare an antibody solution; and mixing the antibody solution with a protective solution to obtain the ANA profile quality control product. The main objective of this application is to provide an ANA profile composite quality control product whose analytical performance and stability meet the needs of clinical testing, and which is compatible with mainstream manufacturers' immunoblotting reagent platforms. It can cover 15 detection items of the ANA profile, and subsequent detection can be performed efficiently and quickly using immunoblotting to achieve the purpose of quality control of the ANA profile composite items.

Description

Technical Field

[0001] This application belongs to the field of in vitro diagnostic technology, specifically relating to an antinuclear antibody profile quality control product, its preparation method, and its application. Background Technology

[0002] Antinuclear antibodies (ANA), also known as antinuclear antigen antibodies, are a series of antibodies targeting components of the cell nucleus, collectively referred to as the antinuclear antibody spectrum. The cell nucleus plays a crucial regulatory role in cellular physiological activities, while ANA can damage DNA, RNA, proteins, and other substances within the nucleus, thereby affecting cellular physiological functions and potentially inducing various autoimmune-related diseases. The antinuclear antibody spectrum includes antibodies against double-stranded DNA, Sm, SSA, Ro-52, nucleosomes, histones, ribonucleoprotein 70, SS-B, Jo-1, Scl-70, ribosomal P protein, centromere, proliferating cell nuclear antigens, PM-Scl, and mitochondrial M2 antibodies, among others.

[0003] Traditional techniques for preparing quality control products involve processing plasma samples containing different antibodies against extractable nuclear antigens (ENA), including anti-SSA, anti-SSB, anti-Sm, anti-RNP70, anti-Jo1, and anti-Scl70, to obtain corresponding antibody solutions. Then, different amounts of each antibody are mixed with a protective solution according to the target concentration to obtain a composite quality control product against ENA. Currently, this preparation process only covers six antibody profiles for ENA: SSA, SSB, Sm, RNP70, Jo1, and Scl-70. However, the antinuclear antibody profile includes not only ENA but also multiple other antibody profiles such as Nuc, His, Rib P0, CENP, Ro-52, PCNA, AMA-M2, PM-Scl, and ds-DNA. Therefore, existing quality control products against extractable nuclear antigens cannot cover most of the antinuclear antibody profiles and cannot simultaneously monitor all analytes in a single bottle of quality control product, potentially affecting the accuracy of quality control in the diagnosis of autoimmune diseases.

[0004] Furthermore, the anti-ENA autoantibody composite quality control only reduces interference from impurities such as small organic molecules, fibrin, and lipoproteins; it does not increase the antibody concentration in the plasma sample. If the concentration of a certain antibody in the obtained plasma sample is low, and the quality control needs to be composited with 15 items, this processing method will have significant limitations, resulting in insufficient coverage of antinuclear antibody profiles. As clinical reagent selection increasingly favors reagents with higher composite specifications to obtain more comprehensive and abundant diagnostic indicators, the current composite quality control is insufficient to meet clinical quality control needs.

[0005] Secondly, the stability of antinuclear antibody profile quality control in traditional technologies is generally poor, with stability after opening less than 30 days and freeze-thaw cycles less than 3 times. The sample volume required for immunoblotting is also relatively small, only 10 μL. Therefore, the shelf life of quality control is crucial and is an urgent market demand.

[0006] Finally, the antinuclear antibody profile control products in traditional technologies all need to be used with specific reagents and cannot be used with third-party testing reagents outside of the matching platform. These issues have imposed certain limitations on clinical use. Summary of the Invention

[0007] Based on this, one embodiment of this application provides a method for preparing antinuclear antibody profile quality control material. The antinuclear antibody profile quality control material prepared by this method has high accuracy and coverage, and can be adapted to multiple platforms.

[0008] This application provides a method for preparing an antinuclear antibody profile quality control, comprising:

[0009] Using plasma samples containing antinuclear antibodies as the raw material to be concentrated; the raw material to be concentrated is subjected to ultrafiltration to prepare an antibody solution; and

[0010] The antibody solution was mixed with a protective solution to prepare an antinuclear antibody profile quality control.

[0011] In one embodiment, the process further includes screening for co-positive raw materials to be concentrated before ultrafiltration concentration. In one embodiment, the process further includes a filtration step before ultrafiltration concentration.

[0012] The filtration process involves passing the raw material to be concentrated sequentially through filter screens with diameters of 2.8 μm to 3.2 μm, 1 μm to 1.4 μm, 0.6 μm to 1 μm, and 0.4 μm to 0.5 μm.

[0013] In one embodiment, the conditions for ultrafiltration concentration include:

[0014] Ultrafiltration was performed using an ultrafiltration vessel of 90kd to 110kd at a temperature of 3.5℃ to 4.5℃ and a rotation speed of 5800rpm to 6200rpm for 18min to 22min. The filtrate was collected and the process was repeated until the volume of the concentrated antibody solution was 1 / 4 to 1 / 6 of the volume of the raw material to be concentrated before filtration.

[0015] In one embodiment, the protective liquid comprises:

[0016] 45mM to 55mM MOPS buffer and 0.85% (w / v) to 0.95% (w / v) neutral salt, stabilizer and preservative.

[0017] The neutral salt includes one or both of sodium chloride and sodium sulfate;

[0018] The stabilizers include 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12 mM to 14 mM sodium EDTA-2, 6% (w / v) to 10% (w / v) mannitol, 5% (w / v) to 2.5% (w / v) trehalose, 0.05% (w / v) to 0.15% (w / v) Triton X-100, and 0.8% (w / v) to 2% (w / v) AEP-HBC; and / or

[0019] The preservatives include 0.04% (w / v) to 0.06% (w / v) PC300 and 0.04% (w / v) to 0.06% (w / v) gentamicin sulfate.

[0020] In one embodiment, the following steps are also included:

[0021] The antinuclear antibody profile quality control sample was freeze-dried to obtain a dry powder quality control sample.

[0022] In one embodiment, the freeze-drying process includes:

[0023] Cool to -12℃ to -8℃ in 4-6 minutes and hold for 40-50 minutes; cool to -55℃ to -45℃ in 4-6 minutes and hold for 85-90 minutes; cool to -30℃ to -20℃ in 4-6 minutes and hold for 25-35 minutes; cool to -60℃ to -50℃ in 4-6 minutes and hold for 380-420 minutes; and

[0024] Heat to -35℃ to -25℃ for 45 to 55 minutes, hold for 1300 to 1400 minutes, and maintain a vacuum of 10 Pa to 12 Pa.

[0025] Heat to -15℃ to -5℃ for 45-55 minutes, hold for 380-400 minutes, vacuum degree 8 Pa to 10 Pa; heat to 5℃ to 15℃ for 45-55 minutes, hold for 400-500 minutes, vacuum degree 8 Pa to 10 Pa.

[0026] In one embodiment, the antinuclear antibody includes anti-double-stranded DNA antibody, anti-Sm antibody, anti-SSA antibody, anti-Ro-52 antibody, anti-nucleosome antibody, anti-histone antibody, anti-ribonucleoprotein 70 antibody, anti-SS-B antibody, anti-Jo-1 antibody, anti-Scl-70 antibody, anti-ribosomal P protein antibody, anti-centromere antibody, anti-proliferating cell nuclear antigen antibody, anti-PM-Scl antibody, and anti-mitochondrial antibody M2 type antibody.

[0027] In another aspect, this application provides an antinuclear antibody profile quality control material, which is prepared according to the above-described method for preparing antinuclear antibody profile quality control materials.

[0028] This application also provides an antinuclear antibody detection kit, the kit comprising an immunoblotting detection reagent and the aforementioned antinuclear antibody profile quality control.

[0029] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is the process flow for quality control products; among them, the hexagon represents a 100,000-level production workshop, the rhombus represents a 10,000-level production workshop, and the rectangle represents a general production area;

[0032] Figure 2 For quality control samples, use YHLO reagent to test the bands;

[0033] Figure 3 For quality control samples, use Euromon reagent to test the bands;

[0034] Figure 4 Test strips are used on the new platform for quality control products. Detailed Implementation

[0035] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] the term

[0038] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0039] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0040] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0042] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0043] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0044] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0045] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0046] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0047] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0048] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0049] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0050] The main purpose of this application is to provide an antinuclear antibody profile composite quality control product that meets the needs of clinical testing in terms of analytical performance and stability, and is compatible with the immunoblotting reagent platforms of mainstream manufacturers. It can cover 15 detection items of antinuclear antibody profile, and subsequent detection can be carried out efficiently and quickly by immunoblotting, so as to achieve the purpose of quality control of antinuclear antibody profile composite items.

[0051] This application provides a method for preparing an antinuclear antibody profile quality control, comprising:

[0052] Using plasma samples containing antinuclear antibodies as the raw material to be concentrated; the raw material to be concentrated is subjected to ultrafiltration to prepare an antibody solution; and

[0053] The antibody solution was mixed with a protective solution to prepare an antinuclear antibody profile quality control.

[0054] This application innovatively applies ultrafiltration concentration to the concentration of serum raw materials, effectively increasing the concentration of serum antibodies. It also determines the optimal ultrafiltration conditions and methods based on the characteristics of antinuclear antibody raw materials, solving the problem that some raw materials cannot reach the required concentration.

[0055] In one specific example, the process of ultrafiltration concentration includes screening for co-positive raw materials.

[0056] In one specific example, a filtration step is included before ultrafiltration concentration;

[0057] The filtration process involves passing the raw material to be concentrated sequentially through filter screens with diameters of 2.8 μm to 3.2 μm, 1 μm to 1.4 μm, 0.6 μm to 1 μm, and 0.4 μm to 0.5 μm.

[0058] To ensure that the intrinsically controlled samples closely resemble clinical samples in terms of reaction consistency during testing, some items use human-derived positive materials as raw materials. The human-derived positive materials corresponding to each item in the antinuclear antibody spectrum are filtered sequentially in the order of 3μm-1.2μm-0.8μm-0.45μm to remove precipitates and large particulate impurities from the positive serum.

[0059] In a specific example, the conditions for ultrafiltration concentration include:

[0060] Ultrafiltration was performed using an ultrafiltration vessel of 90kd to 110kd at a temperature of 3.5℃ to 4.5℃ and a rotation speed of 5800rpm to 6200rpm for 18min to 22min. The filtrate was collected and the process was repeated until the volume of the concentrated antibody solution was 1 / 4 to 1 / 6 of the volume of the raw material to be concentrated before filtration.

[0061] The filtered positive material was concentrated using an ultrafiltration tube (100 kDa). Analysis of the subclassified liquid revealed no target antibody, indicating that the 100 kDa ultrafiltration tube effectively retained the target antibody. The ultrafiltration conditions were 4°C and 6000 rpm. After 20 minutes, the ultrafiltered positive material was collected and mixed. This ultrafiltration process was repeated until the volume of the concentrated positive material was 1 / 4 to 1 / 6 of its original volume. This method effectively increased the concentration of the positive material by 2-5 times. In contrast, the common method of pretreatment of raw materials, such as lyophilization to remove moisture, results in significant losses, and the concentration increase after concentration is only about 1.5 times. Furthermore, reconstitution is difficult, which may affect the quality of subsequent quality control products.

[0062] Secondly, ultrafiltration concentration improves the usability of positive materials and reduces the feed ratio of low-concentration positive materials for some items, making it possible to perform multiple composites. The concentrated antinuclear antibody spectrum of human positive materials was serially diluted and verified on three mainstream immunoblotting reagent platforms. The acceptable standard is that after a 30-fold dilution, the gray value of the membrane strip measured on each manufacturer's immunoblotting reagent platform is more than three times the weak positive threshold.

[0063] Optionally, the protective solution includes: 45mM to 55mM MOPS buffer and 0.85% (w / v) to 0.95% (w / v) of neutral salt, stabilizer and preservative.

[0064] The neutral salt includes one or both of sodium chloride and sodium sulfate. Neutral salts can increase the surface charge of protein molecules, enhance the interaction between protein molecules and water molecules, and increase the solubility of proteins in aqueous solutions through the salt-dissolving effect.

[0065] Further optionally, the stabilizer includes 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12 mM to 14 mM sodium EDTA-2, 6% (w / v) to 10% (w / v) mannitol, 1.5% (w / v) to 2.5% (w / v) trehalose, 0.05% (w / v) to 0.15% (w / v) Triton X-100, 0.8% (w / v) to 1.2% (w / v) AEP-HBC; and / or

[0066] The preservatives include 0.04% (w / v) to 0.06% (w / v) PC300 and 0.04% (w / v) to 0.06% (w / v) gentamicin sulfate.

[0067] This application uses MOPS sodium salt as a buffer system; EDTA-2 sodium reduces interference from other cations in the system and provides specific binding; bovine serum albumin serves as a support for the lyophilized product and also mimics the effect of human serum matrix; mannitol and trehalose help fix the morphology of the quality control, effectively preventing the lyophilized product from collapsing and resulting in a loose crystalline structure after lyophilization; PC300 and gentamicin sulfate are used as dual preservatives. Since the single-use volume of this quality control product is only 10 μL and most of the antibodies are plasma products, the addition of two preservatives greatly improves the long-term stability of the quality control product.

[0068] In a specific example, the step further includes: freeze-drying the liquid quality control sample to obtain a dry powder quality control sample.

[0069] Optionally, the freeze-drying process includes:

[0070] Cool to -12℃ to -8℃ in 4-6 minutes and hold for 40-50 minutes; cool to -55℃ to -45℃ in 4-6 minutes and hold for 85-90 minutes; cool to -30℃ to -20℃ in 4-6 minutes and hold for 25-35 minutes; cool to -60℃ to -50℃ in 4-6 minutes and hold for 380-420 minutes; and

[0071] Heat to -35℃ to -25℃ for 45-55 minutes, hold for 1300-1400 minutes, vacuum degree 10 Pa to 12 Pa; heat to -15℃ to -5℃ for 45-55 minutes, hold for 380-400 minutes, vacuum degree 8 Pa to 10 Pa; heat to 5℃ to 15℃ for 45-55 minutes, hold for 400-500 minutes, vacuum degree 8 Pa to 10 Pa.

[0072] This application optimizes the freeze-drying process. Because the raw materials involved in this study are numerous and complex in composition, problems such as freeze-drying collapse or unqualified reconstitution stability are likely to occur. The optimized freeze-drying process further ensures the appearance and performance of the product.

[0073] In the improved freeze-drying process, extending the sublimation drying time effectively avoids the problems of localized product concentration and poor solubility; extending the desorption drying time reduces the moisture content of the freeze-dried quality control products. Lowering the heating temperature and increasing the vacuum degree of the freeze-drying chamber prevents the product from shrinking and bubbling. The improved freeze-drying process maintains the biological activity and physicochemical properties of the quality control products, resulting in a loose crystalline structure that is beneficial for long-term preservation.

[0074] In a specific example, the antinuclear antibody includes anti-double-stranded DNA antibody, anti-Sm antibody, anti-SSA antibody, anti-Ro-52 antibody, anti-nucleosome antibody, anti-histone antibody, anti-ribonucleoprotein 70 antibody, anti-SS-B antibody, anti-Jo-1 antibody, anti-Scl-70 antibody, anti-ribosomal P protein antibody, anti-centromere antibody, anti-proliferating cell nuclear antigen antibody, anti-PM-Scl antibody, and anti-mitochondrial antibody M2 type antibody.

[0075] In another aspect, this application provides an antinuclear antibody profile quality control material, which is prepared according to the method for preparing the antinuclear antibody profile quality control material.

[0076] This application also provides an antinuclear antibody detection kit, the kit comprising an immunoblotting detection reagent and the aforementioned antinuclear antibody profile quality control material.

[0077] Increasing the number of antibody types can improve coverage, but it also presents several challenges, such as the initial collection and processing of raw materials, co-positive interference between different items, how to ensure the analytical performance, stability, and compatibility of all items, and how to ensure the product's appearance meets requirements when using multiple raw materials. Ensuring product performance is a problem that needs to be addressed.

[0078] Based on this, the antinuclear antibody profile quality control product 15S (immunoblotting method) prepared in this application overcomes the difficulties of insufficient serum raw material concentration or excessively high feed ratios for some items by using ultrafiltration pretreatment of raw materials. Simultaneously, it utilizes co-positive reactions between raw materials during preparation, enabling the composite quality control to cover 15 antibody items in the antinuclear antibody profile: anti-double-stranded DNA antibody, anti-Sm antibody, anti-SSA antibody, anti-Ro-52 antibody, anti-nucleosome antibody, anti-histone antibody, anti-ribonucleoprotein 70 antibody, anti-SS-B antibody, anti-Jo-1 antibody, anti-Scl-70 antibody, anti-ribosomal P protein antibody, anti-centromere antibody, anti-proliferating cell nuclear antigen antibody, anti-PM-Scl antibody, and anti-mitochondrial antibody M2 type antibody. This application provides a more comprehensive testing indicator for the clinical quality control of the antinuclear antibody profile.

[0079] Furthermore, the diluent selected in this application uses MOPS sodium salt as a buffer system. EDTA-2 sodium, bovine serum albumin, mannitol, and trehalose are added to provide support and protection for the analytes. PC300 and gentamicin sulfate are added as dual preservatives to ensure the long-term stability of the quality control samples. The addition of the surfactant TRITON™ X-100 and the protein protectant AEP-HBC effectively improves the reconstitution stability of the quality control samples. The diluent composition of this application ensures the stability of the quality control samples under different conditions.

[0080] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0081] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0082] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0083] Example 1

[0084] One embodiment of this application provides a method for preparing an antinuclear antibody profile quality control sample. The complete preparation and testing steps are as follows:

[0085] 1. Pretreatment of raw materials

[0086] The antinuclear antibody (ANA) positive material was filtered sequentially in the order of 3μm-1.2μm-0.8μm-0.45μm to remove impurities and ensure a clear and transparent appearance. The filtered positive material was then serially diluted (starting at 30-fold) to confirm co-positivity and concentration. Materials with concentrations lower than the standard (more than three times the weak positive threshold value measured on the membrane strip grayscale test platform after 30-fold dilution) were subjected to ultrafiltration concentration. The ultrafiltration conditions were 4℃ and 6000rpm. After 20 minutes, the ultrafiltered positive material was collected and mixed thoroughly. This ultrafiltration process was repeated until the volume of the concentrated positive material was 1 / 4 to 1 / 6 of the original volume, completing the concentration process.

[0087] 2. Preparation of protective solution

[0088] Sodium chloride, sodium EDTA-2, PC300, gentamicin sulfate, bovine serum albumin, AEP-HBC, mannitol, trehalose, and Triton X-100 were added sequentially to MOPS buffer with a pH range of 7.4 ± 0.1.

[0089] The components contained in the following amounts: 50 mM MOPS buffer, 0.9% (w / v) sodium chloride, 4% (w / v) bovine serum albumin, 13 mM sodium EDTA-2, 8% (w / v) mannitol, 2% (w / v) trehalose, 0.1% (w / v) Triton X-100, 1% (w / v) AEP-HBC; 0.05% (w / v) PC300, and 0.05% (w / v) gentamicin sulfate.

[0090] 3. Preparation and freeze-drying of quality control products

[0091] Mix the positive material solution from step 1 with the protective solution obtained in step 2 in a specific ratio, and preferentially add the positive material solution with multiple co-positives to ensure that the gray value measured on the YHLO immunoblotting reagent platform is more than three times the weak positive threshold. For items that do not meet this standard, supplement with single positive raw materials until all items meet the requirements.

[0092] The prepared ANA quality control sample was dispensed into lyophilized bottles and freeze-dried. The procedure included:

[0093] Cool to -10℃ in 5 minutes and hold for 45 minutes; cool to -50℃ in 5 minutes and hold for 90 minutes; cool to -25℃ in 5 minutes and hold for 30 minutes; cool to -55℃ in 5 minutes and hold for 400 minutes; and

[0094] Heat to -30℃ in 50 minutes, hold for 1350 minutes, vacuum degree 10pa~12pa;

[0095] Heat to -10℃ in 50 minutes and hold for 390 minutes, with a vacuum of 8 Pa to 10 Pa; heat to 10℃ in 50 minutes and hold for 450 minutes, with a vacuum of 8 Pa to 10 Pa.

[0096] After the freeze-drying process is completed and the samples are collected, the moisture content of the quality control samples is analyzed using a cassette moisture analyzer.

[0097] 4. Testing

[0098] The prepared quality control samples were tested using an antinuclear antibody profiling kit 15S (immunoblotting method) matched with the YHLO Automated Immunoblotting Analyzer, and detection systems from Kexin and Euromon. The acceptable standard for comparing the test results from different manufacturers' immunoblotting reagent platforms was that the membrane strip grayscale value was more than three times the weak positive cutoff value.

[0099] Example 2

[0100] 1. Pretreatment of raw materials

[0101] This application uses ultrafiltration to concentrate positive materials, improving the utilization rate of low-concentration raw materials. To ensure that the intrinsic control samples more closely resemble clinical samples in terms of reaction consistency during testing, some items use human-derived positive materials. The human-derived positive materials corresponding to each item in the antinuclear antibody spectrum are filtered sequentially in the order of 3μm-1.2μm-0.8μm-0.45μm to remove precipitates and large particulate impurities from the positive serum.

[0102] The filtered positive material was concentrated using an ultrafiltration tube (100 kDa). Analysis of the subclassified liquid revealed no target antibody, indicating that the 100 kDa ultrafiltration tube effectively retained the target antibody. The ultrafiltration conditions were 4°C and 6000 rpm. After 20 minutes, the ultrafiltered positive material was collected and mixed thoroughly. This ultrafiltration process was repeated until the volume of the concentrated positive material was 1 / 4 to 1 / 6 of its original volume. This method effectively increased the concentration of the positive material by 2–5 times. In contrast, the common method of pretreatment of raw materials, such as lyophilization to remove moisture, results in significant losses, and the concentration increase after concentration is only about 1.5 times. Furthermore, reconstitution is difficult, which may affect the quality of subsequent quality control products.

[0103] Ultrafiltration concentration improves the usability of positive materials, reduces the feed ratio of low-concentration positive materials for some items, and makes it possible to perform multiple composite tests. The concentrated antinuclear antibody spectrum of human positive materials was serially diluted and validated on three mainstream immunoblotting reagent platforms. The acceptable standard is that after a 30-fold dilution, the gray value of the membrane strip measured on each manufacturer's immunoblotting reagent platform is more than three times the weak positive threshold.

[0104] 2. Screening of quality control matrix formulations: Improving the stability of quality control.

[0105] Positive control matrix solution: MOPS sodium salt is used as a buffer system; EDTA-2 sodium reduces interference from other cations in the system and provides specific binding; bovine serum albumin serves as both a support for the lyophilized product and a matrix that mimics human serum; mannitol and trehalose help fix the morphology of the control, effectively preventing collapse of the lyophilized product and resulting in a loose crystalline structure after lyophilization; PC300 and gentamicin sulfate are used as dual preservatives. Since the single-use volume of this control is only 10 μL and most of the antibodies are plasma products, the addition of two preservatives greatly improves the long-term stability of the control.

[0106] To further improve the stability of the quality control products, four formulations were validated.

[0107] Formula 1 is a positive control matrix solution without the addition of TRITON(TM)X-100 and protein protectant AEP-HBC.

[0108] Formula 2 is a positive control matrix solution with added TRITON(TM)X-100.

[0109] Formula 3 is a positive control matrix solution with added protein protectant AEP-HBC.

[0110] Formula 4 is a positive control matrix solution containing both TRITON(TM)X-100 and the protein protectant AEP-HBC.

[0111] The specific steps are as follows:

[0112] The matrix solution consisted of 50 mM Mops sodium salt buffer, 0.9% (w / v) sodium chloride, and stabilizers: 4% (w / v) bovine serum albumin, 13 mM EDTA-2 sodium, 8% (w / v) mannitol, 2% (w / v) trehalose, 0.1% (w / v) Triton X-100, and 1% (w / v) AEP-HBC.

[0113] Preservatives: 0.05% (w / v) PC300, 0.05% (w / v) gentamicin sulfate, weighed according to the content of each component.

[0114] First, add the Mops buffer, sodium chloride, and 800 mL of pure water. After the materials dissolve, measure the pH of the solution. The pH should be within the range of 7.4 ± 0.5. Then, add PC300, gentamicin sulfate, bovine serum albumin, sodium EDTA-2, mannitol, trehalose, TRITON™ X-100, and protein protectant AEP-HBC sequentially. Make up the volume to 1 L with pure water, stir well, and measure the pH of the solution. The final pH should be within the range of 7.4 ± 0.1. The stability verification comparisons of different formulations are shown in Tables 1 to 4.

[0115] Table 1

[0116]

[0117]

[0118] Table 2

[0119]

[0120]

[0121] Table 3

[0122]

[0123]

[0124] Table 4

[0125]

[0126]

[0127] Experimental results show that the simultaneous addition of TRITON(TM)X-100 and the protein protectant AEP-HBC can effectively improve the reconstitution stability of the quality control samples, ensuring that the 30-day reconstitution stability of all test items meets the requirements (grayscale value deviation from the control is less than 10%). TRITON(TM)X-100 is a surfactant that can maintain protein stability by decomposing proteases, while the addition of the protein protectant AEP-HBC inhibits protein aggregation and precipitation, thereby protecting its structure and function.

[0128] Positive control materials: Prioritize the addition of positive materials with multiple co-positive items. Since the degree of co-positivity varies for different items, after adding co-positive raw materials, ensure that the grayscale measurement value on the Yahuilong immunoblotting reagent platform is more than three times the weak positive threshold. For items that do not meet this standard, supplement with single-positive raw materials until all items meet the requirements. By utilizing the co-positivity of raw materials, the utilization of raw materials can be maximized while meeting performance requirements, thereby reducing costs.

[0129] Negative control: Human serum containing antinuclear antibody (ANA) profiles that are negative for all analytes.

[0130] 3. Freeze-dried quality control products:

[0131] Optimization of the freeze-drying process:

[0132] In the improved freeze-drying process, extending the sublimation drying time effectively avoids the problems of localized product concentration and poor solubility; extending the desorption drying time reduces the water content of the freeze-dried quality control samples. Lowering the heating temperature and increasing the vacuum level of the freeze-drying chamber prevents product shrinkage and bubbling. The improved freeze-drying process maintains the biological activity and physicochemical properties of the quality control samples, resulting in a loose crystalline structure that is beneficial for long-term preservation. The specific freeze-drying process is shown in Table 5.

[0133] Table 5: Novel and Conventional Freeze-Drying Procedures for Quality Control Products

[0134]

[0135] The prepared ANA quality control sample was dispensed into low-borosilicate brown glass vials using an electric pipette. The vials were sealed with pore-filled rubber stoppers and lyophilized using a novel lyophilization program in a freeze dryer. After lyophilization, the vials were capped under vacuum. After inspecting the appearance of the lyophilized quality control sample and confirming no issues, the moisture content was measured using a cassette moisture analyzer. The moisture content should be below 2%. The ANA quality control sample was reconstituted and then analyzed using the YHLO antinuclear antibody spectrum assay kit 15S (immunoblotting) on ​​an automated immunoblotting instrument to determine the grayscale value. The loss rate after lyophilization was calculated, and the specific results are shown in Table 6. A loss rate below 20% indicates that the lyophilization requirements are met.

[0136] Table 6: Verification of Freeze-Drying Loss Rate of Quality Control Products

[0137]

[0138] 4. Quality control product value assignment:

[0139] Samples were reconstituted and assigned values ​​in a standard workshop. The assignment steps were as follows: Three different models of immunoblotting instruments were prepared, and the ANA-positive quality control samples were tested using all three instruments for five consecutive days, four times per day. Outliers were removed according to the Grubbs criterion, with the number of outliers not exceeding 2.5%. After removing outliers, the mean (X) and standard deviation (SD) of the remaining data were calculated to determine the target range of the grayscale value for the quality control samples (X ± 3SD).

[0140] 5. Quality control of semi-finished products, assembly, finished product inspection, and warehousing:

[0141] After the value is assigned, a semi-finished product inspection is performed to check the accuracy and repeatability of the quality control product. If the semi-finished product inspection passes, the product is assembled and the finished product is inspected. If the finished product inspection passes, the finished product is put into storage; otherwise, it is treated as a defective product.

[0142] 6. Stability verification:

[0143] To simulate the performance of antinuclear antibody (ANA) profile quality control products after high-temperature aging, this application conducted accelerated stability verification of the product at 37°C for 14 days, with products stored at 2°C–8°C serving as controls. To verify the reconstituted stability of the ANA profile quality control products, this application stored the reconstituted product at 2°C–8°C, -20°C, and -80°C for 30, 60, and 90 days, respectively, with unreconstituted lyophilized products stored at 2°C–8°C serving as controls. Using the YHLO antinuclear antibody profile detection kit (immunoblotting method), the relative deviation of the grayscale values ​​of the ANA profile quality control products under different conditions compared to the control group was within ±10%, indicating that the ANA profile quality control products have good stability and can meet clinical needs.

[0144] 7. Multi-platform compatibility

[0145] like Figures 2-4 As shown, the product is compatible not only with the YHLON reagent platform, but also with the Euromon and Kexin reagent platforms. Figure 2 For quality control samples, use YHLO reagent to test the bands. Figure 3 The test strips were prepared using Euromon reagents for quality control. Figure 4 Test strips are used on the new platform for quality control products.

[0146] The embodiments described above merely illustrate several implementation methods of this application to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a quality control sample for antinuclear antibody spectrometry, characterized in that, include: Plasma samples containing antinuclear antibodies were used as the raw material for concentration. The raw material to be concentrated is subjected to ultrafiltration to prepare an antibody solution; as well as The antibody solution was mixed with a protective solution to prepare an antinuclear antibody profile quality control. The conditions for ultrafiltration concentration include: using an ultrafiltration vessel of 90kd~110kd at a temperature of 3.5℃~4.5℃, a rotation speed of 5800rpm~6200rpm, ultrafiltration for 18min~22min, collecting the filtrate, repeating the process until the volume of the concentrated antibody solution is 1 / 4~1 / 6 of the volume of the raw material to be concentrated before filtration, thus completing the ultrafiltration concentration. The protective liquid includes: 45mM~55mM MOPS buffer and 0.85% (w / v)~0.95% (w / v) neutral salt, stabilizer and preservative; The neutral salt includes one or both of sodium chloride and sodium sulfate; The stabilizers include 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12 mM to 14 mM sodium EDTA-2, 6% (w / v) to 10% (w / v) mannitol, 1.5% (w / v) to 2.5% (w / v) trehalose, 0.05% (w / v) to 0.15% (w / v) Triton X-100, and 0.8% (w / v) to 1.2% (w / v) AEP-HBC; The preservatives include 0.04% (w / v) to 0.06% (w / v) PC300 and 0.04% (w / v) to 0.06% (w / v) gentamicin sulfate; The antinuclear antibody profile quality control sample was freeze-dried to obtain a dry powder quality control sample; The freeze-drying process includes: Cool to -12℃ to -8℃ in 4-6 minutes and hold for 40-50 minutes; cool to -55℃ to -45℃ in 4-6 minutes and hold for 85-90 minutes; heat up to -30℃ to -20℃ in 4-6 minutes and hold for 25-35 minutes; cool to -60℃ to -50℃ in 4-6 minutes and hold for 380-420 minutes; and Heat to -35℃ to -25℃ for 45 to 55 minutes, hold for 1300 to 1400 minutes, and maintain a vacuum of 10 Pa to 12 Pa. Heat to -15℃ to -5℃ for 45-55 minutes, hold for 380-400 minutes, vacuum degree 8 Pa to 10 Pa; heat to 5℃ to 15℃ for 45-55 minutes, hold for 400-500 minutes, vacuum degree 8 Pa to 10 Pa. The antinuclear antibodies include: anti-dsDNA antibody, anti-nucleosome antibody, anti-histone antibody, anti-SmD1 antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-proliferating cell nuclear antigen antibody, anti-SSA / Ro60kD antibody, anti-SSA / Ro52kD antibody, anti-SSB / La antibody, anti-centromere B protein antibody, anti-Scl70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, and anti-mitochondrial M2 antibody.

2. The method for preparing the antinuclear antibody profile quality control product according to claim 1, characterized in that, Before ultrafiltration concentration, the process also includes screening for co-positive raw materials to be concentrated.

3. The method for preparing the antinuclear antibody profile quality control product according to claim 1, characterized in that, Ultrafiltration concentration also includes a filtration step; The filtration process involves passing the raw material to be concentrated sequentially through filter screens with diameters of 2.8 μm to 3.2 μm, 1 μm to 1.4 μm, 0.6 μm to 1 μm, and 0.4 μm to 0.5 μm.

4. A quality control product for antinuclear antibody profiles, characterized in that, The antinuclear antibody spectral control material was prepared according to any one of claims 1 to 3.

5. An antinuclear antibody detection kit, characterized in that, The kit includes an immunoblotting assay reagent and the antinuclear antibody profile quality control as described in claim 4.

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