Antinuclear antibody spectrum quality control product as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to an antinuclear antibody spectrum quality control product as well as a preparation method and application thereof. The preparation method of the antinuclear antibody spectrum quality control product comprises the following steps: providing a plasma sample containing an antinuclear antibody as a raw material, and screening a co-positive raw material as a to-be-concentrated raw material; performing ultrafiltration concentration on the to-be-concentrated raw material to prepare an antibody solution; and mixing the antibody solution with a protective solution to obtain the antinuclear antibody spectrum quality control product. The invention mainly aims to provide the antinuclear antibody spectrum composite quality control product with analysis performance and stability meeting clinical examination requirements, the antinuclear antibody spectrum composite quality control product is adaptive to an immunoblotting reagent platform of a mainstream manufacturer, 15 detection items of an antinuclear antibody spectrum can be covered by the antinuclear antibody spectrum composite quality control product, subsequent efficient and rapid detection can be performed through an immunoblotting method, and the detection efficiency is greatly improved. Therefore, the aim of controlling the quality of the antinuclear antibody spectrum composite project is fulfilled.

Description

Technical Field

[0001] The present application belongs to the field of in vitro diagnostic technology, and specifically relates to an antinuclear antibody spectrum quality control product and a preparation method and application thereof. Background Art

[0002] Antinuclear antibodies, also known as anti-nucleic acid antigen antibodies, are composed of a series of antibodies against cell nuclear components, collectively referred to as the antinuclear antibody spectrum. The cell nucleus plays an important regulatory role in the physiological activities of the cell, and antinuclear antibodies can damage DNA, RNA, proteins and other substances in the cell nucleus, thereby affecting the physiological functions of the cell, and may subsequently induce various autoimmune-related diseases. The antinuclear antibody spectrum includes anti-double-stranded DNA antibodies, anti-Sm antibodies, anti-SSA antibodies, anti-Ro-52 antibodies, anti-nucleosome antibodies, anti-histone antibodies, anti-ribonucleoprotein 70 antibodies, anti-SS-B antibodies, anti-Jo-1 antibodies, anti-Scl-70 antibodies, anti-ribosomal P protein antibodies, anti-centromere antibodies, anti-proliferating cell nuclear antigen antibodies, anti-PM-Scl antibodies, anti-mitochondrial antibodies M2 antibodies, etc.

[0003] In the preparation process of related quality control products, the traditional technology uses plasma samples of different antibodies including anti-extractable nuclear antigen antibodies, including anti-SSA antibodies, anti-SSB antibodies, anti-Sm antibodies, anti-RNP70 antibodies, anti-Jo1 antibodies and anti-Scl70 antibodies, to obtain the corresponding antibody solutions, and then take different amounts of each according to the target concentration and mix them with the protective solution to obtain the anti-ENA autoantibody composite quality control products. The current anti-ENA antibody spectrum of this preparation process only covers six antibody items, including SSA, SSB, Sm, RNP70, Jo1 and Scl-70. In addition to the anti-ENA antibody spectrum, the anti-nuclear antibody spectrum also includes Nuc, His, Rib P0, CENP, Ro-52, PCNA, AMA-M2, PM-Scl and ds-DNA. Therefore, the existing anti-extractable nuclear antigen antibody quality control products cannot cover most of the items of the anti-nuclear antibody spectrum, and it is impossible to monitor all analytes simultaneously with one bottle of quality control products, which may affect the accuracy of the quality control of autoimmune disease diagnosis.

[0004] Moreover, the anti-ENA autoantibody composite quality control only reduces the interference of organic small molecules, fibrin, lipoprotein and other impurities in the treatment of antibodies, and does not increase the concentration of antibodies in plasma samples. If the concentration of a certain antibody in the plasma sample is low and the quality control product needs to be composited with 15 items, this treatment method will have great limitations and insufficient coverage of antinuclear antibody spectrum items. Since the selection of clinical reagents will also tend to choose reagents with higher composite specifications in order to obtain more comprehensive and rich test indicators for diagnosis, the existing quality control composite degree is difficult to meet the clinical quality control needs.

[0005] Secondly, the stability of antinuclear antibody spectrum quality control in traditional technologies is relatively poor. The stability after opening is less than 30 days, the number of freeze-thaw cycles is less than 3 times, and the amount of sample used for testing by immunoblotting is relatively small, only 10 μL. Therefore, the shelf life of quality control is extremely important and is also an urgent need of the market.

[0006] Finally, the antinuclear antibody spectrum quality control products in traditional technologies must be used in conjunction with specific reagents and cannot be adapted to third-party detection reagents outside the supporting platform. These problems have imposed certain restrictions on clinical use. Summary of the invention

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

[0008] On the one hand, the present application provides a method for preparing an antinuclear antibody spectrum quality control product, comprising:

[0009] Using a plasma sample containing antinuclear antibodies as a raw material to be concentrated; performing ultrafiltration concentration on the raw material to be concentrated to prepare an antibody solution; and

[0010] The antibody solution is mixed with a protective solution to prepare an antinuclear antibody spectrum quality control product.

[0011] In one embodiment, the ultrafiltration and concentration step further includes screening the co-positive raw materials to be concentrated. In one embodiment, the ultrafiltration and concentration step further includes filtering;

[0012] The filtration comprises passing the raw material to be concentrated through filter screens 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 in sequence.

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

[0014] Use a 90kd to 110kd ultrafiltration container at a temperature of 3.5°C to 4.5°C and a rotation speed of 5800rpm to 6200rpm for ultrafiltration for 18min to 22min, collect the filtrate, and repeat the process until the volume of the antibody solution after concentration is 1 / 4 to 1 / 6 of the volume of the raw material to be concentrated before filtration.

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

[0016] 45 mM to 55 mM 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 stabilizer comprises 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12mM to 14mM EDTA-2 sodium, 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) TritonX-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 spectrum quality control product is freeze-dried to obtain a dry powder quality control product.

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

[0023] Cool down to -12℃~-8℃ in 4min~6min, keep it for 40min~50min; cool down to -55℃~-45℃ in 4min~6min, keep it for 85min~90min; cool down to -30℃~-20℃ in 4min~6min, keep it for 25min~35min; cool down to -60℃~-50℃ in 4min~6min, keep it for 380min~420min; and

[0024] Heat up to -35℃~-25℃ in 45min~55min, maintain for 1300min~1400min, vacuum degree 10Pa~12Pa;

[0025] Take 45min to 55min to increase the temperature to -15℃~-5℃, maintain it for 380min to 400min, and the vacuum degree is 8pa~10pa; take 45min to 55min to increase the temperature to 5℃~15℃, maintain it for 400min to 500min, and the vacuum degree is 8Pa~10Pa.

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

[0027] On the other hand, the present application provides an antinuclear antibody spectrum quality control product, which is prepared according to the above-mentioned method for preparing the antinuclear antibody spectrum quality control product.

[0028] On the other hand, the present application also provides an antinuclear antibody detection kit, which includes an immunoblotting detection reagent and the above-mentioned antinuclear antibody spectrum quality control product.

[0029] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0031] Figure 1 It is the process flow of quality control product preparation; among them, the hexagon is a 100,000-level production workshop, the diamond is a 10,000-level production workshop, and the rectangle is the ordinary production area;

[0032] Figure 2 For quality control, the strips were tested with the Yahuilong reagent;

[0033] Figure 3 For quality control, test the strips with Euromon reagent;

[0034] Figure 4 For quality control products, use the Kexin platform test strips. DETAILED DESCRIPTION

[0035] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

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

[0037] the term

[0038] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0039] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0040] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0041] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0042] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

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

[0044] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0045] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0046] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0047] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0048] In the present 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 documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0050] The main purpose of this application is to provide an antinuclear antibody spectrum composite quality control product whose analytical performance and stability meet the needs of clinical testing, and is compatible with the immunoblotting reagent platform of mainstream manufacturers. It can cover 15 detection items of the antinuclear antibody spectrum, and can subsequently be tested efficiently and quickly by immunoblotting to achieve the purpose of quality control of the antinuclear antibody spectrum composite items.

[0051] On the one hand, the present application provides a method for preparing an antinuclear antibody spectrum quality control product, comprising:

[0052] Using a plasma sample containing antinuclear antibodies as a raw material to be concentrated; performing ultrafiltration concentration on the raw material to be concentrated to prepare an antibody solution; and

[0053] The antibody solution is mixed with a protective solution to prepare an antinuclear antibody spectrum quality control product.

[0054] This application innovatively applies ultrafiltration concentration to the concentration of serum raw materials, effectively improving the serum antibody concentration, and determines the optimal ultrafiltration conditions and methods based on the characteristics of the antinuclear antibody spectrum raw materials, solving the difficulty that some raw materials cannot reach the feed concentration.

[0055] In a specific example, the ultrafiltration concentration further includes screening of co-positive raw materials.

[0056] In a specific example, the ultrafiltration concentration further includes a filtering step;

[0057] The filtration comprises passing the raw material to be concentrated through filter screens 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 in sequence.

[0058] In order to ensure that the intrinsic control products are closer to the consistency of the reactions of clinical samples during testing, the raw materials used in some projects are human positive materials. The human positive materials corresponding to each item of the antinuclear antibody spectrum are filtered in the order of 3μm-1.2μm-0.8μm-0.45μm to remove the precipitation and large particle impurities in the positive serum.

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

[0060] Use a 90kd to 110kd ultrafiltration container at a temperature of 3.5°C to 4.5°C and a rotation speed of 5800rpm to 6200rpm for ultrafiltration for 18min to 22min, collect the filtrate, and repeat the process until the volume of the antibody solution after concentration is 1 / 4 to 1 / 6 of the volume of the raw material to be concentrated before filtration.

[0061] The filtered positive material was ultrafiltered and concentrated using an ultrafiltration tube (100kd). After testing the supernatant, no target antibody was found, indicating that the ultrafiltration tube (100kd) can effectively retain the target antibody. The temperature condition of ultrafiltration is 4°C and the speed is 6000rpm. After 20 minutes, the positive material after ultrafiltration is collected and mixed. After mixing, the above ultrafiltration operation is repeated until the volume of the concentrated positive material is 1 / 4 to 1 / 6 before filtration. This method can effectively increase the concentration of the positive material by 2-5 times. Compared with the general method of pretreatment of raw materials, removing water by freeze-drying will cause greater losses. The concentration of the raw material after concentration is only about 1.5 times, and it is difficult to re-dissolve, which may affect the quality of the quality control product in the future.

[0062] Secondly, ultrafiltration concentration improves the availability of positive materials and reduces the feed ratio of low-concentration positive materials collected from some projects, providing the possibility for multiple complexes. The concentrated human positive materials of the antinuclear antibody spectrum were verified by gradient dilution on three mainstream immunoblotting reagent platforms. The acceptable standard was that the grayscale measurement of the membrane strip on the immunoblotting reagent platform of each manufacturer after dilution 30 times was more than three times the weak positive critical value.

[0063] Optionally, the protective solution includes: 45 mM to 55 mM 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 salt can increase the surface charge of protein molecules, enhance the interaction between protein molecules and water molecules, and increase the solubility of protein in aqueous solution through salt dissolution effect.

[0065] Further optionally, the stabilizer includes 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12mM to 14mM EDTA-2 sodium, 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 the interference of other cations in the system and provides specific binding; bovine serum albumin plays a role in supporting the lyophilized product on the one hand, and simulates the human serum matrix effect on the other hand; mannitol and trehalose help fix the morphology of the quality control, which can effectively prevent the collapse of the lyophilized product and make it a loose crystalline structure after lyophilization; PC300 and gentamicin sulfate are used as dual preservatives. Since the single use amount of the quality control product is only 10 μL, and most 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 method further includes the following steps: freeze-drying the liquid quality control product to obtain a dry powder quality control product.

[0069] Optionally, the freeze-drying procedure comprises:

[0070] Cool down to -12℃~-8℃ in 4min~6min, keep it for 40min~50min; cool down to -55℃~-45℃ in 4min~6min, keep it for 85min~90min; cool down to -30℃~-20℃ in 4min~6min, keep it for 25min~35min; cool down to -60℃~-50℃ in 4min~6min, keep it for 380min~420min; and

[0071] Heat up to -35℃~-25℃ in 45min~55min, keep it for 1300min~1400min, and the vacuum degree is 10Pa~12Pa; heat up to -15℃~-5℃ in 45min~55min, keep it for 380min~400min, and the vacuum degree is 8Pa~10Pa; heat up to 5℃~15℃ in 45min~55min, keep it for 400min~500min, and the vacuum degree is 8Pa~10Pa.

[0072] This application optimizes the freeze-drying procedure. Since the raw materials in this study involve many projects and have complex ingredients, it is easy to have problems such as freeze-drying collapse or unqualified appearance or re-dissolution stability. The appearance and performance of the product are further guaranteed by the optimized freeze-drying process.

[0073] In the improved freeze-drying procedure, by extending the time of sublimation drying, the problem of local concentration and poor solubility of the product is effectively avoided; by extending the time of analytical drying, the water content of the freeze-dried quality control product is reduced. By lowering the heating temperature and increasing the vacuum degree of the freeze-drying chamber, the product is prevented from shrinking and bubbling; the improved freeze-drying procedure maintains the biological activity and physical and chemical properties of the quality control product, and the appearance is a loose crystal structure, which is conducive to the long-term storage of the quality control product.

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

[0075] On the other hand, the present application provides an antinuclear antibody spectrum quality control product, which is prepared according to the preparation method of the antinuclear antibody spectrum quality control product.

[0076] The present application also provides an antinuclear antibody detection kit, which includes an immunoblotting detection reagent and the antinuclear antibody spectrum quality control product.

[0077] Increasing the number of antibody types can improve coverage, but there are also a variety of problems, such as the early collection and processing of raw materials, co-positive interference between projects, how to ensure the analytical performance, stability and adaptability of all projects, how to ensure the qualified appearance of the product when multiple raw materials are added, etc. Ensuring product performance is a problem that needs to be solved.

[0078] Based on this, the antinuclear antibody spectrum quality control product 15S (Western blotting method) prepared by the present application, through ultrafiltration pretreatment of raw materials, overcomes the problems of low serum raw material concentration in some projects that cannot be fed or too high feeding ratio, and at the same time utilizes the co-positivity between raw materials during deployment, so that the composite quality control can cover 15 antibody projects of the antinuclear antibody spectrum: 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, anti-mitochondrial antibody M2 type antibody. The present application provides a more comprehensive test index for the clinical quality control of the antinuclear antibody spectrum.

[0079] Furthermore, the diluent selected in this application uses MOPS sodium salt as a buffer system, provides support and protection for the analyte by adding EDTA-2 sodium, bovine serum albumin, mannitol and trehalose, and adds PC300 and gentamicin sulfate as double preservatives to ensure the long-term stability of the quality control product. The addition of surfactant TRITON(TM) X-100 and protein protectant AEP-HBC effectively improves the redissolution stability of the quality control product. The diluent components of this application ensure the stability of quality control under different conditions.

[0080] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0081] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0082] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

[0083] Example 1

[0084] An embodiment of the present application provides a method for preparing an antinuclear antibody spectrum quality control product, and the complete preparation and testing steps are as follows:

[0085] 1. Pretreatment of raw materials

[0086] The antinuclear antibody spectrum positive materials were filtered in the order of 3μm-1.2μm-0.8μm-0.45μm to remove impurities and ensure a clear and bright appearance. The filtered positive materials were gradient diluted (the starting multiple was 30 times), the co-positivity and concentration of the raw materials were confirmed, and the raw materials with a concentration lower than the standard (the grayscale measurement of the membrane strip on the immunoblotting reagent platform of each manufacturer after dilution was more than three times the weak positive critical value) were ultrafiltered and concentrated. The ultrafiltration temperature was 4°C and the speed was 6000rpm. After 20 minutes, the ultrafiltered positive materials were collected and mixed. After mixing, the above ultrafiltration operation was repeated until the volume of the concentrated positive material was 1 / 4 to 1 / 6 of the volume before filtration.

[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 in the range of 7.4±0.1.

[0089] The contents of each component are: 50mM MOPS buffer, 0.9% (w / v) sodium chloride, 4% (w / v) bovine serum albumin, 13mM EDTA-2 sodium, 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, 0.05% (w / v) gentamicin sulfate.

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

[0091] The positive material solution in step 1 is mixed with the protective solution obtained in step 2 in a specific ratio, and the positive material solution with multiple co-positives is added preferentially to ensure that the grayscale measurement value on the Invitrogen immunoblotting reagent platform is more than three times the weak positive critical value. For items that do not meet the standard, single positive raw materials are used for supplementary addition until all items meet the requirements.

[0092] The prepared ANA quality control product is divided into freeze-dried bottles and freeze-dried. The procedure includes:

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

[0094] Heat to -30℃ in 50min, maintain for 1350min, vacuum degree 10Pa~12Pa;

[0095] Heat up to -10℃ in 50min, maintain for 390min, vacuum degree 8Pa~10Pa; heat up to 10℃ in 50min, maintain for 450min, vacuum degree 8Pa~10Pa.

[0096] After the freeze-drying process is completed, the water content of the quality control product is analyzed using a Karl Fischer moisture analyzer.

[0097] 4. Testing

[0098] The quality control products were tested using the antinuclear antibody spectrum detection kit 15S (immunoblotting method) matched with the detection systems of Yahuilong immunoblotting automatic analyzer, Kexin and Oumen. The acceptable standard for the comparison results of the immunoblotting reagent platforms of various manufacturers is that the gray value of the membrane strip is more than three times the weak positive critical value.

[0099] Example 2

[0100] 1. Pretreatment of raw materials

[0101] This application conducts ultrafiltration concentration on positive materials to improve the utilization rate of low-concentration raw materials. In order to ensure that the essential control products are closer to the reaction consistency of clinical samples during testing, the raw materials used in some projects are human positive materials. The human positive materials corresponding to each item of the antinuclear antibody spectrum are filtered in the order of 3μm-1.2μm-0.8μm-0.45μm to remove the precipitate and large particle impurities in the positive serum.

[0102] The filtered positive material was ultrafiltered and concentrated using an ultrafiltration tube (100kd). After testing the supernatant, no target antibody was found, indicating that the ultrafiltration tube (100kd) can effectively retain the target antibody. The temperature condition of ultrafiltration is 4°C and the speed is 6000rpm. After 20 minutes, the positive material after ultrafiltration is collected and mixed. After mixing, the above ultrafiltration operation is repeated until the volume of the concentrated positive material is 1 / 4 to 1 / 6 before filtration. This method can effectively increase the concentration of the positive material by 2 to 5 times. Compared with the general method of pretreatment of raw materials, removing water by freeze-drying will cause greater losses. The concentration of the raw material after concentration is only about 1.5 times, and it is difficult to re-dissolve, which may affect the quality of the quality control product in the future.

[0103] Ultrafiltration concentration improves the availability of positive materials and reduces the feed ratio of low-concentration positive materials collected from some projects, making it possible for multiple complexes. The concentrated human positive materials of the antinuclear antibody spectrum were verified by gradient dilution on three mainstream immunoblotting reagent platforms. The acceptable standard was that the grayscale measurement of the membrane strip on the immunoblotting reagent platform of each manufacturer after dilution 30 times was more than three times the weak positive critical value.

[0104] 2. Screening of quality control matrix formula: improving the stability of quality control

[0105] Positive quality control matrix liquid: MOPS sodium salt is used as the buffer system; EDTA-2 sodium reduces the interference of other cations in the system and provides specific binding; bovine serum albumin plays the role of supporting the lyophilized product on the one hand, and simulates the effect of human serum matrix on the other hand; mannitol and trehalose help fix the shape of the quality control, which can effectively prevent the collapse of the lyophilized product and make it a loose crystalline structure after lyophilization; PC300 and gentamicin sulfate are used as dual preservatives. Since the single use amount of this quality control product is only 10μL and most antibodies are plasma products, the addition of two preservatives greatly improves the long-term stability of the quality control product.

[0106] In order to further improve the stability of quality control products, four formulations were verified.

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

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

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

[0110] Formula 4 is a positive quality control matrix solution to which TRITON(TM) X-100 and protein protectant AEP-HBC are added simultaneously.

[0111] The specific operations are as follows:

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

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

[0114] First add Mops buffer, sodium chloride and 800mL pure water, and measure the solution pH after the material is dissolved. The pH should be within the range of 7.4±0.5. Then add PC300, gentamicin sulfate, bovine serum albumin, EDTA-2 sodium, mannitol, trehalose, TRITON(TM)X-100, and protein protectant AEP-HBC in sequence, and fill to 1L with pure water. After stirring and mixing, measure the solution pH. The final pH should be within the range of 7.4±0.1. The stability verification comparison of different formulas is 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] The experimental results show that the simultaneous addition of TRITON(TM)X-100 and protein protectant AEP-HBC can effectively improve the re-dissolution stability of the quality control product, so that the 30-day re-dissolution stability of all test items meets the requirements (the gray value deviation is less than 10% compared with the control). TRITON(TM)X-100 is a surfactant that can maintain the stability of the protein by decomposing proteases, and supplements the protein protectant AEP-HBC to inhibit protein aggregation and precipitation, thereby protecting its structure and function.

[0128] Positive quality control products: Positive materials with more co-positive items will be given priority. Due to the different co-positive degrees of different items, after the raw materials with co-positive are fed, it is guaranteed that the grayscale measurement value on the Invitrogen immunoblotting reagent platform is more than three times the weak positive critical value. For items that do not meet the standard, single-positive raw materials will be used to supplement the feeding until all items meet the requirements. By utilizing the co-positivity of the raw materials, the raw materials can be utilized to the greatest extent while meeting the performance requirements, thereby achieving the goal of reducing costs.

[0129] Negative control: Human serum containing antinuclear antibodies that test negative for each analyte.

[0130] 3. Freeze-drying of quality control products:

[0131] Optimization of freeze-drying process:

[0132] In the improved freeze-drying procedure, by extending the time of sublimation drying, the local concentration of the product and the problem of poor solubility are effectively avoided; the time of analytical drying is extended to reduce the water content of the freeze-dried quality control product. The heating temperature is lowered and the vacuum degree of the freeze-drying box is increased to avoid shrinkage and bubbling of the product; the improved freeze-drying procedure maintains the biological activity and physical and chemical properties of the quality control product, and the appearance is a loose crystal structure, which is conducive to the long-term storage of the quality control product. The specific freeze-drying procedure is shown in Table 5:

[0133] Table 5: New and conventional lyophilization procedures for quality control products

[0134]

[0135] The prepared ANA quality control product was dispensed into low borosilicate brown glass bottles with an electric pipette, and the bottle mouth was covered with a rubber stopper with air holes, and freeze-dried in a freeze dryer using a new freeze-drying program. After freeze-drying, the lid was pressed under vacuum, and after the freeze-dried quality control was inspected for appearance problems, the water content of the sample was measured with a calorimeter, and the water content should be less than 2%. After the ANA quality control product was re-dissolved, the gray value was determined on a fully automatic immunoblotting instrument using the Yahuilong antinuclear antibody spectrum detection kit 15S (immunoblotting method), and the loss rate after freeze-drying was calculated. The specific calculation results are shown in Table 6. If it is less than 20%, it means that the freeze-drying requirements are met.

[0136] Table 6: Verification of freeze-drying loss rate of quality control products

[0137]

[0138] 4. Assignment of quality control products:

[0139] The samples were re-dissolved and assigned in the general workshop. The assignment steps are as follows: prepare 3 different types of immunoblotting analyzers, use 3 instruments to measure ANA positive quality control products for 5 consecutive days, and test 4 times a day. The test results are eliminated according to the Grubbs criterion, and the number of outliers eliminated shall not exceed 2.5%. After eliminating the outliers, calculate the mean X and standard deviation SD of the remaining data to determine the target value range of the gray value of the quality control product (X±3SD).

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

[0141] After the assignment, semi-finished product inspection is carried out to check the accuracy and repeatability of the quality control products. If the semi-finished product inspection passes, product assembly and finished product inspection are carried out. After the finished product inspection passes, the finished product is put into storage, otherwise it will be treated as a defective product.

[0142] 6. Stability verification:

[0143] In order to simulate the performance of the antinuclear antibody spectrum quality control product after high-temperature aging, this application places the product at 37°C for accelerated stability verification, heat-treated at 37°C for 14 days, and stored at 2°C to 8°C as a control; to verify the stability of the antinuclear antibody spectrum quality control product after reconstitution, this application stores the product at 2°C to 8°C, -20°C and -80°C for 30 days, 60 days and 90 days after reconstitution, and stores the unreconstituted lyophilized product at 2°C to 8°C as a control. The Yahuilong antinuclear antibody spectrum detection kit (immunoblotting method) was used for testing. Compared with the control group, the grayscale values ​​of the antinuclear antibody spectrum quality control products under different conditions had relative deviations within ±10%, indicating that the antinuclear antibody spectrum quality control product has good stability and can meet clinical use needs.

[0144] 7. Multi-platform adaptation

[0145] like Figure 2 to Figure 4 As shown, the essential control products can be adapted to the reagent platforms of Yahuilong as well as those of Oumeng and Kexin. Figure 2 For quality control products, use the Yahuilong reagent test strips. Figure 3 For quality control, use Euromon reagent to test the strips. Figure 4 For quality control products, use the Kexin platform test strips.

[0146] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for preparing an antinuclear antibody spectrum quality control product, characterized in that: include: The plasma sample containing antinuclear antibodies is used as the raw material to be concentrated; Performing ultrafiltration and concentration on the raw material to be concentrated to prepare an antibody solution; as well as The antibody solution is mixed with a protective solution to prepare an antinuclear antibody spectrum quality control product.

2. The method for preparing the antinuclear antibody spectrum 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 spectrum quality control product according to claim 1, characterized in that: The ultrafiltration concentration also includes a filtration step; The filtration comprises passing the raw material to be concentrated through filter screens 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 in sequence.

4. The method for preparing the antinuclear antibody spectrum quality control product according to claim 3, characterized in that: The conditions for ultrafiltration concentration include: Use a 90kd to 110kd ultrafiltration container at a temperature of 3.5°C to 4.5°C and a rotation speed of 5800rpm to 6200rpm for ultrafiltration for 18min to 22min, collect the filtrate, and repeat the process until the volume of the antibody solution after concentration is 1 / 4 to 1 / 6 of the volume of the raw material to be concentrated before filtration.

5. The method for preparing the antinuclear antibody spectrum quality control product according to any one of claims 1 to 4, characterized in that: The protective solution comprises: 45 mM to 55 mM MOPS buffer and 0.85% (w / v) to 0.95% (w / v) neutral salt, stabilizer and preservative; The neutral salt includes one or both of sodium chloride and sodium sulfate; The stabilizer comprises 3.5% (w / v) to 4.5% (w / v) bovine serum albumin, 12mM to 14mM EDTA-2 sodium, 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; and / or 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.

6. The method for preparing an antinuclear antibody spectrum quality control product according to any one of claims 1 to 4, characterized in that: The following steps are also included: The antinuclear antibody spectrum quality control product is freeze-dried to obtain a dry powder quality control product.

7. The method for preparing the antinuclear antibody spectrum quality control product according to claim 6, characterized in that: The freeze drying procedure includes: Cool down to -12℃~-8℃ in 4min~6min, keep it for 40min~50min; cool down to -55℃~-45℃ in 4min~6min, keep it for 85min~90min; cool down to -30℃~-20℃ in 4min~6min, keep it for 25min~35min; cool down to -60℃~-50℃ in 4min~6min, keep it for 380min~420min; and Heat up to -35℃~-25℃ in 45min~55min, maintain for 1300min~1400min, vacuum degree 10Pa~12Pa; Take 45min to 55min to increase the temperature to -15℃~-5℃, maintain it for 380min to 400min, and the vacuum degree is 8pa~10pa; take 45min to 55min to increase the temperature to 5℃~15℃, maintain it for 400min to 500min, and the vacuum degree is 8Pa~10Pa.

8. The method for preparing the antinuclear antibody spectrum quality control product according to claim 7, characterized in that: The antinuclear antibodies include anti-double-stranded DNA antibodies, anti-Sm antibodies, anti-SSA antibodies, anti-Ro-52 antibodies, anti-nucleosome antibodies, anti-histone antibodies, anti-ribonucleoprotein 70 antibodies, anti-SS-B antibodies, anti-Jo-1 antibodies, anti-Scl-70 antibodies, anti-ribosomal P protein antibodies, anti-centromere antibodies, anti-proliferating cell nuclear antigen antibodies, anti-PM-Scl antibodies and anti-mitochondrial antibodies M2 type antibodies.

9. An antinuclear antibody spectrum quality control product, characterized in that: It is prepared according to the method for preparing the antinuclear antibody spectrum quality control product according to any one of claims 1 to 8.

10. An antinuclear antibody detection kit, characterized in that: The kit comprises an immunoblotting detection reagent and the antinuclear antibody spectrum quality control product according to claim 9.

Images