Preparation method of anti-dsDNA antibody reference reagent as well as product and application thereof

Abstract

The invention discloses a preparation method of an anti-dsDNA (double-stranded deoxyribonucleic acid) antibody reference reagent, which is characterized by comprising the following steps: mixing 5000-6000 IU freeze-dried thrombin powder and plasma exchange liquid according to a ratio of 50IU: 1ml, lightly stirring to be uniform, and standing at 37 DEG C for 1-2 hours; transferring to 4 DEG C to 5 DEG C, placing for 48 to 72 hours, and centrifuging at the speed of 5,000 to 6,000 revolutions per minute for 10 to 20 minutes; collecting supernate, performing sterile filtration, subpackaging and freeze-drying, so that the plasma replacement liquid is strongly positive for resisting double-stranded DNA antibodies, namely positive for ELISA (enzyme-linked immuno sorbent assay) and indirect immunofluorescence, positive for antinuclear antibodies (ANA) and with the titer of 1: 500-700. The invention also discloses a product and application thereof. The invention prepares a reference reagent of an anti-double-stranded DNA antibody. The reference reagent shows good stability and consistency in multiple quality control detections. As a deviation calibration product or a quality control reference product of a detection result of a method for detecting an anti-double-stranded DNA antibody, the kit is used for judging the activity of systemic lupus erythematosus (SLE) disease and monitoring the curative effect.

Description

Technical Field

[0001] The present invention relates to the field of manufacturing double-stranded DNA antibody reference reagents, and in particular to a preparation method and product of an anti-dsDNA antibody reference reagent and application thereof. Background Art

[0002] Patients with systemic lupus erythematosus (SLE) should follow international recommendations and perform quantitative testing of anti-double-stranded DNA antibodies (anti-dsDNA) regularly (every 3-6 months). Anti-double-stranded DNA antibodies are one of the immunological markers of SLE and are of great significance for diagnosis. Changes in their quantitative measurements can also reflect the activity of the disease and the effectiveness of treatment.

[0003] However, there are great differences in the actual application of quantitative detection methods for anti-double-stranded DNA antibodies. The detection methods provided by different manufacturers are different. For example, the Farr method (RIA) of radioimmunoassay has been gradually eliminated due to biohazard issues; enzyme-linked immunosorbent assay (ELISA) is increasingly popular due to its simple operation and moderate cost, but there are 6 different brands available in the Chinese market; in addition, new technologies such as more sensitive chemiluminescence immunoassay (CLIA), magnetic barcode immunofluorescence assay (MIA) and magnetic bead flow fluorescence immunoassay (MBFFI) are also emerging. The widespread application of these methods will provide more possibilities for the monitoring and treatment of SLE. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, the object of the present invention is to provide a method for preparing an anti-dsDNA antibody reference reagent, a product and an application thereof.

[0005] In order to achieve the purpose of the invention, the technical solution adopted is:

[0006] A method for preparing an anti-dsDNA antibody reference reagent, comprising:

[0007] Mix 5000-6000 IU of freeze-dried thrombin powder with plasma exchange solution at a ratio of 50 IU:1 ml, stir gently until uniform, and let stand at 37°C for 1-2 hours;

[0008] Move to 4°C-5°C and place for 48-72 hours, then centrifuge at 5,000-6,000 rpm for 10-20 minutes;

[0009] The supernatant is collected, sterile filtered, packaged and freeze-dried. The plasma replacement fluid is a plasma replacement fluid that is strongly positive for anti-double-stranded DNA antibodies, that is, positive for both ELISA and indirect immunofluorescence assays, and positive for antinuclear antibodies (ANA) with a titer of 1:500-700.

[0010] In a preferred embodiment of the present invention, the freeze-dried thrombin powder is 5000 IU.

[0011] In a preferred embodiment of the present invention, the centrifugal speed is 5,000 rpm.

[0012] In a preferred embodiment of the present invention, the freeze-drying after packaging is to package the solution into glass reagent bottles in an amount of 500 microliters per bottle for freeze-drying.

[0013] In a preferred embodiment of the present invention, the plasma exchange fluid is a plasma exchange fluid that is strongly positive for anti-double-stranded DNA antibodies, that is, positive for both ELISA and indirect immunofluorescence assay, and positive for antinuclear antibodies (ANA) with a titer of 1:640.

[0014] An anti-dsDNA antibody reference reagent is a lyophilized product obtained by the above preparation method.

[0015] An application of an anti-dsDNA antibody reference reagent, wherein the anti-dsDNA antibody reference reagent is used as a deviation calibrator or quality control reference for the detection results of a method for detecting anti-double-stranded DNA antibodies, for determining the disease activity of systemic lupus erythematosus (SLE) and monitoring the therapeutic effect.

[0016] The beneficial effects of the present invention are:

[0017] A reference reagent for anti-double-stranded DNA antibody was prepared and showed good stability and consistency in multiple quality control tests.

[0018] The launch of this local reference reagent may facilitate data sharing and standardization, improve test accuracy, and better interpret results.

[0019] For SLE patients, regular quantitative testing of anti-dsDNA antibodies, combined with the use of this validated reference reagent, can more accurately assess disease status and treatment efficacy, thereby formulating more effective treatment plans.

[0020] In general, although there are various detection methods, the use of standardized reference reagents can ensure the accuracy and comparability of the test results, providing strong support for the diagnosis and treatment of SLE patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the indirect immunofluorescence results of Crithidia spp., source of the anti-double-stranded DNA antibody reference reagent sample.

[0022] Figure 2Schematic diagram of the indirect immunofluorescence results of anti-nuclear antibodies derived from the anti-double-stranded DNA antibody reference reagent sample.

[0023] Figure 3 Schematic diagram of the finished product of anti-double-stranded DNA antibody reference reagent.

[0024] Figure 4 Schematic diagram of the performance validation results of anti-double-stranded DNA antibody reference reagents for 38 laboratories. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following structure, the description of the known structure and technology is omitted to avoid unnecessary confusion of the concept of the present invention.

[0026] 1. Preparation of Anti-double-stranded DNA Antibody Reference Reagent

[0027] 1.1 Patient and biomaterial characteristics

[0028] The patient was an 18-year-old female who was admitted to our hospital in 2023 due to a severe attack of lupus with thrombotic microangiopathy (TMA: microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury). Her autologous antibody laboratory test showed strong positive anti-double-stranded DNA antibodies (both ELISA and indirect immunofluorescence were positive, see Figure 1 ), ANA positive (1:640, homogeneous type, see Figure 2 The patient was treated with plasma exchange, and a total of 8 plasma exchange volumes (13.5 L in total) were collected.

[0029] 1.2 Preparation of serum from plasma and serum freeze-drying

[0030] Freeze-dried thrombin powder (5000 IU, Zhejiang Hangkang Pharmaceutical Co., Ltd., China) was mixed with the patient's plasma exchange solution at a ratio of 50 IU:1 ml, gently stirred until uniform, and allowed to stand at 37°C for 1 hour, then moved to 4°C for 72 hours. After centrifugation at 5,000 rpm for 15 minutes, the supernatant was collected, filtered through a sterile filter, and dispensed into glass reagent bottles at 500 μl per bottle (see Figure 3 The freeze-drying was carried out strictly according to the standard operating procedures of the vacuum freeze dryer (Ningbo Xinzhi Freeze Drying Equipment Co., Ltd., Ningbo, China), and different batches of freeze-dried samples were obtained through the preparation of different batches.

[0031] 2. Detection of anti-double-stranded DNA antibody reference reagent using ELISA

[0032] The anti-double-stranded DNA antibody ELISA kit of Euroimmun (Germany) was used as an operating reference:

[0033] 2.1 Prepare all necessary reagents, buffers and diluents according to the instructions provided by Euroimmun. Ensure that all equipment and materials are sterile and properly labeled.

[0034] 2.2 Preparation: Allow all reagent components to equilibrate at room temperature for at least 30 minutes.

[0035] 2.3 Sample addition: Prepare standards and samples according to the kit instructions. Samples should be diluted in a specific ratio (1:201) and 100 μl of the diluted sample should be added to the corresponding wells. At the same time, add negative and positive controls as instructed. Set up duplicate wells for all samples, standards, and controls.

[0036] 2.4 Incubation: Incubate at room temperature (18-25°C) for 30 minutes.

[0037] 2.5 Washing: Discard the liquid in the wells. Dilute the washing solution with distilled water at a volume ratio of 1:100, and then wash each well with 300 μl of the diluted washing solution. Repeat this step three times.

[0038] 2.6 Enzyme conjugate incubation: After discarding the washing solution, gently invert and tap the microplate to dry. Add 100 μl of enzyme-labeled secondary antibody to each well and incubate at room temperature (18-25°C) in the dark for 30 minutes.

[0039] 2.7 Washing: Repeat the washing step in step 2.5.

[0040] 2.8 Add substrate: Add substrate solution (3,3',5,5'-tetramethylbenzidine, TMB) to each well and incubate at room temperature until a color change occurs. The intensity of the color reaction is proportional to the amount of the corresponding antibody or antigen in the sample.

[0041] 2.9 Add stop solution: Add stop solution (5% dilute sulfuric acid solution) to each well to stop the color reaction.

[0042] 2.10 Reading: Within a specified time range (e.g., within 30 minutes) after adding the stop solution, measure the absorbance at 450 nm using an ELISA reader. Use the absorbance value for qualitative or quantitative analysis.

[0043] result:

[0044] We evaluated the performance of the reference reagents using commercial anti-double-stranded DNA antibody ELISA kits produced by Shanghai Kexin Biotechnology Co., Ltd. These reagents were stored at 4°C and showed excellent uniformity in three consecutive tests, with the coefficient of variation (CV) of the detection values ​​of the inter-group and intra-group data being less than 5%, as shown in Table 1, which shows that the reference reagents of the present invention have good uniformity in response to repeatability tests.

[0045] Table 1 Homogeneity study of anti-double-stranded DNA antibody reference reagents

[0046]

[0047] To further verify its stability, the detection time was extended to 3 months, and the stability study of the reference reagent was performed at 4 °C.

[0048] The results showed that the CV of the detection value remained within 5%, as shown in Table 2.

[0049] Table 2: Real stability study of anti-dsDNA antibody reference reagent

[0050]

[0051] In the accelerated destabilization experiment, after the reference reagent was placed at 37°C for 24 hours or 72 hours, its CV% was also kept within 5%, as shown in Table 3.

[0052] Table 3 Accelerated destructive study of anti-double-stranded DNA antibody reference reagents

[0053]

[0054] In addition, through the inter-batch stability analysis of 10 batches of reference reagents, it was found that their CV% were all less than 5%, as shown in Table 4 for details.

[0055] Table 4 Batch stability study of anti-double-stranded DNA antibody reference reagent

[0056]

[0057]

[0058] After 9 months of storage at different temperatures, the titer of the anti-double-stranded DNA antibody reference reagent remained above 90%, as shown in Table 5, indicating that it can be effectively stored.

[0059] Table 5

[0060]

[0061] Thirty-eight laboratories participated in the evaluation of the performance of the reference reagents for antibodies against double-stranded DNA. Details of the methods and a breakdown of the reagents used are given in Table 6.

[0062] Laboratory No. Manufacturer Methodology 1 Shanghai Technology Co., Ltd. Flow cytometry 2,3 Guangzhou Biotechnology Co., Ltd. Chemiluminescence 4 Hangzhou Medical Laboratory Diagnostics Co., Ltd. ELISA 5,6 Hangzhou Medical Laboratory Diagnostics Co., Ltd. ELISA 7,8,9 A Shanghai Biotechnology Co., Ltd. ELISA 10-21 Shenzhen Biotechnology Co., Ltd. Chemiluminescence 22 Hangzhou Medical Diagnostic Technology Co., Ltd. Chemiluminescence 23,24,25 A Sichuan Biotechnology Co., Ltd. Chemiluminescence 26-31 A biopharmaceutical company in Jiangsu Chemiluminescence 32 A diagnostic company Chemiluminescence 33-37 Zhuhai Reagent Co., Ltd. Magnetic barcode immunofluorescence luminescence 38 Self-built methodology Chemiluminescence

[0063] The parallel test results of the reference reagent of the present invention and the specific serum sample were used to calculate the potency ratio. The results showed that most of the potency ratios fell within the range of 1.0-1.4. Figure 3 .

[0064] This illustrates the normal distribution of the data indicating that the reference reagents are helpful in accurately estimating the reference values ​​of different methods. The basic principles and main features of the invention and the advantages of the invention are shown and described above.

[0065] Those skilled in the art should understand that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing an anti-dsDNA antibody reference reagent, characterized in that: include: Mix 5000-6000 IU of freeze-dried thrombin powder with plasma exchange solution at a ratio of 50 IU:1 ml, stir gently until uniform, and let stand at 37°C for 1-2 hours; Move to 4°C-5°C and place for 48-72 hours, then centrifuge at 5,000-6,000 rpm for 10-20 minutes; The supernatant is collected, sterile filtered, packaged and freeze-dried. The plasma replacement fluid is a plasma replacement fluid that is strongly positive for anti-double-stranded DNA antibodies, that is, positive for both ELISA and indirect immunofluorescence assays, and positive for antinuclear antibodies (ANA) with a titer of 1:500-700.

2. The method for preparing an anti-dsDNA antibody reference reagent according to claim 1, characterized in that: The freeze-dried thrombin powder is 5000 IU.

3. The method for preparing an anti-dsDNA antibody reference reagent according to claim 1, characterized in that: The centrifugal speed was 5,000 rpm.

4. The method for preparing an anti-dsDNA antibody reference reagent according to claim 1, characterized in that: The freeze-drying after subpackaging is to subpack into glass reagent bottles in an amount of 500 microliters per bottle for freeze-drying operation.

5. The method for preparing an anti-dsDNA antibody reference reagent according to claim 1, characterized in that: The plasma replacement fluid is a plasma replacement fluid that is strongly positive for anti-double-stranded DNA antibodies, that is, positive for both ELISA and indirect immunofluorescence assays, and positive for antinuclear antibodies (ANA) with a titer of 1:

640.

6. An anti-dsDNA antibody reference reagent, which is a lyophilized product obtained by the preparation method according to any one of claims 1 to 5.

7. An application of an anti-dsDNA antibody reference reagent, characterized in that: The application is to use the anti-dsDNA antibody reference reagent as described in claim 6 as a deviation calibrator or quality control reference for the detection results of the anti-double-stranded DNA antibody detection method, for judging the disease activity of systemic lupus erythematosus (SLE) and monitoring the efficacy.

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