Rheumatoid arthritis marker substance control product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a rheumatoid arthritis-based marker substance control substance as well as a preparation method and application thereof. The invention provides a rheumatoid arthritis marker substance control product which comprises rheumatoid factors, an anti-cyclic citrullinated peptide antibody, an anti-citrullinated waveform protein antibody, an anti-keratin antibody and C-reactive protein as markers, and a preparation method and application of the rheumatoid arthritis marker substance control product. The quality control material is a multi-marker composite quality control material, solves the problem that stability, sensitivity and specificity cannot be achieved due to large difference of properties of markers, can meet the quality control requirements of multiple items by one-time sample adding of a client, is suitable for detection platforms such as a light-emitting platform and a biochemical platform, can reduce laboratory operation, and improves the detection efficiency. The sensitivity and the specificity of experimental detection are improved, the efficiency is high, the cost is saved, and the judgment of rheumatoid arthritis is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a control product based on a rheumatoid arthritis marker substance and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic connective tissue disease. It is an inflammatory response to joints caused by the immune system's mistaken attack on normal joint tissue. It is characterized by chronic, destructive, and progressive multi-joint dysfunction, with the small joints of the limbs being the most frequently affected. Patients experience joint stiffness in the morning, and the duration of morning stiffness is closely related to the degree of disease activity. It is accompanied by symptoms such as joint pain, swelling, and deformity. Daily activities such as dressing, washing, and walking may be affected to varying degrees, and even affect multiple organ systems such as blood, nerves, heart, and lungs, seriously reducing the patient's quality of life. RA is a global disease with a global incidence of about 1.0%. There are about 5 million RA patients in my country. In recent years, affected by multiple factors such as the environment, people's lifestyle, and dietary structure, the incidence of RA has increased year by year, and has become the main cause of loss of labor and disability in the Chinese population. RA has an insidious onset and repeated procrastination, which can cause permanent damage to the joints. Therefore, early diagnosis and treatment are of great significance to improving the rehabilitation effect of patients. RA has no obvious clinical manifestations in the early stages, and the accuracy of diagnosis based on physical examination, joint X-rays, etc. is not high, and misdiagnosis and missed diagnosis are very likely to occur, causing patients to miss the best time for treatment, resulting in delayed disease progression, increased difficulty in clinical treatment, and affecting patient prognosis.

[0003] Rheumatoid factor (RF) is a commonly used detection indicator for RA. It has high sensitivity but low specificity, and its application effect in early diagnosis and treatment is not ideal.

[0004] Positive anti-CCP antibodies are an important sign of erosive joint damage. They are mainly secreted by B cells in RA patients and have strong specificity. When they show high titer positivity, it is very likely to be RA. They can be used to monitor disease progression and judge prognosis, but this indicator has low sensitivity and a high risk of missed diagnosis.

[0005] Vimentin is a ubiquitous citrullinated protein that can be observed in the rheumatoid synovial tissue of RA patients. It is a natural human protein. Vimentin can bind to RA-specific antibodies more effectively. Huge amounts of anti-citrullinated vimentin antibodies can be found in RA patients. Therefore, citrullinated vimentin antibodies have high sensitivity and specificity for the diagnosis of rheumatoid arthritis and are closely related to the condition and prognosis of rheumatoid arthritis.

[0006] AKA is an anti-keratin antibody, an important antibody for diagnosing rheumatoid arthritis, which is related to the severity and activity of the disease and is one of the indicators for early diagnosis and prognosis of rheumatoid arthritis. The sensitivity of AKA for RA diagnosis is about 40%, which is much lower than that of anti-CCP antibodies, while the specificity is as high as 95%-99%. The AKA level is not only related to the activity of RA disease, but also can make up for the deficiency of RF in RA diagnosis to a certain extent, especially for RA patients with negative RF, which has a high diagnostic significance.

[0007] CRP is an important laboratory indicator of rheumatic disease activity. Serum CRP increases significantly during the active phase of RA and decreases significantly during the remission phase of RA. Among many test items, CRP is the most commonly used auxiliary diagnostic item in clinical practice. Although it has no specificity, it has certain value in differential diagnosis and prognosis of diseases.

[0008] At present, relevant quality control products are provided for rheumatoid arthritis disease monitoring both at home and abroad, such as single rheumatoid factor (RF) quality control products, Michael's rheumatoid multiple quality control products, including anti-cyclic citrullinated peptide antibody (Anti-CCP), rheumatoid factor (RF), rheumatoid factor IgM (RF-IgM), rheumatoid factor IgA (RF-IgA), Mindray rheumatism three-item (ASO / CRP / RF) composite quality control products, which are only provided in the form of single items or two or three items, and fail to effectively combine sensitivity and specificity. In addition, most quality control product matrices are provided in the form of buffer solutions, which introduce matrix effects and are insufficient in applicability and applicability. At the same time, the sensitivity index of the test is not effectively combined with the specificity index. Users need to perform multiple operations to complete the quality control of the project, which is inconvenient for customers to operate and has a poor user experience. Summary of the invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a stable rheumatoid arthritis marker quality control product and a preparation method thereof. The quality control product covers a variety of detection markers, has high sensitivity, strong specificity, and is resistant to long-term storage.

[0010] The present invention provides a quality control matrix solution, which includes human serum and the following components:

[0011] 5~200mM TRIS buffer with pH 7.2~7.6, 0.1wt%~1wt% Bro, 0.02wt%~0.3wt% NaN 3 , 0.2wt%~5wt%ADP, 20~100U / mL aprotinin and 0.2wt%~5wt% AEP-HBC.

[0012] Compared with the prior art, the matrix liquid of the present invention is added with human serum matrix, which is closer to clinical samples, reduces matrix effect, adds TRIS buffer, adjusts pH to 7.2-7.6, and is closer to the human body environment; at the same time, aprotinin, ADP, and AEP-HBC are added to further improve the ability of the matrix liquid to maintain the stability of the marker, and at the same time, it has the characteristics of sterilization, which can ensure that the quality control product will not be reduced in stability or turbid in appearance due to contamination after liquid or freeze-dried reconstitution. At the same time, since sensitivity markers and specific markers are generally difficult to combine together, the difficulty lies in the difference in the properties of each marker, and a suitable stabilizer is required to ensure its stability. The matrix liquid provided by the present invention can provide a stable environment for compounding sensitivity markers with specific markers, and its compatibility is strong, thereby obtaining a more accurate technical effect.

[0013] In some embodiments, it comprises human serum and the following components:

[0014] 10mM TRIS buffer, pH 7.4, 0.2wt% Bro, 0.2wt% NaN 3 , 2wt% ADP, 50U / mL aprotinin and 2wt% AEP-HBC.

[0015] Experiments show that within the above-mentioned concentration range, the quality control products obtained by compounding have higher sensitivity, stronger specificity and higher stability, thus achieving more accurate technical results.

[0016] The present invention provides application of the quality control product matrix solution in preparing a rheumatoid arthritis marker substance control product.

[0017] The invention provides a quality control product of a rheumatoid arthritis marker, which consists of a rheumatoid arthritis marker and the quality control product matrix liquid.

[0018] In some embodiments, the rheumatoid arthritis markers include at least one of rheumatoid factor, anti-cyclic citrullinated peptide antibodies, anti-citrullinated vimentin antibodies, anti-keratin antibodies, and C-reactive protein.

[0019] In some embodiments, it includes a low level control, a mid level control, and a high level control, wherein:

[0020] The low-level quality control products include 21-39 U / mL rheumatoid factor, 10.5-19.5 U / mL anti-cyclic citrullinated peptide antibodies, 13-21 U / mL anti-citrullinated vimentin antibodies, 5-11 U / mL anti-keratin antibodies, and 1.8-4.2 mg / L C-reactive protein;

[0021] The mid-level quality control products include 31.5~58.5 U / mL rheumatoid factor, 17.5~32.5 U / mL anti-cyclic citrullinated peptide antibody, 22~29 U / mL anti-citrullinated vimentin antibody, 13~24 U / mL anti-keratin antibody and 3~7 mg / L C-reactive protein;

[0022] The high-level quality control products include 49~91 U / mL rheumatoid factor, 24.5~45.5 U / mL anti-cyclic citrullinated peptide antibodies, 31~42 U / mL anti-citrullinated vimentin antibodies, 22~35 U / mL anti-keratin antibodies and 6~14 mg / L C-reactive protein.

[0023] In some specific embodiments,

[0024] The low-level quality control products include 30 U / mL rheumatoid factor, 15 U / mL anti-cyclic citrullinated peptide antibody, 16 U / mL anti-citrullinated vimentin antibody, 8 U / mL anti-keratin antibody and 3 mg / L C-reactive protein;

[0025] The mid-level quality control products include 40 U / mL rheumatoid factor, 22 U / mL anti-cyclic citrullinated peptide antibody, 25 U / mL anti-citrullinated vimentin antibody, 18 U / mL anti-keratin antibody and 5 mg / L C-reactive protein;

[0026] The high-level quality control includes 70 U / mL rheumatoid factor, 30 U / mL anti-cyclic citrullinated peptide antibody, 36 U / mL anti-citrullinated vimentin antibody, 30 U / mL anti-keratin antibody and 10 mg / L C-reactive protein.

[0027] The present invention provides a method for preparing the rheumatoid arthritis marker substance control product, comprising taking the rheumatoid arthritis marker and the quality control product matrix solution, mixing and freeze-drying to obtain the rheumatoid arthritis marker substance control product.

[0028] In some embodiments, the lyophilized rheumatoid arthritis marker control is stored at 2-8°C.

[0029] The present invention provides use of the quality control material matrix solution, the rheumatoid arthritis marker substance control material or the rheumatoid arthritis marker substance control material prepared by the preparation method in preparing a reagent and / or a kit for detecting rheumatoid arthritis.

[0030] The present invention provides a reagent and / or a kit for detecting rheumatoid arthritis, comprising the quality control substance matrix solution, the rheumatoid arthritis marker substance control substance or the rheumatoid arthritis marker substance control substance prepared by the preparation method.

[0031] The present invention provides a method for detecting rheumatoid arthritis, comprising using the reagent and / or kit for detecting rheumatoid arthritis to detect a sample.

[0032] The present invention provides a rheumatoid arthritis marker material control product comprising rheumatoid factor, anti-cyclic citrullinated peptide antibody, anti-citrullinated vimentin antibody, anti-keratin antibody and C-reactive protein as markers, and a preparation method and application thereof. The rheumatoid arthritis marker material control provided by the present invention is a multi-marker composite quality control product, which solves the problem that stability, sensitivity and specificity cannot be achieved due to large differences in the properties of the markers. The customer can complete the quality control requirements of multiple projects by adding samples once. At the same time, it is applicable to detection platforms such as luminescence platforms and biochemical platforms, which can reduce laboratory operations, improve the sensitivity and specificity of experimental detection, have high efficiency and save costs, and is helpful for the judgment of rheumatoid arthritis. DETAILED DESCRIPTION

[0033] The present invention provides a rheumatoid arthritis marker substance control product and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0034] The preparation method of the present invention is to add rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (Anti-CCP), anti-citrullinated vimentin antibody (MCV), anti-keratin antibody (AKA), C-reactive protein (CRP) and other materials into a serum matrix liquid containing a buffer, a stabilizer, an excipient, and a preservative, and then divide or freeze-dry, or obtain a stable quality control product in a liquid form.

[0035] The present invention is a multi-item composite quality control product. The composite quality control product usually contains multiple markers. As the basis for judging rheumatoid arthritis, the properties of these markers may be different. For example, rheumatoid factor RF has an unstable property of 2-8℃. If normal serum is stored at 2-8℃ for more than 16 hours, the detection of RF may change from negative to positive, causing the quality control product to be unable to be used for a long time after re-dissolving. Based on this, the patent adds aprotinin to form aprotinin-protease complex through the serine active part on the enzyme to achieve an inhibitory effect, thereby improving the stability of RF. The present invention improves the stability of anti-cyclic citrullinated peptide antibodies (Anti-CCP), anti-citrullinated vimentin antibodies (MCV), anti-keratin antibodies (AKA), and C-reactive protein (CRP) by adding aprotinin, ADP, and AEP-HBC; at the same time, it has a bactericidal property, which can ensure that the quality control product will not be reduced in stability or turbid in appearance due to bacterial contamination after liquid or freeze-dried re-dissolution. Existing quality control is difficult to apply to both luminescence platforms and biochemical platforms at the same time. This patent uses human serum matrix + natural antibodies to meet the detection needs of multiple platforms at the same time.

[0036] The test materials used in the present invention are all common commercial products, all purchased from Zhengzhou Iminuo Biotechnology Co., Ltd.

[0037] The present invention will be further described below in conjunction with the embodiments.

[0038] Example 1

[0039] The invention relates to a method for preparing a quality control product for rheumatoid arthritis.

[0040] The present invention is obtained by following steps:

[0041] Step 1: Prepare the matrix solution (the following percentages are by mass):

[0042] Formula 1: Human serum + 10mmol / L TRIS + 0.2% BRO (PH7.4)

[0043] Formula 2: Human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN 3 (PH7.4)

[0044] Formula 3: Human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN 3 +2% ADP(PH7.4)

[0045] Formula 4: Human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN 3 +2% ADP+50U / mLAprotinin(PH7.4)

[0046] Formula 5: Human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN 3 +2% ADP+50U / mLAprotinin(PH7.4)+2% AEP-HBC;

[0047] Formula 6: Human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN 3 +2% ADP+100U / mLAprotinin(PH7.4)+2% AEP-HBC;

[0048] Formula 7: human serum + 10mmol / L TRIS + 0.2% BRO + 0.2% NaN3 + 2% ADP + 50U / mL Aprotinin (PH7.4) + 4% AEP-HBC;

[0049] Step 2: Add different concentrations of rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (Anti-CCP), anti-citrullinated vimentin antibody (MCV), anti-keratin antibody (AKA), and C-reactive protein (CRP) to obtain high, medium and low level quality control products, including:

[0050] The low-level controls included 30 U / mL rheumatoid factor, 15 U / mL anti-cyclic citrullinated peptide antibody, 16 U / mL anti-citrullinated vimentin antibody, 8 U / mL anti-keratin antibody, and 3 mg / L C-reactive protein;

[0051] The mid-level quality control includes 40 U / mL rheumatoid factor, 22 U / mL anti-cyclic citrullinated peptide antibody, 25 U / mL anti-citrullinated vimentin antibody, 18 U / mL anti-keratin antibody, and 5 mg / L C-reactive protein;

[0052] High-level quality control products include 70 U / mL rheumatoid factor, 30 U / mL anti-cyclic citrullinated peptide antibody, 36 U / mL anti-citrullinated vimentin antibody, 30 U / mL anti-keratin antibody, and 10 mg / L C-reactive protein;

[0053] Step 3: Pack and freeze-dry the prepared product;

[0054] Step 4: Store at 2-8°C.

[0055] Effect Example 1

[0056] The quality control products prepared from formulas 1 to 7 of Example 1 were placed at 2-8°C for 14 days (redissolution stability) and 37°C for 14 days (accelerated stability). The specific test steps included:

[0057] Reconstitution stability: After the quality assessment product is opened and reconstituted, it should be sealed and stored at 2℃~8℃ for 14 days. The newly opened bottle and the quality assessment product at the end of the stability period should be tested simultaneously, and the relative deviation of the results should be calculated according to the following formula;

[0058] Accelerated stability: Place the quality control products within the validity period at 37°C for 14 days. Use the corresponding test kit to test the quality control products that are normally stored and opened for the first time and at the end of the thermal stability period. Calculate the relative deviation B of the results according to the following formula.

[0059] Calculation formula:

[0060] Where:

[0061] B----Relative deviation;

[0062] X --- the mean of the test results at the end of the stable period;

[0063] X 0 ----The average of the initial test results after opening a new bottle.

[0064] The product stability data are shown in Tables 1 and 2 below.

[0065] Table 1 2-8℃ 14-day reconstitution stability

[0066]

[0067] Table 2 37℃ 14 days accelerated stability

[0068]

[0069] The data show that the quality control product has good stability when placed at 2-8°C for 14 days and at 37°C for 14 days, and the three-level quality control product prepared with formula five has better stability.

[0070] Effect Example 2

[0071] The sensitivity and specificity of the three-level quality control product prepared by formula five in Example 1 were further verified.

[0072] The samples were clinical samples collected by the company's Zhengzhou Yihe Hospital, including 68 known rheumatoid arthritis (RA) patients and 80 non-RA patients, including 10 systemic lupus erythematosus, 20 Sjögren's syndrome, 21 osteoarthritis, and 29 connective tissue diseases. Roche Cobas8000 was used to detect anti-CCP, Beckman Coulter AU680 was used to detect CRP and RF, and the AKA detection method was immunofluorescence staining. The kit was the AKA kit of Omen Company. ELISA was used to detect serum anti-MCV antibody levels. The kit used for the test was purchased from ORGENTECDiagnostika GmbH.

[0073] The product sensitivity and specificity data are shown in Table 3 and Table 4. Sensitivity = true positive / (true positive + false negative); specificity = true negative / (true negative + false positive)

[0074] Table 3 Results of positive and negative test

[0075]

[0076] Table 4 Sensitivity and specificity results

[0077]

[0078] The data showed that the five-item composite of quality control products significantly enhanced the sensitivity and specificity in detecting rheumatoid arthritis.

[0079] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quality control matrix solution, characterized in that: It includes human serum and the following components: 5~200mM TRIS buffer with a pH of 7.2~7.6, 0.1wt%~1wt% Bro, 0.02wt%~0.3wt% NaN3, 0.2wt%~5wt% ADP, 20~100U / mL aprotinin and 0.2wt%~5wt% AEP-HBC.

2. The quality control matrix solution according to claim 1, characterized in that: It includes human serum and the following components: 10 mM TRIS buffer at pH 7.4, 0.2 wt % Bro, 0.2 wt % NaN3, 2 wt % ADP, 50 U / mL aprotinin, and 2 wt % AEP-HBC.

3. Use of the quality control matrix solution according to claim 1 or 2 in the preparation of a control product for a marker substance of rheumatoid arthritis.

4. A control product for a marker substance of rheumatoid arthritis, characterized in that: The method comprises a rheumatoid arthritis marker and the quality control matrix liquid according to claim 1 or 2.

5. The rheumatoid arthritis marker substance control product according to claim 4, characterized in that: The rheumatoid arthritis marker comprises at least one of rheumatoid factor, anti-cyclic citrullinated peptide antibody, anti-citrullinated vimentin antibody, anti-keratin antibody and C-reactive protein.

6. The rheumatoid arthritis marker substance control product according to claim 4 or 5, characterized in that: It includes low-level quality control products, medium-level quality control products and high-level quality control products, among which: The low-level quality control products include 21-39 U / mL rheumatoid factor, 10.5-19.5 U / mL anti-cyclic citrullinated peptide antibodies, 13-21 U / mL anti-citrullinated vimentin antibodies, 5-11 U / mL anti-keratin antibodies, and 1.8-4.2 mg / L C-reactive protein; The mid-level quality control products include 31.5~58.5 U / mL rheumatoid factor, 17.5~32.5 U / mL anti-cyclic citrullinated peptide antibody, 22~29 U / mL anti-citrullinated vimentin antibody, 13~24 U / mL anti-keratin antibody and 3~7 mg / L C-reactive protein; The high-level quality control products include 49~91 U / mL rheumatoid factor, 24.5~45.5 U / mL anti-cyclic citrullinated peptide antibodies, 31~42 U / mL anti-citrullinated vimentin antibodies, 22~35 U / mL anti-keratin antibodies and 6~14 mg / L C-reactive protein.

7. The method for preparing the rheumatoid arthritis marker substance control product according to any one of claims 4 to 6, characterized in that: The method comprises taking the rheumatoid arthritis marker and the quality control product matrix solution, mixing and freeze-drying to obtain the rheumatoid arthritis marker material control product.

8. The preparation method according to claim 7, characterized in that: The freeze-dried rheumatoid arthritis marker substance control product is stored at 2-8°C.

9. Use of the quality control matrix solution according to claim 1 or 2, the rheumatoid arthritis marker substance control product according to any one of claims 4 to 6, or the rheumatoid arthritis marker substance control product prepared by the preparation method according to claim 7 or 8 in the preparation of reagents and / or kits for detecting rheumatoid arthritis.

10. A reagent and / or kit for detecting rheumatoid arthritis, characterized in that: It comprises the quality control substance matrix liquid as described in claim 1 or 2, the rheumatoid arthritis marker substance control substance as described in any one of claims 4 to 6, or the rheumatoid arthritis marker substance control substance prepared by the preparation method as described in claim 7 or 8.

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