CN113049814A - Application of rheumatoid arthritis marker detection reagent in serum and diagnostic kit - Google Patents
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- 206010039073 rheumatoid arthritis Diseases 0.000 title claims abstract description 57
- 210000002966 serum Anatomy 0.000 title claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 16
- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 10
- 239000003550 marker Substances 0.000 title claims description 17
- 238000001514 detection method Methods 0.000 title abstract description 17
- 102000000344 Sirtuin 1 Human genes 0.000 claims abstract description 39
- 108010041191 Sirtuin 1 Proteins 0.000 claims abstract description 39
- 230000014509 gene expression Effects 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 4
- 239000013641 positive control Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 abstract description 17
- 238000011160 research Methods 0.000 abstract description 5
- 102000011990 Sirtuin Human genes 0.000 description 15
- 108050002485 Sirtuin Proteins 0.000 description 15
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- 230000002072 anti-mutant effect Effects 0.000 description 4
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- 230000035945 sensitivity Effects 0.000 description 4
- 210000005048 vimentin Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 3
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- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
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- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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Abstract
The invention relates to the technical field of diagnosis or detection of rheumatoid arthritis related markers, in particular to application of a detection reagent for rheumatoid arthritis markers in serum and a diagnostic kit. By carrying out quantitative detection on four markers, namely SIRT1, anti-CCP, anti-MCV and RF, research results show that the diagnosis of RA by SIRT1, anti-CCP, anti-MCV and RF has clinical significance, but the specificity of RA diagnosis by SIRT1 is superior to other indexes, and the RA diagnosis level can be obviously improved by combined detection of the SIRT1 and the anti-CCP, so that the SIRT1 can be used as a new index for diagnosing RA, and has wide clinical application and research prospects.
Description
Technical Field
The invention relates to the technical field of diagnosis or detection of rheumatoid arthritis related markers, in particular to application of a detection reagent for rheumatoid arthritis markers in serum and a diagnostic kit.
Background
Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease. It is a synovial inflammation caused by abnormal activation of the immune system, and is characterized by the production of a large number of inflammatory cells and cytokines in synovial fluid and the production of various antibodies in serum. Because RA is hidden and the clinical manifestations lack specificity, patients often cannot diagnose and adopt effective treatment in time. In recent years, biomarkers for RA diagnosis have been increasingly developed, but the levels of sensitivity and specificity of examination indices vary, and therefore, diagnosis of RA in clinical work is still insufficient. It has been found in the literature that SIRT1, a member of the family of Sirtuins, is a key regulator of RA pathogenesis, and inhibits the aggressiveness and inflammatory response of RA-like synoviocytes by inhibiting the NF- κ B pathway. The SIRT1 is suggested to be closely related to the occurrence and development of RA, but the SIRT1 is not sufficient to be independently applied to clinic.
Disclosure of Invention
In order to overcome the defects, the invention provides the application of the detection reagent of the rheumatoid arthritis marker in serum and a diagnostic kit containing the marker, which can be used for rheumatoid arthritis diagnosis and curative effect monitoring.
In order to achieve the purpose, the invention adopts the following technical scheme: the application of the test reagent for the rheumatoid arthritis marker in serum is specifically the application of the test reagent for the rheumatoid arthritis marker in serum in preparing a product for diagnosing rheumatoid arthritis, wherein the marker is selected from any one of the following combinations:
(1) sirtuin (1SIRT1), anti-cyclic citrullinated peptide antibody (anti-CCP);
(2) sirtuin (1SIRT1), human anti-mutant citrullinated vimentin antibody (anti-MCV);
(3) sirtuin (1SIRT1), Rheumatoid Factor (RF);
(4) sirtuin (1SIRT1), anti-cyclic citrullinated peptide antibody (anti-CCP), human anti-mutant citrullinated vimentin antibody (anti-MCV);
(5) sirtuin (1SIRT1), anti-cyclic citrullinated peptide antibody (anti-CCP), Rheumatoid Factor (RF);
(6) sirtuin (1SIRT1), human anti-mutant citrullinated vimentin antibody (anti-MCV), Rheumatoid Factor (RF); (7) sirtuin (1SIRT1), anti-cyclic citrullinated peptide antibody (anti-CCP), human anti-mutant citrullinated vimentin antibody (anti-MCV), Rheumatoid Factor (RF).
Further, an increased level of expression of each marker selected from the combination as compared to a healthy control indicates that the subject has rheumatoid arthritis.
Still further, the subject is a human.
Furthermore, the product for diagnosing rheumatoid arthritis is a diagnostic kit.
Further, the diagnostic kit comprises the following components: the kit comprises the marker, a negative control and a positive control, wherein the positive control is serum of a rheumatoid arthritis patient, and the negative control is serum of a healthy person.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a marker of rheumatoid arthritis in serum and a novel rheumatoid arthritis detection method, which improve the sensitivity and specificity of detection by detecting a plurality of markers and provide possibility for screening and auxiliary diagnosis of rheumatoid arthritis in clinic; the operation is convenient and simple, the cost is low, and the harm to patients is low by taking the serum as a detection sample. The invention has good application prospect.
Drawings
FIG. 1 is a ROC curve for candidate markers in diagnosing RA.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
This study demonstrated that SIRT1, anti-CCP, anti-MCV and RF were significantly elevated in the serum of RA patients. Among them 70.9% (100 cases) of RA patients gave positive SIRT1 results, suggesting that RA cannot be accurately diagnosed based on serum SIRT1 levels alone. However, only 3.5% (8 cases) of non-RA and healthy controls showed positive results in SIRT1, and therefore, the detection of SIRT1 as an exclusive detection index has significant meaning in the differential diagnosis of RA. While serum SIRT1 levels were significantly elevated (P >0.05) in patients with RA, Ankylosing Spondylitis (AS) and Osteoarthritis (OA) relative to healthy controls, suggesting the presence of SIRT1 overexpression in erosive arthropathies, especially in patients with RA, whether it is a contributing factor to the destruction of articular bone mass has yet to be confirmed by further studies.
1. Sources of materials
1.1 study subjects: the subjects were hospitalized and outpatient confirmed patients or health examiners in the second affiliated hospital of Nanchang university from 1 month in 2019 to 9 months in 2020. 141 patients with RA, 36 men and 105 women, age 22-82 years, mean (52.7 +/-15.8) years, and inclusion criteria all meet the latest RA diagnosis criteria of European Union of rheumatism and arthritis (EULAR). The non-RA group was 144 cases, of which Ankylosing Spondylitis (AS) group was 40 cases, 11 men, 29 women, aged 28-78 years, and mean (52.9 ± 13.9) years; osteoarthritis (Osteoarthritis, OA) group 48 cases, 14 men, 34 women, age 34-76 years, mean (55.1 ± 12.2); sjogren's syndrome, SS group 56, 15 men, 41 women, age 31-81 years, mean (58.7 + -15.0). The healthy control groups were 88, 25 men and 63 women, aged 26-79 years, and the mean (52.6 + -15.2) years, all were healthy subjects in the hospital physical examination center. All non-RA patients met relevant disease guideline diagnostic criteria and were confirmed by relevant department physicians. All specimens were informed consent was obtained and approved by the ethical committee of the hospital of the second subsidiary hospital of southern chang university.
1.2 inclusion criteria: the research objects voluntarily participate in the research and sign an informed consent; second, the diagnosis of the patient is clear, and the related image examination and clinical medical record data are complete; and each system of the health physical examiners has no abnormity in routine examination.
1.3 exclusion criteria: combining abnormal metabolic diseases; ② combining other autoimmune diseases and tumor diseases; ③ severe liver, kidney, heart or lung system diseases; fourthly, the women in pregnancy and lactation; children in growth and development stage.
2. Method of producing a composite material
2.1 specimen collection and processing: collecting 3ml of fasting venous blood of all the study objects by using a separation gel vacuum coagulation-promoting blood collection tube, standing for 30min, centrifuging for 15min at 1026 Xg, and taking upper serum for later use.
2.2 instruments, reagents and methods: SIRT1 is measured by ELISA method reagent produced by Heifeier biotechnology Limited liability company; the anti-CCP is measured by an iFlash3000-C type chemiluminescence immunoassay analyzer and a matched reagent of Shenzhen Shenhuilong Biotechnology corporation; the anti-MCV is measured by adopting an ELISA method reagent produced by Xiugeng biotechnology development Limited company in Tianjin; CRP (C-reactive protein) and RF are determined by a rate-scattering turbidimetry by adopting an IMMAGE 800 type full-automatic specific protein analysis system and a matched reagent of Beckmann company of America; ESR (erythrocyte sedimentation rate) is measured by the Weissen method by using a full-automatic rapid sedimentation analyzer Roller 20 and a matched reagent of ALIFAX Italy. The operation is strictly carried out according to the specification and the quality management standard of the second subsidiary hospital of Nanchang university.
3. Results
3.1 expression of candidate markers in serum of patients with rheumatoid arthritis
As shown in Table 1, SIRT1, anti-CCP and anti-MCV were significantly elevated in the serum of RA patients.
TABLE 1 expression of molecular markers of candidate proteins
Note: in patients # RA, the differences were statistically significant (P < 0.05). Differences were statistically significant (P <0.01) compared to normal controls.
3.2 diagnostic value of candidate markers for rheumatoid arthritis
The potential utility of serum proteins as RA biomarkers was assessed using the area under the curve (AUC) of the ROC curve.
As a result: as shown in Table 2 and FIG. 1, in this study, the anti-CCP specificity was 95.7%, and only after SIRT1, the result of the ROC curve also shows that the AUC of the serum level of SIRT1 to RA diagnosis was 0.87, and the diagnosis accuracy was high.
TABLE 2 clinical evaluation of the results of detection of RA by candidate markers
Note: AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value; + LR, positive likelihood ratio; -LR, negative likelihood ratio.
3.3 diagnostic value of candidate marker combinations for rheumatoid arthritis
At least 2 candidate markers are combined together for diagnosis, the series connection and the parallel connection are included, the series connection and the parallel connection combination and clinical evaluation are shown in table 3, after indexes such as SIRT1, anti-CCP, anti-MCV, RF and the like are detected in parallel, the sensitivity is obviously increased, particularly when SIRT1 and anti-CCP are detected in parallel, the sensitivity and the specificity are both more than 90%, and YDI can reach 0.85. This suggests that the combined detection of SIRT1 and anti-CCP has important significance for the diagnosis of RA.
3.4 correlation of candidate markers with each other
The correlation between SIRT1 and anti-MCV, anti-CCP, RF and the like is verified through Pearson correlation analysis, and the results are shown in Table 4, wherein SIRT1 is in positive correlation with anti-MCV, anti-CCP, RF and the like, and SIRT1 is in highest correlation with CRP (C-reactive protein) and in second order with ESR (erythrocyte sedimentation rate). While ESR and CRP serve as important indicators for assessing RA activity, the close association of SIRT1 suggests that SIRT1 may be related to disease activity in RA.
TABLE 4 correlation of SIRT1 with anti-MCV, anti-CCP, RF and CRP
In conclusion, the SIRT1, anti-CCP, anti-MCV and the like have clinical significance on the diagnosis of RA, but the specificity of SIRT1 for diagnosing RA is superior to other indexes, and the combined detection of the SIRT1 and the anti-CCP can obviously improve the diagnosis level of RA. Therefore, the SIRT1 can be used as a new index for diagnosing RA, and has wide clinical application and research prospects.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. The application of the test reagent for the rheumatoid arthritis marker in serum is characterized in that the application is specifically the application of the test reagent for the rheumatoid arthritis marker in serum in preparing a product for diagnosing rheumatoid arthritis, and the marker is selected from any one of the following combinations:
(1)SIRT1，anti-CCP；
(2)SIRT1，anti-MCV；
(3)SIRT1，RF；
(4)SIRT1，anti-CCP，anti-MCV；
(5)SIRT1，anti-CCP，RF；
(6)SIRT1，anti-MCV，RF；
(7)SIRT1，anti-CCP，anti-MCV，RF。
2. the use according to claim 1, wherein an elevated level of expression of each marker selected from the combination of claim 1, as compared to a healthy control, is indicative of rheumatoid arthritis in the subject.
3. The use of claim 2, wherein the subject is a human.
4. The use according to claim 2, wherein the product for diagnosing rheumatoid arthritis is a diagnostic kit.
5. A diagnostic kit comprising the marker of claim 1, wherein the diagnostic kit comprises the following components: the marker of claim 1, a negative control and a positive control, wherein the positive control is serum of a rheumatoid arthritis patient, and the negative control is serum of a healthy person.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114594266A (en) * | 2022-03-02 | 2022-06-07 | 安徽中医药大学第一附属医院(安徽省中医院) | Application of M1 and M2 type macrophage factor combined as biomarker in diagnosis and treatment monitoring of rheumatoid arthritis |
| CN116148466A (en) * | 2023-04-23 | 2023-05-23 | 北京大学人民医院 | Multi-serum marker combination for rheumatoid arthritis diagnosis |
| CN116891893A (en) * | 2023-07-27 | 2023-10-17 | 上海市同仁医院 | Primer combination and diagnosis model for detecting seronegative rheumatoid arthritis |
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| CN116148466A (en) * | 2023-04-23 | 2023-05-23 | 北京大学人民医院 | Multi-serum marker combination for rheumatoid arthritis diagnosis |
| CN116148466B (en) * | 2023-04-23 | 2023-11-21 | 北京大学人民医院 | Multi-serum marker combination for rheumatoid arthritis diagnosis |
| CN116891893A (en) * | 2023-07-27 | 2023-10-17 | 上海市同仁医院 | Primer combination and diagnosis model for detecting seronegative rheumatoid arthritis |
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