Application of reagent for detecting pyroglutamate aminopeptidase in preparation of rheumatoid arthritis diagnostic product

Abstract

The invention relates to the field of biological detection, and discloses application of a reagent for detecting pyroglutamic acid aminopeptidase in preparation of a rheumatoid arthritis diagnostic product. The reagent assists in identifying RA patients by detecting the expression level of pyroglutamic acid aminopeptidase in a subject sample, can be used for treatment response evaluation and prognosis judgment, and can also effectively distinguish RA from osteoarthritis. Experiments prove that the expression of pyroglutamic acid aminopeptidase in serum of RA patients is remarkably improved, and the pyroglutamic acid aminopeptidase also shows a relatively high expression level in RA patients with rheumatism factor (RF) negative and anti-cyclic citrullinated peptide antibody (CCP) negative. In addition, the expression level of pyroglutamic acid aminopeptidase is in positive correlation with RA score, and is gradually reduced along with the improvement of the illness state after treatment. The reagent disclosed by the invention is high in sensitivity and good in specificity, and can be used for early diagnosis of RA and dynamic monitoring of an illness state.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to the application of a reagent for detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnosis product. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic, systemic, autoimmune disease. RA is characterized by synovial inflammation and progressive destruction of joint structure, which may lead to joint deformity and loss of function. The pathogenesis of RA is complex and is closely related to genetic susceptibility, environmental factors and abnormal immune regulation. The main pathological characteristics of the disease are synovial tissue hyperplasia, neovascularization and massive infiltration of immune cells, accompanied by excessive release of inflammatory factors. Early diagnosis and intervention are of great significance in delaying disease progression and improving the quality of life of patients. However, due to the diverse clinical manifestations of RA and the lack of a single specific diagnostic criterion, the diagnosis and progression assessment of the disease still face great challenges.

[0003] At present, the clinical diagnosis of RA mainly relies on comprehensive medical history, imaging examination and laboratory tests. The common detection methods are described as follows. ① Serological markers: Anti-cyclic citrullinated peptide antibodies (CCP): have high specificity and sensitivity, but there is a problem that some patients are negative. ② Rheumatoid factor (RF): It is the earliest marker used in the diagnosis of RA, but its specificity is low and it is easily interfered by other diseases or physiological states. ③ Inflammatory indicators: C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have certain value in reflecting inflammatory activity, but lack RA specificity. ④ Imaging examination: X-ray, ultrasound or MRI are used to detect joint destruction, but it is often difficult to find specific changes in the early stage of the disease. Although the above methods play an important role in the diagnosis and monitoring of RA, there are still many problems, including: ① Existing indicators cannot evaluate the degree of progression and recovery of RA, and cannot guide medication; ② About 20%-30% of RA patients have seronegative (that is, both RF and anti-CCP antibodies are negative at the time of diagnosis, but they still belong to rheumatoid arthritis, which is medically defined as seronegative rheumatoid arthritis). Therefore, there is an urgent need to discover and develop new diagnostic markers to further improve the diagnostic accuracy of RA and guide the grading of RA.

[0004] Rheumatoid arthritis (RA) and osteoarthritis (OA) are two common joint diseases, but their pathological mechanisms and treatment strategies are significantly different. RA is an autoimmune disease characterized by synovial inflammation, immune cell infiltration and joint destruction, while OA is a degenerative disease characterized by articular cartilage degeneration and osteophyte formation. Clinically, the symptoms of RA and OA may overlap in the early stages of the disease, such as joint pain, stiffness and dysfunction. When osteoarthritis appears on the fingers, it may be misdiagnosed as rheumatoid arthritis, which poses a challenge to accurate diagnosis. Therefore, finding a method that can effectively distinguish and diagnose rheumatoid arthritis and osteoarthritis is an urgent problem to be solved. Summary of the invention

[0005] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a reagent for detecting pyroglutamate aminopeptidase for use in preparing a product for diagnosing rheumatoid arthritis.

[0006] The technical solution adopted in this application is: The first aspect of the present invention provides a use of a reagent for quantitatively detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product.

[0007] Preferably, the reagent for quantitatively detecting pyroglutamate aminopeptidase includes a reagent for quantitatively detecting pyroglutamate aminopeptidase gene mRNA and / or a reagent for quantitatively detecting pyroglutamate aminopeptidase protein.

[0008] Preferably, the rheumatoid arthritis includes at least one of serum rheumatoid factor (RF)-positive rheumatoid arthritis, serum anti-cyclic citrullinated antibody (anti-CCP antibody)-positive rheumatoid arthritis, and serum RF and anti-CCP antibody-negative rheumatoid arthritis.

[0009] Preferably, the above detection is achieved by the following method, which includes at least one of enzyme-linked immunosorbent assay, protein immunoblotting, immunohistochemistry, fluorescent quantitative PCR or mass spectrometry analysis.

[0010] Preferably, the above-mentioned product includes at least one of a kit, a chip, a test paper, or a high-throughput sequencing platform.

[0011] Preferably, the rheumatoid arthritis diagnostic product is used to diagnose and determine rheumatoid arthritis in chronic joint diseases including rheumatoid arthritis and osteoarthritis; and / or, the rheumatoid arthritis diagnostic product is used for graded diagnosis of rheumatoid arthritis.

[0012] A second aspect of the present invention provides a detection system for diagnosing rheumatoid arthritis, comprising: (1) Sample collection module: used to collect biological samples from subjects; (2) Sample detection module: used to quantitatively detect the expression level of pyroglutamate aminopeptidase in vitro; (3) Data analysis module: used to compare the test results with the diagnostic threshold and generate a diagnostic report.

[0013] Preferably, the sample includes at least one of whole blood, serum, plasma, and tissue fluid.

[0014] Preferably, the diagnostic threshold is a cut off value; in some embodiments, the cut off value is 57.28 ng / mL, that is, when the expression level of pyroglutamate aminopeptidase in the test result is greater than or equal to 57.28 ng / mL, the patient can be preliminarily identified as RA.

[0015] The third aspect of the present invention provides a rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product, which comprises a reagent for detecting the expression level of pyroglutamate aminopeptidase in a sample.

[0016] Preferably, the product includes at least one of an ELISA detection kit, a Western Blot detection kit, an IHC staining kit, and an immunochromatography test strip.

[0017] In some of the embodiments, the data analysis module further combines the test results of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (CCP) to generate a comprehensive diagnostic conclusion.

[0018] A method for evaluating the disease activity of rheumatoid arthritis, which determines the disease activity by detecting the changing trend of the expression level of pyroglutamate aminopeptidase in patients before and after treatment.

[0019] In some of these embodiments, the patient's inflammatory markers (CRP, ESR, etc.) and imaging data are combined to further assess the severity of the disease.

[0020] A method for formulating an individualized treatment plan for rheumatoid arthritis, which selects appropriate drugs, dosages and treatment cycles based on the dynamic changes in the expression level of pyroglutamate aminopeptidase in the subjects.

[0021] In some of these embodiments, the treatment strategy is optimized by combining the pyroglutamate aminopeptidase test results with the patient's immune indicators (such as IL-6, TNF-α levels, etc.).

[0022] The invention discloses a reagent for detecting pyroglutamate aminopeptidase in preparing a product for distinguishing between rheumatoid arthritis and osteoarthritis.

[0023] The beneficial effects of this application are: (1) This study found that the content of glutamate aminopeptidase can be used for the diagnosis of seronegative rheumatoid arthritis, especially for the diagnosis of rheumatoid arthritis patients with negative RF and CCP. The use of pyroglutamate aminopeptidase in the diagnosis of RA can make up for the shortcomings of existing detection indicators and fill the gap in the current field of RA diagnosis.

[0024] (2) This study found that the level of pyroglutamate aminopeptidase is linearly correlated with the progression of RA. By monitoring the level of pyroglutamate aminopeptidase, dynamic assessment of RA disease activity and treatment response can be achieved, providing a scientific basis for formulating individualized treatment plans.

[0025] (3) This study found that the level of pyroglutamate aminopeptidase can effectively distinguish RA and osteoarthritis (OA) with similar clinical symptoms, thereby improving the accuracy of RA diagnosis.

[0026] In summary, the use of pyroglutamate aminopeptidase for the diagnosis of RA can not only make up for the shortcomings of existing detection indicators, but also dynamically evaluate RA disease activity and treatment response, while helping to distinguish RA from OA, providing important support for the diagnosis and precision medicine of RA, and has significant clinical and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the ROC curve of serum pyroglutamate aminopeptidase in RA patients.

[0028] Figure 2 This is a statistical chart detecting the differential expression of pyroglutamate aminopeptidase at the protein level (***P<0.001).

[0029] Figure 3 is the expression level of pyroglutamate aminopeptidase in patients with seronegative rheumatoid arthritis (***P<0.001).

[0030] Figure 4 The level of pyroglutamate aminopeptidase was positively correlated with the clinical score of rheumatoid arthritis. DETAILED DESCRIPTION

[0031] Terminology explanation: Pyroglutamate aminopeptidase (EC:3.4.19.3) catalyzes the removal of L-pyroglutamate from the N-terminus of certain peptides and proteins. Pyroglutamate aminopeptidases are found in almost all life domains, including mammals, birds, fish, plants, protists, and fungi. In addition, pyroglutamate aminopeptidases are also highly conserved and ubiquitously distributed in human tissues, and new physiological functions have been proposed in recent decades. In previous studies, pyroglutamate aminopeptidase was considered to be involved in the absorption of peptides and proteins in the mammalian digestive tract due to its presence in the small intestine and duodenum. In addition, based on the widespread distribution of pyroglutamate aminopeptidase in functionally different tissues, researchers have proposed that pyroglutamate aminopeptidase may be involved in the intracellular breakdown and resynthesis of peptides. Pyroglutamate aminopeptidase is also believed to affect certain disease states (e.g., memory impairment) by regulating the level of free L-pyroglutamate.

[0032] Although pyroglutamate aminopeptidase may be involved in the regulation of certain diseases, the expression of pyroglutamate aminopeptidase in RA is not clear. More importantly, there is a lack of clear research evidence in the existing technology as to whether pyroglutamate aminopeptidase can be used as a diagnostic indicator for RA.

[0033] In this application study, it was found that pyroglutamate aminopeptidase was linearly correlated with the progression of rheumatoid arthritis, which indicates that pyroglutamate aminopeptidase can be used to judge the disease progression of rheumatoid arthritis and evaluate the recovery, which is impossible with existing objective detection indicators (such as RF, CCP). In addition, pyroglutamate aminopeptidase can also be used for the diagnosis of seronegative rheumatoid arthritis, which is a supplement to existing diagnostic indicators (such as RF, CCP) and can make up for the shortcomings of existing methods.

[0034] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0035] In the examples, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0036] Examples 1-3 are data analyses of patient samples. The diagnosis of all RA cases complies with the RA diagnostic criteria revised by the American College of Rheumatology in 1987, that is, the test indicators include: "morning stiffness (lasting more than 1 hour)", "joint symptoms (swollen at least 3 joints)", "rheumatoid nodules (subcutaneous nodules commonly found in areas prone to friction)", "RF (elevated serum levels)", "X-ray (visible bone erosion changes)". All cases of osteoarthritis patients meet the diagnostic criteria for OA proposed by Altam, that is, the tests include: hypertrophy of at least 2 finger joints, bone hypertrophy, bone friction sensation, morning stiffness, etc. The clinical samples used in this application study were provided by the Affiliated Hospital of Xi'an Jiaotong University, and the patients were informed and approved by the Ethics Committee.

[0037] Example 4 is a data analysis of the RA mouse model.

[0038] The pyroglutamic acid aminopeptidase protein content of the sample serum in Examples 1-3 was detected using Human PGPEP1 (Pyroglutamyl-peptidase 1) ELISA Kit. The pyroglutamic acid aminopeptidase protein content of the sample serum in Example 4 was detected using Mouse Pyroglutamyl-peptidase 1 (PGPEP1) ELISA Kit. All sera used in the Elisa test were prepared according to the standard protocol. The serum was diluted and the detection procedure strictly followed the commercial Elisa kit protocol. Example 1

[0039] The pyroglutamate aminopeptidase protein content in the serum of 10 healthy volunteers and 10 RA patients (patients diagnosed with RA, without considering the distinction between positive or negative RF and CCP) was detected, as shown in Table 1. ROC curve analysis was performed in the hope of finding an indicator with high area under the curve (AUC), sensitivity and specificity to determine the optimal screening positive critical value (cutoff value). The closer the AUC is to 1, the better the diagnostic effect.

[0040] Table 1. Pyroglutamate aminopeptidase levels in serum (upper arm venous blood) of RA patients and normal subjects

[0041] like Figure 1As shown, the result is AUC = 0.986, p < 0.0001, indicating that the content of pyroglutamate aminopeptidase can clearly distinguish RA patients from healthy people. When the cut off value = 57.28, the specificity of judging RA is 100% and the sensitivity is 91.67%, that is, when this cut off value is taken, patients without RA can be identified 100% without false positives; at the same time, the detection rate (or true positive rate) is 91.67%, and the high sensitivity can effectively identify RA patients and reduce the missed diagnosis rate. In other words, when ≥57.28 ng / mL, the patient can be preliminarily identified as RA.

[0042] Example 2 In Example 2, 4 RA patients and 4 osteoarthritis patients showed similar external symptoms, namely morning stiffness, hypertrophy of finger joint tissue, bone friction, etc., which are difficult to effectively distinguish only by external joint manifestations. In this example, by detecting the content of pyroglutamate aminopeptidase in the patient's venous blood (the detection method is the same as that in Example 1), using the cutoff value in Example 1 (57.28 ng / mL), RA patients and osteoarthritis patients can be effectively distinguished.

[0043] The results are as follows Figure 2 As shown in the results, the serum pyroglutamate aminopeptidase level in osteoarthritis patients was 46.6 ng / mL, which was lower than the cut-off value (57.28 ng / mL), while the serum pyroglutamate aminopeptidase level in RA patients was 86.9 ng / mL, which was much higher than the cut-off value. The data showed that pyroglutamate aminopeptidase could effectively distinguish between rheumatoid arthritis and osteoarthritis, P<0.001. Example 3

[0044] In Example 3, blood samples of 3 RA patients with seronegative rheumatoid arthritis (RF negative, CCP negative) were obtained from RA patients in the hospital, including 2 females and 1 male, with an average age of (55±8) years and an average disease course of (2±1) years. 3 normal blood samples were obtained from volunteers. The detection method was the same as that in Example 1, and the results are shown in Figure 3 .

[0045] like Figure 3 As shown in the results, in patients with seronegative rheumatoid arthritis, pyroglutamate aminopeptidase was still highly expressed at 96±5 ng / mL, which was much higher than the cut off value (57.28 ng / mL), P<0.001, indicating that pyroglutamate aminopeptidase can be used for the diagnosis of seronegative rheumatoid arthritis. Example 4

[0046] Construction of RA mouse model: 12 male DBA / 1 mice. LPS and Freund's adjuvant were completely emulsified, and 0.2 mg of emulsified LPS and Freund's adjuvant were intradermally injected into the mice on days 0 and 7. Usually, an inflammatory response of the joints will occur on the 10th to 13th day after stimulation. The thickness of the soles was measured using a vernier caliper with an accuracy of 0.01 mm, and the clinical scoring was used for evaluation. The scoring criteria are divided into two aspects: one is the inflammation score of the mouse toes, and the other is the swelling score of the mouse paws. See Table 2 for details.

[0047] Table 2. Inflammatory response in the paw joints of mice

[0048] The score of each mouse was the sum of the inflammation score of the toes and the swelling score of the paws (the maximum score was 14 points). A score of 1.5 was considered a successful model construction. The mice with successful RA model construction were re-randomly grouped to continue the following drug administration experiment.

[0049] Ten mice with successful modeling (all scored 3 points) were selected for further experiments. The experiment was divided into a model control group and a drug administration group, with 5 mice in each group. The mice in the model control group did not receive any treatment. The mice in the drug administration group were orally administered with the compound methotrexate (at a dose of 14 mg / kg / week) by gavage, once a day, for three consecutive weeks. Methotrexate is a cornerstone drug for the treatment of RA. The "2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis" recommends the use of methotrexate alone and as a "cornerstone drug" for combined treatment. After the start of treatment, the mice were scored for CIA every two weeks, and blood was collected from the tail vein to measure the content of pyroglutamyl-peptidase in the blood (detected by Mouse Pyroglutamyl-peptidase 1 (PGPEP1) ELISA Kit). The results showed that the CIA score and pyroglutamyl-peptidase content of the model control group remained basically unchanged (the average CIA score was about 3.0, and the average pyroglutamyl-peptidase content was about 90 ng / mL). In addition, the drug administration group was plotted with CIA score as the horizontal axis and pyroglutamate aminopeptidase content as the vertical axis. Figure 4 As shown in the figure, as the CIA score of the drug-treated group gradually decreased, the pyroglutamate aminopeptidase content also gradually decreased. The regression equation was Y = 23.02X + 20.52, and the goodness of fit R² = 0.9888.

[0050] After RA model mice received methotrexate treatment, the PGP-1 concentration in their serum gradually decreased with the treatment time, and the downward trend was consistent with the improvement trend of the inflammation score, indicating that PGP-1 can be used to reflect the disease progression during the treatment process and has potential prognostic evaluation value.

[0051] In summary, the above experimental data show that the level of pyroglutamate aminopeptidase is positively correlated with the progression of RA, suggesting that serum pyroglutamate aminopeptidase levels can be used to grade RA and evaluate prognosis.

[0052] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of a reagent for quantitatively detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product.

2. The use according to claim 1, characterized in that: The rheumatoid arthritis includes at least one of serum rheumatoid factor-positive rheumatoid arthritis, serum anti-cyclic citrulline antibody-positive rheumatoid arthritis, and serum rheumatoid factor and serum anti-cyclic citrulline antibody-negative rheumatoid arthritis.

3. The use according to claim 1, characterized in that: The reagent for quantitatively detecting pyroglutamate aminopeptidase includes a reagent for quantitatively detecting pyroglutamate aminopeptidase gene mRNA and / or a reagent for quantitatively detecting pyroglutamate aminopeptidase protein.

4. The use according to claim 1 or 3, characterized in that: The detection is achieved by the following methods, which include at least one of enzyme-linked immunosorbent assay, protein immunoblotting, immunohistochemistry, fluorescent quantitative polymerase chain reaction or mass spectrometry.

5. The use according to claim 1, characterized in that: The product includes at least one of a kit, a chip, a test paper, or a high-throughput sequencing platform.

6. The use according to claim 1, characterized in that: The rheumatoid arthritis diagnostic product is used for diagnosing and determining rheumatoid arthritis in chronic joint diseases including rheumatoid arthritis and osteoarthritis; and / or, the rheumatoid arthritis diagnostic product is used for graded diagnosis of rheumatoid arthritis.

7. A detection system for diagnosing rheumatoid arthritis, comprising: (1) Sample collection module: used to collect biological samples from subjects; (2) Sample detection module: used to quantitatively detect the expression level of pyroglutamate aminopeptidase in vitro; (3) Data analysis module: used to compare the test results with the diagnostic threshold and generate a diagnostic report.

8. The detection system according to claim 7, characterized in that: The biological sample includes at least one of whole blood, serum, plasma, and tissue fluid.

9. A rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product, characterized in that: The product comprises a reagent for detecting the expression level of pyroglutamate aminopeptidase in a sample.

10. The rheumatoid arthritis diagnosis product or rheumatoid arthritis prognosis assessment product according to claim 9, characterized in that: The product includes at least one of an ELISA detection kit, a Western Blot detection kit, an IHC staining kit, and an immunochromatography test strip.

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