CN117990903A - Application of anti-PCOLCE antibody in preparation of rheumatoid arthritis supplementary diagnostic product - Google Patents
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Abstract
The invention discloses an application of an anti-PCOLCE antibody in preparing a rheumatoid arthritis supplementing diagnosis product, wherein the anti-PCOLCE antibody is used as a marker, and the product is used for detecting the content of the anti-PCOLCE antibody. According to the invention, compared with the normal healthy people and other common rheumatic immune diseases which are easy to be confused with rheumatoid arthritis, the anti-PCOLCE antibody is obviously increased in serum of a patient suffering from the rheumatoid arthritis, so that the defects that the existing autoantibody commonly used for diagnosis is low in positive rate and 1/3 of patients are negative can be effectively overcome, and the diagnosis value of the rheumatoid arthritis is better supplemented.
Description
Technical Field
The invention relates to the technical field of disease diagnosis, in particular to application of an anti-PCOLCE antibody in preparation of rheumatoid arthritis supplementary diagnosis products.
Background
Rheumatoid arthritis (rheumatoid arthritis, RA) is a chronic, progressive, erosive systemic autoimmune disease that is predominantly represented by symmetric polyarthritis. If the disease can not be diagnosed and treated in time, the disease can be developed into joint stiffness and deformity, thereby seriously affecting the quality of life. The disease progress of RA is rapid, and the disability rate of the treatment is 75% after the treatment, so that the early diagnosis and treatment of RA are important.
Diagnosis of RA relies largely on detection of serum markers, in addition to patient-based clinical manifestations and imaging changes. Currently, the most commonly used diagnostic method in clinic is to detect the anti-citrullinated protein antibody (anti-cyclic citrullinated peptide antibody, ACPA) titres in serum by enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), of which the most commonly used is to detect anti-cyclic citrullinated peptide (cyclic citrullinated peptide, CCP) antibody titres. anti-CCP antibodies are highly specific autoantibodies to RA, but about 2/3 of RA patients antibodies detected positive and currently lack better diagnostic methods for seronegative patients.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a novel rheumatoid arthritis biomarker for supplementary diagnosis of rheumatoid arthritis.
The technical scheme of the invention is as follows:
In a first aspect, the invention provides the use of an anti-PCOLCE antibody as a marker for the preparation of a complementary diagnostic product for rheumatoid arthritis for the detection of anti-PCOLCE antibody content.
PCOLCE, collectively referred to as procollagen C endopeptidase enhancer (procollagen C-endopeptidase enhancer), also known as procollagen hydroxyl end protease enhancer (Procollagen COOH-Terminal Proteinase Enhancer, PCEP), is a glycoprotein comprising 449 amino acids. PCOLCE are involved in heparin binding and metallopeptidase inhibitor activity, and enhance the process of decomposing type I procollagen C propeptide into type III procollagen C propeptide by procollagen C protease, participating in the necessary steps of extracellular matrix major fiber component assembly. Previous studies have shown that PCOLCE is an important determinant of bone mechanical properties, geometry and collagen fiber morphology in mammals.
PCOLCE is expressed in various normal organs such as lung, liver, heart, brain and the like, and partial researches report that the PCOLCE can be related to various diseases such as osteosarcoma, myocardial fibrosis, liver fibrosis, keloid and the like. Through a great deal of researches, the invention discovers that compared with normal healthy people and other common rheumatic immune diseases (such as osteoarthritis, systemic lupus erythematosus and Sjogren syndrome) which are easy to confuse with rheumatoid arthritis, the anti-PCOLCE antibody is obviously increased in serum of RA patients, and has better supplementary diagnosis value for RA diagnosis.
Preferably, the biological sample is blood, plasma, serum or interstitial fluid.
Preferably, the product is a detection reagent or a supplementary diagnostic kit.
In a second aspect, the invention provides a product for the supplementary diagnosis of rheumatoid arthritis, the product comprising reagents for detecting anti-PCOLCE antibodies.
Preferably, the product is a detection reagent or a supplementary diagnostic kit.
Preferably, the reagent for detecting the anti-PCOLCE antibody comprises a coating antigen citrullinated polypeptide, and the amino acid sequence of the citrullinated polypeptide is shown as SEQ ID NO. 1.
The invention has the following beneficial effects:
The anti-PCOLCE antibody provided by the invention is used as a marker, has obvious difference with normal healthy people and other common rheumatic immune patients which are easy to be confused with rheumatoid arthritis in RA patients, has sensitivity of 42.86 percent and specificity of 92.44 percent in RA diagnosis, has positive rate of 27.27-31.34 percent in serum anti-CCP antibodies and/or RF negative RA patients, can effectively overcome the defect of low detection accuracy of the conventional antibodies, and has better supplementary diagnosis value for RA diagnosis.
Drawings
FIG. 1 is a graph showing the relative abundance of anti-PCOLCE antibodies detected by ELISA in serum samples of RA patients, healthy people, and other common rheumatic immune patients who are easily confused with rheumatoid arthritis in example 1;
FIG. 2 is a ROC curve of the serum anti-PCOLCE antibody used in example 2 to distinguish RA patients from healthy people and disease controls;
FIG. 3 is a graph showing the relative abundance of anti-PCOLCE antibodies detected by ELISA in serum samples from seronegative RA patients in example 3;
FIG. 4 is a ROC curve for a healthy population and seronegative RA patients differentiated by anti-PCOLCE antibodies in serum in example 3.
Detailed Description
For a better understanding of the present application, reference will now be made in detail to the present embodiments, examples of which are illustrated in the accompanying drawings, wherein the present application is illustrated in the accompanying drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, shall fall within the scope of the present application. Unless otherwise specified, the instruments and reagents used in the examples were purchased commercially, and the experimental means used were conventional.
EXAMPLE 1 significant increases in serum levels of anti-PCOLCE antibodies in RA patients
The level of anti-PCOLCE antibodies in human serum was specifically probed using a citrullinated BSA polypeptide consisting of 19 amino acids (shanghinode biotechnology limited) using the IEDB website B cell epitope prediction tool to determine PCOLCE epitopes.
Citrullinated polypeptide sequence: K-T-L-P-Cit-G-T-A-K-E-G-Q-G-P-G-P-K-Cit-G (SEQ ID NO. 1), wherein the 5 th and 18 th amino acids in the sequence are citrulline.
In addition to the serum sample tested, the primary reagents used include: the method comprises the steps of binding a coated antigen (the citrullinated polypeptide) with an ELISA plate, detecting an antibody (namely an ELISA secondary antibody, specifically recognizing the coated antigen and binding with horseradish peroxidase), developing a color substrate (namely TMB reagent, catalyzing by horseradish peroxidase, developing color, and presenting a test result according to the color development degree of the substrate), and standardizing serum.
The experimental steps are as follows:
The volume ratio of the serum sample to be tested to the standard serum is 1 by using PBS containing 0.1% BSA and 0.05% Tween-20 as a diluent: dilution of 100.
The coated antigen was diluted to 10. Mu.g/mL with PBS at pH 7.2 and then incubated overnight at 4℃with the addition of 96-well ELISA plates (100. Mu.L/well).
Plates were washed with 300. Mu.L of 0.05% Tween-20 PBST soak 2 min times for a total of 4 times.
Mu.L of PBS containing 2% BSA and 0.05% Tween-20 was added to each well, and the wells were blocked at room temperature for 2 hours.
Plates were washed with 300. Mu.L of 0.05% Tween-20 PBST soak 2 min times for a total of 4 times.
100. Mu.L of diluted serum samples were added to each well and incubated for 2 hours at room temperature.
Plates were washed with 300. Mu.L of 0.05% Tween-20 PBST soak 2 min times for a total of 4 times.
Detection antibody 1:10000 was diluted with PBS containing 0.5% BSA and 0.05% Tween-20 as a diluent, 100. Mu.L was added to each well, and incubated at room temperature for 1 hour.
Plates were washed with 300. Mu.L of 0.05% Tween-20 PBST soak 2 min times for a total of 4 times.
100. Mu.L of TMB reagent was added to each well and incubated at room temperature in the dark for 10-30 min.
The reaction was quenched by the addition of 100. Mu.L of 2M sulfuric acid solution and the absorbance (OD) was read on a microplate reader at 450 nm and 570 nm.
Sample: 119 patients with established Rheumatoid Arthritis (RA) (RA classification: 2010 ACR/EULAR rheumatoid arthritis classification), 41 patients with Osteoarthritis (OA), 45 patients with Systemic Lupus Erythematosus (SLE), 38 patients with Sjogren's Syndrome (SS); healthy Control (HC) 82.
Serum from each sample was collected and detected by enzyme-linked immunosorbent assay (ELISA) using citrullinated polypeptides as coating antigens.
The results of the measurements are shown in the following table and the following graph (Table 1, FIG. 1). The results show that the expression level of anti-PCOLCE antibodies in RA population is significantly higher than in disease control group (osteoarthritis, systemic lupus erythematosus, sjogren syndrome) and healthy control group.
Table 1: expression level of anti-PCOLCE antibodies in RA, OA, SLE, SS and HC populations
Example 2, anti-PCOLCE antibodies have good diagnostic value in RA
The sensitivity and specificity of the anti-PCOLCE antibody were calculated based on the results of example 1.
AU value (Arbitrary units) represents fluorescence intensity, and the calculation formula is: AU= [ OD Peptides -OD Non-specific background ] test serum/[ OD Peptides -OD Non-specific background ] positive serum×100.
The ROC curve was plotted with the true positive rate (sensitivity) as the ordinate and the false positive rate (1-specificity) as the abscissa, and the area of the ROC curve for the anti-PCOLCE antibody was 0.724 (FIG. 2).
Cut-off value: the AU value at which the about log index is maximum, i.e. 19.98, is selected.
The AU value of the serum to be detected is positive when the AU value is higher than the cut-off value.
The AU value at which the about step index is maximum, 19.98, is selected as the cut-off value. Based on this value, the positive rate of anti-PCOLCE antibody in RA patients (42.86%) was significantly higher than in Osteoarthritis (OA) patients (2.44%), systemic lupus erythematosus (systemic lupus erythematosus, SLE) patients (8.88%), sjogren's syndrome, SS) patients (10.53%) and healthy controls (healthy control, HC) (6.10%). The anti PCOLCE antibody has the diagnostic sensitivity and specificity of RA of 42.86 percent and 92.44 percent respectively, and has better diagnostic value.
Example 3 anti-PCOLCE antibodies have better supplementary diagnostic value in seronegative RA
Some RA patients were negative for RA-specific antibodies (RF, anti-CCP antibodies), i.e. seronegative RA. This fraction of patients presents diagnostic difficulties, and we explored the complementary diagnostic value of anti-PCOLCE antibodies in seronegative RA patients.
The serum sample was selected from 137 seronegative RA patients with confirmed diagnosis. ELISA procedure was as in example 1.
The expression levels of anti-PCOLCE antibodies in seronegative RA populations are shown in figure 3 and table 2.
The ROC curve was plotted with the true positive rate (sensitivity) as the ordinate and the false positive rate (1-specificity) as the abscissa, and the area of the ROC curve for the anti-PCOLCE antibody was 0.678 (FIG. 4).
Table 2: expression level of anti-PCOLCE antibodies in established and seronegative RA populations
The anti PCOLCE antibody has good diagnostic value in seronegative RA diagnosis in that the anti CCP antibody is negative RA, RF negative RA and anti CCP antibody is negative, and the positive rate in RF negative RA is 31.34%, 30.00% and 27.27% respectively.
Specific examples are set forth herein to illustrate the invention in detail, and the description of the above examples is only for the purpose of aiding in understanding the core concept of the invention. It should be noted that any obvious modifications, equivalents, or other improvements to those skilled in the art without departing from the inventive concept are intended to be included in the scope of the present invention.
Claims (5)
1. Use of an anti-PCOLCE antibody in the manufacture of a rheumatoid arthritis complement diagnostic product, wherein the anti-PCOLCE antibody is used as a marker, the product comprising reagents for detecting the anti-PCOLCE antibody for detecting the anti-PCOLCE antibody content;
The reagent for detecting the anti-PCOLCE antibody comprises a coating antigen citrullinated polypeptide, and the amino acid sequence of the citrullinated polypeptide is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the level of anti-PCOLCE antibodies in the biological sample of rheumatoid arthritis patients is significantly increased relative to normal healthy people, osteoarthritis patients, systemic lupus erythematosus patients and sjogren's syndrome patients.
3. The use according to claim 2, wherein the biological sample is blood, plasma, serum or interstitial fluid.
4. The use according to claim 2, wherein the product is a detection reagent or a supplementary diagnostic kit.
5. A product for the supplemental diagnosis of rheumatoid arthritis, said product comprising reagents for detecting an anti-PCOLCE antibody;
The reagent for detecting the anti-PCOLCE antibody comprises a coating antigen citrullinated polypeptide, and the amino acid sequence of the citrullinated polypeptide is shown as SEQ ID NO. 1.
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| CN1796997A (en) * | 2004-12-22 | 2006-07-05 | 上海富纯中南生物技术有限公司 | Detection kit for diagnosing RA, preparating kit, and method for completing standard of quality detection |
| CN106526196A (en) * | 2016-10-08 | 2017-03-22 | 北京大学人民医院 | Application of SR-A as diagnostic marker and intervention target for rheumatoid arthritis |
| CN110018156A (en) * | 2019-03-27 | 2019-07-16 | 北京大学人民医院(北京大学第二临床医学院) | Application of the anti-CarP antibody as systemic lupus erythematosus diagnosis marker |
| CN111868073A (en) * | 2019-05-31 | 2020-10-30 | 广州市雷德生物科技有限公司 | Specific polypeptide related to rheumatoid arthritis and application thereof |
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-
2024
- 2024-04-07 CN CN202410407445.5A patent/CN117990903A/en active Pending
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| CN1796997A (en) * | 2004-12-22 | 2006-07-05 | 上海富纯中南生物技术有限公司 | Detection kit for diagnosing RA, preparating kit, and method for completing standard of quality detection |
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