Diagnostic marker for rheumatoid arthritis and detection chip and application thereof

Abstract

The application provides a diagnostic marker for rheumatoid arthritis, a detection chip and application thereof, and belongs to the technical field of disease diagnosis.The diagnostic marker for rheumatoid arthritis provided by the application comprises at least one of the following antibodies: an anti-ANAPC15 antibody, an anti-LSP1 antibody, an anti-APBB1 antibody, an anti-parathymosin antibody and an anti-UBL7 antibody.The specific antibody for rheumatoid arthritis provided by the application is used as a diagnostic marker based on the above scheme, and the target antibody in the serum to be detected is detected by using the specific binding property of the antigen and the antibody.The immune chip provided by the application is simple to operate, and can complete the detection of the diagnostic marker with high throughput, high efficiency and high accuracy, has the characteristics of low cost and rapid detection, and is suitable for market promotion and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of disease diagnosis, and particularly relates to a diagnostic marker for rheumatoid arthritis, and a detection chip and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis and progressive joint destruction. Anti-cyclic citrullinated peptide / protein antibodies (anti-CCP / ACPA) and rheumatoid factor (RF) are currently the biomarkers with high diagnostic ability applied in RA. However, ACPA-negative RA patients still have disease progression due to delayed diagnosis and treatment, and screening of novel diagnostic autoantibodies is crucial for improving early diagnosis and timely treatment of RA, especially for ACPA-negative and RF-negative RA.

[0003] About one-third of RA patients are ACPA-negative. In addition to RF and ACPAs, there are various autoantibodies in the serum of RA patients, but whether these antibodies are specific for diagnosing RA patients (including ACPA-negative RA patients and RF-negative RA patients) has not been reported. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a diagnostic marker for rheumatoid arthritis, which improves the diagnostic accuracy of rheumatoid arthritis by screening specific antibodies present in early RA or ACPA-negative patients as diagnostic markers.

[0005] The present application provides a diagnostic marker for rheumatoid arthritis, which comprises at least one antibody selected from the group consisting of:

[0006] anti-ANAPC15 antibody, anti-LSP1 antibody, anti-APBB1 antibody, anti-parathymosin antibody, and anti-UBL7 antibody.

[0007] The present application provides an immune chip for diagnosing rheumatoid arthritis, which is coated with antigens specifically recognized by antibodies in the diagnostic marker.

[0008] Preferably, the antigens specifically recognized by antibodies in the diagnostic marker are labeled with GST.

[0009] The present application provides a kit for diagnosing rheumatoid arthritis, which comprises the immune chip and a marker-modified anti-human IgG antibody.

[0010] Preferably, the marker in the marker-modified anti-human IgG antibody comprises one of the following fluorescent dyes: Alexa-647 and Cy5.

[0011] Preferably, the positive standard also includes a mouse anti-GST antibody and a label-modified anti-mouse IgG antibody.

[0012] The positive standard includes a mouse anti-GST antibody and a label-modified anti-mouse IgG antibody.

[0013] The present application provides use of the immunochip in preparation of a kit for diagnosing rheumatoid arthritis.

[0014] Preferably, the immunochip is combined with one or more of the following in preparation of a kit for diagnosing rheumatoid arthritis: a label-modified anti-human IgG antibody, a mouse anti-GST antibody and a label-modified anti-mouse IgG antibody.

[0015] The present application provides use of an anti-parathymosin antibody in preparation of a drug for preventing and treating bone erosion.

[0016] The present application provides use of an anti-parathymosin antibody in preparation of a reagent or kit for diagnosing bone erosion or evaluating prognosis of bone erosion.

[0017] The present application provides a diagnostic marker for rheumatoid arthritis, comprising at least one of the following antibodies: an anti-ANAPC15 antibody, an anti-LSP1 antibody, an anti-APBB1 antibody, an anti-parathymosin antibody and an anti-UBL7 antibody. The present application uses HuProt array to screen RA autoantibodies, and determines five potential diagnostic markers of RA through preliminary screening and subsequent verification, which are expressed in early stage of disease and ACPA-negative patients.

[0018] The present application also provides an immunochip for diagnosing rheumatoid arthritis, which is arrayed with antigens specifically recognized by antibodies in the diagnostic marker. The present application uses specific antibodies for rheumatoid arthritis provided in the above scheme as diagnostic markers, and detects target antibodies in a serum to be tested by using the property of specific binding between antigens and antibodies. The immunochip provided by the present application is simple to operate, and can complete detection of diagnostic markers with high throughput, high efficiency and high accuracy, has the characteristics of low cost and rapid detection, and is suitable for market promotion and application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Linear correlation and reproducibility results of the chip;

[0020] Figure 2 KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of signaling pathways of 51 antigens;

[0021] Figure 3 Fluorescence intensity box plot of LSP1 (BC001785.1);

[0022] Figure 4 Box plot of fluorescence intensity for PTMS (NM_002824);

[0023] Figure 5 Box plot of fluorescence intensity for ANAPC15 (NM_014042);

[0024] Figure 6 Box plot of fluorescence intensity for APBB1 (NM_145689);

[0025] Figure 7 Box plot of fluorescence intensity for UBL7 (NM_032907);

[0026] Note: RA represents rheumatoid arthritis, OA represents osteoarthritis, SLE represents systemic lupus erythematosus, pSS represents primary Sjogren's syndrome, SpA represents seronegative spondyloarthritis, HC represents healthy control, Control represents control;

[0027] Figure 8 Relationship of 5 autoantibodies with clinical characteristics of RA; the heat map shows the results of correlation analysis of 5 autoantibodies with clinical and immunological characteristics of RA, and the filling color represents the size of correlation coefficient r value; negative correlation is represented by blue color, and positive correlation is represented by red color; **P<0.01, *P<0.05; the 5 antibodies include: anti-ANAPC15 (NM_014042), anti-LSP1 (BC001785.1), anti-APBB1 (NM_145689), anti-PTMS (NM_002824) and anti-UBL7 (NM_032907). DETAILED DESCRIPTION

[0028] The present application provides a diagnostic marker for rheumatoid arthritis, which comprises at least one of the following antibodies: anti-ANAPC15 antibody, anti-LSP1 antibody, anti-APBB1 antibody, anti-PTMS antibody and anti-UBL7 antibody.

[0029] In the present application, the diagnostic marker is screened from 51 disease-related autoantigens, and the above-mentioned 5 antibodies involved have the characteristics of strong antigen specificity and high antigen sensitivity, with a sensitivity of 39.6% to 46.2% and a specificity of 90% to 95%, which ensures the accuracy of diagnosis. One antibody in the diagnostic marker can diagnose whether rheumatoid arthritis is developed, and the combination of 2 or more antibodies increases the positive rate of diagnosis.

[0030] The present application provides an immune chip for diagnosing rheumatoid arthritis, which is arrayed with antigens specifically recognized by the antibodies in the diagnostic marker.

[0031] In the present application, the antigen is preferably a recombinantly expressed antigen. The antigen preferably includes the following: ANAPC15 (NM_014042), LSP1 (BC001785.1), APBB1 (NM_145689), Parathymosin (PTMS, NM_002824) (NM_002824), and UBL7 (NM_032907).

[0032] In the present application, the antigen specifically recognized by the antibody in the diagnostic marker is preferably GST-labeled. The nucleotide sequence of the coding gene of the GST is shown in SEQ ID NO: 1, and the amino acid sequence of the GST is shown in SEQ ID NO: 2. The labeling method is preferably entrusted to Beijing Huada Protein Research and Development Center Co., Ltd.

[0033] The present application does not have special restrictions on the preparation method of the immunochip, and the preparation method of the immunochip known in the art can be used, for example, the recombinantly expressed labeled antigen is dissolved and added dropwise to the chip with a coating solution, washed, blocked, washed again and dried, and vacuum packaged. In the embodiments of the present application, the immunochip is entrusted to Beijing Huada Protein Research and Development Center Co., Ltd.

[0034] The present application provides a kit for diagnosing rheumatoid arthritis, comprising the immunochip and the labeled anti-human IgG antibody.

[0035] In the present application, the label in the labeled anti-human IgG antibody preferably includes various fluorescent dyes, such as AF-647 or Cy5.

[0036] In the present application, the kit further comprises a positive standard. The positive standard preferably includes a mouse anti-GST antibody and a labeled anti-mouse IgG antibody.

[0037] In the present application, the detection principle of the kit is preferably as follows: the serum to be detected or the diluted serum is added dropwise to the immunochip, the target antibody in the test serum specifically reacts with the coated antigen, and after adding the labeled anti-human IgG antibody, a ternary complex of coated antigen-target antibody-labeled anti-human IgG antibody is formed. After detecting the signal of the label, the detection signal is the same as that of the positive standard, it is judged that the test serum is likely to be a patient with rheumatoid arthritis, and a comprehensive judgment is made in combination with other medical detection indexes.

[0038] The present application provides the use of the immunochip in the preparation of a kit for diagnosing rheumatoid arthritis.

[0039] In the present application, the immune chip is preferably combined with one or more of the following reagents for preparing a kit for diagnosing rheumatoid arthritis: a marker-modified anti-human IgG antibody, a mouse anti-GST antibody, and a marker-modified anti-mouse IgG antibody.

[0040] The present application provides use of an anti-parathymosin (PTMS) antibody in the preparation of a drug for preventing and treating bone erosion.

[0041] The present application provides use of an anti-parathymosin antibody in the preparation of a reagent or kit for diagnosing bone erosion or evaluating the prognosis of bone erosion.

[0042] In the present application, Spearman correlation analysis method is used to analyze the correlation of the above-mentioned five diagnostic markers with clinical correlation, and the results show that the anti-parathymosin antibody is correlated with disease activity score 28 (DAS28) and swollen joint count, suggesting that the anti-parathymosin antibody may be involved in the pathogenesis of RA inflammation. The anti-parathymosin antibody is negatively correlated with joint x-ray progression, suggesting that it may have a protective effect on RA bone erosion and be used for predicting the outcome of bone erosion.

[0043] The diagnostic marker for rheumatoid arthritis, the detection chip thereof, and the application thereof provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0044] Example 1

[0045] In the phase I study, 51 of 19,275 human proteome proteins (HuProt) were identified as reacting with RA serum IgG.

[0046] Human Proteome Microarray (HuProt TM V3.1 chip: composed of about 19,275 full-length human proteins, constructed by CDI company. This microarray contains 48 modules arranged in a 25x32 array layout. Each sample has 2 replicates, and the control probes include blank buffer, human IgG, human IgM, biotin-labeled BSA, BSA, GST (glutathione S-transferase). All recombinant human proteins are expressed by a Saccharomyces cerevisiae expression system and have an N-terminal GST (SEQ ID NO: 1) label. Mouse anti-GST antibody and anti-mouse IgG antibody are used to detect the quality of the chip.

[0047] HuProt TM (Human Proteome Microarray v3.1, CDI Labs, Mayaguez, PR) contains more than 19,275 complete and individually purified human proteins, covering more than 75% of the proteome. Simply put, HuProt is blocked with a blocking solution (5% BSA / TBS-T) at room temperatureTM Array 1 hour, then incubated overnight at 4°C with serum samples (diluted 1:1000). The array was then washed 3 times for 10 minutes with 1xTBS-T, detected with Alexa- 647 labeled anti-human IgG (Jackson Immuno Research, West Grove, PA) for 1 hour at room temperature, then washed 3 times for 10 minutes with 1xTBS-T, then spun to dry, then scanned. Slides were scanned on a GenePix 4200A microarray scanner (Axon Instruments). To identify positive samples on the array, two replicate protein spots must agree, but not be present in the blank or secondary-only control. TM Array 1 hour, then incubated overnight at 4°C with serum samples (diluted 1:1000). The array was then washed 3 times for 10 minutes with 1xTBS-T, detected with Alexa- 647 labeled anti-human IgG (Jackson Immuno Research, West Grove, PA) for 1 hour at room temperature, then washed 3 times for 10 minutes with 1xTBS-T, then spun to dry, then scanned. Slides were scanned on a GenePix 4200A microarray scanner (Axon Instruments). To identify positive samples on the array, two replicate protein spots must agree, but not be present in the blank or secondary-only control.

[0048] Results:

[0049] In the phase I study, 10 RA patients in early stage of disease (duration less than 2 years) were recruited. The 10 RA cases included 5 anti-CCP positive and 5 anti-CCP negative. The sera of these RA patients were screened on a high-throughput proteome microarray containing 19,275 human proteins (HuProt TM Array 1 hour, then incubated overnight at 4°C with serum samples (diluted 1:1000). The array was then washed 3 times for 10 minutes with 1xTBS-T, detected with Alexa- 647 labeled anti-human IgG (Jackson Immuno Research, West Grove, PA) for 1 hour at room temperature, then washed 3 times for 10 minutes with 1xTBS-T, then spun to dry, then scanned. Slides were scanned on a GenePix 4200A microarray scanner (Axon Instruments). To identify positive samples on the array, two replicate protein spots must agree, but not be present in the blank or secondary-only control. Figure 1 ). The autoantibodies against 51 RA-associated candidate autoantigens were identified with p-value <0.05 and fold change >1.3 (see Table 1).

[0050] To clarify the potential biological pathways of the 51 autoantigens recognized by RA autoantibodies, we performed KEGG enrichment analysis (see Table 2). The top 2 enriched pathways were "Endoplasmic reticulum protein processing" and "Metabolic pathways" Figure 2 ). The results suggest that these RA autoantibodies might interfere with these two biological processes and be involved in the pathogenesis of RA.

[0051] Table 1 List of 51 antigens

[0052]

[0053] Table 2 KEGG pathway analysis of 51 antigens

[0054]

[0055]

[0056] Example 3

[0057] A method for preparing and detecting an immunochip

[0058] 1. Preparation method of fusion protein

[0059] Fifty-one autoantigens from Example 1 were selected, and a GST-coding gene (SEQ ID NO: 2) was linked to the N-terminus of each antigen-coding gene. After gene synthesis, encoding genes for 51 GST-antigen fusion proteins were obtained. These 51 encoding genes for GST-antigen fusion proteins were then recombinantly expressed in yeast. The recombinant proteins were isolated and purified to obtain the GST-antigen fusion proteins. The specific procedures were outsourced to Beijing BGI Protein Research Center Co., Ltd.

[0060] 2. Preparation and detection methods of immunochips

[0061] HuProt was blocked at room temperature with a blocking solution (TBS-T solution containing 5% BSA). TM The custom-designed chip was incubated for 1 hour, then overnight at 4°C with serum samples (diluted 1:1000). The array was then washed three times with 1×TBS-T for 10 minutes each time, followed by incubation at room temperature for 1 hour with Alexa-647-labeled anti-human IgG (Jackson ImmunoResearch, West Grove, PA), then washed three times with 1×TBS-T for 10 minutes each time, rotated to dry, and then scanned. Positive standards were also tested simultaneously by adding mouse anti-GST antibody to the chip, incubating, washing, and then adding Alexa-647-labeled anti-mouse IgG, followed by washing. The chip was scanned on a GenePix 4200A microarray scanner (Axon Instruments) and compared to the original GenePix Array List file. All spotted proteins on the chip array were replicated (double-duplicate wells). The values ​​for each of the 51 candidate antigens were read individually.

[0062] The detection results of ANAPC15 (NM_014042), LSP1 (BC001785.1), APBB1 (NM_145689), parathymosin (PTMS, NM_002824) and UBL7 (NM_032907) are shown in [the table below]. Figures 3-7 .

[0063] Example 3

[0064] Screening methods for non-AMPAs as diagnostic biomarkers for RA

[0065] In the phase II study, 51 selected autoantigens were synthesized and diluted by coating solution to be spotted in the immunochip prepared in Example 2 in an array manner. The validation population included 182 RA patients and 261 controls (103 healthy controls and 158 disease controls) (see Table 3 for the population information). The serum of the population to be tested was detected by the method in Example 2.

[0066] The area under curve (AUC) value between 0.7 and 0.9 is considered to have good discrimination ability. There are 22 autoantibodies with AUC greater than 0.7 (see Table 4). Among them, the diagnostic specificity of anti-CHAC2 (NM_001008708), anti-ANAPC15 (NM_014042), anti-GDE1 (NM_016641), anti-TSR2 (NM_058163), anti-CCDC32 (NM_052849), anti-RDH16 (NM_003708), and anti-EXTL3 (NM_001440) are all higher than 90%. The ROC analysis gives AUC values of 0.713-0.755 (see Table 4). Anti-ANAPC15 antibody (NM_014042) is considered to be the most specific RA diagnostic biomarker (sensitivity of 41.8% and specificity of 91.5%, see Table 4).

[0067] Meanwhile, as can be seen from the data in Table 4, anti-ANAPC15 antibody, anti-LSP1 antibody, anti-APBB1 antibody, anti-parathymosin antibody, and anti-UBL7 antibody can all detect ACPA-negative RA patients, and the detection rate of anti-ANAPC15 antibody is the highest, reaching 20.8%. This shows that the above-mentioned 5 autoantibodies provided by the present application not only have high sensitivity and specificity, but also can be used for the diagnosis of ACPA-negative RA patients.

[0068] Table 3 Information of the screening population

[0069]

[0070] Table 4 Sensitivity and specificity of 22 antigens

[0071]

[0072]

[0073] Example 4

[0074] Clinical relevance of representative non-modified protein antigens (non-AMPAs)

[0075] The clinical symptoms of the above-mentioned 5 ACPA-negative RA high-specificity autoantibodies and non-AMPAs were analyzed for correlation by Spearman.

[0076] Figure 8 The clinical significance of five kinds of ACPA-negative RA high-specificity autoantibodies was analyzed and summarized. The results show that the five kinds of autoantibodies are positively correlated with erythrocyte sedimentation rate (ESR), IgG and rheumatoid factor (RF). Anti-parathymus antibody (NM_002824) is correlated with disease activity score 28 (DAS28) and swollen joint count (r=0.170, P=0.031, r=0.184, P=0.020), indicating that the anti-parathymus antibody may be involved in the pathogenesis of RA inflammation.

[0077] Meanwhile, the anti-parathymus antibody is negatively correlated with joint x-ray progression (Sharp-van der Heijde score, r=-0.175, P=0.047), indicating that it may have a protective effect on RA bone erosion and be used for predicting the outcome of bone erosion.

[0078] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of an antigen specifically recognized by an antibody of a diagnostic marker for rheumatoid arthritis, which is at least one of an anti-ANAPC15 antibody, an anti-APBB1 antibody, an anti-parathymosin antibody, or a combination of at least one of the anti-ANAPC15 antibody, the anti-APBB1 antibody, the anti-parathymosin antibody, and an anti-LSP1 antibody and an anti-UBL7 antibody, in the preparation of a kit for diagnosing rheumatoid arthritis.

2. Use of an immunochip for diagnosing rheumatoid arthritis in the manufacture of a kit for diagnosing rheumatoid arthritis, characterized in that, The immunochip array is coated with an antigen specifically recognized by an antibody of the diagnostic marker used in claim 1.

3. Use according to claim 2, characterized in that, The antigen specifically recognized by an antibody of the diagnostic marker is GST-labeled.

4. Use according to claim 2, characterized in that, The immunochip array is used in the preparation of a kit for diagnosing rheumatoid arthritis in combination with one or more of a label-modified anti-human IgG antibody, a mouse anti-GST antibody, and a label-modified anti-mouse IgG antibody.

5. Use according to claim 4, characterized in that, The label in the label-modified anti-human IgG antibody comprises one of a fluorescent dye, a phalloidin, and a cyanine dye.

6. Use according to claim 4 or 5, characterized in that, The kit further comprises a positive standard: The positive standard comprises a mouse anti-GST antibody and a label-modified anti-mouse IgG antibody.

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