Application of an RNAP III specific polypeptide antigen in a kit for detecting systemic sclerosis

Abstract

The present invention relates to an application of an RNAP III specific polypeptide antigen in a kit for detecting systemic sclerosis, belonging to the field of biotechnology detection technology, and solving at least one of the problems of low detection specificity and low positive rate of existing kits in the prior art. The RNAP III specific polypeptide antigen includes polypeptides with amino acid sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3. The RNAP III specific polypeptide antigen of the present invention has good application value in the detection of serum diagnosis of systemic sclerosis patients, because the purity of the RNAP III specific polypeptide antigen is high, which can reach more than 95%, the impurity content is small, the antigen specificity is strong, the result is stable in the detection, the specificity of the detection is improved, and the specificity is close to 100%, and the production cost of the RNAP III specific polypeptide of the present invention is low.

Description

Technical Field

[0001] The invention relates to the field of biotechnology detection technology, and in particular to an application of an RNAP III specific polypeptide antigen in a kit for detecting systemic sclerosis. Background Art

[0002] Systemic sclerosis (SSc) is a connective tissue disease of unknown cause, characterized by localized or diffuse skin thickening and fibrosis. The disease also affects internal organs such as the lungs, digestive tract, kidneys, and cardiovascular system. Due to abnormal immune activation, a variety of autoantibodies (such as anti-scl-70 antibodies, anti-CENP antibodies, etc.) can be detected in the serum of SSc patients. In 1993, Okano et al. discovered anti-RNA polymerase III (RNAP III) using radioimmunoprecipitation technology and found it to be associated with SSc for the first time. 60% of anti-RNAP III antibody positivity was found in patients with negative anti-scl-70 and anti-CENP antibodies, indicating that anti-RNAP III antibody positivity can better detect SSc patients. Anti-RNAP III antibody detection can significantly improve the diagnostic effect of SSc, and improving the positive rate of anti-RNAP III antibody detection has great clinical application value.

[0003] In addition, anti-RNAP III antibodies are autoantibodies associated with kidney disease in SSc patients. SSc patients will develop kidney disease, and the 5-year survival rate of SSc patients with kidney disease is only about 50%. Patients with SSc kidney disease may develop malignant hypertension, anuria, and renal failure, serious complications that exacerbate the progression of the disease and lead to death in some cases. SSc patients with positive anti-RNAP III antibodies have a higher risk of kidney disease than those with negative antibodies, with an OR of 17.8 and a 95% CI of 5.1-6.2. In SSc patients, the incidence of renal crisis was 6.4%, which increased to 42.9% in SSc patients with positive anti-RNAP III antibodies, suggesting that anti-RNAP III antibodies have a good predictive value for kidney disease.

[0004] Early diagnosis and risk assessment of SSc are extremely important for improving the survival rate of patients. Anti-RNAP III antibodies are included in the latest classification and diagnostic criteria for SSc, which are of great value for the early diagnosis of SSc and the assessment of renal involvement. Therefore, there is an urgent need for products to detect anti-RNAP III antibodies in clinical practice.

[0005] At present, the detection of anti-RNAP III antibodies usually adopts the immunoblotting method, which is time-consuming and cannot be quantitative. In addition, the existing ELISA method can quantitatively detect anti-RNAP III antibodies, but the detection of anti-RNAP III antibody ELISA kits is prone to problems such as non-specific binding and unstable results. Summary of the invention

[0006] In view of the above analysis, the present invention aims to provide an application of RNAP III specific polypeptide antigen in a kit for detecting systemic sclerosis, so as to solve at least one of the problems of the existing kits, such as low detection specificity and low positive rate.

[0007] In a first aspect, the present invention provides a use of an RNAP III-specific polypeptide antigen in a kit for detecting systemic sclerosis, wherein the RNAP III-specific polypeptide antigen comprises polypeptides with amino acid sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.

[0008] Furthermore, the polypeptides with amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 are used in combination.

[0009] Furthermore, the kit includes the RNAP III specific polypeptide antigen, coating diluent, blocking solution, washing solution, sample diluent, horseradish peroxidase-labeled goat anti-human IgG, substrate solution, stop solution and physiological saline.

[0010] Furthermore, a cysteine ​​residue was added to the C-terminus of the polypeptides of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the polypeptides with the cysteine ​​residue connected to the C-terminus were coupled to BSA using succinimide 3-(2-pyridyldisulfide)-propionate, respectively, and then coated with coating diluent and added to different wells of the kit for detection.

[0011] Furthermore, the coating diluent is a 0.05 mol / L sodium carbonate-sodium bicarbonate buffer solution, and the pH of the coating diluent is 9.6.

[0012] Furthermore, the blocking solution is fetal bovine serum with a mass fraction of 10%.

[0013] Furthermore, the washing solution is a PBST buffer with a pH of 7.4.

[0014] Furthermore, the sample diluent is a PBS buffer solution with a pH of 7.4.

[0015] Furthermore, the dilution range of the horseradish peroxidase-labeled goat anti-human IgG is 1:5000-1:100000.

[0016] Furthermore, the substrate solution is TMB-urea hydrogen peroxide solution.

[0017] Furthermore, the stop solution is a H2SO4 solution with a concentration of 2 mol / L.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0019] (1) Compared with the prior art, the present invention uses polypeptide antigens to detect anti-RNAP III antibodies, providing a new detection method for detecting serum diagnosis of systemic sclerosis patients. The test kit of the present invention has a high positive detection rate, and the RNAP III-specific polypeptide antigen has good application value in detecting serum diagnosis of systemic sclerosis patients;

[0020] (2) The RNAP III specific polypeptide antigen molecule of the present invention is relatively small, and it is easier to obtain high purity than protein synthesis, the purity can reach more than 95%, the impurity content is small, the antigen specificity is strong, and the problems of non-specific binding and unstable results can be effectively avoided. The results are stable during the detection, and the specificity of the detection is improved, and the specificity is close to 100%. In addition, the production cost of the RNAP III specific polypeptide of the present invention is low.

[0021] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are only used for the purpose of illustrating specific embodiments and are not to be considered as limiting the present invention. In the entire drawings, the same reference symbols represent the same components;

[0023] Figure 1 This is a graph showing the results of detecting anti-RNAP III antibodies in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy people (HC) using the method of Comparative Example 1;

[0024] Figure 2 This is a graph showing the results of detecting anti-RNAP III antibodies in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy people (HC) using the method of Comparative Example 2;

[0025] Figure 3 This is a graph showing the results of detecting anti-RNAP III antibodies in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy people (HC) using the method of Comparative Example 3;

[0026] Figure 4 This is a graph showing the results of detecting anti-RNAP III antibodies in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy people (HC) using the method of Comparative Example 4;

[0027] Figure 5 This is a graph showing the results of detecting anti-RNAP III antibodies in systemic sclerosis (SSc) and healthy people (HC) using the method in Comparative Example 5. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0029] A specific embodiment of the present invention discloses the use of an RNAP III-specific polypeptide antigen in a kit for detecting systemic sclerosis, wherein the RNAP III-specific polypeptide antigen comprises a polypeptide having an amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3. The RNAP III-specific polypeptide antigen of the present invention can detect anti-RNAP III antibodies, and can further be used to detect systemic sclerosis (SSc).

[0030] In a specific embodiment, the polypeptides with amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 are used in combination.

[0031] Any one of the polypeptides in the kit of the present invention that is detected positive is considered positive.

[0032] In a specific embodiment, the kit includes the RNAP III specific polypeptide antigen, coating diluent, blocking solution, washing solution, sample diluent, horseradish peroxidase-labeled goat anti-human IgG, substrate solution, stop solution and physiological saline.

[0033] In a specific embodiment, a cysteine ​​residue is added to the C-terminus of the polypeptides of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the polypeptides with the cysteine ​​residue connected to the C-terminus are coupled to BSA using succinimidyl 3-(2-pyridyldisulfide)-propionate, respectively, and then coated with a coating diluent and added to different wells of the kit for detection.

[0034] Preferably, the wells on the kit are evenly divided into three groups, and the polypeptides of the amino acid sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 are respectively in each group. Specifically, the three groups are the first group, the second group and the third group, the polypeptide of the amino acid sequence shown in SEQ ID No.1 is added to the first group, the polypeptide of the amino acid sequence shown in SEQ ID No.2 is added to the second group, and the polypeptide of the amino acid sequence shown in SEQ ID No.3 is added to the third group.

[0035] In a specific embodiment, the coating diluent is 0.05 mol / L sodium carbonate-sodium bicarbonate buffer, and the pH of the coating diluent is 9.6.

[0036] Specifically, the sodium carbonate-sodium bicarbonate buffer solution is obtained by the following method: 0.15 g of sodium carbonate, 0.29 g of sodium bicarbonate, and 0.02 g of sodium azide are added with double distilled water to 100 mL, and the pH is adjusted to 9.6.

[0037] In a specific embodiment, the blocking solution is 10% fetal bovine serum by mass.

[0038] Specifically, 10% fetal bovine serum is prepared by dissolving 10 mL of fetal bovine serum in 90 mL of 1×PBS (pH=7.4).

[0039] In a specific embodiment, the washing solution is PBST buffer with a pH of 7.4.

[0040] Specifically, the PBST buffer was obtained by the following method: NaCl 0.8 g, KH2PO4 0.02 g, Na2HPO4 0.12 g, H2O 0.29 g, KCl 0.02 g, Tween 20 0.05 ml, sodium azide 0.01 g, double distilled water was added to 100 ml, and the pH was adjusted to 7.4.

[0041] In a specific embodiment, the sample diluent is a PBS buffer with a pH of 7.4.

[0042] Specifically, the PBS buffer solution is obtained by the following method: NaCl 0.8g, KH2PO4 0.02g, Na2HPO4 0.12g, H2O 0.29g, KCl 0.02g, sodium azide 0.01g, double distilled water is added to 100ml, and the pH is adjusted to 7.4.

[0043] In a specific embodiment, the dilution range of goat anti-human IgG labeled with horseradish peroxidase is 1:5000-1:100000.

[0044] In a specific embodiment, the substrate solution is TMB-hydrogen peroxide urea solution.

[0045] Specifically, the TMB-urea hydrogen peroxide solution is prepared by mixing substrate solution A and substrate solution B in a volume ratio of 1:1.

[0046] The substrate solution A is composed of 20 mg TMB, 10 ml anhydrous ethanol, and double distilled water to 100 ml.

[0047] Substrate solution B is 0.1 mol / L citric acid-0.2 mol / L sodium dihydrogen phosphate buffer, pH 5.0-5.4.

[0048] The specific preparation method of substrate solution B is as follows: take 1.46 g of Na2HPO4, 0.933 g of citric acid, 0.64 ml of 0.75% urea hydrogen peroxide, add triple distilled water to 100 ml, and adjust to pH 5.0-5.4.

[0049] In a specific embodiment, the stop solution is a H2SO4 solution with a concentration of 2 mol / L.

[0050] The stop solution is prepared by taking 200 ml of double distilled water, 34 ml of concentrated sulfuric acid (slowly adding dropwise with constant stirring), and adding double distilled water to 300 ml.

[0051] Specifically, the method of using the kit is as follows:

[0052] (1) Dilute the RNAP III specific polypeptide antigen to an appropriate concentration (1 μg / mL) with coating diluent, add 100 μL of RNAP III specific polypeptide antigen to each well of the ELISA plate, place at 4°C for 12 hours, and discard the liquid in the well;

[0053] (2) Blocking the enzyme-labeled reaction wells: Block the reaction wells with 10% fetal bovine serum at 37°C for 60 min. Fill the reaction wells with the blocking solution and remove the bubbles in the wells. After blocking, wash the wells three times with washing solution, each time for 3 min.

[0054] The washing method is as follows: aspirate the reaction solution in the wells, fill the wells with the washing solution, place for 2 minutes, shake slightly, aspirate the solution in the wells, pour off the liquid, and pat dry on absorbent paper;

[0055] (3) Add the sample to be tested (establish an appropriate concentration gradient): dilute the sample with sample diluent at a dilution of 1:400 during the test, add the diluted sample to the enzyme-labeled reaction well, add at least two wells for each sample, 100 μL per well, place at room temperature for 120 min, and wash the wells three times with washing solution, each time for 3 min;

[0056] (4) Add enzyme-labeled antibody: The enzyme-labeled antibody is diluted at 1:10000, 100 μL is added to each well, incubate at room temperature for 60 min, and then wash the well three times with washing solution, each time for 3 min;

[0057] (5) Add substrate solution (prepared immediately before use): 100 μL per well, place at room temperature away from light for 10-15 min, and add stop solution for color development;

[0058] (6) Termination reaction: Add 50 μL of stop solution to each well to terminate the reaction and measure the test results within 20 min.

[0059] The RNAP III specific polypeptide of the present invention is synthesized by a solid phase synthesis method, specifically:

[0060] (1) Using polystyrene-phenylene glycol cross-linked resin, the target peptide is synthesized from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus) according to the amino acid sequence of the target peptide;

[0061] (2) After the synthesis is completed, the peptide is cleaved and cleaved from the resin, and the protecting group of the peptide is removed with piperidine-CH2CL2 to obtain the target peptide.

[0062] It should be noted that in step (1), the amino acid is fixed by a covalent bond formed between the carboxyl group and the reactive group of the resin, and 9-fluorenylmethoxycarbonyl is used as an α-amino protecting group to protect the amino and carboxyl groups that are not temporarily involved in the formation of the amide bond.

[0063] Preferably, a cysteine ​​residue is added to the C-terminus of the target polypeptide for linking to the coupling agent via its thiol group.

[0064] Specifically, the target polypeptide with a cysteine ​​residue connected to the C-terminus is coupled with BSA using succinimidyl 3-(2-pyridyldisulfide)-propionate to obtain the RNAP III specific polypeptide. The final purity of the RNAPIII specific polypeptide prepared by the method of the present invention is >95%.

[0065] The polypeptides of the present invention whose sequences are SEQ ID No. 1 to 3 are respectively denoted as P1 to P3, and the specific sequences are as follows:

[0066] P1: KKTRDKYGINDNGTTE ;

[0067] P2: LQEIKKFIKGVSEKIK;

[0068] P3: MEGKDEPLEFKRVLDN.

[0069] The technical solution of the present invention is further explained below in conjunction with specific embodiments.

[0070] Preparation Example 1

[0071] This preparation example prepares a coupled P1 polypeptide, and the specific method is as follows:

[0072] (1) Using polystyrene-phenylene glycol cross-linked resin, the peptide was synthesized from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus) according to the amino acid sequence of the P1 polypeptide;

[0073] (2) After the synthesis is completed, the peptide is cleaved and cleaved from the resin, and the protecting group of the peptide is removed with piperidine-CH2CL2 to obtain the P1 peptide;

[0074] (3) A cysteine ​​residue was added to the C-terminus of the P1 polypeptide, and the polypeptide with the cysteine ​​residue connected to the C-terminus was coupled with BSA using succinimidyl 3-(2-pyridyldisulfide)-propionate to obtain the coupled P1 polypeptide.

[0075] Preparation Example 2

[0076] In this example, the coupled P2 polypeptide was prepared by the same method as in Example 1.

[0077] Preparation Example 3

[0078] In this example, the coupled P3 polypeptide was prepared by the same method as in Example 1.

[0079] During the study, the inventors designed a variety of other polypeptides, see Preparation Examples 4 to 6 for details.

[0080] Preparation Example 4

[0081] In this example, the coupled P4 polypeptide was prepared. The sequence of the P4 polypeptide was MLSQEEKKQFLDYLKR (SEQ ID No. 4). The specific method was the same as that in Example 1.

[0082] Preparation Example 5

[0083] In this example, the coupled P5 polypeptide was prepared. The sequence of the P5 polypeptide was YLQKRGLKKK (SEQ ID No. 5). The specific method was the same as that in Example 1.

[0084] Preparation Example 6

[0085] In this example, the coupled P6 polypeptide was prepared. The sequence of the P6 polypeptide was AQLDKDDDAD (SEQ ID No. 6). The specific method was the same as that in Example 1.

[0086] Example 1

[0087] A method for performing ELISA detection on SSc patient samples using a kit, specifically comprising the following steps:

[0088] (1) Coating: The coupled P1, P2 and P3 polypeptides prepared in Preparation Examples 1-3 were diluted to an appropriate concentration (5 μg / mL) with a coating diluent to obtain three coated polypeptides. The wells in the ELISA plate were divided into three groups according to the number. 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No. 1 was added to each well of the first group, 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No. 2 was added to each well of the second group, and 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No. 3 was added to each well of the third group. The plates were placed at 4° C. for 24 h, and the liquid in the wells was discarded. The coating diluent was 0.05 mol / L sodium carbonate-sodium bicarbonate buffer, and the pH of the coating diluent was 9.6. The sodium carbonate-sodium bicarbonate buffer was obtained by the following method: 0.15 g of sodium carbonate, 0.29 g of sodium bicarbonate, and 0.02 g of sodium azide were added to 100 mL of double distilled water and adjusted to pH = 9.6;

[0089] (2) Blocking the enzyme-labeled reaction wells: Use blocking solution to block for 60 min. Fill each reaction well with blocking solution and remove bubbles in each well. After blocking, wash the wells three times with washing solution, each time for 3 min.

[0090] The washing method is as follows: aspirate the reaction solution in the wells, fill the wells with the washing solution, place for 2 minutes, shake slightly, aspirate the solution in the wells, pour off the liquid, and pat dry on absorbent paper;

[0091] The blocking solution was 10 mL of fetal bovine serum dissolved in 90 mL of 1× PBS (pH = 7.4);

[0092] (3) Add the sample to be tested (establish an appropriate concentration gradient): dilute the sample with sample diluent at a dilution of 1:400 during the test, add the diluted sample to the enzyme-labeled reaction well, add at least two wells for each sample, 100 μL per well, place at room temperature for 120 min, and wash the wells three times with washing solution, each time for 3 min;

[0093] The sample diluent is a PBS buffer with a pH of 7.4, and the PBS buffer is obtained by the following method: NaCl 0.8g, KH2PO4 0.02g, Na2HPO4 0.12g, H2O 0.29g, KCl 0.02g, sodium azide 0.01g, double distilled water is added to 100ml, and the pH is adjusted to 7.4;

[0094] (4) Add enzyme-labeled antibody: horseradish peroxidase-labeled goat anti-human IgG at a dilution of 1:10,000, add 100 μL to each well, incubate at room temperature for 60 min, and wash the wells three times with washing solution, each time for 3 min;

[0095] (5) Add substrate solution (prepared immediately before use): 100 μL per well, place at room temperature away from light for 5 min, and add stop solution for color development;

[0096] The substrate solution is a mixture of substrate solution A and substrate solution B in a volume ratio of 1:1. The substrate solution A is specifically 20 mg of TMB, 10 ml of anhydrous ethanol, and double distilled water is added to 100 ml. The specific preparation method of substrate solution B is as follows: 1.46 g of Na2HPO4, 0.933 g of citric acid, 0.64 ml of 0.75% urea hydrogen peroxide, triple distilled water is added to 100 ml, and the pH is adjusted to 5.0-5.4 to obtain;

[0097] (6) Termination reaction: Add 50 μL of stop solution to each well to terminate the reaction and measure the test results within 20 min.

[0098] The washing solution in this embodiment is a PBST buffer with a pH of 7.4, which is obtained by the following method: NaCl 0.8g, KH2PO4 0.02g, Na2HPO4 0.12g, H2O 0.29g, KCl 0.02g, Tween20 0.05ml, sodium azide 0.01g, double distilled water is added to 100ml, and the pH is adjusted to 7.4.

[0099] The stop solution is 200 ml of double distilled water and 34 ml of concentrated sulfuric acid (slowly add dropwise with constant stirring), and add double distilled water to 300 ml to obtain.

[0100] Comparative Example 1

[0101] The detection method of this comparative example is the same as that of Example 1, except that only the coupled P1 polypeptide prepared in Preparation Example 1 is added to each well in the kit.

[0102] Comparative Example 2

[0103] The detection method of this comparative example is the same as that of Example 1, except that only the coupled P2 polypeptide prepared in Preparation Example 2 is added to each well in the kit.

[0104] Comparative Example 3

[0105] The detection method of this comparative example is the same as that of Example 1, except that only the coupled P3 polypeptide prepared in Preparation Example 3 is added to each well in the kit.

[0106] Comparative Example 4

[0107] The method for performing ELISA detection on SSc patient samples in the kit of this comparative example is the same as that in Example 1, except that a protein produced by GenScript Biotech is added to each well in the kit.

[0108] Comparative Example 5

[0109] The present comparative example uses a commercially available kit to perform ELISA testing on the serum samples of SSc patients. The commercially available kit in the present comparative example is Human Anti-RNA Polymerase III Antibody (Pol III-Ab) ELISA Kit, brand: Abbexa, item number: abx055783-96 tests. The test is performed according to the instructions of the kit.

[0110] Test Example 1

[0111] The kits of Comparative Examples 1-3 and Comparative Example 4 were used to perform ELISA tests on the serum of patients with systemic sclerosis (SSc), other rheumatic diseases (rheumatoid arthritis (RA), systemic lupus erythematosus (SLE)) and healthy controls (HC) (according to the method of Example 1). The results are shown in Figure 1-4 Comparative Example 5: ELISA test was performed on the serum of systemic sclerosis (SSc) and healthy controls (HC) according to the test method in the specification. The results are shown in Figure 5 .

[0112] Among them, the samples of patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) in this trial were collected and provided by outpatient clinics and wards, and the samples of healthy controls (HC) came from the healthy physical examination population.

[0113] (1) Positive rate check:

[0114] There were 131 SSc patients, including 16 males and 115 females with an average age of (53.48±14.22) years.

[0115] Specifically, ELISA was performed on the serum of SSc patients using the kits of Comparative Examples 1-4 and Example 1. After calculation, it was found that the positive rate of P1 (Comparative Example 1) in SSc was 7.63% (10 / 131), P2 (Comparative Example 2) was 5.34% (7 / 131), and P3 (Comparative Example 3) was 1.53% (2 / 131).

[0116] Since any one of the three polypeptides P1-P3 (the kit of Example 1) is positive, the positive rate of Example 1 using the three polypeptides P1-3 in SSc is 9.92%, the positive rate of the kit of Comparative Example 4 in SSc is 8.40%, and the positive rate of the kit of Comparative Example 5 in SSc is 7.63%. The positive rate of the kit using three polypeptides at the same time (Example 1) is higher than that of the kits of Comparative Examples 1-5.

[0117] (2) Specific detection:

[0118] A total of 96 patients with other rheumatic diseases (CTD) were included; 48 patients with rheumatoid arthritis, including 8 males and 40 females with an average age of (55.7±15.27) years; 48 patients with systemic lupus erythematosus, including 6 males and 42 females with an average age of (35.72±15.43) years;

[0119] There were 68 healthy controls (HC), including 16 males and 52 females with an average age of (49.51±15.12).

[0120] The kits of Comparative Examples 1-4 were used to detect anti-RNAP III antibodies in the sera of the above-mentioned other rheumatic disease patients and healthy controls (HC). It was calculated that P1 (Comparative Example 1), P2 (Comparative Example 2) and P3 (Comparative Example 3) were not detected in other rheumatic disease patients and healthy controls (HC), and only systemic sclerosis (SSc) patients were detected.

[0121] Since the combined detection of any one of the polypeptides P1-P3 (the kit of Example 1) is positive, Example 1 was not detected in other rheumatic disease patients and healthy controls (HC), and the specificity was 100%, indicating that the specificity of the three polypeptides P1-P3 of Example 1 is 100%.

[0122] The comparative example 4 kit was used to detect anti-RNAP III antibodies in the serum of patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus) and healthy controls (HC). The results showed that the positive rate of the protein used in the comparative example 4 kit in other rheumatic diseases was 5.2%, and the positive rate in healthy controls was 1.47%.

[0123] The anti-RNAP III antibodies in the serum of healthy controls (HC) were detected using the kit of Comparative Example 5. The results showed that no anti-RNAP III antibodies were detected in the healthy controls (HC), and the specificity was 100%.

[0124] The detection positive rate of the P1-P3 three polypeptides (Example 1) kit in patients with systemic sclerosis (9.92%) was higher than that of the kit in Comparative Example 4 (8.40%) and Comparative Example 5 (7.63%). At the same time, the kit in Comparative Example 4 still had a certain positive rate in other rheumatic diseases and healthy controls, and its specificity (96.3%) was lower than that of the P1-P3 three polypeptides (specificity 100%).

[0125] Test Example 2

[0126] The kits of Comparative Examples 1-4 were used to detect anti-RNAP III antibody positive serum of systemic sclerosis (SSc), and the results are shown in Table 1.

[0127] Specifically, the coupled P1, P2 and P3 polypeptides in Example 1 were replaced with the coupled P4-6 polypeptides prepared in Preparation Examples 4-6, and ELISA was performed on the positive serum. The results are shown in Table 1, that is, only 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No.4 was added to each well of the first group, the second group and the third group (referred to as the P4 group), only 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No.5 was added to each well of the first group, the second group and the third group (referred to as the P5 group), and only 100 μL of the polypeptide with the amino acid sequence shown in SEQ ID No.6 was added to each well of the first group, the second group and the third group (referred to as the P6 group).

[0128] Table 1

[0129]

[0130] The kits of comparative examples 1-4 can distinguish positive serum from negative serum, while the readings of groups P4, P5 and P6 are all lower than 0.1, and there is basically no difference between positive serum and negative serum, that is, there are obvious differences in the detection results of polypeptides with different sequences. In the kit of the present invention, three polypeptides P1-P3 are selected, which can clearly distinguish positive serum from negative serum.

[0131] Based on the above analysis, it can be seen that the combined use of polypeptides P1-P3 in the present invention can specifically detect anti-RNAP III antibodies in SSc patients. The RNAP III specific polypeptide antigen and ELISA kit provided by the present invention have good application value in serological diagnosis.

Claims

1. Use of an RNAP III specific polypeptide antigen in the preparation of a kit for detecting systemic sclerosis, characterized in that: The RNAP III specific polypeptide antigen is prepared by adding a cysteine ​​residue to the C-terminus of the polypeptides of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and coupling the polypeptides connected to the cysteine ​​residue at the C-terminus with BSA using succinimide 3-(2-pyridyldisulfide)-propionate. The RNAP III specific polypeptide antigen is coated with a coating diluent and then added to the wells of the kit for detection.

2. The use according to claim 1, characterized in that: The polypeptides with amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 are used in combination.

3. The use according to claim 1, characterized in that: The kit comprises the RNAP III specific polypeptide antigen, coating diluent, blocking solution, washing solution, sample diluent, goat anti-human IgG labeled with horseradish peroxidase, substrate solution, stop solution and physiological saline.

4. The use according to claim 1, characterized in that: The coating diluent is a 0.05 mol / L sodium carbonate-sodium bicarbonate buffer solution, and the pH of the coating diluent is 9.

6.

5. The use according to claim 3, characterized in that: The blocking solution is fetal bovine serum with a mass fraction of 10%.

6. The use according to claim 3, characterized in that: The washing solution is PBST buffer with a pH of 7.

4.

7. The use according to claim 3, characterized in that: The sample diluent is a PBS buffer solution with a pH of 7.

4.

8. The use according to claim 3, characterized in that: The dilution range of the horseradish peroxidase-labeled goat anti-human IgG is 1:5000-1:100000.

9. The use according to claim 3, characterized in that: The substrate solution is TMB-hydrogen peroxide urea solution.

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