CN109725158B - Application of polypeptide SLE2018-V001 in kit for diagnosing systemic lupus erythematosus - Google Patents

Abstract

The invention discloses application of a polypeptide SLE2018-V001 in a kit for diagnosing systemic lupus erythematosus. The amino acid sequence of the polypeptide SLE2018-V001 is as follows: YEHAMYRSAVLL are provided. The invention adopts the enzyme-linked immunosorbent assay (ELISA) technology widely used in clinic and uses an indirect method to qualitatively detect the level of the IgG antibody of the anti-YEHAMYRSAVLL polypeptide in human serum. The kit provided by the invention can be used as a means for assisting the early diagnosis of the systemic lupus erythematosus, and can greatly improve the specificity and the sensitivity of the early diagnosis of the systemic lupus erythematosus, wherein the specificity is 74 percent, and the sensitivity is 88 percent.

Description

Application of polypeptide SLE2018-V001 in kit for diagnosing systemic lupus erythematosus

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a polypeptide SLE2018-V001 in a kit for diagnosing systemic lupus erythematosus.

Background

Systemic Lupus Erythematosus (SLE) is an autoimmune-mediated autoimmune disease that presents as clinical multiple system lesions, often causing irreversible damage to multiple organ systems, severely affecting the life span and quality of life of patients. The incidence rate of systemic Lupus erythematosus is in the range of 0.02% -0.07%, the incidence rate of urban population is higher, the age of patients is mainly distributed in the range of 15-45 years, and female patients are more, and the incidence rate of male and female patients is about 1:9(Lupus 2006.15(5): 308-) -318.). The study of the prevalence of systemic lupus erythematosus, the pathological mechanism and its biomarkers is three important aspects of systemic lupus erythematosus research. The clinical manifestations of systemic lupus erythematosus are diverse, and the serological and immunological indexes are very variable. The autoimmune disease involves multiple organs, including skin, kidney, brain, often with joint damage in addition to these organs, so the immune response is systemic. The component of the self-tissue that elicits the autoimmune response is called autoantigen, and includes hidden autoantigen (which has never been contacted with autologous lymphocytes during the embryonic period and cannot be recognized as self-material by the body) and modified autoantigen (which is formed as autoantigen by the conformational change of the self-tissue under the influence of infection, drugs, burns, ionizing radiation, etc.). When the body produces self-antigen, it recognizes it as a heterozygote, and produces corresponding self-antibody to resist it. These self-antigens or self-antibodies are often produced in the early stages of disease phenotype development, and the expression level usually shows a certain trend along with the evolution of disease condition. According to this feature, these autoantigens or autoantibodies can be used as biomarkers for diagnosing diseases.

Biomarkers are a measurement tool that researchers use to diagnose the occurrence, recurrence, prognosis of disease and for dynamic assessment of treatment efficacy. The timely diagnosis of systemic lupus erythematosus is still a great challenge, and the early and accurate diagnosis of the biomarkers of systemic lupus erythematosus is a key point. Good systemic lupus erythematosus biomarkers should have the conditions: can accurately diagnose the systemic lupus erythematosus; the diagnosis process has no adverse reaction to patients. Currently, disease biomarkers mainly include gene level markers, cell level markers, serum level markers, and the like. For systemic lupus erythematosus, known immune targets (or autoantigens) are primarily nuclear components, including ds-DNA chromosome-associated proteins, Ro protein (SSA), La protein (SSB), and Sm protein. For the diagnosis of SLE, the clinical diagnosis standard of SLE has been well established since the American College of Rheumatology, ACR (American Rheumatism Association, ARA, 1988) established for the first time in 1971. The first serological indicators (antinuclear antibodies, anti-ds-DNA antibodies and anti-Sm antibodies) were added in the 1982 revision, and the biometric analysis was used in the formulation process, which was verified for clinical diagnosis. The criteria "anti-cardiolipin antibody positive and lupus anticoagulant positive" were added as revised in 1997, and although not validated, serological markers have been the focus of research. (J Nephrol Dially transfer [ J ] Vol.22No.2Apr.2013:153-157.)

Serum proteomics is one of effective means for searching systemic lupus erythematosus. In the process of the occurrence and development of autoimmune diseases, the change of the expression quantity of the autoantibodies influences the composition of a serum proteome, and the autoantibodies have important value in the discovery of serum biomarkers. Therefore, more and more researchers are focusing on the research of serum protein markers, and striving to search for biomarkers in the proteomics level and establish a multi-parameter model for diagnosis, prognosis and drug effect evaluation.

Currently available markers for systemic lupus erythematosus include antinuclear antibodies (ANAs), anti-ds-DNA antibodies, anti-Sm antibodies, and the like. Antinuclear antibodies are commonly found in patients as markers of the disease, and in the diagnosis standard, the standard of antinuclear antibodies refers to that under the condition of not using drugs to induce drug lupus, the immunofluorescence or other experiment antinuclear antibody titer equivalent to the method is abnormal; serum levels of anti-dsDNA antibodies significantly correlated with disease activity and impaired renal function, recurrence of disease can be predicted by detecting anti-dsDNA antibody levels, and higher levels of anti-dsDNA antibodies can be detected before disease progression. (medical Recapillarate [ J ], Aug.2015, Vol.21, No.16: 2956-; anti-dsDNA antibodies and anti-Sm antibodies, while more specific in diagnosing systemic lupus erythematosus, are not commonly present in patient serum (Arthritis & Rheumatism Vol.47, No.5, October 15,2002: 546-555). Autoantibodies such as antinuclear antibodies can predict disease onset, but lack of specificity prevents them from being useful predictive biomarkers. Autoantibodies remain a hot spot for the early diagnosis of autoimmune diseases due to their central role in the pathogenesis of autoimmune diseases. Researchers hope to find autoantibodies with remarkable specificity and high sensitivity as biomarkers, so that the development of diseases can be detected, and the severity of the diseases can be distinguished.

The pursuit of accurate and efficient biomarkers has prompted the development of a variety of biomarker screening methods. In this era of continuous development of high throughput technology, researchers have sought to identify chemical molecules with significant differences in the serum of patients and healthy persons among a large number of candidates by way of library construction. In 2013, Jiexia Quan et al screened compound markers with specificity of 97.5% and sensitivity of 70% for systemic lupus erythematosus (Journal of Immunological Methods 402(2014) 23-34.) by synthesizing peptide libraries as a substitute for autoantigens. In addition to chemically synthesized peptidomimetics, proteins and polypeptides are candidates.

Since the advent of protein chips, protein chips have become a powerful tool for exploring biomolecule-protein interactions and screening biomarkers, which is to spot and fix a large number of protein molecules on the surface of a solid-phase carrier according to a preset arrangement sequence in a certain way to form a microarray, incubate a sample to be analyzed with the chip, wash off components which cannot be bound with proteins on the chip, incubate with a fluorescence-labeled antibody, and finally read the fluorescence signal values of the spots under a fluorescence scanner. The content of protein in serum is very heterogeneous, the abundance of autoantibodies is very low, the problem of huge difference of autoantibody expression in different samples is difficult to solve by common mass spectrometry, but the defects of conventional mass spectrometry can be overcome by the characteristics of global property, unbiased property and high flux of the protein chip. The protein chip can be used for determining the difference between a patient and a healthy person in a short time and efficiently searching for serum markers.

The polypeptide display library has various technologies, and from the perspective of expression vectors, there are phage display libraries, bacterial display libraries, yeast display libraries, cell display libraries, and the like; as the type of the substance to be expressed, there are cDNA library, mRNA library, polypeptide library, and the like. The greatest advantage of phage display is high throughput and diversity compared to other display technologies. Phage display libraries were a high throughput screening technology developed in the 80's of the last century. Since the 21 st century, with the advent of high throughput sequencing technologies, phage display library technology has met with a new surge of development. Phage display libraries study protein-protein, protein-polypeptide, and protein-DNA interactions by linking genetic information to proteins. Currently available phage display libraries include omic peptide libraries and random peptide libraries, with parallel analysis of thousands of samples having been achieved using phage display libraries. In 2010, H Benjamin Larman et al constructed a first human proteome phage display library by expressing human proteomes in T7 phage, and screened the autoantibodies of paraneoplastic neurological diseases by the display library (Nature Biotechnology. Vol 29, No.6JUNE 2011: 535-541). After that, phage display libraries have completed many serological exploration tasks with their high throughput advantages coupled with the currently developed second generation sequencing technologies. The panning of random peptide library and the second generation sequencing technology can be used for determining epitopes and also can be used for determining the interaction condition of other immune related proteins such as autoantigens. In 2015, Christiansen A et al probed epitopes for IgE interaction with autoantigens in serum of peanut allergic patients by combining phage display libraries with secondary sequencing (Sci Rep.12913 (2015)). Similar work is still much, but the screening of biomarkers of systemic lupus erythematosus through a random peptide library is not reported, so that the high-throughput phage display library technology is used for screening the polypeptides with high discrimination in patients and normal people so as to obtain the early diagnosis marker more effective on the systemic lupus erythematosus.

Disclosure of Invention

Aiming at the existing technical problems and the requirement of more accurate serum marker discovery of the systemic lupus erythematosus, the invention provides the application of the polypeptide SLE2018-V001 (with the polypeptide sequence of YEHAMYRSAVLL) in a kit for diagnosing the systemic lupus erythematosus, so as to qualitatively detect the level of an IgG antibody of the polypeptide in human serum, and as a means for assisting the diagnosis of the systemic lupus erythematosus, the sensitivity and the specificity of early diagnosis of the systemic lupus erythematosus are expected to be greatly improved.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention provides a polypeptide SLE2018-V001, the amino acid sequence of which is: YEHAMYRSAVLL are provided.

In a second aspect, the invention provides an application of the polypeptide SLE2018-V001 in preparing a composition for diagnosing systemic lupus erythematosus.

The method comprises the following steps: connecting synthesized polypeptide SLE2018-V001 antigen to Bovine Serum Albumin (BSA) through cyclohexane-1-carboxylic acid succinimide ester (SMCC) to coat a microporous plate to prepare a solid-phase antigen, sequentially adding serum to be detected into the microporous plate coated with the antigen, adding an enzyme labeling reagent containing Horse Radish Peroxidase (HRP) labeled anti-Human IgG antibody,forming a polypeptide SLE 2018-V001-antibody-enzyme labeled secondary antibody compound, and adding an enzyme substrate solution 3,3',5,5' -Tetramethylbenzidine (TMB) for color development after thorough washing. TMB is converted to blue by HRP enzyme catalysis and to the final yellow by acid, and then the reaction is terminated by OD₄₅₀Values detect levels of IgG antibodies in the sample that specifically recognize the anti-polypeptide SLE 2018-V001.

Preferably, the polypeptide SLE2018-V001 is panned from M13 phage random polypeptide library.

In a third aspect, the invention provides a diagnostic kit for diagnosing systemic lupus erythematosus, which comprises the polypeptide SLE 2018-V001.

Preferably, the polypeptide SLE2018-V001 is coupled to BSA via cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to form an SMCC-BSA-polypeptide coupling product.

Preferably, the kit further comprises a standard substance, a coating buffer solution, a blocking solution, a sample diluent, a stop solution, an enzyme labeling reagent, an enzyme substrate solution and a washing solution.

Preferably, the standard comprises standard serum 1 with a concentration of 0U/mL of IgG antibodies against the polypeptide SLE2018-V001 and standard serum 2 with a concentration of 100U/mL of IgG antibodies against the polypeptide SLE 2018-V001; the standard serum 1 is normal human serum, and the standard serum 2 is serum with a positive SLE2018-V001 antibody;

preferably, the polypeptide SLE2018-V001 antigen is diluted by using a coating buffer which is 0.05 +/-0.005M, pH 9.6.6 +/-0.05 carbonate buffer, namely 1.59g of Na is contained in 1L of solution₂CO₃，2.93g NaHCO₃；

Preferably, the blocking solution is 0.01 + -0.005M, pH 7.4.4 + -0.05 phosphate-NaCl buffer solution (PBS) containing 0.5% bovine serum albumin, i.e., 5g Bovine Serum Albumin (BSA), 8g NaCl, 0.2g KH per 1L₂PO₄，2.9g Na₂HPO₄·12H₂O，0.2g KCl。

Preferably, in step C, the enzyme substrate solution comprises: color-developing agent A: 500mL of solution contains 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30% hydrogen peroxide; display deviceColor agent B: 500mL of solution contains 350mg of TMB, 20mL of DMSO, and citric acid & H₂O 5.1g。

Preferably, in step B, the standard and the serum sample to be tested are diluted by a sample diluent, wherein the sample diluent is 0.01M phosphate-NaCl buffer solution (PBS) with pH 7.4;

the washing solution adopted by the washing is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution (PBST) containing 0.05 percent Tween-20, namely, 8g of NaCl and 0.2g of KH are contained in each 1 liter of solution₂PO₄，2.9g Na₂HPO₄·12H₂O，0.2g KCl，0.5mL Tween-20；

The stop solution is 2 +/-0.1M H₂SO₄A solution;

the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase.

Preferably, each reagent employed in the kit further comprises a preservative to facilitate preservation.

In a fourth aspect, the invention provides a method for qualitatively detecting IgG antibodies against the polypeptide SLE2018-V001 in human serum, comprising the steps of:

A. coupling the polypeptide SLE2018-V001 to BSA via SMCC;

B. diluting the coupled polypeptide, coating the diluted polypeptide in micropores on an ELISA plate to prepare a solid phase antigen, and adding a confining liquid;

C. diluting the standard substance and a serum sample to be detected, adding the diluted standard substance and the serum sample to be detected into respective antigen determination holes, incubating, and adding an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase into each hole to form an SLE 2018-V001-antibody-enzyme-labeled secondary antibody compound;

D. after the treatment of step C, thoroughly washing, adding an enzyme substrate solution for color development, adding a stop solution to stop the reaction, and passing through OD₄₅₀The value is the level of IgG antibodies against the polypeptide SLE2018-V001 in the sample.

Preferably, in step a, the step of coupling the polypeptide SLE2018-V001 to BSA via SMCC specifically comprises:

a1, adding cyclohexane-1-carboxylic acid succinimide ester (SMCC) into buffer solution PBS containing BSA, uniformly mixing, and reacting at 25 ℃ for 1h to obtain a BSA-SMCC solution;

a2, adding a BSA-SMCC solution into the polypeptide SLE2018-V001 solution, uniformly mixing, standing at 25 ℃ for 4-6 hours to obtain a coupling product BSA-SMCC-polypeptide SLE 2018-V001.

More preferably, in step a1, the mass ratio of SMCC to BSA is 1: 5;

the concentration of the BSA-SMCC solution is 4 mg/mL.

The invention develops a set of technology for rapidly obtaining disease serum markers by utilizing the advantages of high throughput and rapid analysis of phage random polypeptide display library and next-generation sequencing. By analyzing 200 parts of systemic lupus erythematosus serum (100 patients and 100 healthy patients), the reactivity difference between the serum IgG of the patients and the serum IgG of the healthy patients is compared in a short time, and the polypeptide SLE2018-V001 serving as the serum marker is screened out, and is expected to be used for early diagnosis of the systemic lupus erythematosus.

Compared with the prior art, the invention has the following beneficial effects:

1. the specificity of the serum marker provided by the invention is 74%, the sensitivity is 88%, and the serum marker has the characteristics of high specificity and high sensitivity.

2. The invention provides a sensitive, safe, reliable and easily-operated commercialized kit, which is used for qualitatively determining the level of an IgG antibody of the anti-polypeptide SLE2018-V001 in human serum and is beneficial to assisting in early diagnosis of systemic lupus erythematosus.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the results of the electrophoretic validation of PBS-conjugated polypeptides prepared in example 1 of the present invention;

FIG. 2 is a ROC curve obtained in example 1 of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1

1. Preparation of the polypeptide and coupling of the polypeptide to BSA

1) Polypeptide SLE2018-V001 (amino acid sequence: YEHAMYRSAVLL) is a polypeptide having cysteine attached to the C-terminus, which is synthesized by Gill Biochemical (Shanghai) Co., Ltd, by HPLC. SMCC (cyclohexane-1-carboxylic acid succinimidyl ester) (cat # 22360) was purchased from Thermo. Dissolved in DMSO (dimethyl sulfoxide) to a final concentration of 100mg/mL, and stored at-20 ℃. The shelf life is one month.

2) 10mg of BSA was weighed and added to 1mL of PBS, to prepare a 10mg/mL BSA solution.

3) 2mg of SMCC (1: 5 relationship with BSA) was added to the carrier protein solution, mixed well and reacted at 25 ℃ for 1 hour.

4) Dialyzed overnight in a 10kD dialysis bag.

5) The dialyzed carrier protein solution was placed in a fresh centrifuge tube, the primary coupling buffer was added, and the activated BSA-SMCC concentration was diluted to 4 mg/mL.

6) The polypeptide SLE 2018-V0011 mg was taken in an Eppendorf tube, and 10. mu.L of DMSO was added to dissolve the polypeptide. Adding 200 mu L PBS to obtain a polypeptide SLE2018-V001 solution, and measuring the pH value of the solution within 7.0-7.5.

1. To the polypeptide was added 200. mu.L of activated BSA-SMCC (concentration 4mg/mL), mixed well and then left to stand at 25 ℃ for 4 to 6 hours.

2. After the coupling was complete, PBS was added to bring the volume to 0.8mL, at which time the solution concentration was 1 mg/mL.

3. The coupling was verified by electrophoresis.

The results are shown in FIG. 1, and the protein bands are shown as follows from left to right: marker, BSA, BSA-SMCC, BSA-SMCC-polypeptide SLE 2018-V001. It can be seen that the molecular weight of BSA after coupling to SMCC shows about 20kD change, the molecular weight of BSA-SMCC after coupling to polypeptide shows smaller change, and the polypeptide consists of 12 amino acids, so the molecular weight change is not large, but the successful coupling of the polypeptide can be still seen.

2. Preparation of serum samples:

placing the whole blood sample at room temperature for 2 hours or overnight at 4 ℃, centrifuging at 1000g for about 20 minutes, and taking the supernatant to immediately detect; or subpackaging, and storing at-20 deg.C or-80 deg.C, but avoiding repeated freeze thawing. The thawed sample should be centrifuged again and then examined. The sample to be detected cannot contain NaN₃Due to NaN₃Inhibiting activity of Horse Radish Peroxidase (HRP).

3. ELISA method various buffers and reagents preparation method:

(1) coating buffer solution: 0.05M Na pH 9.6₂CO₃-NaHCO₃The compositions are shown in table 1 below.

TABLE 1

(2) Sample diluent: pH 7.4PBS solution, the composition is shown in Table 2 below.

TABLE 2

(3) Washing liquid: PBST solution pH 7.4, composition as shown in table 3 below.

TABLE 3

(4) Sealing liquid: 0.5% BSA in PBS at pH 7.4, with the composition shown in Table 4 below.

TABLE 4

(5) Enzyme substrate solution: color-developing agents A and B (ready-to-use), the compositions of which are shown in tables 5 and 6 below.

TABLE 5 color reagent A

TABLE 6 color reagent A

(6) Stopping liquid: 2mol/L H₂SO₄The composition of the solution (concentrated sulfuric acid was slowly added dropwise to distilled water during the preparation, and mixed uniformly) is shown in table 7 below.

TABLE 7

4. The ELISA method is used for determining the concentration of IgG antibody resisting the polypeptide SLE2018-V001 in serum so as to assist in diagnosing the systemic lupus erythematosus, and the specific operation steps are as follows:

(1) coating: diluting the purified coupling product BSA-SMCC-polypeptide SLE2018-V001 to 1 mu g/mL by using a coating buffer solution, adding the diluted coupling product BSA-SMCC-polypeptide SLE2018-V001 into a 96-well enzyme label plate, wherein each well is 100 mu L, and coating at 37 ℃ for 2 hours or overnight at 4 ℃; the plate was washed with washing liquid 1 time and spin-dried.

(2) And (3) sealing: adding 200 mu L of confining liquid, and keeping the temperature at room temperature for 2 hours; the plate was washed with washing liquid 1 time and spin-dried.

(3) Diluting and loading the standard substance and the sample: the standard substance (the concentration of IgG antibody resisting the polypeptide SLE2018-V001 is 0U/mL standard serum 1 and the concentration of IgG antibody resisting the polypeptide SLE2018-V001 is 100U/mL standard serum 2; the standard serum 1 is normal human serum, and the standard serum 2 is serum positive to the antibody of SLE 2018-V001) and the serum sample 1:100 to be detected are diluted to 100 mu L by using a sample buffer solution, and the diluted sample solution and the sample solution are added into respective antigen determination pore plates. And (3) paying attention to no air bubbles, adding the sample to the bottom of the hole of the quincunx standard plate in a sample adding mode, keeping the sample from touching the hole wall as far as possible, slightly shaking and uniformly mixing, and covering or laminating a film on the ELISA plate. If the serum samples to be detected are more, a multi-tube micro liquid adding device is recommended to be used for sample adding. The standard substance and the sample to be detected are prepared within 15 minutes before use, are discarded after use, and the freshly prepared standard substance is used for next detection.

(4) And (3) incubation: placing the enzyme label plate at 37 ℃ for reaction for 120 minutes, completely throwing liquid in the hole, and washing for 6 times.

(5) Adding an enzyme: and adding 100 mu L of enzyme labeling reagent containing horseradish peroxidase labeled anti-Human IgG antibody into each well, and forming SLE 2018-V001-antibody-enzyme labeled secondary antibody compound at 37 ℃ for 60 minutes. And (5) completely throwing off the liquid in the holes, and patting the liquid for 6 times with the upper washing plate.

(6) Color development: after the mixture is patted dry, 50 mu L of color developing agent A is firstly dropped into each hole, 50 mu L of color developing agent B is added, the mixture is gently shaken and uniformly mixed, and the mixture is shaded at 37 ℃ for developing for 15 minutes.

(7) And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well in sequence. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. The stop solution should be added as soon as the substrate reaction time is reached.

(8) And (4) judging a result:

i. the optical density (OD value) of each well was measured sequentially at a wavelength of 450nm using an enzyme-linked analyzer.

Unit value (U/mL) ═ (a450< sample > -a450< standard serum 1>)/(a450< standard serum 2> -a450< standard serum 1>) × 100

A450 is an abbreviation for absorbance at 450 nm.

At present, the antibodies such as polypeptide and the like have no international current reference standard, so that the detection result is calibrated by using relative units.

Determination of the value of anti-polypeptide SLE2018-V001 in serum

The unit value is more than or equal to 100U/mL: can preliminarily diagnose the patient as the patient with the systemic lupus erythematosus

Unit value < 100U/mL: the patient cannot be diagnosed as systemic lupus erythematosus

Quality control

Each test result must meet the following criteria:

a450 of standard serum 1: less than or equal to 0.100

A450 of standard serum 2: not less than 0.700

If the above criteria are not met, the result is deemed invalid and must be retested.

interpretation of test results

The above reference values were established by ROC analysis of 50 healthy human sera, 50 patient sera.

Specificity and sensitivity detection: the diagnostic kit of the present invention was subjected to specificity and sensitivity detection using 100 parts of serum from patients associated with autoimmune diseases (50 parts of systemic lupus erythematosus patients, 50 parts of healthy persons). Detecting the light absorption value OD₄₅₀Then, an ROC curve was obtained using SPSS 17.0 (as shown in FIG. 2, the abscissa shows 1-specificity and the ordinate shows sensitivity). The specificity of the diagnosis kit for assisting in diagnosing the systemic lupus erythematosus is 74%, the sensitivity is 88%, and AUC is 0.81, so that the indexes of systemic lupus erythematosus diagnosis in the prior art are improved.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims (9)

1. A polypeptide SLE2018-V001, wherein the amino acid sequence of said polypeptide is: YEHAMYRSAVLL are provided.

2. Use of the polypeptide SLE2018-V001 according to claim 1 for the preparation of a composition for the diagnosis of systemic lupus erythematosus.

3. A diagnostic kit for diagnosing systemic lupus erythematosus comprising the polypeptide SLE2018-V001 of claim 1.

4. The diagnostic kit for diagnosing systemic lupus erythematosus of claim 3, wherein the kit further comprises a standard substance, a coating buffer solution, a blocking solution, a sample diluent, a stop solution, an enzyme labeling reagent, an enzyme substrate solution and a washing solution.

5. The diagnostic kit for diagnosing systemic lupus erythematosus of claim 4, wherein the standards comprise a standard serum 1 with a concentration of 0U/mL of IgG antibodies against the polypeptide SLE2018-V001 and a standard serum 2 with a concentration of 100U/mL of IgG antibodies against the polypeptide SLE 2018-V001; the standard serum 1 is normal human serum, and the standard serum 2 is serum with a positive SLE2018-V001 antibody;

the polypeptide SLE2018-V001 antigen is diluted by adopting a coating buffer solution which is a carbonate buffer solution of 0.05 +/-0.005M, pH 9.6.6 +/-0.05;

the confining liquid is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution containing 0.5 percent of bovine serum albumin.

6. The diagnostic kit for diagnosing systemic lupus erythematosus of claim 4, wherein the enzyme substrate solution includes: color-developing agent A: 500mL of solution contains 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30% hydrogen peroxide; and a color developing agent B: 500mL of solution contains 350mg of TMB, 20mL of DMSO, and citric acid & H₂O 5.1g。

7. The diagnostic kit for diagnosing systemic lupus erythematosus of claim 4, wherein the standard substance is diluted with a sample diluent, which is a phosphate-NaCl buffer solution of 0.01 ± 0.005M pH 7.4;

the washing solution is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution containing 0.05 percent of Tween-20;

the stop solution is 2 +/-0.1M H₂SO₄A solution;

the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase.

8. The diagnostic kit for diagnosing systemic lupus erythematosus according to claim 4, wherein each reagent used in the kit further comprises a preservative.

9. A method for the non-diagnostic therapeutic qualitative detection of IgG antibodies against the polypeptide SLE2018-V001 in human serum comprising the steps of:

A. coupling the polypeptide SLE2018-V001 to BSA via SMCC; the amino acid sequence of the polypeptide is as follows: YEHAMYRSAVLL, respectively;

B. diluting the coupled polypeptide by coating buffer solution, then coating the polypeptide in micropores on an ELISA plate to prepare solid phase antigen, and adding confining liquid;

C. diluting the standard substance and a serum sample to be detected by using a sample diluent, adding the diluted sample and the diluted sample into respective antigen measuring holes, incubating, and adding an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase into each hole to form an SLE 2018-V001-antibody-enzyme-labeled secondary antibody compound;

D. after the treatment of step C, thoroughly washing, adding an enzyme substrate solution for color development, adding a stop solution to stop the reaction, and passing through OD₄₅₀The value is the level of IgG antibodies against the polypeptide SLE2018-V001 in the sample.

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