阅读说明：本技术 诊断试剂盒及mak16在制备系统性红斑狼疮早期诊断试剂中的应用 (Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus ) 是由 叶冬青 凌华志 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种诊断试剂盒及MAK16在制备系统性红斑狼疮(SLE)早期诊断试剂中的应用,该诊断试剂盒,可用于系统性红斑狼疮疾病的早期诊断,试剂盒包括酶标板、作为抗原的人蛋白质MAK16、包被液、标准血清、酶标试剂、封闭液、样品稀释液,将人蛋白质MAK16包被于酶标板上,通过测定血清中抗人蛋白质MAK16的IgG抗体的光密度,以辅助诊断系统性红斑狼疮。本发明提供了一种灵敏、安全、可靠、易操作的商品化试剂盒,通过定性检测人血清中抗蛋白质MAK16的IgG抗体的水平,有助于辅助诊断和鉴别诊断SLE；提供的试剂盒用于SLE疾病筛查或初步诊断的特异性为88.12％,敏感性为48.98％；用于SLE疾病鉴别诊断的特异性为86.50％,敏感性为39.50％,可以作为现有SLE诊断标记物的有益补充。(The invention discloses a diagnostic kit and an application of MAK16 in preparation of an early diagnostic reagent of Systemic Lupus Erythematosus (SLE), wherein the diagnostic kit can be used for early diagnosis of systemic lupus erythematosus diseases, and comprises an enzyme-labeled plate, human protein MAK16 serving as an antigen, coating liquid, standard serum, an enzyme-labeled reagent, confining liquid and sample diluent, the human protein MAK16 is coated on the enzyme-labeled plate, and the diagnosis of the systemic lupus erythematosus is assisted by measuring the optical density of an IgG antibody of the human protein MAK16 in the serum. The invention provides a sensitive, safe, reliable and easy-to-operate commercialized kit, which is beneficial to auxiliary diagnosis and differential diagnosis of SLE by qualitatively detecting the level of IgG antibody of anti-protein MAK16 in human serum; the provided kit has 88.12% of specificity and 48.98% of sensitivity when used for SLE disease screening or primary diagnosis; the specificity for the differential diagnosis of SLE diseases is 86.50%, the sensitivity is 39.50%, and the SLE diagnostic marker can be used as a beneficial supplement of the prior SLE diagnostic marker.)

1. A diagnostic kit is used for diagnosing systemic lupus erythematosus and is characterized by comprising an enzyme label plate, human protein MAK16 serving as an antigen, coating liquid, standard serum, an enzyme labeling reagent, confining liquid and sample diluent, wherein the human protein MAK16 is coated on the enzyme label plate, and the diagnostic kit is used for assisting in diagnosing the systemic lupus erythematosus by measuring the optical density of an IgG antibody of the human protein MAK16 in the serum.

2. The diagnostic kit of claim 1, wherein said human protein MAK16 is overexpressed from saccharomyces cerevisiae, affinity purified, and has a concentration of 50 μ g/mL.

3. The diagnostic kit of claim 1, wherein the enzyme labeling reagent comprises 0.8mg/mL of anti-human IgG antibody labeled with HRP.

4. The diagnostic kit of claim 1, wherein the standard sera comprise positive standard sera and negative standard sera, the negative standard sera being human healthy sera that are negative for known MAK16 antibodies; the positive standard serum is the serum which is known to be positive by the MAK16 antibody.

5. The diagnostic kit of claim 1, wherein the blocking solution is a 0.01mol/L phosphate-NaCl buffer solution with pH7.4 containing 3% BSA, i.e., 1 liter solution containing 30g BSA, 8g NaCl, 0.2g KH₂PO₄、2.9gNa₂HPO₄·12H₂O and 0.2g KCl.

6. The diagnostic kit of claim 1, wherein the sample diluent is 0.01mol/LpH7.4 PBS solution, i.e. 1 liter solution contains the following components: 10gBSA, 8gNaCl, 0.2gKH₂PO₄、2.9gNa₂HPO₄·12H₂O、0.2gKCl、1mLTween-20、10mL FBS、0.4mL ProClin300。

7. The diagnostic kit of claim 1, wherein the diagnostic kit employs ELISA technology widely used in clinical practice, and uses indirect method to qualitatively detect IgG antibody against protein MAK16 in human serum, specifically: coating a microporous plate with purified MAK16 protein to prepare a solid phase antigen, sequentially adding serum to be detected into the coated microporous plate, combining with an anti-human IgG antibody marked by HRP to form an antigen-antibody-enzyme labeled antibody compound, completely washing, adding a substrate TMB for color development, converting the TMB into blue under the catalysis of the HRP enzyme, and finally converting the TMB into yellow under the action of acid, wherein the shade of the color is in positive correlation with the level of the IgG antibody of the anti-protein MAK16 in a sample.

The application of MAK16 in preparing an early diagnosis reagent for systemic lupus erythematosus.

Technical Field

The invention belongs to the field of bioscience, and particularly relates to a diagnostic kit and application of MAK16 in preparation of an early diagnosis reagent for systemic lupus erythematosus.

Background

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that seriously threatens human health and may damage multiple systems and organs throughout the body, and has a large difference in diseases of different genders, races and regions, among which the incidence of asia and americans is high (Rees F, et al: rheumatology (oxford),2017,56(11): 1945:1961), the average incidence of diseases is about 17/10-48/10 ten thousand in the world, the incidence of diseases is about 40/10-70/10 ten thousand in china, the ratio of men and women is about 1:9, and the disease is difficult to be clinically cured, and may cause irreversible damage to each organ or system of the body along with the continuation or repeated outbreak of the disease course, among which the more common injuries are skin mucosa, kidney, blood system, nervous system, joint (Wu H, et al: Autoimmiture reviews,2017,16(4):391-397), other lesions are also found in almost all organs and systems throughout the body, such as the liver, lungs, eyes, heart, brain, etc.

It is currently believed that Systemic Lupus Erythematosus (SLE) occurs as a result of the combined action of genetic, environmental, hormonal, and other factors (Paley MA, et al: Curr Opin Rheumatotol, 2017,29(2): 178-. Because the pathogenesis of SLE is complex, the early state of the disease is hidden, the related organs are damaged a lot, the early clinical diagnosis has great difficulty, and the accurate and reliable golden standard is lacked, so that the comprehensive diagnosis needs to be carried out by combining clinical symptoms, physical signs, medical history and laboratory examination. In the past decades, the diagnosis, treatment and prognosis of systemic lupus erythematosus are continuously improved, however, systemic lupus erythematosus patients still bear heavy burden of diseases and seriously affect the life quality of the patients due to the reasons that the course of disease is easily delayed and repeated and cannot be predicted, and the patients still cannot be completely cured clinically.

The clinical manifestations of systemic lupus erythematosus are various, and the serological and immunological indexes have large variability (Apostolopoulos D, et al: Aust Fam Physician,2013,42(10): 696-700). In view of the fact that the generation of a large amount of autoantibodies attacks the corresponding target antigen, or binds with the target antigen to form immune complexes, and the deposition of the autoantibodies on various tissues of the body is a direct cause of the onset of SLE, researchers at home and abroad are always dedicated to the discovery of new autoantibodies with high specificity and sensitivity. Abnormal elevations of various immunoglobulins or antibodies can be found in patient sera by laboratory examination. Therefore, the discovery of the autoantibody diagnosis marker of the systemic lupus erythematosus has important clinical application value.

The ideal systemic lupus erythematosus diagnosis marker meets the following conditions: (1) the sensitivity is high; (2) the specificity is high; (3) is present in body fluids, particularly blood, and is easy to detect. In recent years, a review of the literature has reported that over 100 autoantibodies have been found to be abnormally highly expressed in SLE patients (Yaniv G, et al: Autoimmun Rev,2015,14(1): 75-79). However, none of the numerous serum immunological markers currently available has been used alone to achieve high sensitivity and specificity for early diagnosis of clinical SLE. Currently, guidelines recommend and clinically commonly used autoantibodies are: ANA, anti-dsDNA, anti-Sm, aPL, anti-rib-P, anti-DNP, AHA, AnuA, ANCA, etc. Among the commonly used clinical autoantibodies, ANA reacts with the nuclei of all animals and can be detected in most mixed connective tissue diseases (MTCD) such as SLE, and is the primary serological indicator of SLE; anti-dsDNA antibodies can appear in the early stage of SLE onset, are closely related to SLE, rise and fall with different activity degrees of diseases, and can be detected in 40% -70% of SLE patients; the positive detection rate of the anti-Sm antibody in the SLE patient is about 30 percent; anti-phospholipid antibodies (aPL) were detectable in 30-40% of SLE patients; the positive rate of the anti-ribosomal P protein antibody (anti-rib-P) in SLE patients is 15% -35%, and is related to neuropsychiatric symptom complications in SLE (Viewa VT, et al: Lupus,2017,26(5): 453-462); anti-deoxyribonuclein antibodies (anti-DNPs) have some correlation with SLE patient disease activity; anti-neutrophil cytoplasmic antibodies (ANCA) are often associated with SLE activity, with a positive rate of about 21% -25% in SLE patients (Weiner M, et al: Autoimmun Rev,2016,15(10): 978-982). Analysis of the application value of the common autoantibodies shows that the common autoantibodies play a certain role in diagnosis and differential diagnosis of SLE, occurrence and development of diseases, appearance of various clinical characteristics and the like, but have some defects, and further have important clinical significance in exploring unknown SLE highly-related autoantibodies from the whole proteome level.

Disclosure of Invention

The first purpose of the present invention is to provide a diagnostic kit to solve the technical problem that the prior art systemic lupus erythematosus diagnostic kit cannot realize early diagnosis with high sensitivity and high specificity on SLE.

The second objective of the present invention is to provide an application of MAK16 in the preparation of an early diagnosis reagent for systemic lupus erythematosus, so as to solve the technical problem that the systemic lupus erythematosus diagnosis kit in the prior art cannot realize early diagnosis with high sensitivity and high specificity on SLE.

In order to solve the problems, the technical scheme of the invention is as follows:

a diagnostic kit is used for early diagnosis of systemic lupus erythematosus, and comprises an enzyme label plate, human protein MAK16 used as an antigen, coating liquid, standard serum, an enzyme labeling reagent, confining liquid and sample diluent, wherein the human protein MAK16 is coated on the enzyme label plate, and the diagnostic kit is used for assisting in diagnosis of systemic lupus erythematosus by measuring the concentration of IgG antibody of human protein MAK16 in the serum.

Preferably, the human protein MAK16 is over-expressed from Saccharomyces cerevisiae and affinity purified, and the concentration is 50 μ g/mL.

Preferably, the enzyme labeling reagent contains 0.8mg/mL of HRP-labeled anti-human IgG antibody, wherein the anti-human IgG antibody is prepared by an affinity purification goat method.

Preferably, the standard serum comprises a positive standard serum and a negative standard serum, wherein the negative standard serum is a healthy human serum negative to the known MAK16 antibody; the positive standard serum is the serum which is known to be positive by the MAK16 antibody.

Preferably, the blocking solution is 0.01mol/L phosphate-NaCl buffer solution (PBS) containing 3% BSA, pH7.4, i.e., 30g BSA (bovine serum albumin), 8g NaCl, 0.2g KH in 1 liter solution₂PO₄、2.9gNa₂HPO₄·12H₂O and 0.2g KCl.

Preferably, the sample diluent is 0.01mol/L PBS solution with pH7.4, namely 1 liter solution comprises the following components: 10gBSA, 8gNaCl, 0.2gKH₂PO₄、2.9gNa₂HPO₄·12H₂O, 0.2gKCl, 1mLTween-20, 10mLFBS (fetal bovine serum), 0.4mLProClin300。

Preferably, the diagnostic kit adopts the ELISA technology widely used in clinic at present, and uses an indirect method to qualitatively detect the IgG antibody of the anti-protein MAK16 in human serum, specifically: coating a microporous plate with purified MAK16 protein to prepare a solid phase antigen, sequentially adding serum to be detected into the coated microporous plate, combining with an HRP-labeled anti-human IgG antibody to form an antigen-antibody-enzyme-labeled antibody compound, completely washing, adding a TMB solution for color development, converting TMB into blue under the catalysis of HRP enzyme, and finally converting the TMB into yellow under the action of acid, wherein the shade of the color is in positive correlation with the level of the IgG antibody of the anti-protein MAK16 in a sample.

The invention also provides application of the MAK16 in preparation of an early diagnosis reagent for systemic lupus erythematosus, and the diagnosis reagent is used for determining the optical density of an IgG antibody of anti-MAK 16 in serum so as to assist in diagnosing the systemic lupus erythematosus.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects:

1. the invention provides a sensitive, safe, reliable and easy-to-operate commercialized kit, which is helpful for auxiliary diagnosis and differential diagnosis of SLE by qualitatively detecting the level of IgG antibody of anti-protein MAK16 in human serum.

2. The specificity of the provided serum biomarker diagnostic detection kit for SLE disease screening or preliminary diagnosis is 88.12%, and the sensitivity is 48.98%; the specificity for the differential diagnosis of SLE diseases is 86.50%, the sensitivity is 39.50%, and the SLE diagnostic marker can be used as a beneficial supplement of the prior SLE diagnostic marker.

Drawings

FIG. 1 is a box plot of SLE patient serum versus healthy human control serum optical density values;

FIG. 2 is a subject work curve comparing SLE patients to healthy human control serum optical density values;

FIG. 3 is a boxplot of SLE patients compared to disease control serum optical density values;

figure 4 is a subject work curve comparing SLE patients to disease control serum optical density values.

Detailed Description

The following will explain in detail the diagnostic kit and the use of MAK16 in the preparation of an early diagnostic reagent for systemic lupus erythematosus according to the present invention with reference to the accompanying drawings and the specific examples. Advantages and features of the present invention will become apparent from the following description and from the claims.

MAK16 is called RNA binding protein 13 (RBM 13), and is localized in the nucleus, and is involved in key processes such as RNA splicing, transport, localization, editing, and mRNA posttranscriptional regulation (Andersen JS, et al: Current Biology Cb,2002,12(1): 1-11). As observed by a cryoelectron microscope, the researchers found that MAK16 is involved in the formation and stabilization of ribosomal 60S large subunit precursor (Kater L, et al: Cell,2017,171(7):1599-1610e 14); it has also been suggested by researchers to participate in the maturation of Saccharomyces cerevisiae 25S and 5.8S rRNAs by related studies on Saccharomyces cerevisiae (Pellett S, et al: Yeast,2006,23(7): 495-506). In recent years, it has been discovered that MAK16 is a protein containing nuclear localization signal and playing an important role in the cell cycle of eukaryotic cells and the formation of ribosomal 60S subunit, and is involved in the transport of nucleic acid protein in some parasites (Crossomo-Vazzezez Mdel P, et: Korean J Parasitol,2014,52(4):429-33), and the rest has little research on the functions of MAK16 gene and protein.

At present, no report of using the MAK16 test cassette as a SLE biomarker is found.

The invention provides a diagnostic kit for early diagnosis of systemic lupus erythematosus, which comprises an enzyme label plate, human protein MAK16 as an antigen, coating liquid, standard serum, an enzyme labeling reagent, confining liquid and sample diluent, wherein the human protein MAK16 is coated on the enzyme label plate, and the diagnostic kit is used for assisting in diagnosis of the systemic lupus erythematosus by measuring the concentration of IgG antibody of the human protein MAK16 in the serum.

Preferably, the human protein MAK16 is overexpressed from Saccharomyces cerevisiae and affinity purified at a concentration of 50. mu.g/mL.

Preferably, the enzyme labeling reagent contains 0.8mg/mL of horseradish peroxidase (HRP) -labeled anti-human IgG antibody, wherein the anti-human IgG antibody is prepared by an affinity purification goat method.

Preferably, the standard serum comprises a positive standard serum and a negative standard serum, wherein the negative standard serum is a healthy human serum negative to the known MAK16 antibody; the positive standard serum is the serum which is known to be positive by the MAK16 antibody.

Preferably, the enzyme substrate display solution is also included, the enzyme substrate display solution is a TMB solution, and the TMB solution is a single-component display solution.

Preferably, the blocking solution is a 3% BSA in 0.01mol/L phosphate-NaCl buffer solution (PBS) pH7.4, i.e., 1 liter solution containing 30g BSA (bovine serum albumin), 8g NaCl, 0.2g KH₂PO₄、2.9gNa₂HPO₄·12H₂O and 0.2g KCl.

Preferably, the sample dilution is 0.01mol/L PBS solution with pH7.4, i.e. 1 liter solution contains the following components: 10gBSA, 8gNaCl, 0.2gKH₂PO₄、2.9gNa₂HPO₄·12H₂O, 0.2gKCl, 1mLTween-20, 10mLFBS (fetal bovine serum), 0.4mLPROClin 300.

Preferably, the kit further comprises a washing solution and a stop solution, wherein the washing solution is 0.01mol/L PBST solution (phosphate-NaCl buffer solution) with pH7.4, and the PBST solution contains 0.1% Tween-20, namely 1 liter of solution contains 8g of NaCl and 0.2gKH₂PO₄、2.9gNa₂HPO₄·12H₂O, 0.2gKCl and 1 mLTween-20; the stop solution is 2mol/L H₂SO₄And (3) solution.

Preferably, the diagnostic kit adopts the ELISA technology widely used in clinic at present, and uses an indirect method to qualitatively detect the IgG antibody of the anti-protein MAK16 in human serum, specifically: coating a microporous plate with purified MAK16 protein to prepare a solid phase antigen, sequentially adding serum to be detected into the coated microporous plate, combining with an HRP-labeled anti-human IgG antibody to form an antigen-antibody-enzyme-labeled antibody compound, completely washing, adding a TMB solution for color development, converting TMB into blue under the catalysis of HRP enzyme, and finally converting the TMB into yellow under the action of acid, wherein the shade of the color is in positive correlation with the level of the IgG antibody of the anti-protein MAK16 in a sample.

The invention also provides application of the MAK16 in preparation of an early diagnosis reagent for systemic lupus erythematosus, and the diagnosis reagent is used for determining the optical density of an IgG antibody of anti-MAK 16 in serum so as to assist in diagnosing the systemic lupus erythematosus.

The specific method steps for preparing the diagnostic kit and using the diagnostic kit are as follows:

1. preparation of serum samples

The whole blood specimen is placed at room temperature for 2 hours or at 4 ℃ overnight and then centrifuged at 3500rpm for 5-10 minutes, the supernatant is taken for immediate detection, or subpackaged, and the specimen is stored at-20 ℃ or-80 ℃, but repeated freeze thawing is avoided. The thawed sample should be centrifuged again and then examined. The sample to be detected cannot contain NaN₃Due to NaN₃Inhibit the (HRP) activity of horseradish peroxidase.

2. The preparation method of various buffers and reagents in the ELISA method comprises the following steps:

(1) coating liquid: 0.05M Na pH9.6₂CO₃-NaHCO₃

(2) Sealing liquid: 0.01mol/L PBS solution of 3% BSA at pH7.4

(3) Sample diluent: pH7.4 PBST containing BSA and FBS

(4) Washing liquid: 0.01mol/L pH7.4 PBST solution (ready for use)

(5) Stopping liquid: 2mol/L H₂SO₄Solution (when dispensing, concentrated sulfuric acid is slowly dropped into distilled water and mixed with the distilled water)

1. ELISA method to determine the Optical Density (OD) value of the anti-protein MAK16 IgG antibody in serum to aid in the diagnosis of SLE: the specific operation steps are as follows:

(1) antigen coating: diluting 50 mu g/mL human protein MAK16 antigen to 1 mu g/mL by using a coating solution, adding the diluted antigen into an enzyme label plate by using a multi-channel pipettor, vibrating the plate for about 1 minute on a plate vibrating device, checking whether the coating solution is contained in each hole or not, judging whether the adding amount of the coating solution is consistent or not, and whether bubbles are contained at the bottom of each hole or not, defoaming by using the pipettor if the bubbles are contained, and drying the coating at room temperature (humidity of 20 percent and 25 ℃) for overnight until the liquid is dried;

(2) washing the plate: adding a washing solution with the concentration of 300 mu L/hole into the ELISA plate by using a multi-channel pipettor for washing, standing for more than 30 seconds, and then throwing the washing solution in the ELISA plate into a waste liquid tank; repeating the step for 3 times, and finally, placing on absorbent paper for drying;

(3) and (3) sealing: adding 300 mu L/hole of confining liquid into the ELISA plate by using a multi-channel pipette, and confining for 3-5 hours at 37 ℃;

(4) and (3) drying treatment: throwing off the sealing liquid and patting dry; drying for 2-3 hours at room temperature (humidity 20%, 25 ℃);

(5) dilution of standards and samples (preparation during blocking): taking out a serum sample from a refrigerator at minus 80 ℃, placing the serum sample in a chromatography cabinet at 4 ℃ for freeze thawing for about 2 hours (the specific freeze thawing time is determined according to the sample volume), and centrifuging the serum sample for 30 minutes on a high-speed freezing centrifuge at 10000-12000 rpm and 4 ℃ after the freeze thawing is finished. After centrifugation is finished, diluting the serum sample to be detected and the standard substance by a sample diluent according to the ratio of 1: 500;

(6) sample adding: respectively arranging blank holes (the blank control holes are not added with a sample and an enzyme labeling reagent, and the rest steps are the same), sample holes to be detected, positive control holes and negative control holes, adding the diluted serum into respective antigen determination pore plates, and adding 100 mu L of the diluted serum into each hole;

(7) and (3) incubation: placing a sealing film plate at the rear part and incubating for 30 minutes at 37 ℃;

(8) washing: carefully tearing the opening plate film, throwing off liquid, patting to dry, filling 300 mu L of washing liquid into each hole, standing for 30 seconds, discarding, repeatedly washing for 5 times, and patting to dry;

(9) adding an enzyme standard reagent: diluting an enzyme-labeled reagent by sample diluent according to the proportion of 1:10000, and adding 100 mu L of the diluted enzyme-labeled reagent into each hole except for blank holes; wherein the enzyme labeling reagent contains 0.8mg/mL of anti-human IgG antibody labeled by HRP enzyme;

(10) and (3) incubation: placing a sealing film plate at the rear part and incubating for 30 minutes at 37 ℃;

(11) washing: carefully tearing the opening plate film, throwing off liquid, patting to dry, filling 300 mu L of washing liquid into each hole, standing for 30 seconds, discarding, repeatedly washing for 6 times, and patting to dry;

(12) color development: adding 100 mu L of TMB solution into each hole, shaking gently and mixing uniformly, and developing for 10 minutes at 37 ℃ in a dark place;

(13) and (4) terminating: adding 50 mu L of stop solution into each hole, and stopping the reaction;

(14) and (3) reading measurement: the optical density (OD value) of each well is measured sequentially at a wavelength of 450nm/620nm with the blank well as zero, and the final OD value is the difference between the OD values of the two wavelengths. Note that: the measurement should be performed within 15 minutes after the addition of the stop solution.

(15) And (4) interpretation of results:

a. when the experiment is used for SLE disease screening or preliminary diagnosis, the cutoff value is 0.104, when the OD value is more than 0.104, the SLE is judged to be positive, and when the OD value is less than or equal to 0.104, the SLE is judged to be negative;

b. when the experiment is used for differential diagnosis between SLE and other autoimmune diseases, the cutoff value is 0.142, the SLE is judged to be positive when the OD value is more than 0.142, and the SLE is judged to be negative when the OD value is less than or equal to 0.142.

(16) Quality control:

each test result must meet the following criteria:

the positive standard serum OD value is more than or equal to 3.500;

the OD value of the negative standard serum is less than or equal to 0.080;

if the standard is not met, the result is regarded as invalid and needs to be detected again.

Interpretation of test results:

ROC analysis of 294 SLE patient sera, 261 health examiner sera established cutoff values for SLE disease screening or preliminary diagnosis; ROC analysis of the sera of 200 patients with SLE and 200 other autoimmune diseases established cutoff values for differential diagnosis between SLE and other autoimmune diseases.

FIG. 1 shows the optical density values of the antibody MAK16 in the serum of SLE patients and the serum of healthy control groups when the kit is used, which shows that the optical density values of SLE patients are obviously higher than those of healthy control groups, thus the kit can be used for SLE disease screening or preliminary diagnosis; FIG. 3 shows the blood serum of SLE patients and the blood serum of control groups with other diseases, and the optical density value of the antibody of MAK16 in the kit is obviously higher than that of the control groups with other diseases, which shows that the kit can be used for differential diagnosis between SLE diseases and other autoimmune diseases.

The above-mentioned enzyme-labeled plate used in the preparation of the diagnostic kit was purchased from Thermo FisherScientific, usa, human protein MAK16 was purchased from CDI Laboratories, usa, and HRP enzyme was purchased from jackson immunoresearch, usa; TMB solution was purchased from invitro hu.

4. Specificity and sensitivity detection:

the diagnostic test kit of the invention is used for specific and sensitive detection by 755 parts of serum (294 parts of SLE patients, 200 parts of other autoimmune disease patients and 261 parts of health examination people) (as shown in figure 2 and figure 4). The kit provided by the invention is used for early diagnosis or differential diagnosis of SLE patients with other diseases, the specificity of the diagnostic detection kit for SLE disease screening or primary diagnosis is 88.12%, the sensitivity is 48.98% (as shown in figure 2, AUC represents the working curve area of a subject); the specificity for differential diagnosis of SLE disease was 86.50% and the sensitivity was 39.50% (as shown in figure 4, AUC represents the subject working curve area). Compared with the existing SLE serum diagnostic marker, the SLE serum diagnostic marker can be used as a beneficial supplement of the existing diagnostic marker.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.