CN110687285B - Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus - Google Patents
Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus Download PDFInfo
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- CN110687285B CN110687285B CN201911039849.9A CN201911039849A CN110687285B CN 110687285 B CN110687285 B CN 110687285B CN 201911039849 A CN201911039849 A CN 201911039849A CN 110687285 B CN110687285 B CN 110687285B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
Abstract
The invention discloses a diagnostic kit and an application of MAK16 in preparing a Systemic Lupus Erythematosus (SLE) early diagnosis reagent, wherein the diagnostic kit can be used for early diagnosis of systemic lupus erythematosus diseases, and comprises an enzyme-linked 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-linked 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 the specificity of 88.12 percent and the sensitivity of 48.98 percent 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.
Description
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 can damage multiple systems and organs throughout the body and seriously threaten human health, with wide variation in the incidence of diseases of different genders, races and regions, among which asia and americans are high (Rees F, et al: rheumatology (Oxford), 2017,56 (11): 1945-1961), which has a global average incidence of 17/10 to 48/10 ten thousand, and has a chinese incidence of 40/10 to 70/10 thousand, and has a man-woman ratio of about 1, and is clinically difficult to cure, and may cause irreversible damage to various organs or systems of the body with the progression or recurrence of the disease, among which the more common injuries are mucosal, kidney, blood system, nervous system, joint (Wu H, et al: autoimmune diseases, 391, 2017, 391 (2017) and almost all other organs visible in the liver, lungs, and the like.
Systemic Lupus Erythematosus (SLE) is currently thought to occur as a result of the combined action of many factors, including genetics, environment, hormones, etc. (Paley MA, et al: curr Opin Rheumatotol, 2017,29 (2): 178-186). 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 great 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, there have been reports of literature reviews 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 autoantibodies commonly found in clinical use, ANA reacts with cell nuclei of all animals, can be detected in most mixed connective tissue diseases (MTCD) such as SLE, and is a primary serological indicator for SLE; anti-dsDNA antibodies can be present in SLE early onset, and SLE is more closely related, with the activity of the disease changes, can be in 40% -70% SLE patients detection; 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 relevance to 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 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 concentration of IgG antibody of the 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 mug/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 serum which is known to be positive for the MAK16 antibody.
Preferably, the blocking solution is a 3% BSA-containing 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 KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O 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: 10g BSA, 8g NaCl, 0.2g KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O, 0.2gKCl, 1mLTween-20, 10mLFBS (fetal bovine serum), 0.4mLPRoClin300.
Preferably, the diagnostic kit adopts the ELISA technology widely used in clinical practice 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 anti-human IgG antibody marked by HRP 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 an application of the MAK16 in preparing an early diagnosis reagent of the systemic lupus erythematosus, and the diagnosis reagent is used for measuring the optical density of the anti-MAK 16 IgG antibody 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 beneficial to 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 diagnosis 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;
figure 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 diagnosis 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 (RBM13), is located in nucleus, and is involved in key processes such as RNA splicing, transport, location, editing, and mRNA post-transcriptional regulation (Andersen JS, et al: current Biology Cb,2002,12 (1): 1-11). It has been found by the researchers through observation with a cryoelectron microscope that MAK16 is involved in the formation and stabilization of the 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 a nuclear localization signal and playing an important role in the cell cycle of eukaryotic cells and the formation of ribosomal 60S subunits, and is involved in the transport of nucleic acid proteins in some parasites (Crossomo-Vazquez Mdel P, et al: korean J Parasitol,2014,52 (4): 429-33), and the rest of the research on the functions of the MAK16 gene and protein is very little.
At present, the MAK16 detection box is not found to be a report of SLE biomarker.
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 diagnosing 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 over-expressed from Saccharomyces cerevisiae, purified by affinity, and has 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 includes a positive standard serum and a negative standard serum, the negative standard serum being a healthy human serum negative to the known MAK16 antibody; the positive standard serum is 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-containing 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 KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O and 0.2g KCl.
Preferably, the sample dilution is a 0.01mol/L PBS solution with pH7.4, i.e. 1 liter solution contains the following components: 10g BSA, 8g NaCl, 0.2g KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O、0.2gKCl, 1mLTween-20, 10mLFBS (fetal bovine serum), 0.4mLPROClin300.
Preferably, the kit further comprises a washing solution and a stop solution, wherein the washing solution is 0.01mol/L of PBST solution (phosphate-NaCl buffer solution) with pH7.4, the content of the PBST solution is 0.1% Tween-20, i.e., 8g NaCl and 0.2g KH in 1L of the solution 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O, 0.2gKCl and 1mLTween-20; the stop solution is 2mol/L H 2 SO 4 And (3) solution.
Preferably, the diagnostic kit adopts the ELISA technology widely used in clinic at present, and applies 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 anti-human IgG antibody marked by HRP 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 the application of the MAK16 in the preparation of an early diagnosis reagent of the systemic lupus erythematosus, and the diagnostic reagent is used for measuring the optical density of an IgG antibody of the 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 out and can be immediately detected or subpackaged, and the specimen is stored at-20 ℃ or-80 ℃, but repeated freezing and thawing are avoided. The thawed sample should be centrifuged again and then examined. The sample to be detected cannot contain NaN 3 Due to NaN 3 Inhibit 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 pH9.6 2 CO 3 -NaHCO 3
(2) Sealing liquid: 3% of 0.01mol/L PBS solution of BSA, 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 H 2 SO 4 Solution (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) values of the anti-protein MAK16 IgG antibodies 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 if so, defoaming by using the pipettor, and drying the coating at room temperature (humidity 20%,25 ℃) overnight until the liquid is dried;
(2) Washing the plate: adding a 300 mu L/hole washing solution into the ELISA plate by using a multi-channel pipette for washing, standing for more than 30 seconds, and then throwing the washing solution in the ELISA plate away from 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 depends on the sample volume), and centrifuging the serum sample for 30 minutes on a high-speed freezing centrifuge at 10000-12000rpm and 4 ℃ after the freeze thawing is finished. After the centrifugation is finished, diluting the serum sample to be detected and the standard substance by using a sample diluent according to the ratio of 1;
(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 labeling reagent: diluting an enzyme-labeled reagent by using a sample diluent according to the proportion of 1 to 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 membrane sealing 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 blank hole is set to be zero, the optical density (OD value) of each hole is measured sequentially at the wavelength of 450nm/620nm, and the final OD value is the difference of 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 disease and other autoimmune diseases, the cutoff value is 0.142, SLE is judged to be positive when the OD value is more than 0.142, and 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 criteria are not met, the result is deemed invalid and must be retested.
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 MAK16 antibody in the serum of the SLE patient and the serum of the healthy control group when the kit is used, which shows that the optical density values of the SLE patient are obviously higher than those of the healthy control group, and indicates that the kit can be used for SLE disease screening or preliminary diagnosis; FIG. 3 shows the blood serum of SLE patient and the blood serum of contrast group with other diseases, and the optical density value of the antibody of MAK16 in the kit is obviously higher than that of contrast group with other diseases, which shows that the kit can be used for differential diagnosis between SLE disease and other autoimmune diseases.
The above-mentioned ELISA plate required for preparing the diagnostic kit was purchased from Thermo Fisher Scientific, inc., USA, human protein MAK16 was purchased from CDI Laboratories, inc., 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 of SLE patients or differential diagnosis of SLE patients and other diseases, the specificity of the diagnostic detection kit for SLE disease screening or primary diagnosis is 88.12%, and 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.
Claims (7)
- The application of the MAK16 in the preparation of the early diagnosis reagent for the systemic lupus erythematosus is characterized in that the diagnosis reagent comprises human protein MAK16 serving as an antigen and is contained in a kit, the kit comprises an enzyme label plate, a coating solution, standard serum, an enzyme labeling reagent, a confining solution and a sample diluent, wherein the human protein MAK16 is coated on the enzyme label plate, and the diagnosis 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 use of MAK16 of claim 1 in the preparation of an early diagnostic reagent of Systemic Lupus Erythematosus (SLE), wherein the human protein MAK16 is obtained by overexpression and affinity purification from Saccharomyces cerevisiae at a concentration of 50 μ g/mL.
- 3. The use of MAK16 of claim 1 in the preparation of an early diagnostic reagent of systemic lupus erythematosus, wherein the enzyme-labeled reagent contains 0.8mg/mL of HRP enzyme-labeled anti-human IgG antibody.
- 4. The use of MAK16 in the preparation of an early diagnostic reagent for systemic lupus erythematosus, according to claim 1, wherein the standard serum comprises a positive standard serum and a negative standard serum, and the negative standard serum is a healthy human serum negative to the known MAK16 antibody; the positive standard serum is serum which is known to be positive by the MAK16 antibody.
- 5. Use of the MAK16 of claim 1 in the preparation of an early diagnostic reagent for systemic lupus erythematosus wherein the blocking solution is a 3% BSA 0.01mol/L pH7.4 phosphate-NaCl buffer solution, i.e., 1 liter solution containing 30g BSA, 8g NaCl, 0.2g KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O and 0.2g KCl.
- 6. The use of MAK16 of claim 1 for preparing an early diagnosis reagent of systemic lupus erythematosus, wherein the sample diluent is 0.01mol/L PBS solution with pH7.4, namely 1 liter of the solution contains the following components: 10g BSA, 8g NaCl, 0.2g KH 2 PO 4 、2.9gNa 2 HPO 4 ·12H 2 O、0.2gKCl、1mLTween-20、10mL FBS、0.4mL ProClin300。
- 7. The use of MAK16 in the preparation of an early diagnosis reagent of systemic lupus erythematosus as claimed in claim 1, wherein the diagnosis reagent kit employs an ELISA technique widely used in clinical practice, and uses an indirect method to qualitatively detect IgG antibodies 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.
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| CN104655852A (en) * | 2013-11-21 | 2015-05-27 | 苏州中赢医疗科技有限公司 | Diagnostic marker for systemic lupus erythematosus |
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