CN117471100A - Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity - Google Patents
Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity Download PDFInfo
- Publication number
- CN117471100A CN117471100A CN202210865715.8A CN202210865715A CN117471100A CN 117471100 A CN117471100 A CN 117471100A CN 202210865715 A CN202210865715 A CN 202210865715A CN 117471100 A CN117471100 A CN 117471100A
- Authority
- CN
- China
- Prior art keywords
- antibody
- cell
- sle
- cells
- cell suspension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000009266 disease activity Effects 0.000 title claims abstract description 29
- 238000003745 diagnosis Methods 0.000 title claims abstract description 19
- 201000000596 systemic lupus erythematosus Diseases 0.000 title claims description 6
- 238000011156 evaluation Methods 0.000 title description 6
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims description 88
- 239000006285 cell suspension Substances 0.000 claims description 30
- 239000008055 phosphate buffer solution Substances 0.000 claims description 28
- 210000005259 peripheral blood Anatomy 0.000 claims description 26
- 239000011886 peripheral blood Substances 0.000 claims description 26
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 18
- 238000010186 staining Methods 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 13
- 201000010099 disease Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 210000003743 erythrocyte Anatomy 0.000 claims description 9
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000007796 conventional method Methods 0.000 claims description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical group O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 4
- 230000009390 immune abnormality Effects 0.000 claims description 4
- 230000007246 mechanism Effects 0.000 claims description 4
- 210000000601 blood cell Anatomy 0.000 claims description 3
- 230000006037 cell lysis Effects 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 230000002934 lysing effect Effects 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000013592 cell lysate Substances 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 4
- 230000006044 T cell activation Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 101150013553 CD40 gene Proteins 0.000 description 2
- 102000016918 Complement C3 Human genes 0.000 description 2
- 108010028780 Complement C3 Proteins 0.000 description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 206010013142 Disinhibition Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005211 surface analysis Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of detecting Th40 cell subgroup in preparing diagnosis assisting and SLE disease activity evaluating kit. The invention is based on the research result obtained by the inventor that the expression of Th40 cell subgroup in SLE patient with disease activity is obviously increased for the first time, and the higher the disease activity is, the higher the Th40 cell subgroup ratio is. Therefore, the Th40 cell subgroup can be used as one of laboratory immune related detection indexes for assisting in diagnosing SLE, and simultaneously, important reference data is provided for evaluating the disease activity of SLE patients.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of Th40 cell subsets in preparing a reagent kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity.
Background
Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is an autoimmune disease formed by pathogenic autoantibodies, and lymphocyte subset dysfunction plays an important role in SLE pathogenesis, wherein CD8+ T cell and NK cell dysfunction, loss of inhibition of CD4 + T cell capacity such that it continuously stimulates B cell activation to produce autoantibodies, resulting in a sustained autoimmune response. At present, the indexes for clinically evaluating the activity degree and the curative effect of SLE diseases are single, and no immune marker for effectively predicting the onset of SLE is available.
At present, the disease severity degree and the disease activity degree are mainly evaluated through SLEDAI-2000, but the SLEDAI-2000 scores are completely different due to different organs due to obvious SLE heterogeneity, and the SLE disease activity degree evaluation tool is single and lacks a comprehensive, accurate and objective laboratory index to evaluate the disease activity degree. The SLE patient has T cell immune imbalance, and the research discovers that some immune indexes related to T cell activation, especially the detection of the immune indexes related to the T cell activation, not only discusses the mechanism of the T cell immune abnormality of the SLE patient from the research field, but also can be used as clinical indexes for assisting in diagnosing SLE and judging the disease degree of the SLE patient. At present, most of research on T cell activation is conducted by discussing the mechanism of T cell immune abnormality of SLE patients, and no characteristic change of immune related indexes of SLE patients is seen yet, and no immune indexes of T cell activation for diagnosing and judging disease activity are available. Therefore, the difference of T cell immunity change of SLE patients, especially heavy SLE and light SLE patients, is comprehensively known, and has good application prospect in the aspect of auxiliary diagnosis of severe SLE of clinicians in the future.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide the application of Th40 cell subpopulations in preparing a kit for assisting diagnosis and evaluation of SLE disease activity.
It is another object of the invention to provide a kit for aiding in the diagnosis and assessment of SLE disease activity.
It is a further object of the invention to provide a method for detecting a subpopulation of Th40 cells.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the use of Th40 cell subsets in the preparation of kits for aiding diagnosis and assessment of SLE disease activity is based on the first discovery by the inventors of the present invention that Th40 (CD 4) in peripheral blood of SLE patients (SLE patients in this specification include both light and heavy patients) + CD40 + T) cell subpopulation ratio is significantly elevated in SLE patients, especially most significantly in heavy SLE patients.
The kit comprises components, operation rules and judgment standards which can be used for detecting the Th40 cell subgroup;
the components of the monoclonal antibodies which can be used for detecting the Th40 cell subgroup are different fluorescent markers: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies;
the operation procedure comprises the following steps:
1) Treating a peripheral blood sample to be detected to form single cell suspension;
2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;
3) Washing cells, adding PBS (phosphate buffer solution) to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation;
the judgment criteria are as follows: when the Th40 cell subgroup is in CD4 + The median of the proportion of T cells is 13.30%, and the probability of SLE disease activity is high, wherein Th40 cells are sub-Group CD4 + The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
A kit for aiding in the diagnosis and assessment of SLE disease activity comprising components useful for detecting Th40 cell subsets, such as the following different fluorescently labeled monoclonal antibodies: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies.
The fluorescent label of the anti-CD 3 antibody is preferably FITC.
The fluorescent label of the anti-CD 4 antibody is preferably APC-H7.
The fluorescent label of the anti-CD 40 antibody is preferably PE-cy7.
The kit for assisting in diagnosing and evaluating SLE disease activity also comprises instructions, and the operation procedure and the judgment standard of the kit are recorded.
The operation procedure comprises the following steps:
1) Treating a peripheral blood sample to be detected to form single cell suspension;
2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;
3) And (3) after washing the cells, adding PBS to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation.
The specific steps for treating the peripheral blood sample to be detected in the step 1) are as follows: and (3) performing whole blood cell lysis treatment on the peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS, and adding a cell staining buffer solution into precipitate obtained by centrifugation to resuspend to form single cell suspension.
The volume of the peripheral blood sample is preferably 200. Mu.L/tube.
The amount of the cell staining buffer is preferably 50. Mu.L/tube.
The amount of anti-CD 3 antibody added in step 2) is preferably 1. Mu.L of anti-CD 3 antibody per 50. Mu.L of single cell suspension.
The amount of anti-CD 4 antibody added in step 2) is preferably 1. Mu.L of anti-CD 4 antibody per 50. Mu.L of single cell suspension.
The amount of anti-CD 40 antibody added in step 2) is preferably 1. Mu.L of anti-CD 40 antibody per 50. Mu.L of single cell suspension.
The specific operation of the light-shielding incubation in the step 2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way; preferably incubated for 20 minutes at room temperature in the absence of light.
The room temperature is 5-35 ℃; preferably 20 to 30 ℃; more preferably 24 to 26 ℃.
The washing described in step 3) is performed using a cell staining buffer.
The PBS in the step 3) is preferably PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M; more preferably, PBS with a pH of 7.4 and a concentration of 0.01M.
The judgment criteria are as follows: when the Th40 cell subgroup is in CD4 + The median of the proportion of T cells was 13.30% and there was a greater likelihood of SLE, where the Th40 cell subpopulation was CD4 + The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
The kit also comprises a red blood cell lysate, a cell staining buffer solution and a Phosphate Buffer Solution (PBS) for lysing peripheral red blood cells.
A detection method of Th40 cell subgroup for non-disease diagnosis or treatment can be applied to the above kit for detection, and comprises the following steps:
(1) Treating a peripheral blood sample to be detected to form single cell suspension;
(2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step (1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;
(3) And (3) after washing the cells, adding PBS to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation.
The specific steps for treating the peripheral blood sample to be detected in the step (1) are as follows: and (3) performing whole blood cell lysis treatment on the peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS, and adding a cell staining buffer solution into precipitate obtained by centrifugation to resuspend to form single cell suspension.
The amount of the peripheral blood sample is preferably 200. Mu.L/tube.
The amount of the cell staining buffer is preferably 50. Mu.L/tube.
The amount of the anti-CD 3 antibody added in the step (2) is preferably 1. Mu.L of the anti-CD 3 antibody per 50. Mu.L of the single cell suspension.
The fluorescent label of the anti-CD 3 antibody is preferably FITC.
The amount of the anti-CD 4 antibody added in the step (2) is preferably 1. Mu.L of the anti-CD 4 antibody per 50. Mu.L of the single cell suspension.
The fluorescent label of the anti-CD 4 antibody is preferably APC-H7.
The amount of anti-CD 40 antibody added in step (2) is preferably 1. Mu.L of anti-CD 40 antibody per 50. Mu.L of single cell suspension.
The fluorescent label of the anti-CD 40 antibody is preferably PE-cy7.
The specific operation of the light-shielding incubation in the step (2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way; preferably incubated for 20 minutes at room temperature in the absence of light.
The room temperature is 5-35 ℃; preferably 20 to 30 ℃; more preferably 24 to 26 ℃.
The washing in step (3) is performed using a cell staining buffer.
The PBS in the step (3) is preferably PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M; more preferably, PBS with a pH of 7.4 and a concentration of 0.01M.
The detection method of the Th40 cell subgroup for non-disease diagnosis or treatment is used for researching the mechanism of T cell immune abnormality of the Th40 cell subgroup in SLE patients.
The application specifically comprises the following steps: and carrying out statistical analysis on the expression proportion of the Th40 cell subgroup, and when the expression proportion of the Th40 cell subgroup is detected to be higher than 13.30%, indicating that SLE patients are more likely, and further analyzing the activity degree of SLE diseases.
The statistical analysis is preferably a rank sum test analysis.
Compared with the prior art, the invention has the following advantages and effects:
(1) The inventors have found for the first time that the proportion of Th40 cells in the peripheral blood of SLE patients is significantly increased in SLE patients, especially in heavy SLE. Can be used as one of laboratory immune related detection indexes for assisting in diagnosing SLE, and also provides important reference data for evaluating the disease activity of SLE patients.
(2) The invention provides a detection method of a Th40 cell subgroup, and the phenotype of the T cell subgroup can be quantitatively counted by the detection method, so that the detection method has a very wide application prospect in the aspects of auxiliary diagnosis of SLE and evaluation of the disease activity of SLE patients.
Drawings
FIG. 1 is a flow cytometry analysis of the proportion of peripheral blood Th40 cell functional sub-populations of SLE patients to healthy controls; wherein A is a healthy control group and B is a SLE patient.
FIG. 2 is a graph showing the proportion of peripheral blood Th40 cell functional sub-population of SLE patients and healthy control groups.
FIG. 3 is a graph of SLE patient Th40 cell function subpopulation ratio versus SLEDAI-2000.
FIG. 4 is a graph of the proportion of Th40 cell functional sub-populations in SLE patients versus complement C3.
FIG. 5 is a graph showing the change in proportion of Th40 cell functional sub-populations before and after treatment of SLE patients.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The anti-CD 3 antibody is fluorescence labeled antibody CD3-FITC, namely FITC labeled mouse anti-human CD3 (clone number: HIT3a, purchased from Biolegend);
the anti-CD 4 antibody is a fluorescence labeled antibody CD4-APC-H7, namely, the APC-H7 labeled mouse anti-human CD4 (clone number: RPA-T4, available from BD Pharmingen);
the anti-CD 40 antibody is a fluorescence labeled antibody CD40-PE-Cy7, namely PE-Cy7 labeled mouse anti-human CD40 (clone number: 5C3, available from Biolegend);
cell staining buffer (cell staining buffer, available from BD Biosciences);
erythrocyte lysate (Red cell lysing buffer, available from BD Biosciences).
Example 1
The proportion of Th40 to T cells was measured using a Flow Cytometer (FCM):
(1) Blood was taken with informed consent signed by the patient, and all specimens were taken from the early morning fasting venous EDTA anticoagulation. A total of 24 peripheral blood samples of SLE were collected, with follow-up collecting peripheral blood samples 1 month after treatment of 15 patients. At the same time 24 healthy human samples were collected, and this part of the study protocol had been passed by the ethics committee of the unit. Clinical data for SLE patients are shown in table 1.
(2) The collected peripheral blood of healthy persons and SLE patients was subjected to erythrocyte lysis using erythrocyte lysate. 2mL of erythrocyte lysate is mixed with every 200 mu L of peripheral blood, the mixture is lysed for 10min at room temperature, a straw is used for gentle blowing and mixing once during the lysis, then centrifugation is carried out for 5min at the speed of 350g, the supernatant is discarded, 1 XPBS is added to 2mL, centrifugal washing is carried out at the speed of 350g, the supernatant is discarded, and 50 mu L of cell staining buffer is added for resuspension to form single cell suspension.
(3) Flow cytometry detects the proportion of Th40 cell subsets.
3.1 preparing 2 flow tubes for each sample, wherein 1 tube is set as a tube to be detected, 1 tube is set as a tube of the same type, and each tube is single cell suspension prepared in the method of step (2).
3.2 adding 1 mu L of corresponding surface analysis fluorescent antibodies, including anti-CD 3 antibodies and anti-CD 4 antibodies, into the tube to be detected and the tube of the same type respectively; the tube to be tested was added with 1. Mu.L of anti-CD 40 antibody, gently mixed, and incubated at room temperature for 20min in the dark.
3.3 cells were washed with cell staining buffer at 350g for 5min.
3.4 after centrifugation the supernatant was removed, cells were resuspended in 200. Mu.L PBS and analyzed using a flow analyser (FACSVerse) to obtain data, the raw data obtained was analyzed using FlowJo Software, the analysis data were pooled, the median of each set of data was calculated using SPSS23.0, and the P value was calculated.
Flow cytometry results are shown in figure 1.
Correlation analysis of Th40 cell occupancy and disease activity and efficacy: and (3) collecting SLEDAI-2000 scores and laboratory indexes of the patients suffering from the initial SLE, and analyzing the correlation between the Th40 cell proportion and the activity degree and curative effect of the diseases. Of the 24 patients with initial SLE (table 2), 15 patients were followed 1 month after treatment, and were found by analysis: the median of the Th40 cell proportion of 24 patients with primary SLE was 13.30%. As shown in fig. 2, the ratio of Th40 cells in SLE patients was significantly greater than that in healthy control group, and the difference was statistically significant (p=0.000); as shown in FIG. 3, the higher the SLEDAI-2000 score, the more Th40 cells are, and SLE disease and Th40 cell proportion have a positive correlation; complement C3 is an indicator of SLE disease activity, and reduction of C3 has a certain correlation with SLE disease activity, whereas Th40 cell proportion is inversely correlated with C3 as shown in fig. 4; as shown in fig. 5, the proportion of Th40 cells after treatment was significantly reduced, and the difference of Th40 cells before (before) and after (after) treatment of the same patient was statistically significant (p=0.005). The method provided by the invention can detect the proportion of Th40 cells, and the proportion of Th40 cells can be used as one of laboratory immune related detection indexes for assisting in diagnosing SLE, and simultaneously, important reference data is provided for evaluating the disease activity of SLE patients.
TABLE 1 clinical data for SLE patients and healthy controls
TABLE 2 proportion of Th40 cells in patients with initial SLE
| Numbering device | Sex (sex) | Age of | Hb(g/L) | PLT(10 9 /L) | WBC(10 9 /L) | ANA(10 9 /L) | Th40(％) |
| SLE1 | F | 23 | 112 | 80 | 2.32 | 1.38 | 5.66 |
| SLE2 | F | 25 | 100.2 | 237.6 | 2.89 | 1.46 | 1.62 |
| SLE3 | F | 25 | 73 | 60 | 1.54 | 1.12 | 14.2 |
| SLE4 | F | 38 | 106 | 166 | 2.93 | 2.07 | 2.99 |
| SLE5 | F | 26 | 59 | 163 | 8.22 | 5.38 | 76 |
| SLE6 | F | 56 | 79 | 267 | 3.73 | 3.09 | 23 |
| SLE7 | M | 22 | 118 | 85 | 2.19 | 1.6 | 27.3 |
| SLE8 | F | 26 | 115 | 208 | 3.01 | 2 | 25.2 |
| SLE9 | F | 33 | 60.7 | 243.2 | 4.28 | 3.76 | 34.3 |
| SLE10 | F | 26 | 65 | 146 | 4.45 | 3.3 | 3.93 |
| SLE11 | F | 27 | 107.6 | 266.4 | 2.7 | 1.48 | 6.95 |
| SLE12 | F | 21 | 76 | 172 | 2.41 | 1.46 | 38 |
| SLE13 | F | 24 | 98 | 310 | 2.92 | 1.8 | 12.4 |
| SLE14 | F | 54 | 77.6 | 100.2 | 3.82 | 2.83 | 4.41 |
| SLE15 | F | 22 | 67 | 149 | 3.71 | 2.63 | 34.9 |
| SLE16 | F | 20 | 83.1 | 512.2 | 18.25 | 16.62 | 8.23 |
| SLE17 | F | 23 | 112.1 | 73.6 | 10.34 | 7.93 | 33.1 |
| SLE18 | M | 31 | 127.2 | 89.6 | 3.89 | 2.72 | 15.7 |
| SLE19 | F | 35 | 131 | 255 | 6.51 | 3.22 | 39.8 |
| SLE20 | F | 24 | 81.1 | 269 | 6.49 | 4.98 | 9.47 |
| SLE21 | F | 22 | 92 | 122 | 3.1 | 1.87 | 10.5 |
| SLE22 | F | 28 | 91 | 175 | 3.27 | 1.68 | 6.99 |
| SLE23 | M | 20 | 105.4 | 29.3 | 0.71 | 16.21 | 3.08 |
| SLE24 | F | 53 | 79 | 106 | 5.4 | 85.4 | 27.07 |
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- Application of the Th40 cell subgroup in preparing a kit for assisting in diagnosing and evaluating systemic lupus erythematosus disease activity.
- 2. The use according to claim 1, characterized in that:the kit comprises components, operation rules and judgment standards which can be used for detecting the Th40 cell subgroup;the components of the monoclonal antibodies which can be used for detecting the Th40 cell subgroup are different fluorescent markers: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies;the operation procedure comprises the following steps:1) Treating a peripheral blood sample to be detected to form single cell suspension;2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;3) Washing cells, adding PBS (phosphate buffer solution) to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation;the judgment criteria are as follows: when the Th40 cell subgroup is in CD4 + The median of the proportion of T cells was 13.30% and there was a greater likelihood of SLE, a Th40 cell subsetIn CD4 + The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
- 3. A kit for aiding in the diagnosis and assessment of SLE disease activity, comprising: including components, protocols, and judgment criteria that can be used to detect Th40 cell subsets;the components of the monoclonal antibodies which can be used for detecting the Th40 cell subgroup are different fluorescent markers: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies;the operation procedure comprises the following steps:1) Treating a peripheral blood sample to be detected to form single cell suspension;2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;3) Washing cells, adding PBS (phosphate buffer solution) to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation;the judgment criteria are as follows: when the Th40 cell subgroup is in CD4 + The median of the proportion of T cells is 13.30%, and the activity of SLE disease is more likely, wherein the Th40 cell subgroup is CD4 + The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
- 4. A kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3, wherein:the kit also comprises at least one of a red blood cell lysate, a cell staining buffer solution and a phosphate buffer solution for lysing peripheral red blood cells.
- 5. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:the fluorescent label of the anti-CD 3 antibody is FITC;the fluorescence label APC-H7 of the anti-CD 4 antibody;the fluorescent label PE-cy7 of the anti-CD 40 antibody.
- 6. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:the specific steps for treating the peripheral blood sample to be detected in the step 1) are as follows: and (3) performing whole blood cell lysis treatment on the peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS, and re-suspending with a cell staining buffer solution to obtain single cell suspension.
- 7. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:the volume of the peripheral blood sample is 200 mu L/tube;the dosage of the cell staining buffer is 50 mu L/tube;the addition amount of the anti-CD 3 antibody in the step 2) is 1 mu L of the anti-CD 3 antibody according to the proportion of each 50 mu L of single cell suspension;the addition amount of the anti-CD 4 antibody in the step 2) is 1 mu L of the anti-CD 4 antibody according to the proportion of each 50 mu L of single cell suspension;the amount of anti-CD 40 antibody added in step 2) is 1 mu L of anti-CD 40 antibody per 50 mu L of single cell suspension;the specific operation of the light-shielding incubation in the step 2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way;the washing in step 3) is performed using a cell staining buffer;the PBS in the step 3) is PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.
- 8. Use of a method for detecting a subpopulation of Th40 cells for non-disease diagnosis or treatment purposes, characterized in that: the detection method of the Th40 cell subgroup for non-disease diagnosis or treatment is used for researching the mechanism of T cell immune abnormality of the Th40 cell subgroup in SLE patients;the method comprises the following steps:(1) Treating a peripheral blood sample to be detected to form single cell suspension;(2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step (1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;(3) And (3) after washing the cells, adding PBS to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation.
- 9. The use according to claim 8, characterized in that:the specific steps for treating the peripheral blood sample to be detected in the step (1) are as follows: performing whole blood erythrocyte lysis treatment on a peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS (phosphate buffer solution), and respectively adding 50 mu L of cell staining buffer solution into precipitates obtained by centrifugation to resuspend to form single cell suspension;the volume of the peripheral blood sample is 200 mu L/tube;the dosage of the cell staining buffer is 50 mu L/tube;the addition amount of the anti-CD 3 antibody in the step (2) is 1 mu L of the anti-CD 3 antibody according to the proportion of each 50 mu L of single cell suspension;the addition amount of the anti-CD 4 antibody in the step (2) is 1 mu L of the anti-CD 4 antibody according to the proportion of each 50 mu L of single cell suspension;the amount of anti-CD 40 antibody added in step (2) was 1. Mu.L of anti-CD 40 antibody per 50. Mu.L of single cell suspension.
- 10. The use according to claim 8, characterized in that:the specific operation of the light-shielding incubation in the step (2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way;the washing in step (3) is performed using a cell staining buffer;the PBS in the step (3) is PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210865715.8A CN117471100A (en) | 2022-07-22 | 2022-07-22 | Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210865715.8A CN117471100A (en) | 2022-07-22 | 2022-07-22 | Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117471100A true CN117471100A (en) | 2024-01-30 |
Family
ID=89629823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210865715.8A Pending CN117471100A (en) | 2022-07-22 | 2022-07-22 | Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117471100A (en) |
-
2022
- 2022-07-22 CN CN202210865715.8A patent/CN117471100A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Urrechaga et al. | Role of leucocytes cell population data in the early detection of sepsis | |
| Dolff et al. | Urinary CD8+ T-cell counts discriminate between active and inactive lupus nephritis | |
| Erskine et al. | Effect of infection with bovine leukosis virus on lymphocyte proliferation and apoptosis in dairy cattle | |
| SE1050373A1 (en) | procedure and kit for cancer diagnosis | |
| CN106501530A (en) | A kind of biomarker of diagnosing tubercle bacillus infection and its related kit | |
| CN109187941A (en) | Application of the CD4+CD70+T cell subsets in preparation auxiliary diagnosis pole aplastic anaemia kit | |
| Farina et al. | Links between inflammation and immune functioning with cognitive status among older Americans in the Health and Retirement Study | |
| CN103823069B (en) | Specificity IgE biologic activity detection method and the test kit used thereof | |
| CN109781987B (en) | Application of terminal effector T cell subset in preparation of kit for auxiliary evaluation of aplastic anemia disease degree | |
| CN109752548B (en) | Combined reagent and system for evaluating prognosis of chronic lymphocytic leukemia | |
| CN112098646A (en) | Kit for quantitatively detecting lymphocyte subpopulation and detection method thereof | |
| CN114636826B (en) | Application of CD177+ neutrophils in preparation of detection product for neonatal necrotizing enterocolitis | |
| CN117471100A (en) | Application of Th40 cell subset in preparing kit for assisting diagnosis and evaluation of systemic lupus erythematosus disease activity | |
| CN112708668B (en) | HSP70 as molecular marker for detecting thalassemia and application of molecular marker in preparation of diagnostic kit | |
| CN115775624A (en) | Immunosenescence evaluation method | |
| CN111856007A (en) | Application of late effector T cell subset in preparation of kit for auxiliary evaluation of aplastic anemia disease degree | |
| Katsoulos et al. | Urine protein‐to‐creatinine ratio in cattle with subclinical renal disease | |
| Iddles et al. | Evaluation of the immature granulocyte count in the diagnosis of sepsis using the sysmex XE-2100 analyser | |
| Triyani et al. | The relation of acid fast bacilli with Ziehl Neelsen staining and histopathologic examination of biopsy specimens in extrapulmonary TB suspected patients | |
| KR102576597B1 (en) | Composition and kit for evaluating immune aging | |
| WO2023046111A1 (en) | Basophil activation detection method and application thereof | |
| US11085912B2 (en) | Methods of diagnosing Clostridium difficile infection or recurrence in a subject | |
| Fonseca et al. | Detection of Chlamydia trachomatis antigen by enzyme immunoassay: importance of confirmatory testing. | |
| Aoki et al. | The Clinical Performance of Cell Population Data for Diagnosis of Bloodstream Infection in Cancer Patients | |
| CN115197322A (en) | Antibody composition for detecting minimal residual disease of chronic lymphocytic leukemia and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |