CN112114126B - Diagnostic marker for systemic lupus erythematosus and application thereof - Google Patents
Diagnostic marker for systemic lupus erythematosus and application thereof Download PDFInfo
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- CN112114126B CN112114126B CN202010813838.8A CN202010813838A CN112114126B CN 112114126 B CN112114126 B CN 112114126B CN 202010813838 A CN202010813838 A CN 202010813838A CN 112114126 B CN112114126 B CN 112114126B
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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
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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
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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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 marker of systemic lupus erythematosus and application thereof, relates to the technical field of biomedical detection, and discloses that a semaphorin 4A (Sema 4A) membrane bound type and a soluble type have higher diagnostic efficacy on systemic lupus erythematosus patients, can be used for evaluating disease state activity and organ injury, and can well identify the systemic lupus erythematosus and rheumatoid arthritis, so that the Sema4A can be used as the diagnostic marker of the systemic lupus erythematosus. And the Sema4A has higher sensitivity to the systemic lupus erythematosus, and the detection of the Sema4A and the autoantibody is helpful for improving the detection rate of the early stage of the disease, which makes up the defect of low diagnosis sensitivity of the current clinical laboratory to a certain extent.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a diagnostic marker for systemic lupus erythematosus and application thereof.
Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against nuclear components and the appearance of multiple clinical manifestations from multiple systemic, multiple organ involvement. SLE is clinically manifested in complex and diverse forms with atypical early symptoms, leading to difficulties in early diagnosis of the disease.
Diagnosis of SLE requires a combination of patient symptoms, physical examination, and laboratory examination. However, the early clinical manifestations of SLE are atypical, and the high-efficiency and accurate laboratory detection indexes become important diagnosis bases for early diseases. Currently, common laboratory diagnostic indicators for SLE include Antinuclear Antibodies (ANA) and extractable nuclear antigen Antibodies (ENA), such as pathogenic to extracellular nuclear antigen Antibodies (Antibodies), anti-dsDNA Antibodies (Antibodies), and characteristic Antibodies, anti-Sm Antibodies (Antibodies). These antibodies all have high specificity for SLE, but their sensitivity is low, leading to a high rate of missed diagnosis in early SLE patients.
In addition, some patients with SLE have finger joint swelling pain as the first symptom, while patients with RA have joint symptoms as the main clinical manifestation. Meanwhile, ANA positive, RF positive, ESR and CRP increase can be generated in both laboratory tests, so that the ANA positive, the RF positive, the ESR and the CRP increase are difficult to distinguish. There is therefore a continuing need to find new diagnostic markers to meet the clinical needs of early disease diagnosis, disease assessment and efficacy monitoring.
Disclosure of Invention
The invention aims to: aiming at the problems that indexes for SLE diagnosis in the prior art are low in sensitivity and easy to cause missed detection, the invention provides a high-sensitivity systemic lupus erythematosus diagnostic marker and application thereof.
The technical scheme adopted by the invention is as follows:
a diagnostic marker of systemic lupus erythematosus, the amino acid sequence of the diagnostic marker is shown as SEQ ID NO:1 to SEQ ID NO: 4; the specific sequence is as follows:
SEQ ID NO：1
MetAlaLeuProAlaLeuGlyLeuAspProTrpSerLeuLeuGlyLeuPheLeuPheGlnLeuLeuGlnLeuLeuLeuProThrThrThrAlaGlyGlyGlyGlyGlnGlyProMetProArgValArgTyrTyrAlaGlyAspGluArgArgAlaLeuSerPhePheHisGlnLysGlyLeuGlnAspPheAspThrLeuLeuLeuSerGlyAspGlyAsnThrLeuTyrValGlyAlaArgGluAlaIleLeuAlaLeuAspIleGlnAspProGlyValProArgLeuLysAsnMetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluThrGlnCysPheAsnPheIleArgValLeuValSerTyrAsnValThrHisLeuTyrThrCysGlyThrPheAlaPheSerProAlaCysThrPheIleGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：2
MetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：3
MetIleProTrpProAlaSerAspArgLysLysSerGluCysAlaPheLysLysLysSerAsnGluThrGlnCysPheAsnPheIleArgValLeuValSerTyrAsnValThrHisLeuTyrThrCysGlyThrPheAlaPheSerProAlaCysThrPheIleGluLeuGlnAspSerTyrLeuLeuProIleSerGluAspLysValMetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
SEQ ID NO：4
MetGluGlyLysGlyGlnSerProPheAspProAlaHisLysHisThrAlaValLeuValAspGlyMetLeuTyrSerGlyThrMetAsnAsnPheLeuGlySerGluProIleLeuMetArgThrLeuGlySerGlnProValLeuLysThrAspAsnPheLeuArgTrpLeuHisHisAspAlaSerPheValAlaAlaIleProSerThrGlnValValTyrPhePhePheGluGluThrAlaSerGluPheAspPhePheGluArgLeuHisThrSerArgValAlaArgValCysLysAsnAspValGlyGlyGluLysLeuLeuGlnLysLysTrpThrThrPheLeuLysAlaGlnLeuLeuCysThrGlnProGlyGlnLeuProPheAsnValIleArgHisAlaValLeuLeuProAlaAspSerProThrAlaProHisIleTyrAlaValPheThrSerGlnTrpGlnValGlyGlyThrArgSerSerAlaValCysAlaPheSerLeuLeuAspIleGluArgValPheLysGlyLysTyrLysGluLeuAsnLysGluThrSerArgTrpThrThrTyrArgGlyProGluThrAsnProArgProGlySerCysSerValGlyProSerSerAspLysAlaLeuThrPheMetLysAspHisPheLeuMetAspGluGlnValValGlyThrProLeuLeuValLysSerGlyValGluTyrThrArgLeuAlaValGluThrAlaGlnGlyLeuAspGlyHisSerHisLeuValMetTyrLeuGlyThrThrThrGlySerLeuHisLysAlaValValSerGlyAspSerSerAlaHisLeuValGluGluIleGlnLeuPheProAspProGluProValArgAsnLeuGlnLeuAlaProThrGlnGlyAlaValPheValGlyPheSerGlyGlyValTrpArgValProArgAlaAsnCysSerValTyrGluSerCysValAspCysValLeuAlaArgAspProHisCysAlaTrpAspProGluSerArgThrCysCysLeuLeuSerAlaProAsnLeuAsnSerTrpLysGlnAspMetGluArgGlyAsnProGluTrpAlaCysAlaSerGlyProMetSerArgSerLeuArgProGlnSerArgProGlnIleIleLysGluValLeuAlaValProAsnSerIleLeuGluLeuProCysProHisLeuSerAlaLeuAlaSerTyrTyrTrpSerHisGlyProAlaAlaValProGluAlaSerSerThrValTyrAsnGlySerLeuLeuLeuIleValGlnAspGlyValGlyGlyLeuTyrGlnCysTrpAlaThrGluAsnGlyPheSerTyrProValIleSerTyrTrpValAspSerGlnAspGlnThrLeuAlaLeuAspProGluLeuAlaGlyIleProArgGluHisValLysValProLeuThrArgValSerGlyGlyAlaAlaLeuAlaAlaGlnGlnSerTyrTrpProHisPheValThrValThrValLeuPheAlaLeuValLeuSerGlyAlaLeuIleIleLeuValAlaSerProLeuArgAlaLeuArgAlaArgGlyLysValGlnGlyCysGluThrLeuArgProGlyGluLysAlaProLeuSerArgGluGlnHisLeuGlnSerProLysGluCysArgThrSerAlaSerAspValAspAlaAspAsnAsnCysLeuGlyThrGluValAla
further, the amino acid sequence of the marker is the amino acid sequence of SEQ ID NO:1 to SEQ ID NO:4 can express the same function after replacing, deleting and/or replacing one or more bases.
The application of the diagnosis marker in preparing a preparation for detecting systemic lupus erythematosus.
An agent comprising an antibody that specifically binds to the diagnostic marker.
A kit for detecting systemic lupus erythematosus, comprising an antibody capable of specifically binding to the diagnostic marker.
Semaphorin (semaphorin) is a large class of membrane-bound or secreted glycoprotein molecules that have important regulatory roles in a variety of physiological processes, including neurite guidance, vascular growth, bone resorption and remodeling, immunoregulation, and cardiac development. Semaphorins can be divided into 7 classes, with semaphorins from classes 1 and 2 being expressed only in invertebrates, semaphorins from classes 3, 4, 6 and 7 being expressed only in vertebrates, and semaphorins from class 5 being expressed in both. Studies show that semaphorin is widely involved in immune function regulation, can induce maturation and migration of DCs, regulate activation, proliferation and differentiation of T, B cells, and promote generation of inflammatory cytokines. The 4 th-class semaphorin has A, B, C, D subtypes, and Sema4A (including membrane bound and soluble) is found to have important value in the clinical diagnosis of SLE through research.
In the invention, when the membrane-bound Sema4A is taken as a diagnostic marker, the expression of the membrane-bound Sema4A on peripheral blood cells is analyzed by Flow Cytometry (FCM), and the specific steps are as follows:
(1) Collecting a peripheral blood sample;
(2) Adding peripheral blood samples into 4 centrifugal tubes of 5ml, and respectively naming the samples as a No. 1 centrifugal tube, a No. 2 centrifugal tube, a No. 3 centrifugal tube and a No. 4 centrifugal tube;
(3) Adding an anti-human CD100-Alexa 647 antibody, an anti-human CD4-PE antibody and an anti-human CD19-FITC antibody into a No. 1 centrifugal tube, and incubating at room temperature in a dark place for 15min; no. 3 centrifuge tube is provided with isotype control; adding an anti-human Sema4A-PerCP-eFluor 710 antibody, an anti-human CD11c-APC antibody, an anti-human CD4-PE antibody and an anti-human CD19-FITC antibody into a No. 2 centrifugal tube, incubating for 15min at room temperature in a dark place, and setting isotype control in the No. 4 centrifugal tube;
(4) Adding erythrocyte lysate into each centrifuge tube, and standing in the dark for 10min until the erythrocytes are completely lysed;
(5) Adding PBS into each tube, mixing uniformly, centrifuging, removing supernatant, and repeating twice;
(6) And (6) performing detection on the machine.
In the invention, when soluble Sema4A is taken as a diagnostic marker, enzyme-linked immunosorbent assay (ELISA) is adopted to detect the concentration of soluble Sema4A in plasma, and the specific steps are as follows:
(1) Preparing a reagent: (1) preparing a standard substance: taking out the Sema4A antibody standard substance from the ELISA kit, centrifuging, dissolving with 1ml of sample diluent, and fully and uniformly mixing to obtain a standard substance S7; then, 7 1.5ml Ep tubes (named as S0-S6 respectively) are sequentially arranged, 250pl sample diluent is added respectively, 250ml S7 standard substance is absorbed and placed in the first Ep tube (S6), 250ml is absorbed from the S6 and placed in the second Ep tube (S5), the standard substance is diluted by times in the same way, and S0 is the sample diluent; (2) preparing a washing working solution: the concentrated wash was washed with deionized water as follows: diluting by 25 times for later use; (3) preparing a biotin labeled antibody working solution: the biotin labeled antibody fluid was expressed as 1: diluting with 100 times of biotin labeled antibody diluent for later use; (4) preparing a horse radish peroxidase labeled avidin working solution: horse radish peroxidase-labeled avidin was added as 1: diluting 100 times with horse radish peroxidase labeled avidin diluent for later use;
(2) The following operations were performed according to the ELISA instructions:
(1) placing various reagents at room temperature for balancing for at least 30min, and preparing the reagents according to the method for later use;
(2) respectively arranging a standard substance hole and a sample hole to be detected, adding 100 mu l of the standard substance or the sample to be detected into each hole, slightly shaking and uniformly mixing, covering a plate, and then incubating for 2h at 37 ℃;
(3) discarding the liquid and spin-drying;
(4) adding biotin labeled antibody working solution into each hole, covering a new plate, and incubating for 1h at 37 ℃;
(5) discarding liquid in the hole, spin-drying, washing and spin-drying again;
(6) adding horseradish peroxidase labeled avidin working solution into each hole, covering a new plate, and incubating for 1h at 37 ℃;
(7) discarding liquid in the hole, spin-drying, washing and spin-drying again;
(8) adding substrate solution into each well, and developing at 37 deg.C in dark for 30min;
(9) adding a termination solution into each hole to terminate the reaction; the optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader within five minutes.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, the membrane-bound and soluble Sema4A are used as the diagnosis markers of the systemic lupus erythematosus, so that the kit has higher diagnosis efficiency, can be used for evaluating the disease activity and has certain value for evaluating organ damage;
2. the systemic lupus erythematosus diagnosis marker Sema4A has higher sensitivity to SLE, is beneficial to improving the detection rate of early stage of diseases by combining detection with the autoantibody, and solves the problems of low index sensitivity and easy detection omission of SLE diagnosis in the prior art, so that the application of the diagnosis marker of the invention has very important significance for early diagnosis of systemic lupus erythematosus which is difficult to diagnose due to atypical early clinical performance;
3. the membrane-bound type and soluble type Sema4A can be used for differential diagnosis of systemic lupus erythematosus and rheumatoid arthritis, and because the membrane-bound type and soluble type Sema4A of systemic lupus erythematosus patients are obviously higher than healthy people, and the membrane-bound type and soluble type Sema4A of rheumatoid arthritis patients are obviously lower than healthy people, the two diseases with similar clinical manifestations can be identified;
4. the application of the invention comprises that the detection reagent of Sema4A is adopted for quantitatively detecting the concentration of soluble Sema4A in plasma or the Sema4A antibody is used for detecting the surface membrane combined Sema4A level of peripheral blood cells by flow cytometry, and the two detection methods are adopted to be used independently or together according to different scenes, so that better diagnosis effect can be obtained.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, not by way of limitation, i.e., the embodiments described are intended as a selection of the best mode contemplated for carrying out the invention, not as a full mode.
Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Flow Cytometry (FCM) analysis of the expression of membrane-bound Sema4A on peripheral blood cells:
(1) Collecting 5ml of peripheral blood sample and placing the peripheral blood sample in an EDTA anticoagulation tube;
(2) Adding the peripheral blood sample into 4 centrifugal tubes of 5ml according to 200 mul per tube, and respectively naming the sample as a No. 1 centrifugal tube, a No. 2 centrifugal tube, a No. 3 centrifugal tube and a No. 4 centrifugal tube;
(3) Adding an anti-human CD100-Alexa 647 antibody, an anti-human CD4-PE antibody and an anti-human CD19-FITC antibody into a No. 1 centrifugal tube, and incubating at room temperature in a dark place for 15min; no. 3 centrifuge tube is provided with isotype control; adding an anti-human Sema4A-PerCP-eFluor 710 antibody, an anti-human CD11c-APC antibody, an anti-human CD4-PE antibody and an anti-human CD19-FITC antibody into a No. 2 centrifugal tube, incubating for 15min at room temperature in a dark place, and setting isotype control in the No. 4 centrifugal tube;
(4) Respectively adding 700 mul of erythrocyte lysate into each centrifuge tube, and placing for 10min in the dark until the erythrocytes are completely lysed;
(5) Adding 2-3 ml PBS into each tube, mixing uniformly, centrifuging for 5min at 1000 r/min, removing supernatant, and repeating twice;
(6) Adding 200 μ l PBS into each tube, mixing, and detecting.
The method is adopted to respectively carry out sampling detection on Systemic Lupus Erythematosus (SLE) patients, rheumatoid Arthritis (RA) patients and healthy people with matched age and gender, and the results are shown in the following table 1:
TABLE 1 Sema4A expression Table on SLE, RA and healthy subjects mDCs
As can be seen from the above table, the concentration of membrane-bound Sema4A in mDCs of SLE patients was significantly higher than that in RA patients (p = 0.000) and healthy patients (p = 0.000). The result shows that the membrane-bound Sema4A can be used for clinical diagnosis of systemic lupus erythematosus and differential diagnosis of rheumatoid arthritis.
Example 2
Enzyme-linked immunosorbent assay (ELISA) detects the concentration of soluble Sema4A in plasma:
(1) Freezing and thawing all reagents in the kit for 30min at room temperature, taking out a plasma sample stored in a refrigerator at minus 80 ℃, standing to room temperature, and centrifuging for later use;
(2) Preparing a reagent: (1) preparing a standard substance: taking a Sema4A antibody standard substance out of the ELISA kit, centrifuging at 10000 r/min for 30 seconds, dissolving with 1ml of sample diluent, repeatedly blowing and beating the bottom of an EP tube by using a gun head to assist the dissolution, and fully and uniformly mixing to obtain a standard substance S7; then, arranging 7 1.5ml Ep tubes (named as S0-S6 respectively) in sequence, adding 250pl of sample diluent respectively, sucking 250ml of S7 standard substance, placing the standard substance in a first Ep tube (S6), lightly blowing, beating and uniformly mixing, sucking 250ml from the S6 into a second Ep tube (S5), lightly blowing, beating and uniformly mixing in the same way, and performing multiple dilution on the standard substance by analogy, wherein S0 is the sample diluent; (2) preparing a washing working solution: the concentrated washing solution is mixed with deionized water according to the ratio of l: diluting by 25 times for later use; (3) preparing a biotin labeled antibody working solution: the biotin labeled antibody fluid was expressed as 1: diluting with 100 times of biotin labeled antibody diluent for later use; (4) preparing a horse radish peroxidase labeled avidin working solution: horse radish peroxidase-labeled avidin was added as 1: diluting 100 times with horse radish peroxidase labeled avidin diluent for later use;
(3) The following operations were performed according to the ELISA instructions:
(1) placing various reagents at room temperature (18-25 deg.C) for balancing for at least 30min, and preparing reagents according to the above method;
(2) respectively arranging a standard substance hole and a sample hole to be detected, adding 100 mu l of the standard substance or the sample to be detected into each hole, slightly shaking and uniformly mixing, covering a plate, and then incubating for 2h at 37 ℃;
(3) discarding the liquid and spin-drying;
(4) adding 100 mul of biotin labeled antibody working solution into each hole, covering a new plate, and incubating for 1h at 37 ℃;
(5) discarding liquid in the holes, spin-drying, washing for 5 times by using an automatic plate washing machine, soaking for 30s each time, and spin-drying at a rate of 200 mul/hole;
(6) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, covering a new plate, and incubating for 1h at 37 ℃;
(7) discarding liquid in the holes, spin-drying, washing for 6 times by using an automatic plate washing machine, soaking for 30s each time, and spin-drying at a rate of 200 mul/hole;
(8) adding 90 μ l of substrate solution into each well, and developing at 37 deg.C in dark for 30min;
(9) adding 50 mu l of termination solution into each hole to terminate the reaction; the optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader within 5 min.
The method is adopted to respectively carry out sampling detection on Systemic Lupus Erythematosus (SLE) patients, rheumatoid Arthritis (RA) patients and healthy patients, and the results are shown in the following table 2:
TABLE 2 SLE, RA and plasma concentrations of soluble Sema4A in healthy subjects
As can be seen from the table above, the expression of soluble Sema4A in the plasma of SLE patients was significantly higher than that of RA patients (p = 0.000) and healthy ones (p = 0.000). The result shows that the secretory Sema4A can be used for clinical diagnosis of systemic lupus erythematosus and also can be used for differential diagnosis with rheumatoid arthritis.
Example 3
Based on the example 1 and the example 2, the correlation between the Sema4A of the SLE patient and the clinical index is further analyzed according to the Pearson correlation coefficient, and the results show that: the concentration of soluble Sema4A in SLE patients is positively correlated with anti-dsDNA antibody levels (p =0.000,r = 0.742); (2) is negatively correlated with the water averages of C3, C4 and Hb (p =0.001,r = -0.583; (3) the concentration of soluble Sema4A in the plasma of SLE patients who were urine protein positive was significantly higher than that of the negative group (p = 0.029).
It can be seen that Sema4A has a strong correlation with anti-dsDNA antibodies, the maturation marker currently used for diagnosing SLE, and therefore can be used as a marker for assessing SLE disease activity (negatively correlated with C3 and C4), and has a certain value for assessing blood system damage (negatively correlated with Hb levels) and kidney damage (the concentration of soluble Sema4A in plasma of SLE patients who are urine protein positive is significantly higher than that in urine protein negative group).
Example 4
On the basis of the example 1 and the example 2, an ROC curve is established to evaluate the diagnosis efficacy of the soluble Sema4A on the SLE through SPSS analysis software, and the area under the curve (AUC) is calculated to be 0.799, the corresponding sensitivity is 0.833, the specificity is 0.696, and the Youden index is 0.529; the ROC curve evaluated the diagnostic efficacy of membrane-bound Sema4A on sleds cells for SLE, calculated as AUC 0.830, corresponding to a sensitivity of 0.864, a specificity of 0.733 and a youden index of 0.597.
Example 5
The diagnostic efficacy of Sema4A was compared to the existing markers on the basis of example 1 and example 2, as shown in table 3 below:
TABLE 3 comparative table of diagnostic efficacy
The Area AUC (Area Under cut) Under the calculated ROC Curve of the marker Sema4A is respectively as follows: soluble form 0.799, membrane bound form 0.830. Compared to other markers available, sema4A has a higher AUC value, second only to ANA. Indicating that Sema4A has better diagnostic efficacy.
Example 6
Based on examples 1 and 2, the sensitivity and specificity of Sema4A was compared to existing markers as shown in table 4 below:
TABLE 4 comparative table of sensitivity and specificity
The anti-dsDNA antibody and the anti-Sm antibody are the indexes with the highest specificity currently used for SLE diagnosis, but the sensitivity of the anti-dsDNA antibody and the anti-Sm antibody is very low, and the missed detection is easily caused. The Sema4A sensitivity of the invention is more than 80% (ROC curve analysis result), which is much higher than the anti-dsDNA antibody and anti-Sm antibody, and can make up the delay of SLE diagnosis and treatment caused by insufficient sensitivity.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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Claims (3)
1. The application of a diagnostic marker of systemic lupus erythematosus in preparing a preparation for distinguishing systemic lupus erythematosus from rheumatoid arthritis is characterized in that the amino acid sequence of the diagnostic marker is shown as SEQ ID NO:1 to SEQ ID NO: 4;
wherein, the membrane bound type and soluble type Sema4A of the systemic lupus erythematosus patient are obviously higher than those of the healthy people, and the membrane bound type and soluble type Sema4A of the rheumatoid arthritis patient are obviously lower than those of the healthy people;
the diagnostic marker is derived from peripheral blood cells.
2. The use of claim 1, wherein the agent comprises an antibody that specifically binds to the diagnostic marker of claim 1.
3. Use of a diagnostic marker for systemic lupus erythematosus in the manufacture of a kit for the differential discrimination between systemic lupus erythematosus and rheumatoid arthritis, the kit comprising an antibody that specifically binds to the diagnostic marker of claim 1.
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