CN104597244A - Method for detecting anti-mt DNA antibody enzyme-linked immunoassay based on epitope antigen peptide and application thereof - Google Patents
Method for detecting anti-mt DNA antibody enzyme-linked immunoassay based on epitope antigen peptide and application thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting anti-mt DNA antibody enzyme-linked immunoassay based on mitochondrial DNA (mt DNA), which can be used for clinical detection of systemic lupus erythematosus, mt DNA sourced from peripheral blood of systemic lupus erythematosus patients is an antigen coating plate, and thereby the detection method for anti-mt DNA antibody enzyme-linked immunoassay can be established. The specialty of the antibody is higher than that of an anti-ds-DNA antibody, so that the method can provide valuable information for detecting clinical diagnosis and treatment of systemic lupus erythematosus by anti-mt DNA antibody.
Description
Technical field
The invention belongs to clinical autoantibody detection field, being specifically related to based on mtDNA is the antibody mtDNA antibody enzyme-linked immunity detection method of antigen and the diagnostic application in systemic loupus erythematosus thereof.
Background technology
Systemic loupus erythematosus (SLE) is autoimmunity mediation, take immune inflammation as the Diffuse Connective Tissue Disease of outstanding behaviours, occurs that the multiple autoantibody in conjunction with DNA/RNA and multisystem are got involved as feature in serum.The DNA nucleoprotein complex that the death of research showed cell discharges afterwards increases or its removing obstacles may start antibody generation and fall ill.Studies have found that SLE patient exists mitochondria dysfunction, Peter Gergely Jr etc. find that the mitochondrial transmembrane potentials of lupus human peripheral lymphocyte and the equal compared with normal control group of mitochondria activity oxygen product and rheumatoid arthritis group raise, thus causing mitochondria dysfunction, ATP generates minimizing, cell death and inflammation and occurs.2004, the people such as Brinkmann, V. defined a kind of new neutrophil leucocyte death pathways " NETosis ", are different from necrosis and apoptosis, and NET sample apoptosis is to discharge immunogenicity self DNA-antibacterial peptide compound for feature.Why being referred to as " NETs ", is that the neutrophil leucocyte of activation, to outside born of the same parents, discharges the unformed DNA of a large amount of mesh-like because in this death pathways.Compared with normal person, the neutrophil apoptosis of SLE patient accelerates and removing obstacles.NETs and SLE activity, lupus nephritis and low complement etc. have correlativity.NETs itself is as autoantigen, and induction SLE patient produces autoantibody, increases the weight of injuries of tissues and organs.DNA in NET had both comprised core DNA and had also comprised mitochondrial DNA, and they all have and potential cause inflammatory.And early stage at neutrophil activation, when cell is dead, the only mode that relied on ROS by NET of mitochondrial DNA, is initiatively released into outside born of the same parents; In late period, cell is dead successively, core DNA and mitochondrial DNA can be detected in NET simultaneously.
Anti-dsDNA antibody (anti-ds-DNA antibody) is one of markup antibody of systemic loupus erythematosus (SLE), thinks that anti-ds-DNA antibody is except assisted diagnosis SLE at present, still can judge the activity of SLE and the mark as assessment curative effect.Prove that anti-ds-DNA antibody and DNA are combined into immune complex, in glomerular basement membrane or multiple organ blood capillary deposition, or anti-ds-DNA antibody directly acts on glomerulus antigen or causes neuropsychic symptom in conjunction with the NMDAR2 of neuronal cell surface, in addition all right activating complement of this antibody, and cause the infringement of SLE patient.But also there are some researchs to think anti-ds-DNA antibody and clinical manifestation inconsistent.
Our research finds that we have detected anti-mtDNA antibody in lupus patient and human normal plasma and Anti-hCG action.We find that anti-mtDNA antibody titer obviously increases (Figure 1A) in SLE patient, and the high patient of anti-mtDNA antibody often also has the Anti-hCG action (Figure 1B) of height.Anti-mtDNA antibody significantly inducing plasma cell sample dendritic cell (pDC) can discharge interferon-' alpha ', and we have carried out SLEDAI scoring to the patient wherein leaving and taking blood sample, and the calculating of interferon-induced gene integration.Interferon integration has been proved the activity can reacting lupus disease to a certain extent.By correlation analysis we find, in SLE patient NET mtDNA content and patient SLEDAI marks (Fig. 2 A) and PERIPHERAL BLOOD MONONUCLEAR CELL interferon integration (Fig. 2 B) positive correlation.Therefore, anti-mtDNA antibody is the labelled antibody of SLE, and is a kind of pathogenic antibody.
Summary of the invention
Goal of the invention of the present invention is the detection method providing a kind of anti-mtDNA antibody ELISA immunity based on epitope antigen peptide, pathogenic between anti-mtDNA antibody, the detection of its alternative anti-ds-DNA antibody, one of biomarker becoming SLE.
Two of object of the present invention is to provide the diagnostic application of this anti-mtDNA antibody enzyme-linked immunity detection method in systemic loupus erythematosus, for judging the critical degree of SLE disease and lapsing to.
Three of the object of the invention is to provide reagent and the kit of anti-mtDNA antibody.
Goal of the invention of the present invention is achieved by the following technical solution:
First in SLE patients serum, anti-mtDNA antibody is have detected in early stage
Based on the anti-mtDNA antibody enzyme-linked immunity detection method of epitope antigen peptide, concrete steps are as follows:
1) mitochondria separating kit (Thermo) extracting mammalian culture cell mitochondria is applied;
2) apply DNA extraction agent box (Qiagen) and extract mitochondrial DNA (mtDNA);
3) DNA gel electrophoresis detection mtDNA integrality (about 16,500bp).NanoDrop
tM1000 spectrophotometers detect mtDNA concentration (pg/ml) and quality (OD260/280>1.8);
4) with DNA coating buffer (Pierce), mtDNA is coated in (Nunc) on elisa plate, wrap by concentration 5 μ g/ml, the 100 every holes of μ L, 4 DEG C are spent the night;
5) 4 DEG C are taken out bag by plate, and PBS washes plate 4 times, and 1% gelatin room temperature closes 1h;
6) empty confining liquid, PBS washes plate 4 times, adds test serum (1:100 dilution), incubated at room 1-2h;
7) empty test serum, PBS washes plate 4 times, adds the anti-human igg two anti-(European Union) of horseradish peroxidase-labeled, incubated at room 1h;
8) empty anti-human igg two to resist, PBS washes plate 5 times, adds substrate, and room temperature lucifuge hatches 10-15min;
9) add stop buffer, microplate reader wavelength 450nm reads OD value.
The antibody detection method of described anti-mtDNA for assessment of the level of mtDNA antibody anti-in Serum in Patients with SLE, the application that the method detects as biomarker at SLE.
The detection method of described based on mtDNA the is anti-mtDNA antibody of antigen, comprises Western blotting, immunoluminescence and colloidal gold method etc.
The reagent of anti-mtDNA antibody and kit are obtained using mitochondrial DNA (mtDNA) as antigen coated elisa plate.
Described mtDNA is for the preparation of diagnosis and evaluating system lupus erythematosus, and quantitative and qualitative analysis detects reagent and the kit of anti-mtDNA antibody in human serum, the information with reference value is provided to the decision-making of clinical diagnosis and treatment, and deoxyribonuclease-1(DNASE1) be the medicine that potential SLE treats.
Accompanying drawing explanation
The ELISA of the anti-mtDNA of Figure 1A and Figure 1B in SLE patient and anti-ds-DNA antibody detects, and with the correlativity of SLE activity points.
The anti-mtDNA antibody immune complex of Fig. 2 A and Fig. 2 B promotes plasmacytoid dendritic cells secretion interferon-' alpha ' (IFN-α), this effect can suppress by DNASE1.
Fig. 3 A show dose relies on experiment and keeps antibody amount constant, and along with the increase of DNA content, interferon-a (IFN-a) secretory volume also rises thereupon, and mtDNA and antibody immune complex thereof all have stronger activation effect.Equally, this situation also shows the DNA in NET.
In Fig. 3 B, NET can stimulate plasmacytoid dendritic cells (pDCs) to secrete a small amount of IFN-a, but after adding antibody, its secretory volume increases greatly.When being mixed with the anti-mtDNA antibody of Isodose, Anti-hCG action and add cultivating system respectively by the NET of same patient, IFN-a secretory volume mtDNA group is 2.5 times of dsDNA group.
Fig. 3 C is the DNA removed with DNase in cultivating system, clock sample acceptor-9(TLR-9 as positive control) activator CpG (be rich in connected by phosphoric acid ester bond cytimidine (C), guanine (G) nucleotide sequence) have obvious pDCs activate effect, but after adding DnaseI, this event resolves.Same situation betides NET+ antibody group, the decline about 90% compared with before not enzyme-added of IFN-a secretory volume.And Dnase is to acting on clock sample acceptor-7(TLR-7) the RNA sequence R837 of acceptor there is no inhibiting effect, and enzyme-added front and back IFN-a measures almost unchanged.The decline of visible IFN-a level is not because DNase1 have impact on cytoactive, but eliminates and can realize with the interactional DNA sequence dna of TLR-9.Visible DNA is the most crucial composition activating pDC in NET, and mtDNA wherein then plays even more important effect.
Fig. 4. in lupus patient's body, how many titre of anti-mtDNA antibody and its albuminuria have good correlativity (scheming B), and meanwhile, and previously study identical, the Anti-hCG action of patient and lupus nephritis have correlativity (scheming A).In order to show the injury of kidney of NETs mediation more intuitively, we wear sample frozen section with kidney and carry out original position Immunofluorescence test.NETs contaminates location confirmation altogether by DNA, mtDNA, neutrophil leucocyte albumen three and generates.Figure C show in Bowman's capsule locate DNA(canescence altogether), mtDNA(white), neutrophil leucocyte myeloperoxidase-MPO(brilliant white), the region display NETs in white box is deposited in Bowman's capsule.
Embodiment
Below in conjunction with accompanying drawing, further illustrate the present invention and how to realize.
Embodiment 1
The anti-endothelin acceptor A antibody enzyme-linked immunity detection method that the present invention is based on epitope antigen peptide is set up
1.1 application mitochondria separating kit (Thermo) extracting mammalian culture cell mitochondrias.
Mitochondrial DNA (mtDNA) is extracted in 1.2 application DNA extraction agent box (Qiagen).
1.3DNA detected through gel electrophoresis mtDNA integrality (about 16,500bp).NanoDrop
tM1000 spectrophotometers detect mtDNA concentration (pg/ml) and quality (OD260/280>1.8).
MtDNA is coated in (Nunc) on elisa plate with DNA coating buffer (Pierce) by 1.4, and wrap by concentration 5 μ g/ml, the 100 every holes of μ L, 4 DEG C are spent the night.
1.54 DEG C are taken out bag by plate, and PBS washes plate 4 times, and 1% gelatin room temperature closes 1h.
1.6 empty confining liquid, and PBS washes plate 4 times, add test serum (1:100 dilution), incubated at room 1h.
1.7 empty test serum, and PBS washes plate 4 times, add the anti-human igg two anti-(European Union) of horseradish peroxidase-labeled, incubated at room 1h.
1.8 empty two resists, and PBS washes plate 5 times, adds substrate, and room temperature lucifuge hatches 15min.
1.9 add stop buffer, and microplate reader wavelength 450nm reads OD value.
Embodiment 2
The relation that mtDNA copy number in SLE patients serum and Lupus activity exponential sum interference scalar product are divided
1. extract the serum 2ml of SLE patient and normal healthy controls, amass SLEDAI according to the clinical manifestation of patient to patient and divide.
2. be separated mtDNA(the same)
3. detect primer and method
Syber-Green method real time quantitative PCR method is used to detect the content of mtDNA in patients serum, forward direction primer cytochrome B-F:5-ATGACCCCAATACGCAAAAT-3, reverse primer cytochrome B-R:5-CGAAGTTTCATCATGCGGAG-3.
4. interferon integration method
Syber-Green method detects 5 interferon-induced genes (people's myxovirus resistance protein 1 (MX1); 2', 5'-oligoadenylate sample synzyme (OASL); 2'5'-oligoadenylate synthetase 1 (OAS1); Inter feron-alpha induced protein 15 (ISG15), lymphocyte antigen 6 complex E (LY6E)) mrna expression, calculate mean value (mutually) and the standard deviation (s) of each destination gene expression level of normal healthy controls group respectively, according to following formulae discovery sI, S=(GeneSLE-GeneHD)/SD (GeneHD).The s value of each sample 5 genes of interest is added, then obtains the interferon integration of this sample.
Embodiment 3
The anti-mtDNA antibody that the present invention obtains is on the impact of plasmacytoid dendritic cells secretion interferon
The anti-mtDNA Positive Sera that 2.1 pairs of detections screen, obtains anti-mtDNA IgG by IgG affinity chromatography
1) take out the EP pipe of the albumen magnetic bead that the peptide containing cell conditioned medium in previous step is hinged, put on EP pipe support;
2) by the G-protein magnetic bead containing 200ul serum, be placed on magnetic frame;
3) treat that magnetic bead comes together in a rear flank of EP pipe, draw and discard clear liquid;
4) draw totally, take out EP pipe and be placed on EP tube sheet;
5) in above-mentioned EP pipe, add 100ul PBS buffer solution and fix magnetic bead-lgG albumen composition 2 times;
6) when the 3rd washing, according to non denatured mode wash-out;
7) non denatured eluent: add 50ul Tris-glycocoll (pH=2.5) in EP pipe Magnetic bead sample, then mixes 2 minutes gently,
8) treat that magnetic bead comes together in a rear flank, draw side clear liquid (containing lgG) to new EP pipe (having added 5ulTris-HCl pH=8.0);
9) non denatured eluent lgG content adopts BCA kit measurement.
The impact of 2.2anti-mtDNA IgG plasmacytoid dendritic cells
1) the anti-mtDNA-immune complex (anti-mtDNA-IC) of mtDNA-and dsDNA-anti-dsDNA-immune complex (anti-dsDNA-IC) extract
(1) blood plasma of SLE patient is stored in-80 DEG C.Anti-mtDNA antibody titer ELISA method detects; The titre radioimmunology of Anti-hCG action detects.
(2) " anti-mtDNA antibody-positive plasma " represents anti-mtDNA antibody titer and represents with OD value and be greater than 1.5 and Anti-hCG action titre is less than 10U/ml; " Anti-hCG action positive blood plasma " represents that Anti-hCG action titre is greater than 50U/ml and anti-mtDNA antibody titer is less than 1.0.
(3) apply HiTrap protein G HP column (GE healthcare) method, anti-mtDNA-IC purifying is from " anti-mtDNA antibody-positive plasma ", and anti-dsDNA-IC purifying is from " the positive blood plasma of Anti-hCG action ".
(4) immune complex BCA Protein Assay Kit (Pierce) after purifying is quantitative and packing is stored in-80 DEG C.
2) be separated and stimulate pDC,
(1) mononuclearcell (PBMC) in normal human peripheral blood is separated with density－gradient centrifuga－tion method.
(2) pDC selects kit Diamond Plasmacytoid Dendritic Cell Isolation Kit(Miltenyi Biotec by the moon) isolate from PBMC.
(3) by CD123 antibody and CD304 antibody (Miltenyi Biotec), dye isolated cell, and flow cytomery cell purity is greater than 98%.
(4) pDCs is resuspended in the RPMI-1640 containing 10%FCS, is laid in 96 orifice plates containing the concentration of 5 × 104 cells by with every hole 200ul, adds different treatment conditions stimulations and spends the night.
(5) different incentive conditions comprises: TLR9 activator CpG-2216 (1ug/ml InvivoGen), TLR7 activator R837 (10 μ g/ml InvivoGen), mtDNA,, dsDNA,, anti-mtDNA-IC (10 μ g/ml), anti-dsDNA-IC (10 μ g/ml), normal human IgG (10 μ g/ml), PMA (10 μ g/ml) post-stimulatory SLE patient's neutrophil leucocyte supernatant (NETs) and Dnase I (6U/ml) (Sigma).
After (6) 12 hours, collect the cell conditioned medium of about 150ul for ELISA method (PBL BiomedicalLaboratories) detection IFN-a level wherein.
3.NETs deposits detection in renal tissue
(1) the fresh kidney of patients with lupus nephritis is worn sample and is immersed in embedding medium
o.C.T
tMcompound (Sakura) is placed in-80 DEG C and freezes.
(2) prepare 5 μm of sections with freezing microtome, be attached on microslide.
(3) with MitoSox Red (5 μMs) coloring line mitochondrial DNA 30min.
(4) fixing section 5min after cold acetone, sheep blood serum closes 60min.
(5) MPO antibody (5 μMs; Abcam), overnight incubation.Thereafter add two anti-and DAPI, hatch 30min.
(6) add and put quencher mounting.Lycra/Cai Si confocal laser scanning microscope.
Analysis of test results
We detect that anti-mtDNA antibody is significantly higher than normal healthy controls group at SLE group positive rate, and with anti-ds-DNA antibody positive correlation (as shown in FIG. 1A and 1B).In serum, the copy number of mtDNA and SLEDAI integration, interference scalar product divide positive correlation (as shown in Figure 2 A and 2 B), under showing at Fig. 3 A the condition keeping antibody amount constant, along with the increase of DNA content, IFN-a secretory volume also rises thereupon, in each gradient, mtDNA and antibody thereof all have stronger activation effect.Equally, this situation also shows the DNA in NET.Show the increase along with antibody dosage in 3B figure, pDCs secretes a small amount of IFN-a amount of secreting and presents dose dependent, and IFN-a secretory volume mtDNA group is 2.5 times of dsDNA group, and there were significant differences for the two.Fig. 3 C is the DNA removed with DNase in cultivating system, but after adding DnaseI, we can find, the TLR-9 activator CpG as positive control activates effect to pDCs and disappears, in NET+ antibody group, the decline about 90% compared with before not enzyme-added of IFN-a secretory volume.And Dnase there is no inhibiting effect to the RNA sequence R837 acting on TLR-7 acceptor.The decline of visible IFN-a level is not because DNase1 have impact on cytoactive, but eliminates and can realize with the interactional DNA sequence dna of TLR-9.Visible DNA is the most crucial composition activating pDC in NET, and mtDNA wherein then plays even more important effect.In the diagram, in lupus patient's body, how many titre of anti-mtDNA antibody and its albuminuria have good correlativity (scheming B), and meanwhile, and previously study identical, the Anti-hCG action of patient and lupus nephritis have correlativity (scheming A).In order to show the injury of kidney of NETs mediation more intuitively, we wear sample frozen section with kidney and carry out original position Immunofluorescence test.NETs contaminates location confirmation altogether by DNA, mtDNA, neutrophil leucocyte albumen three and generates.Left figure Bowman's capsule various DNA, mtDNA and the dyeing of neutrophil leucocyte myeloperoxidase in figure C.In DNA dyeing (DAPI) in upper white portion, upper right is that in white portion, MPO dyes neutrophil leucocyte, in lower be mtDNA dyeing in white portion, bottom right be overlapping dyeing.
To sum up, we think that the NET including mtDNA causes SLE patient to cause a disease the key factor of IFN-a signal path activation, and especially anti-mtDNA antibody can anti-ds-DNA antibody as an alternative, become the mark evaluating the movable and organ injury of SLE.
Claims (4)
1. be an anti-mtDNA antibody enzyme-linked immunity detection method for antigen based on mtDNA, comprise the steps:
1) mitochondria separating kit (Thermo) extracting mammalian culture cell mitochondria is applied;
2) apply DNA extraction agent box (Qiagen) and extract mitochondrial DNA (mtDNA);
3) DNA gel electrophoresis detection mtDNA integrality (about 16,500bp), NanoDrop
tM1000 spectrophotometers detect mtDNA concentration (pg/ml) and quality (OD260/280>1.8);
4) with DNA coating buffer (Pierce), mtDNA is coated in (Nunc) on elisa plate, wrapping by concentration is 5 μ g/ml, the 100 every holes of μ L, and 4 DEG C are spent the night;
5) 4 DEG C are taken out bag by plate, and PBS washes plate 4 times, and 1% gelatin room temperature closes 1h;
6) empty confining liquid, PBS washes plate 4 times, adds the test serum of 1:100 dilution, incubated at room 1-2h;
7) empty test serum, PBS washes plate 4 times, adds the human immunoglobulins G(IgG of horseradish peroxidase-labeled) two resist, incubated at room 1h;
8) empty anti-human igg two to resist, PBS washes plate 5 times, adds substrate, and room temperature lucifuge hatches 10-15min;
9) add stop buffer, microplate reader wavelength 450nm reads OD value.
2. according to claim 1 is the application of the anti-mtDNA antibody enzyme-linked immunity detection method of antigen based on mtDNA, it is characterized in that, the diagnostic application of the method in systemic loupus erythematosus, for the critical degree of decision-making system Erythematosus Disease with lapse to.
3. the anti-mtDNA antibody enzyme-linked immunity detection method being antigen based on mtDNA according to claim 1, comprises Western blotting, immunoluminescence and colloidal gold method.
4. according to claim 1 for anti-mtDNA antibody enzyme-linked immunity detection method reagent or kit, it is characterized in that: obtained using mitochondrial DNA (mtDNA) as antigen coated elisa plate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348104A (en) * | 2001-10-09 | 2002-05-08 | 上海生物芯片有限公司 | Fast test biochip for systemic lupus erythematosus |
| WO2011150316A1 (en) * | 2010-05-28 | 2011-12-01 | Sridharan Rajagopalan | Obtaining analytes from a tissue specimen |
| CN102702326A (en) * | 2012-06-27 | 2012-10-03 | 中国人民解放军军事医学科学院基础医学研究所 | Epitope of systemic lupus erythematosus and application thereof |
| CN102818890A (en) * | 2011-06-10 | 2012-12-12 | 苏州卫生职业技术学院 | Method for detecting autoantibodies of systemic lupus erythematosus patient |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348104A (en) * | 2001-10-09 | 2002-05-08 | 上海生物芯片有限公司 | Fast test biochip for systemic lupus erythematosus |
| WO2011150316A1 (en) * | 2010-05-28 | 2011-12-01 | Sridharan Rajagopalan | Obtaining analytes from a tissue specimen |
| CN102818890A (en) * | 2011-06-10 | 2012-12-12 | 苏州卫生职业技术学院 | Method for detecting autoantibodies of systemic lupus erythematosus patient |
| CN102702326A (en) * | 2012-06-27 | 2012-10-03 | 中国人民解放军军事医学科学院基础医学研究所 | Epitope of systemic lupus erythematosus and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| KHURSHID ALAM ET AL: "Immunogenicity of mitochondrial DNA modified by hydroxyl radical", 《CELLULAR IMMUNOLOGY》 * |
| KHURSHID ALAM ET AL: "Immunogenicity of mitochondrial DNA modified by hydroxyl radical", 《CELLULAR IMMUNOLOGY》, vol. 247, no. 1, 22 August 2007 (2007-08-22) * |
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