CN103728454A - Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension) - Google Patents
Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension) Download PDFInfo
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- CN103728454A CN103728454A CN201210381023.2A CN201210381023A CN103728454A CN 103728454 A CN103728454 A CN 103728454A CN 201210381023 A CN201210381023 A CN 201210381023A CN 103728454 A CN103728454 A CN 103728454A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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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
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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
- G01N2800/12—Pulmonary diseases
Abstract
The invention relates to an enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide; the enzyme-linked immunosorbent assay (ELISA) can be used in clinical tests of CTD-PAH (connective tissue diseases-pulmonary arterial hypertension); four extracellular peptide fragments with different lengths are synthesized in vitro, an epitope peptide fragment in good consistency with full-length ENRA is screened, the epitope peptide fragment is artificially synthesized to use as an antigen peptide package board to establish the enzyme-linked immunosorbent assay (ELISA) based on the anti-ENRA (anti-endothelin receptor A) antibody of the epitope antigen peptide; the epitope antigen peptide is a peptide fragment comprising the following amino acid sequence: DNPERYSTNLSNHVDDFTTFRGTELSFLVTTHQPTNLVLPSNGSMHNYCPQQTKIT; the enzyme-linked immunosorbent assay reduces the cost using full-length eukaryotic-expression endothelin receptor as a substrate, improves the clinical detection practicality, becomes a biomarker of CTD-PAH (especially SLE(systemic lupus erythematosus)-PAH), and provides valuable information for clinical diagnosis and treatment decisions.
Description
Technical field
The invention belongs to clinical autoantibody detection field, be specifically related to the anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide and merge the application in pulmonary hypertension in connective tissue disease (CTD).
Background technology
Pulmonary hypertension (PAH) is the disease of pulmonary circulation that different reasons cause, and finally causes irreversible lung vascular remodeling and right heart failure, and the median time-to-live of natural history is only 2 ~ 3 years.PAH due to connective tissue disease (CTD) (CTD) is the important component part of pulmonary hypertension spectrum of disease, and external report shows that CTD-PAH accounts for 10% of all PAH, and this numeral of domestic data approaches 20%; The data presentation system sclerosis (SSc) in west accounts for definitely leading in CTD-PAH, and China is that systemic loupus erythematosus (SLE) is for causing the first cause (constituent ratio reaches 40%) of CTD-PAH.
In the past in 10 years, along with take endothelin-receptor antagonists, prostacyclin analogs, phosphodiesterase inhibitor as representing the appearance of pulmonary hypertension targeted therapy, the overall prognosis of various PAH is improved really, but CTD-PAH patient's prognosis is still starkly lower than idiopathic PAH patient.Obviously there is distinctive immunopathogenesis mechanism in the CTD-PAH that the SLE of take is representative, and develops the distinctive biomarker of its immunopathogenesis, certainly will contribute to generation and the process of early stage seizure, prediction PAH, for targeting intervention more targetedly provides foundation.
Endothelin receptor path is one of pathogenic path that PAH is generally acknowledged, and its activation has been played key effect to the lung vascular remodeling of PAH.Existing German scholar has found to exist in SSc anti-endothelin acceptor A antibody, and set up ELISA detection method based on endothelin receptor albumen total length (Riemekasten G, Philippe A,
m, et al.Involvement of functional autoantibodies against vascular receptors in systemic sclerosis.Ann Rheum Dis.2011Mar; 70 (3): 530-6.).Its result of study demonstration, there is positive correlation in the being in a bad way property of anti-endothelin acceptor A antibody and SSc; External functional experiment points out this antibody to have the pathogenic function of activation endothelin receptor path.This discovery has proposed series of problems to be needed further to solve: whether whether (1) anti-endothelin acceptor A antibody (anti-ENRA) is present in other CTD (2) anti-ENRA exists associated with CTD-PAH, in other words, whether it can become CTD-PAH(particularly without report, and in the middle of compatriots more common SLE-PAH) biomarker? (3) anti-ENRA is as the autoantibody may with pathogenic function, does it exists provide foundation to the individuation application of endothelin-receptor antagonists? (4) from the technological layer of clinical detection, because endothelin receptor is the g protein coupled receptor of 7 cross-films, the eukaryotic expression of its total length costs dearly, can pass through epitope analysis and then synthetic antigenic peptide, to optimize the also detection method of cost degradation anti-ENRA, improve its clinical detection practicality? these problems are badly in need of being resolved.
Summary of the invention
The deficiency existing for prior art, one of object of the present invention is to provide the anti-ENRA antibody enzyme-linked immunity detection method based on endothelin receptor A epitope antigen peptide, the arm and a leg defect existing to overcome prior art.
Two of object of the present invention is to provide the application that the method detects as biomarker at CTD-PAH.
Three of the object of the invention is to provide endothelin receptor A epitope antigen polypeptide fragment and for the preparation of diagnosis and assessment connective tissue disease (CTD), merges reagent and the kit of anti-endothelin acceptor A antibody in pulmonary hypertension and quantitative and qualitative analysis detection human serum.
Goal of the invention of the present invention is achieved by the following technical solution:
At early stage first alleged occurrence anti-endothelin acceptor A(but not endothelin receptor B in the serum at CTD-PAH) autoantibody, set up the ELISA method of the anti-ENRA based on ENRA total length recombinant protein.And then focus on the SLE-PAH of independent sample, confirmed and surpassed 40% patient and have anti-ENRA(as shown in Figure 1).
An anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide, endothelin receptor A is the g protein coupled receptor of 7 cross-films, 347 amino acid sequences, consists of, and except a segment signal peptide, has the outer peptide section of born of the same parents that 4 length differ.Autoantibody in circulation should be combined by the epitope of peptide section formation outside its born of the same parents.The present invention is exactly by having synthesized the outer peptide section of above-mentioned 4 born of the same parents, screened the epitope peptide section (as shown in Figure 2) good with total length ENRA consistance, by manually synthesizing this epitope peptide section as antigenic peptides wrapper sheet, and set up the anti-ENRA antibody enzyme-linked immunity detection method based on endothelin receptor A epitope antigen peptide at this point.The amino acid sequence that described peptide section is endothelin receptor A is (being 56 amino acid sequences of 21 ~ 76 of endothelin receptor A):
DNPERYSTNLSNHVDDFTTFRGTELSFLVTTHQPTNLVLPSNGSMHNYCPQQTKIT。
Described epitope antigen peptide comprises all amino acid sequences, and puts in order.
Anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide, concrete steps are as follows:
1), coated damping fluid (PH9.60.05M carbonate buffer solution), by 1.59 grams of NaHCO3,2.93 grams of adding distil waters of NaHCO3 are to 1000ml;
2), the antigenic peptides of 5ug/ml is after in carbonic acid buffer, coated 96 orifice plates spend the night, PBS-T(1000mL PBS adds 0.5mL polysorbas20) washing three times;
3), 3% bovine serum albumin(BSA) (or 0.3% gelatin, 10% hyclone) sealing 2 hours, PBS-T washing three times;
4), after patients serum 1:100-1:300 dilution, add under ELISA Plate room temperature and react 1 hour, PBS-T washing three times;
5), add the horseradish peroxidase-anti-human IgG bis-of proper proportion dilution anti-, room temperature reaction 1 hour;
6), adding TMB(Tetramethyl benzidine) substrate reactions is after 10 minutes, by 2M sulfuric acid cessation reaction;
7), microplate reader absorbance 450nM reads plate and surveys absorbance.
Described endothelin receptor A epitope antigen polypeptide fragment merges reagent and the kit of anti-endothelin acceptor A antibody in pulmonary hypertension and quantitative and qualitative analysis detection human serum for the preparation of diagnosis and assessment connective tissue disease (CTD).
Anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide detects for the biomarker of CTD-PAH.
Wherein CTD comprises systemic loupus erythematosus, chorionitis, mixed connective tissue disease, inflammatory myositis, Sjogren syndrome, rheumatoid arthritis, systemic vasculitis, antiphospholipid antibody syndrome.
The present invention is based on the anti-ENRA enzyme-linked immune detection method of epitope antigen peptide, significantly lowered the cost of total length eukaryotic expression endothelin receptor as substrate, improve its clinical detection practicality.The present invention can become particularly SLE-PAH of CTD-PAH() biomarker, the decision-making of clinical diagnosis and treatment is provided to the information with reference value.
Accompanying drawing explanation
The ELISA of Fig. 1 anti-ENRA of ENRA total length recombinant protein in CTD-PAH and SLE-PAH detects.
Fig. 2 ENRA structure mould is built 4 extracellular segments of demonstration.
Fig. 3 be take the ELISA testing result of the anti-ENRA that 21 ~ 76 epitope peptide sections are substrate, and and the consistance contrast of ENRA total length method.(Intraclass correlation coefficient (ICC) for measurement data consistency analysis, alpha coefficient >0.7 thinks that comparison of coherence is high.SLE-PAH group alpha coefficient: 0.821; SLE group alpha coefficient: 0.803; Normal group alpha coefficient: 0.831)
Fig. 4 SLE-PAH positive serum affinity chromatography obtains anti-ENRA IgG can stimulated vascular smooth muscle cell breed, and can strengthen the multiplication effect of endothelin-1 (ET-1) to vascular smooth muscle cell.
Fig. 5 ROC curve.
Embodiment
The present invention is based on the foundation of the anti-endothelin acceptor A antibody enzyme-linked immunity detection method of epitope antigen peptide:
The antigen of 1.15ug/ml is after in carbonic acid buffer, coated 96 orifice plates spend the night, PBS-T washing three times.
1.23% bovine serum albumin(BSA) (or 0.3% gelatin, 10% hyclone) sealing 2 hours, PBS-T washing three times.
After 1.3 patients serum 1:100-1:300 dilutions, add under ELISA Plate room temperature and react 1 hour, PBS-T washing three times.
1.4 add the horseradish peroxidase-anti-human IgG bis-of proper proportion dilution anti-, room temperature reaction 1 hour.
1.5 add TMB(Tetramethyl benzidine) substrate reactions is after 10 minutes, by 2M sulfuric acid cessation reaction.
1.6 microplate reader absorbances 450 are read plate.
Embodiment 2
The impact of the anti-ENRA IgG that the present invention obtains on vascular smooth muscle cell proliferation
2.1 pairs of SLE-PAH positive serums that detection screens, obtain anti-ENRA IgG by IgG affinity chromatography
1) take out the EP pipe of the albumen magnetic bead of the peptide hinge that contains cell conditioned medium in previous step, put to EP pipe support;
2) by the G albumen magnetic bead that contains 200ul cell conditioned medium, 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) after drawing totally, take out EP pipe and be placed on EP tube sheet;
5) to adding fixedly magnetic bead-lgG albumen composition 2 times of 100ul PBS damping fluid washing in above-mentioned EP pipe;
6) when the 3rd washing, according to non-denaturation way wash-out;
7) non-sex change eluent: add 50ul Tris-glycocoll (pH=2.5) in EP pipe magnetic bead sample, then mix gently 2 minutes,
8) treat that magnetic bead comes together in a rear flank, draw a side clear liquid (having contained lgG) to a new EP pipe (having added 5ul Tris-HCl pH=8.0);
9) non-sex change eluent lgG content adopts BCA kit measurement.
The multiplication effect of 2.2Xcelligence Real-Time Monitoring anti-ENRA IgG to vascular smooth muscle cell
1) every hole adds 90ulF-12K nutrient culture media, puts into instrument deck, and whether detection orifice plate contacts good with instrument,
2), according to definite cell density, every hole adds 100ul cell suspension,
3) put into incubator after standing half an hour, then put back to instrument deck,
4) after 24 hour cells are all adherent, change serum free medium and spend the night by cell hunger, make cell synchronization,
5) m seq adds 20% hyclone irritation cell 6 hours, and each organizes the corresponding nutrient solution that adds, or Endothelin 0.25ug/ml and (or) anti-endothelin antibody 0.02ug/ml.
6) every 15 minutes autoscans of instrument once, reading result after 96 hours.
By to detecting the SLE-PAH positive serum screening, by IgG affinity chromatography, obtain anti-ENRA IgG.Experiment in vitro demonstration, anti-ENRA antibody can stimulated vascular smooth muscle cell be bred, and can strengthen endothelin-1 (ET-1) to the multiplication effect of vascular smooth muscle cell (as shown in Figure 4).
Embodiment 3
The kit preparation that anti-DT-56 polypeptide antibody detects
Be if no special instructions conventional method
Kit is prepared required reagent
1.DT-56 polypeptide, after coupling KLH, with the coated high affinity ELISA Plate of 5ug/ml;
2. goat anti-human igg's working fluid concentration of horseradish peroxidase-labeled is 1:10000,12mL/ bottle, 1 bottle.Contain 1% calf serum, 0.1% thimerosal.
3. nitrite ion is tetramethylethylenediamine (TMB) solution, 10mL/ bottle.
4. cleansing solution is 10XPBS-T, contains 0.1% Sodium azide (NaN3), 100mL.
5. the sulfuric acid that stop buffer is 2M, 10mL/ bottle.
6. be coated with damping fluid (PH9.60.05M carbonate buffer solution), by 1.59 grams of NaHCO3,2.93 grams of adding distil waters of NaHCO3 are to 1000ml;
7. confining liquid is 0.5% bovine serum albumin(BSA) PBS solution.
ELISA Plate preparation
1.DT-56 polypeptide is synthesized by company, and connects KLH by glutaraldehyde method strand, and purity > 99%,
2. the carbonic acid buffer that envelope antigen concentration is 5ug/mL,
3. night is spent in the every hole of antigen liquid 100ul coated 4, discards after coating buffer PBS-T washing three times;
4.37 degree are placed in vacuum bag after drying, and 4 degree are long-term to be preserved.
Anti-endothelin antibody biomarker detects
1. the healthy normal person's example of health check-up of collecting age, sex coupling, patients with SLE and connective tissue disease (CTD) merge the patient of pulmonary hypertension, set critical value (mean value+3 standard deviation), i.e. A value.
2. by embodiment 1 step, detect the anti-CTD-PAH body of anti-DT-56.
Calculate positive rate, patient's detected value (P), divided by negative detected value (N), P/N>2.1 is positive.
Analysis of test results
Use the coated indirect ELISA of DT-56-KLH to detect and use the coated anti-ENDRA antibody detection method of ENDRA total length recombinant protein to detect the antibody in patient and normal control patients serum, Fig. 3 result shows, the positive rate that anti-DT-56 antibody merges the patient diagnosis of pulmonary hypertension to connective tissue disease (CTD) is 45.8%, and specificity is 87.5%.To with the coated anti-endothelin antibody recall rate positive rate 40.8% similar with specificity of total length endothelin receptor A, specificity 96.3%.By to data analysis, Fig. 5 shows relatively indifference of ROC curve, p>0.05.Illustrate that two kinds of detection method result consistance are high, anti-DT-56 antibody testing method can substitute the coated indirect elisa method of total length ENDRA.
Anti-ENRA antibody is present in multiple connective tissue disease (CTD) such as comprising systemic loupus erythematosus, systemic sclerosis, mixed connective tissue disease, Sjogren syndrome, inflammatory myopathy and rheumatoid arthritis, and exist associatedly with CTD-PAH, may become particularly SLE-PAH of CTD-PAH() biomarker.Anti-ENRA, as the autoantibody may with pathogenic function, participates in endothelin receptor signal pathway activated and the vascular smooth muscle cell proliferation of PAH.Therefore, to the individuation application of endothelin-receptor antagonists, provide possible clinical foundation.There is stronger clinical practice promotional value.
This patent has covered the included all amino acid sequences of above-mentioned epitope peptide section, for detection of anti-ENRA antibody.This patent relates to this antibody test in the application of all CTD-PAH.
Claims (6)
1. the anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide, it is characterized in that, the outer peptide section of born of the same parents differing by external synthetic 4 length, screen the epitope peptide section good with total length ENRA consistance, by manually synthesizing this epitope peptide section as antigenic peptides wrapper sheet, the anti-endothelin acceptor A antibody enzyme-linked immunity detection method of foundation based on epitope antigen peptide, described epitope antigen peptide is the peptide section that comprises following amino acid sequence:
DNPERYSTNLSNHVDDFTTFRGTELSFLVTTHQPTNLVLPSNGSMHNYCPQQTKIT。
2. a kind of anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide according to claim 1, is characterized in that, concrete steps are as follows:
1), after by the antigenic peptides of 5ug/ml, in carbonic acid buffer, coated 96 orifice plates spend the night, PBS-T washing three times;
2), 3% bovine serum albumin(BSA) or 0.3% gelatin and the sealing of 10% hyclone 2 hours, PBS-T washing three times;
3), after patients serum 1:100-1:300 dilution, add under ELISA Plate room temperature and react 1 hour, PBS-T washing three times;
4), add the horseradish peroxidase-anti-human IgG bis-of proper proportion dilution anti-, room temperature reaction 1 hour;
5), add tmb substrate reaction after 10 minutes, by 2M sulfuric acid cessation reaction;
6), microplate reader absorbance 450nM reads plate and surveys absorbance.
3. a kind of anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide according to claim 1 and 2, is characterized in that, described epitope antigen peptide comprises all amino acid sequences, and puts in order.
4. a kind of anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide according to claim 1 and 2, it is characterized in that, described endothelin receptor A epitope antigen polypeptide fragment merges reagent and the kit of anti-endothelin acceptor A antibody in pulmonary hypertension and quantitative and qualitative analysis detection human serum for the preparation of diagnosis and assessment connective tissue disease (CTD).
5. the anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide according to claim 1 and 2, is characterized in that, the application that the method detects as biomarker at CTD-PAH.
6. the anti-endothelin acceptor A antibody enzyme-linked immunity detection method based on epitope antigen peptide according to claim 5, it is characterized in that, wherein CTD comprises systemic loupus erythematosus, chorionitis, mixed connective tissue disease, inflammatory myositis, Sjogren syndrome, rheumatoid arthritis, systemic vasculitis and anti-phospholipid antibody syndrome.
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Cited By (5)
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| CN105669863A (en) * | 2014-12-05 | 2016-06-15 | 杭州鸿运华宁生物医药工程有限公司 | Antibody capable of being specially combined with human endothelin receptor and application thereof |
| CN106883295A (en) * | 2016-12-27 | 2017-06-23 | 武汉华纪元生物技术开发有限公司 | Human endothelin A receptor immunogenicity peptide fragments and its carrier bacterin |
| WO2017206840A1 (en) * | 2016-05-31 | 2017-12-07 | 杭州鸿运华宁生物医药工程有限公司 | Etar antibody, pharmaceutical composition and use thereof |
| CN107987162A (en) * | 2016-10-27 | 2018-05-04 | 鸿运华宁（杭州）生物医药有限公司 | ETAR antibody, its pharmaceutical composition and its application |
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| CN105669863A (en) * | 2014-12-05 | 2016-06-15 | 杭州鸿运华宁生物医药工程有限公司 | Antibody capable of being specially combined with human endothelin receptor and application thereof |
| US10376581B2 (en) | 2014-12-05 | 2019-08-13 | Gmax Biopharm Llc. | Human endothelin receptor antibody and use thereof |
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| WO2017206840A1 (en) * | 2016-05-31 | 2017-12-07 | 杭州鸿运华宁生物医药工程有限公司 | Etar antibody, pharmaceutical composition and use thereof |
| CN107987162A (en) * | 2016-10-27 | 2018-05-04 | 鸿运华宁（杭州）生物医药有限公司 | ETAR antibody, its pharmaceutical composition and its application |
| CN106883295A (en) * | 2016-12-27 | 2017-06-23 | 武汉华纪元生物技术开发有限公司 | Human endothelin A receptor immunogenicity peptide fragments and its carrier bacterin |
| CN106883295B (en) * | 2016-12-27 | 2020-02-07 | 武汉华纪元生物技术开发有限公司 | Human endothelin A type receptor immunogenic peptide segment and carrier vaccine thereof |
| FR3077574A1 (en) * | 2018-02-07 | 2019-08-09 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | ANTIBODY AGAINST THE SUBTYPE TO ENDOTHELIN RECEPTORS AND USES THEREOF. |
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