CN1525169A - Detection method and reagent box for autoantibody of serum anti alfa1 and AT1 receptor in blood vessel - Google Patents

Detection method and reagent box for autoantibody of serum anti alfa1 and AT1 receptor in blood vessel Download PDF

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Publication number
CN1525169A
CN1525169A CNA03118703XA CN03118703A CN1525169A CN 1525169 A CN1525169 A CN 1525169A CN A03118703X A CNA03118703X A CN A03118703XA CN 03118703 A CN03118703 A CN 03118703A CN 1525169 A CN1525169 A CN 1525169A
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China
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serum
receptor
antibody
receptor autophosphorylation
hypertension
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敏 王
王敏
廖玉华
魏宇淼
黄凯
汪朝晖
程龙献
董继华
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Tongji Medical College of Huazhong University of Science and Technology
Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Abstract

The invention is a blood serum anti-vessel alpha 1- and AT1-receptor self-antibody detecting method and reagent box, its character: the reagent box is coated of antigen coating board of alpha and AT1 antigens, sample diluted liquid, negative contrast blood serum, positive contrast blood serum, lotion, enzyme-labeled bi-antibody, developers an and B and terminating liquid, able to detect the blood serum anti-vessel alpha 1- and AT1-receptor self-antibody.

Description

The anti-angiogenic α 1-of serum and AT1-receptor autophosphorylation antibody detection method and kit
Technical field
The present invention relates to anti-angiogenic alpha 1-receptor autoantibody of medical detectable, particularly serum (anti-α 1-adrenocepter autoantibody) and AT1-receptor autophosphorylation antibody (antiangiotensin II-acceptor 1 autoantibody) detectable.
Background technology
Hypertension is the common disease and the frequently-occurring disease of serious harm human health, in American-European countries, hypertensive morbidity rate is many more than 20%, China also is hypertensive country occurred frequently, according to national part provinces and cities sample census in 1991, total prevalence rate has reached 11.88%, and according to the Health Services investigation in Shanghai City, hypertensive morbidity rate occupies first of various chronic diseases.Hypertension is a long-term chronic disease of progress gradually, hypertension and cerebral apoplexy, left chamber plumpness, heart failure, coronary heart disease, dissection of aorta, chronic renal failure, hypertension eyeground pathological changes are closely related, are the main hazard factors that causes angiocardiopathy death.In China, 70% hyperpietic belongs to light-duty, and disease progression is slow, and about 10% patient belongs to refractory hypertension and severe hypertension, is the main crowd who causes cardiovascular and cerebrovascular death, must strengthen controlling of blood pressure and note early prevention and monitoring.Pathogenesis of hypertension is very complicated, usually be result of various factor comprehensive action, comprise that kidney regulates that blood pressure dysfunction, renin-angiotensin system and stomodaeal nervous system increased activity, blood vessel inner skin cell function are unusual, insulin resistance metabolic syndrome and inherent cause etc.; Wherein, since Ebringe in 1970 and Doyle find to exist among 30% benign hypertension and the accelerated hypertension patient significantly increasing of serum immunoglobulin level, numerous data show in experimental hypertension animal and high blood pressure people and have autoimmune phenomena, the effect of immune factor may be the important mechanisms that development takes place hypertension, does the immunologic dysfunction that exists in the high blood pressure become key areas [the Fu ML.Do immunesystem changes have a role in hypertension of hypertension incidence and the research of hypertension complication generation mechanism? J Hypertens 1995; 13 (11): 1259-65].
In recent years, find that also the high blood pressure philtrum exists the autoantibody at the cardiovascular adjusting acceptor of body, the autoantibody of especially anti-g protein coupled receptor may be the especially key factor of accelerated hypertension morbidity of hypertension.G protein coupled receptor is to regulate the relevant transmembrane receptor molecule superfamily of albumen with GTP, comprises M2-M-ChR, receptor,, alpha-2-adrenoceptor, angiotensin receptor-1 (AT1) etc.G protein coupled receptor is that extracellular signal is conducted to the universal signal molecule in the born of the same parents, in cardiovascular adjusting, has very important effect, its peptide chain structure passes through after birth seven times and forms three cyclic peptide [Hoebeke J.Structural basis of autoimmunity againstG protein coupled membrane receptors.Int J Cardiol 1996,54:103-11.] in outer three cyclic peptide of born of the same parents and the born of the same parents.Discovery at present has anti-M2 and the β1Shou Ti autoantibody that exists among 1/3 the dilated cardiomyopathy patient at outer second cyclic peptide of g protein coupled receptor born of the same parents approximately, and find that anti-M2-receptor autophosphorylation antibody can produce lasting dose dependent negative chronotropic action to neonatal rat myocyte by the M2 acceptor, show as and do not lose responsive M-ChR agonist activity [Wallukat G in time, FuHM, Matsui S, et al.Autoantibodies against M2 muscarinic receptors in patients withcardiomyopathy display non-desensitized agonist-1ike effects.Life Sci 1999,64:465-9.].Anti-β1-Shou Ti autoantibody also has β1-Shou Ti agonist activity [Wallukat G, Morwinski M, Kowal K, et al.Autoantibodies against the beta-adrenergic receptor in humanmyocarditis and dilated cardiomyopathy:beta-adrenergic agonism withoutdesensitization.Eur Heart J 1991,12 (Suppl D): 178.], and can cause cardiac muscle cell's calcium overload and cause cardiac damage [Wallukat G, Morwinski M, Kowal K, et al.Autoantibodies against thebeta-adrenergic receptor in human myocarditis and dilated cardiomyopathy:beta-adrenergic agonism without desensitization.Eur Heart J 1991,12 (SupplD): 178.].The existence of anti-M2-receptor autophosphorylation antibody and anti-β1-Shou Ti autoantibody and pathologic agonist-like activity thereof may be important mechanisms [the Liao YH of the generation of dilated cardiomyopathy morbidity and heart failure, Cheng LX, Tu YS, et al.Mechanism of anti-beta-adrenoceptor antibody mediated myocardial damage in dilatedcardiomyopathy.J Tongji Med University 1997,17:5.]; [Magnusson Y, Wallukat G, Waagstein F et al.Autoimmunity in idiopathic dilated cardiomyopathy:characterrization of antibodies against the β 1-adrenoceptor with positivechronotropic effect.Circulation 1994; 89:2760-7.].
Alpha 1-receptor and AT1-acceptor also belong to the g protein coupled receptor superfamily, and have a very important role in blood pressure regulating.The excitement of alpha 1-receptor can activate Phospholipase C, mobilizes intracellular calcium ion and vasoconstriction, rising blood pressure.The excitement of AT1-acceptor is except that can be by vasoconstriction and the rising blood pressure, the pool sodium Zhu Shui that also can increase kidney is also by stimulating the secretion of adrenal gland aldosterone, strengthen the retention of water sodium and the rising blood pressure, and can cause plumpness [the Pratt RE.Regulation of vascular smooth-muscle cell growth by angiotensin II.Blood Pressure1996 of cardiac muscle and vascular smooth muscle; 2:6-9.].Show the rising that in the accelerated hypertension patient, has anti-alpha 1-receptor and AT1-receptor autophosphorylation antibody titer on evidence, and anti-alpha 1-receptor autoantibody and AT1-receptor autophosphorylation antibody all have agonist-like activity [referring to Fu ML, Herlitz H, Wallukat G, et al.Functional autoimmune epitope on alpha 1-adrenergicreceptors in patients with malignant hypertension.Lancet 1994,344:1660-4.]; [referring to Fu ML, Herlitz H, Wallulat G, Hjalmarson A.Non-desensitized positive chronotropiceffect of anti-angiotensin II receptor autoantibodies in patients with malignanthypertension.Circulation 1996; 94:4046a.]; [referring to Fu ML, Schulze W, Wallulat G etal.Immunohistochemical localization of angiotensin II receptors (AT1) in the heartwith anti-peptide antibodies showing a positive chronotropic effect.Receptors
People such as Fu are [referring to Pratt RE.Regulation of vascular smooth-muscle cell growth byangiotensin II.Blood Pressure 1996; 2:6-9.] find in the accelerated hypertension patient, to exist anti-angiogenic alpha 1-receptor autoantibody, in malignant essential hypertension and malignant secondary hypertension patient, the patient of anti-alpha 1-receptor autoantibody positive reaction accounts for 20% and 40% respectively, and this autoantibody can significantly strengthen the contraction frequency of cultured neonatal rat cardiac myocytes, plays alpha 1-receptor and plays agonist-like activity.People such as Fu also find to remain in the accelerated hypertension the anti-AT1-receptor autophosphorylation antibody in high titre, and immunocytochemical technique finds that the anti-AT1-receptor autophosphorylation of rat source property antibody can play positive chronotropic action to neonatal cardiac myocytes.In addition, [Guatsfsson F.Hypertensivearteiolar necrosis revisited.Blood Press 1997 such as Wallukat; 6:71-7.] find that in the patient of pre-eclampsia, also have the positive reaction of anti-AT1-receptor autophosphorylation antibody, its positive rate is apparently higher than the normal arterial pressure puerpera, this antibody can combine with the ATI-receptor-specific, produces receptor stimulating agent sample activity.Autoantibody may be the key factor that development takes place for accelerated hypertension and pre-eclampsia by the caused pathologic effect of corresponding acceptor.
Accelerated hypertension is a kind of complication due to the serious rising of essential hypertension or secondary hypertension blood pressure, normal with extensive and serious target organ damage, PD is rapid, as active treatment not, cardiovascular and cerebrovascular complication such as most patients die from half a year that the kidney merit declines, cerebral apoplexy, acute myocardial infarction and heart failure.Refractory hypertension-promptly three kinds of effective drug for hypertension sufficient dosages are united use, still can not be with the hypertensive patient of controlling of blood pressure at 140/90mmHg, it is a kind of specific type hypertension, the course of disease is longer, the heart, brain, renal damage take place easily, the generation of refractory hypertension selects not good enough grade relevant with depressor, therefore, is the important topic that current this field faces to the diagnosis of refractory hypertension.
Summary of the invention
The purpose of this invention is to provide the anti-angiogenic α of a kind of detection serum 1Acceptor and AT 1The method of-receptor autophosphorylation antibody.
Another object of the present invention provides a kind of anti-angiogenic α of serum that is used for 1Acceptor and AT 1-receptor autophosphorylation antibody assay kit.
In order to inquire into the relation of refractory hypertension and immunologic dysfunction, we have collected refractory hypertension patient 58 examples altogether, common hypertensive patient's 96 examples and normal arterial pressure contrast 40 examples, the standard that the diagnosis of refractory hypertension proposes by U.S. JNC-VI, promptly three kinds of effective drug for hypertension sufficient dosages are united use, still controlling of blood pressure can not be called refractory hypertension patient [Fu ML to the hypertensive patient of 140/90mmHg, Herlitz H, Wallukat G, et al.Functionalautoimmune epitope on alpha 1-adrenergic receptors in patients with maiignanthypertension.Lancet 1994,344:1660-4.].Two groups of hypertension cases at sex, age, just control aspects such as blood pressure level, hypertension target organ damage situation and be complementary, hypertension case group and normal arterial pressure control group data are in sex, be complementary on the age, found that in refractory hypertension case group, the positive rate of these two kinds of autoantibodies obviously high with other two groups, be respectively 43%, 10.4% and 7.5%, difference has conspicuousness.
We have also observed the antibody positive patient to the different difference that acts on the renin-angiotensin system medicine, but PRELIMINARY RESULTS points out this autoantibody simulated blood vessel nervous plain, exciting angiotensin receptor, have great importance in high blood pressure generation and complication progress thereof, anti-alpha 1-receptor and AT1-receptor antibody are one of uppity latencies of refractory hypertension patient blood pressure.The clinical detection of anti-alpha 1-receptor and AT1-receptor antibody not only helps to seek the pathogenic factors of refractory hypertension, also help to instruct the selection of refractory hypertension patient antihypertensive drugs, the drug for hypertension of specific inhibition alpha 1-receptor and AT1-acceptor may more help this type of refractory hypertension patient's controlling of blood pressure and prevention and treatment of complication.The clinical detection of anti-alpha 1-receptor and AT1-receptor antibody has important clinical application value.
The invention provides the kit that is used to detect anti-angiogenic alpha 1-receptor autoantibody of serum and AT1-receptor autophosphorylation antibody is made up of antigen coated microplate, sample diluting liquid, negative control sera, positive control serum, washing lotion, ELIAS secondary antibody, developer A, developer B, stop buffer.
Antigen coated microplate is coated with α 1, AT 1Antigen, bag is polystyrene board (8 * 12 hole) or bar (12 hole) by plate, polystyrene has the good adsorption performance to protein, antigenic solution can be contacted with polystyrene support, can reach in certain temperature, through the regular hour and wrap the quilt purpose of (process that antigen is incorporated on the solid phase carrier is called the bag quilt). with α 1And AT 1The antigen polypeptide fragment is dissolved in (PH 9-11) in the carbonic acid buffer, with the amount bag quilt of every hole 1 μ g/100 μ l, puts 4 ℃ of refrigerator overnight, adds 1% bovine serum albumin(BSA)+PBS sealing, puts 37 ℃ of temperature and bathes 2 hours, to eliminate nonspecific reaction; It is standby to pat dry, be stored in after the drying 4 ℃ or-20 ℃ of refrigerators; Sample diluting liquid is that the phosphate buffer (PBS) of 0.01M PH7.4 adds 10% cow's serum; Negative control sera is after the serum with the healthy blood donor absorbs with polypeptide antigen, to remove behind the possible cross reaction thing as negative control sera; Positive control serum is that 20 μ l/ prop up packing with the anti-alpha 1-receptor autoantibody of measuring and 30 parts of mixing of serum of AT1-receptor autophosphorylation antibody positive, and-20 ℃ of preservations are standby; Washing lotion is the 0.01M PH7.4 phosphate buffer that contains 0.05% polysorbas20; ELIAS secondary antibody is the anti-people's of rabbit of horseradish peroxidase-labeled an IgG antibody; Developer A (substrate A) is by citric acid 1.26g, sodium acetate 4.64g, and water 400ml adds 30%H 2O 2264ul constitutes; Developer B (substrate B) is dissolved in the 1ml dimethyl sulfoxide with tetramethyl benzidine (TMB) 5mg to constitute; Stop buffer is the sulfuric acid of 2M.
The preparation of each component and composed as follows:
One. the preparation of antigen:
1. with the synthetic α of (Japanese SHIMADZU company) PSMM-8 type Peptide synthesizer with antigenic determinant 1Acceptor and AT 1Outer second cyclic peptide fragment, the i.e. α of-acceptor born of the same parents 1Acceptor 192-218 amino acids residue segment and AT 1Acceptor 165-191 amino acids residue segment.[J.1998 referring to Myocardial injury due to G-protein coupled receptor-autoimmunity.Jpn Heart; 39:261-74]
α 1Acceptor peptide section amino acid sequence is
Gly-Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-Glu-Glu-Ala-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-OH, the C of Val terminates on the carrier.
AT 1-acceptor peptide section amino acid sequence is
Ile-His-Arg-Asn-Val-Phe-Phe-Ile-Glu-Asn-Thr-Asn-Ile-Thr-Val-Cys-Ala-Phe-His-Tyr-Glu-Ser-Glu-Asn-Ser-Thr-Leu-OH, the C of Leu terminates on the carrier.
2. the synthetic aftertreatment of antigen: use washed with methanol 5 times, use degreasing resin (carrier) solvent with polypeptide and resin isolation after the t-J methyl ether cleans 2 times,, use nitrogen (N at last with ether sedimentation, centrifuging 2) drying, it is standby to put-40 ℃ of preservations.
Two. the bag quilt of antigen:
1. with α 1And AT 1The antigen polypeptide fragment is dissolved in (PH 9-11) in the carbonic acid buffer, with the amount bag quilt of every hole 1 μ g/100 μ l, puts 4 ℃ of refrigerator overnight, adds 1% bovine serum albumin(BSA)+PBS sealing, puts 37 ℃ of temperature and bathes 2 hours, to eliminate nonspecific reaction; It is standby to pat dry, be stored in after the drying 4 ℃ or-20 ℃ of refrigerators.
Three. the preparation of other reagent:
(1) dilution of sample liquid: 9 parts of phosphate buffers (PH7.4)+1 part calf serum;
(2) rabbit anti-human igg's antibody of horseradish peroxidase-labeled (magnificent company provides);
(3) developer A (substrate A): citric acid 1.26g, sodium acetate 4.64g, water 400ml adds 30%H 2O 2264ul
(3) developer B (substrate B): 5mg is dissolved in the 1ml dimethyl sulfoxide with tetramethyl benzidine (TMB)
(5) stop buffer: be 2mol/L H 2SO 4
(6) washing lotion: the 0.01mol/L phosphate buffer (PH7.4) that contains 0.05%Tweeu-20.
Use the detecting operation step of detectable of the present invention:
1. take out and be coated with α 1, AT 1The bag of antigen is by plate, with sample diluting liquid dilution test serum, α 1The group serum dilution is 1: 40, AT 1The group serum dilution is 1: 40, and the blood sample 100ul to be measured that dilution has been got well adds bag by in the micropore of plate, places 37 ℃ of temperature bath 2h, washes plate 3 times with washing lotion, promptly adds washing lotion at every turn and fills with micropore, places and pats dry washing lotion after 3 minutes, adds washing lotion again 3 times so repeatedly; Pat dry;
2. every hole adds 1: 2000 horseradish peroxidase-labeled IgG antibody 100 μ l, puts 37 ℃ of temperature and bathes 1h, the same plate of washing;
3. add A liquid 50ul and B liquid (TMB) 50ul temperature again and bathed 10 minutes, add 2mol/L H 2SO 4Cessation reaction;
4. the 450nm wavelength is measured optical density value (OD value) on microplate reader, and the blank positive, negative control are set in the experiment; The result judges: is zero with the blank, with P/N value 〉=2.1 positive (P/N=sample OD value/negative control OD value) [referring to Yin Baiyuan, Wang Renzhi, Li Zhenjia, Xu Yiping shows. " labelled immune " Atomic Energy Press 1998,130～138.].
The preparation of antigen
With the synthetic α of (Japanese SHIMADZU company) PSMM-8 type Peptide synthesizer with antigenic determinant 1Acceptor and AT 1Outer second cyclic peptide fragment, the i.e. α of-acceptor born of the same parents 1Acceptor 192-218 amino acids residue segment and AT 1Acceptor 165-191 amino acids residue segment.[J.1998 referring to Myocardial injury due to G-protein coupled receptor-autoimmunity.Jpn Heart; 39:261-74].
α 1Acceptor peptide section amino acid sequence is:
Gly-Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-Glu-Glu-Ala
-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-OH, the C of Val terminates on the carrier.
AT 1-acceptor peptide section amino acid sequence is:
Ile-His-Arg-Asn-Val-Phe-Phe-Ile-Glu-Asn-Thr-Asn-Ile-Thr-Val-Cys-Ala-Phe-His-Tyr-Glu-Ser-Glu-Asn-Ser-Thr-Leu-OH, the C of Leu terminates on the carrier.
Antigen synthesizes aftertreatment: use washed with methanol 5 times, use degreasing resin (carrier) solvent with polypeptide and resin isolation after the t-J methyl ether cleans 2 times, with ether sedimentation, centrifuging, use nitrogen (N at last 2) drying, it is standby to put-40 ℃ of preservations.
The bag quilt of antigen:
With α 1And AT 1The antigen polypeptide fragment is dissolved in (PH 9-11) in the carbonic acid buffer, with the amount bag quilt of every hole 1 μ g/100 μ l, puts 4 ℃ of refrigerator overnight, adds 1% bovine serum albumin(BSA)+PBS sealing, puts 37 ℃ of temperature and bathes 2 hours, to eliminate nonspecific reaction; It is standby to pat dry, be stored in after the drying 4 ℃ or-20 ℃ of refrigerators.
The preparation of other reagent:
(1) dilution of sample liquid: 9 parts of phosphate buffers (PH7.4)+1 part calf serum;
(2) the anti-human IgG antibody of horseradish peroxidase-labeled (magnificent company provides);
(3) TMB: (preserve by sealing in 1ml dimethyl Asia (DMSO) for the tetramethyl benzidine (TMB) of dissolving 5mg, 4 ℃ of dark places can be preserved half a year), add above-mentioned TMB solution 0.25ml in 12ml 0.1mol/L sodium citrate buffer solution (PH5.5), add 100 μ l 3%H again 2O 2
(4) stop buffer: be 2mol/L H 2SO 4
(5) washing lotion: the 0.01mol/L phosphate buffer (PH7.4) that contains 0.1%Tweeu-20.
Use the detecting operation step of detection kit of the present invention:
1. take out and be coated with α 1, AT 1The bag of antigen is by plate, with sample diluting liquid dilution test serum, α 1The group serum dilution is 1: 40, AT 1The group serum dilution is 1: 20, puts 37 ℃ of temperature and bathes 2h, washes plate 3 times with washing lotion, places 3 minutes at every turn;
2. every hole adds 1: 2000 horseradish peroxidase labeling antibody 100 μ l, puts 37 ℃ of temperature and bathes 1h, the same plate of washing;
3. add substrate (TMB) temperature again and bathed 10 minutes, add 2mol/L H 2SO 4Cessation reaction;
4. the 450nm wavelength is measured optical density value on microplate reader, and the blank positive, negative control are set in the experiment;
5. the result judges: with the blank be zero, and positive with P/N value 〉=2.1.(P/N=sample OD value/negative control OD value).
The detection principle of detection kit of the present invention is: specific antigen bag quilt in solid phase carrier, is added the sample that contains antibody to be measured (human IgG), make it to combine with solid phase antigen, add enzyme and mark anti-human IgG as second antibody; Add substrate A after the reaction again, it is blue that B liquid shows.
Course of reaction is: the IgG antibody of the sample (antibody to be measured is combined with solid antigen) of solid-phase coating (specific antigen is incorporated on the solid phase carrier) → add antibody to be measured (human IgG) → insulation (make antibody to be measured and solid phase antigen at a certain temperature fully in conjunction with) → washing (other material that flush away is not combined with solid phase antigen) → add horseradish peroxidase-labeled (make on specific antigen and the immune complex that antibody to be measured is combined and put on horseradish peroxidase) → insulation (fully in conjunction with) → wash (flush away is not in conjunction with upper unnecessary horseradish peroxidase) → add A, B liquid (A liquid: H2O 2Be hydrogen acceptor, B liquid: reduced form TMB is a hydrogen donor, and horseradish peroxidase at first and H 2O 2Reaction forms active oxygenant, and oxygenant and reduced form TMB reaction generates blue oxidized form TMB then) → insulation (fully reaction solution) → colorimetric.
This method specificity and susceptibility height, easy and simple to handle are for clinical the development detects AT 1-receptor autophosphorylation antibody and anti-α 1-receptor autophosphorylation antibody provides fast, sensitive detecting method, detects the selection that this antibody helps the diagnosis of refractory hypertension and instructs antihypertensive drugs.
We have collected refractory hypertension patient 58 examples altogether, common hypertensive patient's 96 examples, and normal hypertension contrasts 40 examples, two groups of hypertension cases in sex, age, just control aspect such as hypertension level and be complementary.Found that: in the refractory hypertension group, the positive rate of these two kinds of autoantibodies is respectively 43%, 10.4% and 7.5% apparently higher than other two groups, and difference has conspicuousness; Anti-α 1Acceptor and AT 1Receptor antibody is the uppity latency of refractory hypertension patient blood pressure, anti-α 1Acceptor and AT 1The clinical detection of receptor antibody not only helps to instruct the selection of refractory hypertension patient antihypertensive drugs, specific inhibition α 1Acceptor and AT 1The drug for hypertension of acceptor can more help this type of intractable refractory hypertension patient's controlling of blood pressure and prevention and treatment of complication, anti-α 1Acceptor and AT 1The clinical detection of receptor antibody has important clinical application value.
Because autoantibody has higher incidence in accelerated hypertension and pre-eclampsia patient, anti-angiogenic α 1-and AT1-receptor autophosphorylation detection of antibodies can be used for the diagnosis and the evaluation of accelerated hypertension, and help accelerated hypertension patient's medicament selection, as the anti-alpha 1-receptor positive, can select specificity alpha 1-receptor blocking agent, as the AT1-receptor positive, add and use the AT1-receptor antagonist, may more help the control of blood pressure and the control of complication.Though the AT1-receptor antagonist is not used in hypertension of pregnancy patient, AT1-receptor autophosphorylation detection of antibodies has and is beneficial to the prediction that hypertension of pregnancy patient is taken place for pre-eclampsia and eclampsia.Detect anti-angiogenic α 1-and AT1-receptor autophosphorylation antibody capable is offered help to concrete medicament selection.In addition, these two kinds of blood vessel autoantibodies can be used for hypertensive patient's state of illness monitoring, as having occurred the positive reaction of antibody in the development of hypertensive patient's course of disease, point out certain abnormal factors may trigger the generation of body autoimmune response, and might cause accelerated hypertension and refractory hypertension, state of illness monitoring and intervention timely will help the early diagnosis and the control of accelerated hypertension and refractory hypertension, reduce the generation and the development of the cardiovascular and cerebrovascular disease that causes thus, reduce crowd's mortality ratio.The patient that the present invention discloses the anti-alpha 1-receptor autoantibody positive obviously increases than its anti-AT1 receptor autophosphorylation antibody positive rate of patient of this negative antibody, simultaneously, the patient of anti-AT1 receptor autophosphorylation antibody positive has higher anti-alpha 1-receptor autoantibody positive rate than other patients, points out anti-alpha 1-receptor and anti-AT1-receptor autophosphorylation production of antibodies to have certain correlativity.Simultaneously, the patient that immunoglobulin (Ig) raises, its anti-α 1-and AT1-antibody positive rate also are higher than other people.The present invention discloses anti-alpha 1-receptor and there is certain correlativity in AT1-receptor autophosphorylation antibody, may be relevant with the homology of alpha 1-receptor and AT1-acceptor, and may some potential factor be to cause body to produce the reason of multiple autoantibody also.Because it is relevant that the positive situation of these two kinds of autoantibodies exists, detect this two kinds of autoantibodies simultaneously, the diagnosis of refractory hypertension will more be helped, improve specificity and susceptibility that refractory hypertension detects, and can select to provide more valuable evidence to the further medication of refractory hypertension patient, detect anti-α 1-and AT1-receptor autophosphorylation antibody simultaneously, diagnosis, prognostic evaluation and the drug for hypertension of refractory hypertension are selected to have important directive significance.
Fig. 1 detects principle and course of reaction diagram for the present invention.
Embodiment
Embodiment 1:
The preparation of diagnostic kit of the present invention
One, the preparation of antigen:
1. with the synthetic α of (Japanese SHIMADZU company) PSMM-8 type Peptide synthesizer with antigenic determinant 1Acceptor and AT 1Outer second cyclic peptide fragment, the i.e. α of-acceptor born of the same parents 1Acceptor 192-218 amino acids residue segment and AT 1Acceptor 165-191 amino acids residue segment.[J.1998 referring to Myocardial injury due to G-protein coupled receptor-autoimmunity.Jpn Heart; 39:261-74]
α 1Acceptor peptide section amino acid sequence is
Gly-Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-6lu-Glu-Ala-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-OH, the C of Val terminates on the carrier.
AT 1-acceptor peptide section amino acid sequence is
Ile-His-Arg-Asn-Val-Phe-Phe-Ile-Glu-Asn-Thr-Asn-Ile-Thr-Val-Cys-Ala-Phe-His-Tyr-Glu-Ser-Glu-Asn-Ser-Thr-Leu-OH, the C of Leu terminates on the carrier.
2. the synthetic aftertreatment of antigen: use washed with methanol 5 times, use degreasing resin (carrier) solvent with polypeptide and resin isolation after the t-J methyl ether cleans 2 times,, use nitrogen (N at last with ether sedimentation, centrifuging 2) drying, it is standby to put-40 ℃ of preservations.
Two, the bag quilt of antigen:
With α 1And AT 1The antigen polypeptide fragment is dissolved in (PH 9-11) in the carbonic acid buffer, with the amount bag quilt of every hole 1 μ g/100 μ l, puts 4 ℃ of refrigerator overnight, adds 1% bovine serum albumin(BSA)+PBS sealing, puts 37 ℃ of temperature and bathes 2 hours, to eliminate nonspecific reaction; It is standby to pat dry, be stored in after the drying 4 ℃ or-20 ℃ of refrigerators.
Three, the preparation of other reagent:
(1) dilution of sample liquid: 9 parts of phosphate buffers (PH7.4)+1 part calf serum;
(2) the anti-human IgG antibody of horseradish peroxidase-labeled (magnificent company provides);
(3) TMB: (preserve by sealing in 1ml dimethyl Asia (DMSO) for the tetramethyl benzidine (TMB) of dissolving 5mg, 4 ℃ of dark places can be preserved half a year), add above-mentioned TMB solution 0.25ml in 12ml 0.1mol/L sodium citrate buffer solution (PH5.5), add 100 μ l 3%H again 2O 2
(4) stop buffer: be 2mol/L H 2SO 4
(5) washing lotion: the 0.01mol/L phosphate buffer (PH7.4) that contains 0.1%Tweeu-20.
Embodiment 2
Use the detecting operation of detection kit of the present invention:
1. take out be coated with α 1, AT1 antigen bag by plate, with sample diluting liquid dilution test serum, α 1The group serum dilution is 1: 40, AT 1The group serum dilution is 1: 20, puts 37 ℃ of temperature and bathes 2h, washes plate 3 times with washing lotion, places 3 minutes at every turn;
2. every hole adds 1: 2000 horseradish peroxidase labeling antibody 100 μ l, puts 37 ℃ of temperature and bathes 1h, the same plate of washing;
3. add substrate (TMB) temperature again and bathed 10 minutes, add 2mol/L H 2SO 4Cessation reaction;
4. the 450nm wavelength is measured optical density value on microplate reader, and the blank positive, negative control are set in the experiment;
5. the result judges: with the blank be zero, and positive with P/N value 〉=2.1.(P/N=sample OD value/negative control OD value).

Claims (10)

1. anti-α 1-of serum and AT1-receptor autophosphorylation antibody assay kit is characterized in that this detection kit is made up of the antigen coated microplate that is coated with α 1, AT1 antigen, sample diluting liquid, negative control sera, positive control serum, washing lotion, ELIAS secondary antibody, developer A, developer B and stop buffer.
2. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described sample diluting liquid is that the phosphate buffer (PBS) of 0.01M PH7.4 adds 10% cow's serum and forms.
3. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described negative control sera is carried out constituting behind the possible cross reaction thing of absorbing and eliminating with polypeptide antigen by healthy blood donor's serum.
4. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described positive control serum is made of anti-alpha 1-receptor autoantibody and the positive serum mixing of AT1-receptor autophosphorylation antibody.
5. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described washing lotion is the 0.01M PH7.4 phosphate buffer that contains 0.05% polysorbas20.
6. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described ELIAS secondary antibody is the anti-people's of rabbit of horseradish peroxidase-labeled an IgG antibody.
7. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described developer A is citric acid 1.26g, sodium acetate 4.64g, and water 400ml adds 30%H 2O 2264ul constitutes; Developer B is dissolved in the 1ml dimethyl sulfoxide by tetramethyl benzidine (TMB) 5mg and constitutes.
8. anti-α 1-of serum according to claim 1 and AT1-receptor autophosphorylation antibody assay kit is characterized in that described stop buffer is the sulfuric acid of 2M.
9. anti-angiogenic α 1-of serum and AT1-receptor autophosphorylation antibody detection method is characterized in that having used requiring 1 described detection kit.
10. method that detects anti-α 1-of serum and AT1-receptor autophosphorylation antibody is characterized in that may further comprise the steps:
(1) with the bag that is coated with α 1, AT1 antigen by plate, with sample diluting liquid dilution test serum, 1 group of serum dilution of α is 1: 40, AT 1The group serum dilution is 1: 20, puts 37 ℃ of temperature and bathes 2h, washes plate 3 times with washing lotion, places 3 minutes at every turn;
(2) every hole adds 1: 2000 horseradish peroxidase labeling antibody 100 μ l, puts 37 ℃ of temperature and bathes 1h, the same plate of washing;
(3) add substrate (TMB) temperature again and bathed 10 minutes, add 2mol/LH 2SO 4Cessation reaction;
(4) the 450nm wavelength is measured optical density value on microplate reader, and the blank positive, negative control are set in the experiment;
(5) result judges: with the blank be zero, and positive with P/N value 〉=2.1.
CNA03118703XA 2003-02-27 2003-02-27 Detection method and reagent box for autoantibody of serum anti alfa1 and AT1 receptor in blood vessel Pending CN1525169A (en)

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Cited By (3)

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CN108948183A (en) * 2018-06-05 2018-12-07 武汉华纪元生物技术开发有限公司 1 receptor of α and AT1 receptor antigen analogue polypeptide and its antibody assay kit and application
CN111175505A (en) * 2020-01-08 2020-05-19 浙江省肿瘤医院 P53 autoantibody detection kit and application thereof
CN111514291A (en) * 2020-05-05 2020-08-11 华中科技大学同济医学院附属协和医院 Application of lupus erythematosus IgG in preparation of medicine for inhibiting bone damage

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108948183A (en) * 2018-06-05 2018-12-07 武汉华纪元生物技术开发有限公司 1 receptor of α and AT1 receptor antigen analogue polypeptide and its antibody assay kit and application
CN108948183B (en) * 2018-06-05 2022-01-28 武汉华纪元生物技术开发有限公司 Alpha 1 receptor and AT1 receptor antigen analogue polypeptide, antibody detection kit and application thereof
CN111175505A (en) * 2020-01-08 2020-05-19 浙江省肿瘤医院 P53 autoantibody detection kit and application thereof
CN111514291A (en) * 2020-05-05 2020-08-11 华中科技大学同济医学院附属协和医院 Application of lupus erythematosus IgG in preparation of medicine for inhibiting bone damage
CN111514291B (en) * 2020-05-05 2023-05-09 华中科技大学同济医学院附属协和医院 Application of lupus erythematosus IgG in preparation of medicines for inhibiting bone damage

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