CN108948183B - Alpha 1 receptor and AT1 receptor antigen analogue polypeptide, antibody detection kit and application thereof - Google Patents
Alpha 1 receptor and AT1 receptor antigen analogue polypeptide, antibody detection kit and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/321—Arterial hypertension
Abstract
The invention discloses an alpha 1 receptor and AT1 receptor antigen analogue polypeptide, an antibody detection kit and application thereof, wherein the alpha 1 receptor and AT1 receptor antigen analogue polypeptide is included; the test kit comprises a coating plate which respectively coats the alpha 1 receptor antigen analog polypeptide and AT1 receptor antigen analog polypeptide of claim 1. The stability of the two polypeptide coatings is further enhanced, the two polypeptide coatings have the characteristic of easy coupling, and the antibody detection kit prepared by the two polypeptide coatings has higher sensitivity and stronger specificity; the kit can be used for assisting in diagnosing refractory hypertension, malignant hypertension, preeclampsia and hypertensive cerebral apoplexy and assisting in establishing a hypertension treatment scheme for a patient.
Description
Technical Field
The invention relates to the field of medical detection reagents, in particular to alpha 1 receptor and AT1 receptor antigen analogue polypeptide, an antibody detection kit and application thereof.
Background
The pathogenesis of hypertension is very complex, abnormal activation of the sympathetic nervous system, renin angiotensin aldosterone system, immune system is involved, abnormal elevation of vasoactive substances and immune inflammatory factors can lead to vascular remodeling and elevation of blood pressure, causingHypertension and damage to target organs. Domestic and foreign research shows that autoimmune reaction is one of the important mechanisms for the development of hypertension. Alpha of vascular and cardiac distribution1Receptor and AT1The receptors belong to G protein coupled receptors, and the alpha 1 receptor is mainly distributed in vascular smooth muscle, can activate phospholipase C when excited, mobilize intracellular calcium, cause vasoconstriction and increase blood pressure; AT1Receptors are mainly distributed in kidneys, hearts, vascular smooth muscles and the like, and when the receptors are excited, on one hand, the receptors directly contract blood vessels to increase blood pressure, on the other hand, the receptors can stimulate sympathetic nerves to increase release of catecholamine, reduce renal blood flow to stimulate secretion and release of aldosterone, and control water uptake and natriuresis, which are all related to the rise of blood pressure. Anti-alpha1Receptor antibodies and anti-AT1Receptor antibodies are body-produced, autoaggressive antibodies directed against the receptor antigen, acting on alpha1Receptor and AT1Receptors, which activate the receptor, further induce hypertension and hypertension target organ damage. The team of the invention has done a lot of work in this respect over the last 20 years:
1. anti-alpha1Receptor antibodies and anti-AT1Receptor antibody discovery in hypertensive patients: anti-alpha-found in serum of patients from Fu (Lancet. 1994; 344 (8938; 344; 1660: 1660-1660: 3) of Fus) of patients from Fus1An adrenergic receptor autoantibody; the presence of anti-angiotensin II-1 (AT) in the serum of preeclamptic patients was discovered in Wallukat 1999 (J Clin invest, 1999; 103(7):945-52.)1) Receptor autoantibodies (anti-AT for short)1Receptor antibody). In 2000, the team of the invention reported that patients with refractory hypertension had anti-AT1The receptor antibody (JRAAS,2000, 1(1): 57.). We performed ELISA for anti-AT in 194 essential hypertension patients (98 of them are refractory hypertension patients) and 40 normal blood pressure populations1Receptor antibodies and anti-alpha1Receptor antibody, and the positivity of the two antibodies of the essential hypertension patient is respectively 26.8 percent (52/194) and 25.3 percent (49/194), and the positivity of the normal blood pressure patient is respectively (7.5 percent and 5 percent) (p is less than 0.01); refractory hypertension patient anti-AT1Receptor antibodies and anti-alpha1The ratio of the receptor antibody positivity (42.9% (42/98) and 36.7% (36/98), respectively) to the ratioPatients with non-refractory hypertension were significantly elevated (10.4% and 13.5%, respectively) (p <0.01), suggesting that exacerbations of hypertension were associated with immunological abnormalities (Liao Yuhua, et al, hypertens Res 2002; 25: 641-.
2. Anti-alpha1Receptor antibodies and anti-AT1Receptor antibodies are associated with hypertensive stroke recurrence: in the follow-up study of patients with hypertensive cerebral apoplexy for 3.3 years, the team of the invention firstly discovers the alpha resistance1Receptor antibodies and anti-AT1The receptor antibody positive patients increase the recurrence rate and the death rate of the stroke patients (Liao Yuhua, et al. circulation 2005, (exact Supplement): II-346), and the recurrence mechanism and the anti-alpha of the hypertensive stroke are estimated1Receptor antibodies and anti-AT1Receptor antibody mediated vascular remodeling, anti-alpha1Receptor antibodies and anti-AT1The receptor antibody can be used as an immunological marker of refractory hypertension, malignant hypertension and hypertensive cerebral apoplexy. Basic and clinical studies have demonstrated that these two antibodies have agonist effects and can cause vascular remodeling, myocardial hypertrophy, renal damage (Zhou ZH, et al. clinical immunology.2005; 114:164-173.Wang B, et al. Heart and Vessel, 2005; 20(4): 153-8). These have demonstrated that immune dysfunction in hypertensive patients is involved in the development of hypertension.
3. anti-AT1Clinical trials of receptor antibody and hypertension treatment drug selection: to verify anti-AT in a population1The effect of the receptor antibody is significant in the targeted treatment of hypertension, and the group organizes and completes the step treatment of hypertension by candesartan cilexetil and imidapril1Effect of receptor antibodies (SOT-AT)1Study) "clinical trial, SOT-AT1The study included 5 central 512 hypertensive patients and the results demonstrated: anti-AT for hypertensive disorders1The receptor antibody positive patients select an Angiotensin Receptor Blocker (ARB) which has better antihypertensive effect than Angiotensin Converting Enzyme Inhibitor (ACEI) treatment, and provide a new theory and a new method for individualized treatment of hypertension (Wei F, Liao YH, et al, Heart, 2011; 97: 479-.
In conclusion, the anti-alpha was developed1Receptor and anti-AT1The detection of the receptor antibody is not only beneficial to the auxiliary diagnosis of refractory hypertension, malignant hypertension, preeclampsia and hypertensive cerebral apoplexy, but also beneficial to the guidance of the selection of hypertension treatment drugs. Detection of anti-alpha was reported in 20021Receptor and AT1The polypeptide amino acid sequence of the receptor autoantibody (Liao Yuhua, et al hypertens Res 2002; 25: 641-646) and provides sensitivity and specificity of the detection results. Patent numbers: 03118703X patent discloses a serum anti-blood vessel alpha1And AT1A receptor autoantibody detection method and a kit; the antigen used for detecting the autoantibody in the patent is a sequence without any modification of the two receptors, and the clinical application is limited due to the problems of short sequence, low coating efficiency and antigen epitope covering, and insufficient detection sensitivity and specificity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides alpha1Receptor and AT1The two polypeptides are further enhanced in coating stability and have the characteristic of easy coupling, and the antibody detection kit prepared by the two polypeptides has higher sensitivity and stronger specificity; the kit can be used for assisting in diagnosing refractory hypertension, malignant hypertension, preeclampsia and hypertensive cerebral apoplexy and assisting in establishing a hypertension treatment scheme for a patient.
In order to achieve the purpose, the invention designs alpha1Receptor and AT1Receptor antigen analog polypeptides, the alpha 1 receptor and AT1 receptor antigen analog polypeptides including an alpha 1 receptor antigen analog polypeptide and an AT1 receptor antigen analog polypeptide;
wherein, the alpha 1 receptor antigen analogue polypeptide amino acid sequence is as follows (shown as SEQ ID No. 1):
Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-Gl u-Glu-Ala-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-Gly-Gly-Cys；
the AT1The amino acid sequence of the receptor antigen analogue polypeptide is as follows (as shown in SEQ)ID No. 2):
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-Pro-Gly-Gly-Cys。
the invention also provides application of the polypeptide in preparation of an autoantibody detection kit.
The kit for detecting serum anti-alpha 1 receptor and AT1 receptor autoantibody, wherein the kit comprises a coating plate coated with alpha 1 receptor antigen analogue polypeptide of claim 1 and AT1 receptor antigen analogue polypeptide, respectively.
As a preferred scheme, the coated plate coated with the alpha 1 receptor antigen analog polypeptide is formed by coupling the alpha 1 receptor antigen analog polypeptide with carrier protein bovine serum albumin and then coating; the coating plate coated with the AT1 receptor antigen analog polypeptide is formed by coupling the AT1 receptor antigen analog polypeptide with carrier protein bovine serum albumin and then coating.
Or, the preparation method of the coated plate coated with the alpha 1 receptor antigen analogue polypeptide/coated plate coated with AT1 receptor antigen analogue polypeptide comprises the following steps:
1) respectively dissolving alpha 1 receptor antigen analog polypeptide/AT 1 receptor antigen analog polypeptide analogs in a carbonic acid buffer solution, and coating AT the temperature of 2-4 ℃;
2) washing with washing solution, sealing with 1% bovine serum albumin-containing phosphate buffer as sealing solution, incubating, drying, and storing in refrigerator at 4 deg.C or-20 deg.C to obtain corresponding coated plate.
The autoantibody detection kit further comprises a sample diluent, a peroxidase-labeled goat anti-human IgG antibody, a TMB color development solution, a stop solution, a washing solution, negative control serum and positive control serum.
Dilution of the sample: is prepared by mixing 0.1M phosphate buffer solution with pH of 7.4 and calf serum according to the volume ratio of 9: 1;
the negative control serum is healthy human serum;
the positive control serum: human serum positive for anti-alpha 1 receptor or AT1 receptor autoantibodies.
The invention also provides application of the autoantibody detection kit in detection of an anti-alpha 1 receptor antibody and an anti-AT 1 receptor antibody. The detection kit is used for detecting autoantibodies of an anti-alpha 1 receptor and an AT1 receptor, and assisting in diagnosing refractory hypertension, malignant hypertension, preeclampsia and hypertensive cerebral apoplexy and guiding the individuation and targeted treatment of hypertension.
The invention modifies the alpha 1 receptor and AT1 receptor original antigen polypeptide sequence to increase the stability, sensitivity and specificity of the detection method. Compared with the original sequence, the modified polypeptide sequence has the following advantages:
1) the efficiency of the modified alpha 1 receptor and AT1 receptor antigen analogue polypeptide sequence coated in the detection plate is increased, and the sensitivity and specificity of the detection of the autoantibody of the anti-alpha 1 receptor and AT1 receptor are improved.
2) The modified alpha 1 receptor and AT1 receptor antigen analogue polypeptide sequence can be coupled with carrier bovine serum albumin or other carrier proteins, and is used for detecting a sample to be detected after being coated with a detection plate, so that the stability of the coated plate is increased, and the sensitivity and specificity of the detection of the autoantibody of the anti-alpha 1 receptor and the AT1 receptor can be obviously improved.
3) The invention provides a first mature detection kit for autoantibodies of an anti-alpha 1 receptor and an AT1 receptor, which is used for detecting the autoantibodies of the anti-alpha 1 receptor and the AT1 receptor, assisting in diagnosing refractory hypertension, malignant hypertension, preeclampsia and hypertensive cerebral apoplexy and guiding the individualization and the targeted treatment of hypertension.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
EXAMPLE 1 preparation of the kit
Firstly, synthesizing an antigenic peptide:
1. synthesizing antigenic peptides with the same antigenicity as the extracellular second cyclic peptide fragment of the alpha 1 receptor and the AT1 receptor, namely an amino acid residue fragment 193-218 of the alpha 1 receptor and an amino acid residue fragment 165-192 of the AT1 receptor, and simultaneously adding three amino acid sequences GGC (Gly-Gly-Cys) AT the C tail ends of the two peptide segments, wherein the specific sequences are as follows:
(1) the amino acid sequence of the synthesized alpha 1 receptor antigen analogue polypeptide (shown as SEQ ID No. 1):
Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-Gl u-Glu-Ala-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-Gly-Gly-Cys
(2) synthetic AT1 receptor antigen analogue polypeptide amino acid sequence (shown as SEQ ID No. 1):
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-Pro-Gly-Gly-Cys
2. synthesizing the antigen analogue polypeptide analogue by using a PSSM-8 type polypeptide synthesizer solid phase method of SHIMADZU company:
(1) deprotection: fmoc-protected columns and monomers must be freed of the amino protecting group using a basic solvent (piperidine).
(2) Activation and crosslinking: the carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to crosslink, forming a peptide bond. The reaction is driven to completion at this step using a large excess of reagent.
(3) And (3) circulation: these two reactions are repeatedly cycled until the synthesis is complete.
(4) Cleavage and deprotection: the polypeptide is cleaved from the column and the protecting groups are eluted and deprotected with a deprotecting agent (TFA).
3. And (3) separating and purifying after synthesizing the antigen analogue polypeptide:
the washing was performed 5 times with methanol for 1 minute each and 2 times with t-J methyl ether, both of which were aimed at washing away free unbound amino acid. Cutting the synthesized polypeptide from the resin with a resin removing solution (trifluoroacetic acid of sigma company), precipitating with diethyl ether (analytically pure), and centrifuging to separate the resin removing solution (trifluoroacetic acid) from the synthesized polypeptide; the upper layer of the deresination solution was decanted off and finally nitrogen (N) was used2) Drying the precipitated synthesized polypeptide by blowing, and storing in a refrigerator at-40 ℃ for later use.
II, coating of antigen:
respectively dissolving alpha 1 receptor and AT1 receptor antigen analogue polypeptide fragments in two containers filled with pH 9.2 carbonic acid buffer solution, coating with the amount of 1 ug of corresponding polypeptide antigen contained in 100 ul of carbonic acid buffer solution in each hole, or coating after coupling analogue polypeptide fragments with carrier (bovine serum albumin BSA, keyhole limpet hemocyanin KLH, etc.), with the amount of 1 ug of corresponding polypeptide antigen contained in each hole, placing in a 4 ℃ refrigerator for 18 hours, washing with washing solution for 3 times, 3 minutes each time, patting dry, using 1% bovine serum albumin-containing phosphate buffer solution as sealing solution, sealing 150 ul in each hole, incubating AT 37 ℃ for 2 hours to eliminate non-specific reaction, patting dry, and storing in a 4 ℃ or-20 ℃ refrigerator for later use.
Third, the main component of the kit (enzyme linked immunosorbent assay)
TABLE 1 major components of the kit (enzyme linked immunosorbent assay)
Fourth, the main component of the reagent box (enzyme linked immunosorbent assay)
If the alpha 1 receptor and the AT1 receptor antigen analogue polypeptide are coupled with Bovine Serum Albumin (BSA) respectively, the coated plate is a solid phase alpha 1 receptor antigen analogue polypeptide-BSA complex or AT1 receptor antigen analogue polypeptide-BSA complex. The coupling steps are as follows:
1. weighing 2mg of Sulfo-SMCC reagent, adding the reagent into 10mg/ml of 4mg BSA after completely dissolving 200ul of pure water, uniformly mixing, and standing for 30 minutes at room temperature;
2. activated BSA was added to a 10K TFF concentration column, 50mM PBS (containing 1mM EDTA, pH7.2) was added, and 5,000g was centrifuged 3 times to remove free Sulfo-SMCC; the TFF concentration column is provided by Millipore, USA;
3. weighing 2mg of antigen analogue polypeptide, fully dissolving 1ml of 50mM PBS, adding into activated BSA, reacting for 1 hour at room temperature, and lightly shaking once every 20 min;
4. preparing 1L 100mM PBS (containing 1mM EDTA, pH7.2), adding the vaccine mixture into a dialysis bag, dialyzing for 24 hr, and removing free unreacted polypeptide;
5. taking out the obtained product after dialysis, quantifying, and packaging at-80 deg.C for storage, or directly coating.
a. Storage condition and expiration date of kit
The kit is stored at 2-8 ℃ and has the validity period of 12 months. After the kit is unsealed, the kit is stored at 2-8 ℃ for no more than 1 month, and the unused microporous plate bars and the drying agent are sealed together by a self-sealing bag.
The foam box is hermetically transported by adding an ice bag, the transportation time is not more than 1 week, and the transportation temperature is not higher than room temperature.
b. Adapted for instruments
Enzyme mark instrument (wavelength band of 450nm, 600-650 nm)
Fifthly, the detection operation steps of the kit are as follows:
1. sample requirement
1) The sample used in the reagent is human serum.
2) The sample containing sodium azide cannot be detected, and the sodium azide inhibits the activity of horseradish peroxidase; samples containing suspended fibrin or aggregates, with severe hemolysis, could not be detected.
3) The sample should be free of microorganisms. The aseptically separated samples can be stored at 2-8 ℃ for 1 week and below-18 ℃ for three weeks, so that repeated freeze thawing is avoided.
4) Before use, the sample is placed at room temperature (10-30 ℃) for balancing for more than 30 minutes, and before the sample is frozen, the sample is unfrozen to room temperature (10-30 ℃) and mixed evenly.
2. Inspection method
1) Preparing liquid: diluting 1 bottle of concentrated washing solution to 400ml by using distilled water or deionized water;
2) numbering: numbering the micropores corresponding to the sample in sequence, wherein each plate is provided with a negative control hole 3, a positive control hole 2 and a blank hole 1 (the blank hole can not be arranged in the double-wavelength measurement);
3) sample adding: diluting a sample to be detected by using a sample diluent, wherein the dilution gradient is as follows: 1:20, 1:40 and 1:80, and added to the corresponding wells (100. mu.l per well) and blank wells were not added. Respectively adding 100 mul of negative and positive control into corresponding holes, and lightly shaking and mixing;
4) and (3) incubation: sealing the plate by using a sealing plate film, and then putting the plate into a 37 ℃ incubator for incubation for 45 minutes;
5) washing: carefully uncovering the sealing plate film, washing for 5 times (or manually washing the plate) by using a plate washing machine, soaking for 30 seconds each time, filling washing liquid into each hole during washing, and finally drying on absorbent paper as much as possible;
6) adding an enzyme: after shaking up, adding 100 mul of enzyme conjugate into each hole, not adding blank holes, and gently shaking up and mixing evenly;
7) and (3) incubation: placing the plate in a 37 ℃ incubator for incubation for 15 minutes after another sealing plate is used for sealing the plate;
8) washing: the operation is the same as the step 5.
9) Color development: adding 50 mul of color developing agent A into each hole, then adding 50 mul of color developing agent B (including blank holes), and developing for 15 +/-1 minutes at 37 +/-1 ℃ in a dark place;
10) and (3) determination: adding 50 mul of stop solution into each hole to stop reaction, slightly oscillating and uniformly mixing, and respectively measuring the light absorption value A of each hole under the wavelength of 450nm within 5 minutes (a single-wavelength microplate reader needs to zero the blank holes, and a double-wavelength microplate reader does not need to zero the blank holes);
11) and (5) judging a result: the blank control is zero, and the P/N value is more than or equal to 2.1 and is positive. (P/N ═ sample OD value/negative control OD value).
Example 2 kit for detecting patients with refractory hypertension
118 clinically diagnosed intractable hypertension patients and 60 blood pressure normality patients are subjected to verification detection by using the kit so as to test the sensitivity and specificity of the diagnostic kit. The two groups of hypertension cases are matched in the aspects of sex, age, primary treatment hypertension level and the like. The detection result is as follows:
the kit prepared by the two sections of peptide fragments detects that the sensitivity of the anti-alpha 1 receptor antibody is 48.3 percent, and the specificity is 90 percent; the sensitivity of the anti-AT 1 receptor antibody was 53.4%, and the specificity was 93%. Kit prepared from two peptide fragments without GGC at C terminal for detecting anti-alpha1Sensitivity of receptor antibody 36.7%, specificity 84%; anti-AT1Sensitivity of receptor antibody 42.9%, BThe anisotropy was 86%, see Table 2.
By comparison, we found that C-terminal GGC-added anti-alpha1Receptor and AT1The sensitivity and specificity of the detection of the kit prepared by the receptor autoantibody peptide section are higher than those of the kit prepared by the peptide section without GGC.
TABLE 2 comparison of sensitivity and specificity of detection of autoantibodies to alpha 1 receptor and AT1 receptor for refractory hypertension
| Sensitivity (%) | Specificity (%) | |
| Modified alpha 1 receptor antigen sequence | 48.3 | 90 |
| Modified AT1 receptor antigen sequence | 53.4 | 93 |
| Original alpha 1 receptor antigen sequence | 36.7 | 84 |
| Original AT1 receptor antigen sequence | 42.9 | 86 |
Example 3
Effect contrast of reagent kit for detecting essential and refractory hypertension patient
The kit is used for detecting autoantibodies of the anti-alpha 1 receptor and the AT1 receptor of the essential hypertension patients, and the detection results of 226 clinically diagnosed essential hypertension patients (118 cases of the patients are refractory hypertension patients) in the experiment are compared as follows:
after the detection of the anti-alpha 1 receptor autoantibodies and the AT1 autoantibodies are compared with each other, the positive rates of the anti-alpha 1 receptor autoantibodies and the AT1 autoantibodies in the refractory hypertension group are obviously higher than those in the healthy human group (P <0.001) and the essential hypertension group (P <0.01), and the essential hypertension group and the healthy human group have obvious difference (P <0.05), which is shown in Table 3.
TABLE 3 refractory hypertensive patients and control group anti-alpha1Receptor and AT1Receptor autoantibody detection assay
Note: p <0.001 compared to healthy human groups; # P <0.01 compared to essential hypertension group
Example 4
Comparison of effects of antigen analogue polypeptide coated plate and coupled object coated plate kit for detecting hypertension patients
The alpha 1 receptor and AT1 receptor antigen analogue polypeptide is coupled with a carrier bovine serum albumin and then coated on a 96-well plate, and is used for detecting anti-alpha 1 receptor and AT1 receptor autoantibodies in the serum of 118 patients with refractory hypertension and 60 blood pressure normal persons, and the result shows that the detection sensitivity and specificity of the kit are higher than those of the detection without a carrier coupling coating method, wherein the sensitivity and specificity of the kit are 48.3% and 90% respectively for detecting the anti-alpha 1 receptor antibody without the carrier coupling coating method; the sensitivity of the anti-AT 1 receptor antibody was 53.4%, and the specificity was 93%. The coupled coating method detects 56.8% of the sensitivity and 93% of the specificity of the anti-alpha 1 receptor antibody; the sensitivity and specificity of the anti-AT 1 receptor antibody were 59.4% and 96%, respectively, as shown in Table 4.
TABLE 4 comparison of the sensitivity and specificity of detection of antigen-analog polypeptide analog coated plates with conjugate coated plates
Other parts not described in detail are prior art. Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> Wuhan Huayuan Biotechnology development Co., Ltd
<120> alpha 1 receptor and AT1 receptor antigen analogue polypeptide, antibody detection kit and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> PRT
<213> human (Homo sapiens)
<400> 1
Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu
1 5 10 15
Glu Ala Gly Tyr Ala Val Phe Ser Ser Val Gly Gly Cys
20 25
<210> 2
<211> 31
<212> PRT
<213> human (Homo sapiens)
<400> 2
Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn Ile Thr Val Cys
1 5 10 15
Ala Phe His Tyr Glu Ser Glu Asn Ser Thr Leu Pro Gly Gly Cys
20 25 30
Claims (5)
1. An alpha 1 receptor and AT1 receptor antigen analog polypeptide characterized by: the alpha 1 receptor and AT1 receptor antigen analog polypeptides include an alpha 1 receptor antigen analog polypeptide and an AT1 receptor antigen analog polypeptide; wherein, the alpha 1 receptor antigen analogue polypeptide has the following amino acid sequence:
Trp-Lys-Glu-Pro-Val-Pro-Pro-Asp-Glu-Arg-Phe-Cys-Gly-Ile-Thr-Gl u-Glu-Ala-Gly-Tyr-Ala-Val-Phe-Ser-Ser-Val-Gly-Gly-Cys；
the amino acid sequence of the AT1 receptor antigen analog polypeptide is as follows:
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-Pro-Gly-Gly-Cys。
2. use of the polypeptide of claim 1 in the preparation of an autoantibody detection kit.
3. A kit for detecting serum anti-alpha 1 receptor and AT1 receptor autoantibodies, which is characterized in that: the detection kit comprises a coating plate for respectively coating the alpha 1 receptor antigen analog polypeptide and the AT1 receptor antigen analog polypeptide of claim 1; the alpha 1 receptor antigen analogue polypeptide coated plate is formed by coupling alpha 1 receptor antigen analogue polypeptide and carrier protein bovine serum albumin and then coating; the coating plate coated with the AT1 receptor antigen analog polypeptide is formed by coupling AT1 receptor antigen analog polypeptide with carrier protein bovine serum albumin and then coating; the autoantibody detection kit also comprises a sample diluent, a horseradish peroxidase-labeled goat anti-human IgG antibody, a TMB color development solution, a stop solution, a washing solution, negative control serum and positive control serum; the preparation method of the coated plate coated with the alpha 1 receptor antigen analogue polypeptide/the AT1 receptor antigen analogue polypeptide comprises the following steps:
1) respectively dissolving alpha 1 receptor antigen analog polypeptide/AT 1 receptor antigen analog polypeptide in a carbonic acid buffer solution, and coating AT the temperature of 2-4 ℃;
2) washing with washing solution, sealing with 1% bovine serum albumin-containing phosphate buffer as sealing solution, incubating, drying, and storing in refrigerator at 4 deg.C or-20 deg.C to obtain corresponding coated plate.
4. The autoantibody detection kit according to claim 3, characterized in that:
dilution of the sample: is prepared by mixing 0.1M phosphate buffer solution with pH of 7.4 and calf serum according to the volume ratio of 9: 1;
the negative control serum is healthy human serum;
the positive control serum: human serum positive for anti-alpha 1 receptor or AT1 receptor autoantibodies.
5. Use of an autoantibody detection kit according to claim 3 for the detection of anti- α 1 receptor antibodies and anti-AT 1 receptor antibodies.
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