CN114487407A - Angiotensin converting enzyme 2 detection kit - Google Patents
Angiotensin converting enzyme 2 detection kit Download PDFInfo
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- CN114487407A CN114487407A CN202210075288.3A CN202210075288A CN114487407A CN 114487407 A CN114487407 A CN 114487407A CN 202210075288 A CN202210075288 A CN 202210075288A CN 114487407 A CN114487407 A CN 114487407A
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Abstract
The invention discloses a detection kit of angiotensin converting enzyme 2, which comprises a reagent R1, a reagent R2 and an angiotensin converting enzyme 2 calibrator, wherein the detection kit of angiotensin converting enzyme 2(ACE2) is simple in description and easy to operate, can directly adopt a full-automatic biochemical analyzer to carry out sample detection, can meet the requirement of high-throughput rapid sample detection in a clinical laboratory, and can reduce human errors to obviously improve the detection efficiency; the kit adopts a latex immunoturbidimetry method, so that the sensitivity and the accuracy of the reagent are further improved, and the kit has a larger application space clinically and can be widely popularized and applied to most clinical laboratories.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to a detection kit for angiotensin converting enzyme 2.
Background
Angiotensin converting enzyme 2(ACE2) is a pleiotropic monocarboxypeptidase with high homology to ACE found in 2000, and its main function is to directly hydrolyze angiotensin II (Ang II) to generate angiotensin 1-7[ Ang (1-7) ]. The discovery of ACE2 breaks the traditional view that the ACE-Ang ii-AT 1 axis plays a major biological role in the renin-angiotensin system (RAS) metabolic pathway.
ACE2 is a zinc ion dependent metalloprotease consisting of 805 amino acids with a molecular mass of about 120 ku. Its catalytic domain is approximately 42% homologous to ACE and includes 4 portions, an N-terminal signal peptide, a catalytically active extracellular domain, a transmembrane region, and a C-terminal intracellular domain. ACE2 is consistent with the distribution of ACE and acts broadly in vivo to negatively regulate the RAS system. Most tissues with ACE distribution have ACE2 distribution, such as heart, kidney, testis, placenta, colon, small intestine, rat brain, lung, fat, liver, pancreas, retina, etc. However, in contrast to ACE, ACE2 is not expressed as widely as mRNA, and is highly tissue-specific, with a predominant distribution in coronary, renal vascular and tubular endothelium.
ACE2 is an extremely critical cardiac function regulator, and findings suggest that serum ACE2 levels are elevated in patients with Coronary Heart Disease (CHD) and as the severity of coronary stenosis worsens in CHD patients, ACE2 is not an independent contributor to CHD. This shows that CHD promotes the increase of ACE2 level in serum during the development process, and ACE2 level in serum can be used as an index for coronary heart disease prognosis, but not as an index for judging whether coronary heart disease is diseased or not. Studies have also shown that ACE2 plays an important role in acute kidney injury, liver injury, and liver metabolism.
The research on the ACE2 detection method is just in a starting stage, the conventional ACE2 detection technology comprises an enzyme-linked immunosorbent assay (ELISA) method and a PCR method, the traditional ELISA method is applied more generally, but the detection process is complex in operation, long in operation time, low in automation degree, relatively large in repeatability and batch-to-batch difference and not suitable for screening of large-volume samples, and multiple special devices are required to be arranged during application of the method, so that the use cost is increased to a certain extent. The PCR method is complex to operate, has high cost and high requirements on personnel, and is mostly applied to the laboratory stage.
In order to solve the above problems in the prior art, we propose a detection kit for angiotensin-converting enzyme 2.
Disclosure of Invention
The invention aims to provide a detection kit for angiotensin-converting enzyme 2, which is simple, convenient and quick to operate, has good detection stability and repeatability, can be applied to a full-automatic biochemical analyzer, realizes automatic operation and automatic analysis, and relatively reduces the use cost, thereby overcoming the defects in the background art.
In order to achieve the purpose, the invention provides the following technical scheme: a detection kit for angiotensin converting enzyme 2 comprises a reagent R1, a reagent R2 and an angiotensin converting enzyme 2 calibrator; wherein the content of the first and second substances,
the reagent R1 comprises the following components in percentage by weight:
the reagent R2 comprises the following components in percentage by weight:
the pH value of the buffer solution in the reagent R1 and the reagent R2 is 6.0-8.0;
the angiotensin converting enzyme 2 calibrator comprises the following components in parts by weight:
the pH value of the buffer solution in the angiotensin converting enzyme 2 calibrator is 6.0-8.0.
Preferably, the buffer is selected from one or more of Tris-HCL (Tris-HCL) buffer, borate buffer, phosphate buffer, and Good's buffer.
Preferably, the stabilizer is selected from one or more of bovine serum albumin, mannose, mannitol, trehalose, gelatin and sucrose.
Preferably, the inorganic salt is selected from one or more of sodium chloride, potassium chloride and sodium fluoride.
Preferably, the surfactant is selected from one or more of Tween-20, Tween-40 and Triton-100.
Preferably, the coagulant is selected from one or more of polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 and polyethylene glycol 20000.
Preferably, the suspending agent is sodium alginate.
Preferably, the preservative is selected from one or more of sodium azide, Proclin300, sodium citrate and imidazolidinyl urea.
Preferably, the anti-human angiotensin converting enzyme 2(ACE2) antibody is selected from one of a goat anti-human, a mouse anti-human, a rabbit anti-human angiotensin converting enzyme 2(ACE2) antibody.
Preferably, the particle size of the anti-human angiotensin converting enzyme 2(ACE2) antibody coated latex particle is 100-220 nm, and the preparation method comprises the following steps:
(1) adding 1-3 ml of polystyrene latex solution with surface carboxylation and the diameter of 100-220 nm into 10ml of PBS buffer solution to ensure that the final concentration of latex particles is 1.0%;
(2) adding 0.1-1 mg of anti-human angiotensin converting enzyme 2(ACE2) antibody and 20-40 mg of activator 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), incubating at 37 ℃ for 2-4 hours, and chemically crosslinking activated carboxyl and latex particle solution to form a covalent antibody latex compound;
(3) and then adding 10-20 ml of 0.1% BSA solution, sealing for 0.5-2 hours at 37 ℃, sealing surface active groups which are not combined with the antibody on the surface of the latex particles, centrifuging at 12000rpm for 20-50 minutes, removing supernatant, adding 30mmol/L Tris-HCl buffer solution containing 0.1% BSA into the obtained precipitate, washing, and dispersing to obtain the antibody-coated latex solution of the anti-human angiotensin converting enzyme 2(ACE 2).
Compared with the prior art, the invention has the beneficial effects that: the angiotensin converting enzyme 2(ACE2) detection kit provided by the invention is simple in description and easy to operate, can be used for directly detecting samples by adopting a full-automatic biochemical analyzer, can meet the requirement of high-flux rapid detection of samples in a clinical laboratory, and can reduce human errors so as to obviously improve the detection efficiency; the kit adopts a latex immunoturbidimetry method, so that the sensitivity and the accuracy of the reagent are further improved, and the kit has a larger application space clinically and can be widely popularized and applied to most clinical laboratories.
Drawings
FIG. 1 is a graph showing the correlation between the detection results of example 1 of the present invention and the comparative kit;
FIG. 2 is a graph showing the correlation between the detection results of example 2 of the present invention and the comparative kit;
FIG. 3 is a graph showing the correlation between the detection results of example 3 of the present invention and the comparative kit.
Detailed Description
Unless clearly defined, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
Example 1:
reagent R1: the pH value is 6.5
Reagent R2: the pH was 6.5
Calibration products: pH6.5
And adding a corresponding amount of angiotensin converting enzyme 2(ACE2) antigen into the calibrator solution according to the required calibrator concentration to prepare an angiotensin converting enzyme 2(ACE2) calibrator.
In this example, 6 calibrators with different concentrations were prepared, which were: 0ng/L, 200ng/L, 500ng/L, 1000ng/L, 2000ng/L, 4000 ng/L.
A high-value calibrator can be brought during actual delivery, and a client dilutes according to the specified multiple ratio; alternatively, a plurality of calibrators with different concentrations can be prepared and directly placed in the kit, and the 6 points of the calibrators are used for making a calibration curve, and in the example, the measured value is calculated on the calibration curve by the test sample of the instrument.
Example 2:
reagent R1: the pH value is 7.0
Reagent R2: the pH value is 7.0
Calibration products: the pH value is 7.0
And adding a corresponding amount of angiotensin converting enzyme 2(ACE2) antigen into the calibrator solution according to the required calibrator concentration to prepare an angiotensin converting enzyme 2(ACE2) calibrator.
In this example, 6 calibrators with different concentrations were prepared, which were: 0ng/L, 200ng/L, 500ng/L, 1000ng/L, 2000ng/L, 4000 ng/L.
Example 3:
reagent R1: the pH value is 6.5
Reagent R2: the pH value is 6.5
Calibration products: the pH value is 6.5
And adding a corresponding amount of angiotensin converting enzyme 2(ACE2) antigen into the calibrator solution according to the required calibrator concentration to prepare an angiotensin converting enzyme 2(ACE2) calibrator.
In this example, 6 calibrators with different concentrations were prepared, which were: 0ng/L, 200ng/L, 500ng/L, 1000ng/L, 2000ng/L, 4000 ng/L.
In each embodiment, the particle size of the latex particles coated with the anti-human angiotensin converting enzyme 2(ACE2) antibody is 100-220 nm. The preparation method comprises the following steps:
(1) adding a proper amount of polystyrene latex solution with surface carboxylation and the diameter of 100-220 nm into 10ml of PBS buffer solution to ensure that the final concentration of latex particles is 1.0%;
(2) adding a proper amount of an anti-human angiotensin converting enzyme 2(ACE2) antibody and an activator 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), incubating at 37 ℃ for about 3 hours, and chemically crosslinking activated carboxyl and a latex particle solution to form a covalent antibody latex compound;
(3) then, adding a proper amount of 0.1% BSA solution, sealing for 1 hour at 37 ℃, sealing surface active groups which are not combined with the antibody on the surface of the latex particles, then centrifuging for 40 minutes at 12000rpm, removing supernatant, adding the obtained precipitate into 30mmol/L Tris-HCl buffer solution containing 0.1% BSA, washing, and dispersing to obtain the antibody coating latex solution of the anti-human angiotensin converting enzyme 2(ACE 2).
1. The detection method comprises the following steps:
the above examples 1-3 were performed by endpoint method using a fully automated biochemical analyzer of Hitachi LABOSPECT 006, with the detection wavelength of 570nm and the processing parameters shown in Table 1. Purified water was used as a reagent blank before testing the samples.
TABLE 1 reagent assay parameters
And (4) calculating a result:
ACE2 content (ng/L) — (Δ a sample/Δ a calibrator) x calibrator concentration.
2. Repeatability test
The samples of the calibrator and human serum with a standard value of 932ng/L in each example were used to perform a reproducibility test of the kit in each example, the measurement was repeated 10 times according to the required measurement method, and the measurement average value X, the standard deviation SD, and the coefficient of variation CV were calculated to perform the reproducibility test of the kit in each example, and the measurement results are shown in Table 2.
TABLE 2 results of the repeatability tests
As can be seen from the results in Table 2, the CV values of example 1, example 2 and example 3 were all < 5%, with the most desirable results for example 1.
3. Accuracy test
The kit and the calibrator of each embodiment and a control kit (a commercially available enzyme-linked immunosorbent ACE2 detection kit of a certain manufacturer, abbreviated as "ELISA kit") are used for simultaneously testing and detecting 40 human serum samples, the detection results are counted, the correlation coefficients of the two are calculated, linear regression is performed, the correlation analysis is performed on the detection results, so as to perform accuracy investigation on the kits in each example, and the determination results are shown in table 3.
TABLE 3 accuracy test results (ng/L)
As can be seen from the results in Table 3, the correlation coefficients of example 1, example 2 and example 3 are all less than 0.975, which indicates that the kits of the embodiments of the present invention have high accuracy, wherein the correlation of example 1 is 0.9979, and the correlation between the detection result of the kit of example 1 and the ELISA kit of the present invention is the best. The correlation fit curves are shown in FIGS. 1-3.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A detection kit for angiotensin converting enzyme 2 is characterized in that: the detection kit comprises a reagent R1, a reagent R2 and an angiotensin-converting enzyme 2 calibrator; wherein, the first and the second end of the pipe are connected with each other,
the reagent R1 comprises the following components in percentage by weight:
the reagent R2 comprises the following components in percentage by weight:
the pH value of the buffer solution in the reagent R1 and the reagent R2 is 6.0-8.0;
the angiotensin converting enzyme 2 calibrator comprises the following components in parts by weight:
the pH value of the buffer solution in the angiotensin converting enzyme 2 calibrator is 6.0-8.0.
2. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the buffer solution is selected from one or more of tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution, boric acid buffer solution, phosphate buffer solution and Good's buffer solution.
3. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the stabilizer is selected from one or more of bovine serum albumin, mannose, mannitol, trehalose, gelatin and sucrose.
4. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the inorganic salt is selected from one or more of sodium chloride, potassium chloride and sodium fluoride.
5. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the surfactant is one or more selected from Tween-20, Tween-40 and Triton-100.
6. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the coagulant is selected from one or more of polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 and polyethylene glycol 20000.
7. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the suspending agent is sodium alginate.
8. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the preservative is selected from one or more of sodium azide, Proclin300, sodium citrate and imidazolidinyl urea.
9. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the anti-human angiotensin converting enzyme 2 antibody is selected from one of a goat anti-human antibody, a mouse anti-human antibody and a rabbit anti-human angiotensin converting enzyme 2 antibody.
10. The angiotensin converting enzyme 2 assay kit according to claim 1, wherein: the particle size of the latex particles coated with the anti-human angiotensin converting enzyme 2 antibody is 100-220 nm, and the preparation method comprises the following steps:
(1) adding 1-3 ml of polystyrene latex solution with surface carboxylation and the diameter of 100-220 nm into 10ml of PBS buffer solution to ensure that the final concentration of latex particles is 1.0%;
(2) adding 0.1-1 mg of an anti-human angiotensin converting enzyme 2 antibody and 20-40 mg of an activator 1-ethyl- (3-dimethylaminopropyl) carbodiimide, incubating at 37 ℃ for 2-4 hours, and chemically crosslinking activated carboxyl and a latex particle solution to form a covalent antibody latex compound;
(3) and then adding 10-20 ml of 0.1% BSA solution, sealing for 0.5-2 hours at 37 ℃, sealing surface active groups which are not combined with the antibody on the surface of the latex particles, centrifuging at 12000rpm for 20-50 minutes, removing the supernatant, adding 30mmol/L Tris-HCl buffer solution containing 0.1% BSA into the obtained precipitate, washing, and dispersing to obtain the anti-human angiotensin converting enzyme 2 antibody coated latex solution.
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| CN202210075288.3A CN114487407A (en) | 2022-01-22 | 2022-01-22 | Angiotensin converting enzyme 2 detection kit |
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| CN202210075288.3A CN114487407A (en) | 2022-01-22 | 2022-01-22 | Angiotensin converting enzyme 2 detection kit |
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| CN202210075288.3A Pending CN114487407A (en) | 2022-01-22 | 2022-01-22 | Angiotensin converting enzyme 2 detection kit |
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