CN111521830B - BNP detection kit, buffer solution, enzyme working solution and application - Google Patents
BNP detection kit, buffer solution, enzyme working solution and application Download PDFInfo
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Classifications
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- 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
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a BNP detection kit, a buffer solution, an enzyme working solution and application, wherein the BNP detection kit comprises the buffer solution and the enzyme working solution, calf serum and IgG are combined to be used as the buffer solution in the detection kit, and PEG20000 is added into the enzyme working solution, so that interference of a fresh sample on BNP detection can be effectively eliminated, and the sensitivity of a B-type natriuretic peptide sample can be improved.
Description
Technical Field
The invention relates to the technical field of BNP detection, in particular to a BNP detection kit, a buffer solution, an enzyme working solution and application.
Background
Brain natriuretic peptide (Brain Natriuretic Peptide, BNP), also known as type B natriuretic peptide (B-type NatriureticPeptide), brain natriuretic peptide, is a further member of the natriuretic peptide system following the natriuretic peptide (ANP), mainly consisting of a member of the family of natriuretic peptides secreted by the heart, a polypeptide consisting of 32 amino acid residues. It was first isolated from pig brain by Sudoh, japanese scholars, equal to 1988. Can regulate the self-stabilizing balance of blood pressure and blood volume, and has diuretic effect, and in fact, is mainly derived from ventricle. BNP has important pathophysiological significance, can promote sodium discharge and urination, has strong vasodilation effect, can resist the vasoconstriction effect of renin-angiotensin-aldosterone system (RAAS), and is a main endocrine system of human body for resisting overload of capacity and hypertension like ANP. Cardiac dysfunction can greatly activate the natriuretic peptide system, with increased ventricular load resulting in BNP release.
The detection of brain natriuretic peptide mainly comprises kit detection based on chemiluminescence enzyme immunoassay and kit detection based on turbidimetry.
Chemiluminescent enzyme immunoassay (chemiluminescence enzyme immunoassay, CLEIA) is to label an antigen or antibody with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) involved in catalyzing a chemiluminescent reaction, form a solid-phase coated antibody-antigen to be detected-enzyme-labeled antibody complex after immunoreaction with the corresponding antigen (antibody) in a sample to be detected, add a substrate (luminescent agent) after washing, enzymatically catalyze and decompose the substrate to emit light, receive the light signal by a light quantum reading system, convert the light signal into an electric signal and amplify the electric signal, and then transmit the electric signal to a computer data processing system to calculate the concentration of a measured object.
The kit based on chemiluminescent enzyme immunoassay mainly comprises immunomagnetic beads, enzyme working solution, an analysis buffer solution, substrate solution and cleaning solution, wherein a Beckmann full-automatic chemiluminescent immunoassay (ACCESS 2) is adopted for analysis and detection.
In the process of separating serum after blood collection of clinical patients (a kit based on chemiluminescence enzyme immunoassay), interference phenomenon is found when BNP detection reagent detects fresh samples of different individuals, and interference factors are hemoglobin, bilirubin and triglyceride. The buffer solution can reduce the interference phenomenon to a certain extent, but the existing BNP detection kit has poor anti-interference effect.
Disclosure of Invention
The invention aims to provide a BNP detection kit, which solves the problem of poor anti-interference effect of the existing BNP detection kit.
In addition, the invention also provides a buffer solution, an enzyme working solution and application of the BNP detection kit.
The invention is realized by the following technical scheme:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of two animal serums.
The usual buffers for BNP assay are 85% phosphoric Acid (AR), barbituric Acid (AR), calf serum, fetal bovine serum, igG, sheep serum, mouse serum.
The existing BNP detection kit has poor anti-interference effect.
The applicant found through experiments that:
the BNP detection kit has poor anti-interference effect because: the buffer adopts a single component.
The specific experimental process is as follows:
experimental materials:
immunomagnetic beads, enzyme working solution, substrate solution, washing solution, beckmann full-automatic chemiluminescence immunoassay (ACCESS 2), 85% phosphoric Acid (AR), barbituric Acid (AR), calf serum, fetal bovine serum, igG, sheep serum, mouse serum, 1000 μL pipette gun, 150 cases of BNP fresh clinical samples and third-party clinical test report (Beckmann Ai Lier manufacturer) on a daily basis.
The experimental method comprises the following steps:
1) 0.5g barbituric acid was weighed on a daily basis, and 0.5ml calf serum, 0.5ml fetal calf serum, 0.5ml IgG, 0.5ml sheep serum, 0.5ml mouse serum, 0.5ml horse serum, 0.59ml85% phosphoric acid were measured with a 1000. Mu.L pipette;
2) Respectively fixing the weighed and measured buffer substances into purified water in 10ml, enabling the mass fraction of each analysis buffer solution to be 10%, combining with a magnetic bead labeled antibody component and an enzyme working solution component, and randomly combining with buffer systems of 85% phosphoric acid, barbituric acid, calf serum, fetal calf serum, igG, sheep serum, mouse serum and horse serum, wherein the buffer systems are respectively numbered as B, C, D, E, F, G, H and I;
3) BNP assay reagent without added assay buffer material was numbered as group A (control);
4) Detecting 150 clinical fresh samples of BNP with A, B, C, D, E, F, G, H and I groups of reagents on a Beckmann full-automatic chemiluminescence immunoassay analyzer (ACCESS 2);
5) And (3) carrying out statistical data, comparing and analyzing the detection results of the clinical samples of the groups A, B, C, D, E, F, G, H and I with the report results of the third-party clinical test corresponding to the samples, and selecting the most suitable buffer substance. Results are shown in table 1 and table 2:
table 1 15 cases of clinical interference samples 9 kit BNP measurements were compared with Beckmann Mei Ai Lier manufacturer (buffer substance)
Table 2 150 clinical fresh samples 9 kit BNP measurements vs. Beckmann Mei Ai Lier manufacturer statistics (buffer substances)
And analyzing the discrete degree of each group of measured values and Beckmann measured values by using the statistical measured values in the table 1, counting the number of sample interference cases during the test of each group of kits, calculating the interference proportion, selecting the most suitable buffer substance as an analysis buffer solution, and obtaining the statistical data in the table 2.
As can be seen from the statistics in table 2, the anti-interference effect of the above buffer substances alone was not obvious, and the effect of phosphoric acid and barbituric acid organic acid as analysis buffer was worse than that of animal serum and IgG.
Further, the buffer consists of calf serum and IgG.
Further, the volume ratio of calf serum to IgG is 4 or more: 3.
further, the volume ratio of calf serum to IgG is 4:3.
the applicant found through experiments that: when the buffer solution is composed of calf serum and IgG, the buffer effect is optimal, the number of interference cases is obviously reduced along with the increase of the addition amount of the calf serum, and when the volume ratio of the calf serum to the IgG is 4: and 3, the interference of the fresh sample on BNP detection can be effectively eliminated.
Further, PEG20000 was added to the enzyme working fluid.
The applicant found through experiments that: the addition of PEG20000 to the enzyme working fluid can increase the sensitivity of a type B natriuretic peptide sample.
Further, the addition amount of PEG20000 in the enzyme working fluid is 1% -4%, and the addition amount is calculated by the enzyme working fluid.
Further, the addition amount of PEG20000 in the enzyme working fluid was 3%, the addition amount being based on the enzyme working fluid.
A buffer for a BNP detection kit, the buffer consisting of calf serum and IgG, the calf serum and IgG being in a volume ratio of 4:3.
An enzyme working solution for a BNP detection kit, wherein 3% of PEG20000 is added in the enzyme working solution, and the addition amount is calculated by the enzyme working solution.
Use of a BNP detection kit for BNP detection.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the detection kit provided by the invention uses the combination of the calf serum and the IgG as the buffer solution, so that the anti-interference capability can be effectively improved, the number of interference cases is obviously reduced along with the increase of the addition amount of the calf serum, and the volume ratio of the calf serum to the IgG is 4: and 3, the interference of the fresh sample on BNP detection can be effectively eliminated.
2. According to the detection kit, the calf serum and the IgG are combined to serve as a buffer solution, and meanwhile, the PEG20000 is added into an enzyme working solution, so that interference of a fresh sample on BNP detection can be effectively eliminated, and the sensitivity of a B-type natriuretic peptide sample can be improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:
FIG. 1 is a graph showing the trend of luminescence signal change when BNP is detected by adding PEG20000 kit;
FIG. 2 is a graph showing the trend of luminescence signal change when BNP is detected by adding 1% PEG20000 kit;
FIG. 3 is a graph showing the trend of luminescence signal change when BNP is detected by adding 2% PEG20000 kit;
fig. 4 is a graph showing the trend of luminescence signal change when BNP is measured with the 3% peg20000 kit;
fig. 5 is a graph showing the trend of luminescence signal change when BNP was measured with the 4% peg20000 kit.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
Example 1:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 4:3.
example 2:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 1:1.
example 3:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 2:1.
example 4:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 1:2.
example 5:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 3:2.
example 6:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 2:3.
example 7:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 3:4.
the test kits of examples 1-7 were numbered as (6), group (1), group (2), group (3), group (4), group (5), and group (7), respectively, and the 7-and beckmann Ai Lier manufacturers were used to test 15 clinical interference samples, respectively. The test results are shown in tables 3 and 4:
TABLE 3 Table 3
TABLE 4 Table 4
From the data in tables 3 and 4, it can be seen that:
the interference degree of the IgG and the calf serum on the sample is obvious, when 3 parts of IgG and 4 parts of calf serum (the volume ratio of the calf serum to the IgG is 4:3) (group (6)) are used as the preparation analysis buffer solution for measuring the sample, the anti-interference capability of the kit on the sample is optimal, and other component kits also have certain sample anti-interference capability, and the result shows that the analysis cannot reach the expected effect, so that the interference of the sample can be completely eliminated when 3 parts of IgG and 4 parts of calf serum ((group (6)) are used as the analysis buffer component.
Comparative example 1:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of fetal bovine serum and mouse serum, and the weight ratio of the fetal bovine serum to the mouse serum is 5%.
Comparative example 2:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of horse serum and sheep serum, and the ratio of the horse serum to the sheep serum is 5%.
Comparative example 3:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of IgG and calf serum, and the ratio of the IgG to the calf serum is 5%.
Comparative example 4:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of IgG and horse serum, and the ratio of the IgG to the horse serum is 5%.
Comparative example 5:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of phosphoric acid and IgG, and the ratio of the phosphoric acid to the IgG is 5%.
Comparative example 6:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of barbituric acid and IgG, and the ratio of the barbituric acid to the IgG is 5%.
Comparative example 7:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of phosphoric acid and mouse serum, and the ratio of the phosphoric acid to the mouse serum is 5%.
Comparative example 8:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of barbituric acid and sheep serum, and the ratio of the barbituric acid to the sheep serum is 5%.
Comparative examples 1 to 8 were respectively group I, group II, group III, group IV, group V, group VI, group VII and group VIII, and the test results of the Beckmann kit were used as reference groups, and the test results are shown in tables 5 and 6:
TABLE 5
TABLE 6
And analyzing the discrete degree of each group of measured values and Beckmann measured values by using the statistical measured values in the table 5, counting the number of sample interference cases during the test of each group of kits, calculating the interference proportion, selecting the most suitable buffer substance as an analysis buffer solution, and obtaining the statistical data in the table 6.
As shown in the statistics of table 6, most of the effects of the various buffer substances after being combined are poor, the anti-interference effect of the animal serum after being combined in pairs is better than that of other buffer substances, the anti-interference effect of the animal serum after being combined is the best that of IgG and calf serum (group iii), the best ratio of IgG to calf serum combination is carried out, and the mass fraction of the combined buffer solution is still controlled to be 10%.
Example 8:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 4:3, a step of; the addition amount of PEG20000 in the enzyme working fluid is 1%, and the addition amount is calculated by the enzyme working fluid.
Example 9:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 4:3, a step of; the addition amount of PEG20000 in the enzyme working fluid is 2%, and the addition amount is calculated by the enzyme working fluid.
Example 10:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 4:3, a step of; the addition amount of PEG20000 in the enzyme working fluid is 3%, and the addition amount is calculated by the enzyme working fluid.
Example 11:
the BNP detection kit comprises a buffer solution and an enzyme working solution, wherein the buffer solution consists of calf serum and IgG, and the volume ratio of the calf serum to the IgG is 4:3, a step of; the addition amount of PEG20000 in the enzyme working fluid is 4%, and the addition amount is calculated by the enzyme working fluid.
BNP was measured using the kit without PEG20000 as a control group and examples 8 to 11 as a test group, and the trend of the luminescence signal was shown in FIGS. 1 to 5, respectively.
The specific experimental method is as follows:
1) 0.1g,0.2g,0.3g,0.4g PEG20000 are respectively weighed by a ten-thousandth balance;
2) Respectively adding the weighed PEG20000 into 10g of enzyme working solution components, so that the mass fraction of the PEG20000 is respectively 1%,2%,3% and 4%, and respectively numbering the PEG20000, the magnetic bead labeled antibody component and the analysis buffer solution component into a second group, a third group, a fourth group and a fifth group after the PEG20000, the magnetic bead labeled antibody component and the analysis buffer solution component are combined;
3) BNP assay reagent without PEG20000 was numbered as the first group (control group);
4) And (3) detecting a set of BNP calibrator by using the first group, the second group, the third group and the fourth group of reagents and the fifth group of reagents on a Beckmann full-automatic chemiluminescence immunoassay (ACCESS 2) respectively, and comparing and analyzing to obtain a conclusion.
As can be seen from fig. 1 to 5:
when PEG20000 with the content of 3% is added into the enzyme working solution, the photon signal change of the low-value calibrator is sensitive to test, and the linear correlation is good.
To sum up:
the B-type natriuretic peptide assay kit adopts 3 parts of IgG &4 parts of calf serum as an analysis buffer solution, so that sample interference can be eliminated, and on the basis, when 0.3g/mL of PEG20000 is added into an enzyme working solution component, the sensitivity of testing a low-value B-type natriuretic peptide sample can be improved.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (3)
1. The BNP detection kit comprises an analysis buffer solution and an enzyme working solution, wherein the analysis buffer solution is used for participating in sample detection, and is characterized in that a buffer substance of the analysis buffer solution consists of calf serum and IgG; calf serum and IgG volume ratio 4:3, a step of; PEG20000 is added in the enzyme working solution, the addition amount of the PEG20000 in the enzyme working solution is 1% -4%, and the addition amount is calculated by the enzyme working solution.
2. The BNP detection kit of claim 1, wherein said enzyme working solution is added with 3% PEG20000, based on enzyme working solution.
3. Use of a BNP detection kit according to any one of claims 1-2, wherein said detection kit is for BNP detection.
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