CN111912838A - BNP chemical light-emitting detection kit and application thereof - Google Patents
BNP chemical light-emitting detection kit and application thereof Download PDFInfo
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Abstract
The invention relates to a BNP chemical light-emitting detection kit and application thereof, belonging to the technical field of medical detection. According to the invention, the binding sites of the non-crosslinked antibodies on the magnetic beads are sealed by sealing treatment, so that not only is the stronger false positive of the magnetic beads avoided, but also the phenomena of agglomeration and wall attachment in the detection process are reduced, and therefore, the steps of marking the antibodies by the magnetic beads are optimized, and the purpose of improving the performance of the reagent is achieved; the invention also optimizes the components and conditions of the working solution stored by the magnetic beads and prolongs the stability and the storage time of the reagent.
Description
Technical Field
The invention relates to a BNP chemical light-emitting detection kit and application thereof, belonging to the technical field of medical detection.
Background
BNP (brain natriuretic peptide) is a polypeptide neurohormone, has the functions of natriuresis, vasodilation and renin resistance, can be used as an effective marker of myocardial injury, indicates the condition of ventricular pressure increase, and has important values in auxiliary diagnosis and treatment evaluation detection of congestive heart failure and dyspnea complications caused by the congestive heart failure, essential hypertension, left ventricular dysfunction and other cardiovascular related diseases.
After stimulation of the cardiomyocytes, a precursor of the pro-B-type natriuretic peptide (Pre-proBNP) is produced, containing 134 amino acids, followed by the formation of a precursor of BNP (proBNP), containing 108 amino acids, which is cleaved by the action of an endonuclease into biologically inactive NT-proBNP containing 76 amino acids and active B-type natriuretic peptide (BNP) containing 32 amino acids. BNP, NT-proBNP and uncleaved proBNP are released into the peripheral circulation. After entering the peripheral circulation, BNP is effectively cleared, with a half-life of about 20 min.
In the detection process of the BNP chemiluminescence detection reagent, the magnetic bead labeled antibody (particularly after the antibody is added) is easy to agglomerate and attach to the wall, so that the operation time and difficulty are increased, and nonspecific adsorption can be generated when a blank sample is detected, so that the sensitivity is reduced. In addition, the antibody marked by the magnetic beads can be agglomerated, adhered to walls and settled after being stored for a long time, which greatly influences the stability of the reagent.
Disclosure of Invention
Aiming at the existing problems of the detection method, the invention provides the BNP chemical light-emitting detection kit and the application thereof, which solve the problems that magnetic beads mark antibodies are easy to agglomerate and attach to walls, and reduce the operation time and difficulty.
The purpose of the invention is realized by the following technical scheme:
a BNP chemiluminescence detection kit is characterized by comprising: the kit comprises a mixed magnetic bead solution coated with a BNP monoclonal antibody, a BNP calibrator solution, a BNP monoclonal antibody solution labeled with acridinium ester, a cleaning solution and an excitation solution.
The invention provides a BNP chemical light emission detection kit, a preparation method and application thereof. Mixing biological samples (blood serum, blood plasma and whole blood), a BNP monoclonal antibody solution marked by acridinium ester and a BNP monoclonal antibody solution marked by magnetic beads, fixing the mixture of the double-antibody sandwich under the action of a magnetic field, cleaning and discarding the rest antibodies marked by the acridinium ester, adding a light starter, exciting a chemical reaction, and collecting an optical signal.
In the above BNP chemiluminescence detection kit, the preparation method of the magnetic bead mixed solution coated with the BNP monoclonal antibody comprises: activating the crosslinked BNP monoclonal antibody on the magnetic beads, sealing the magnetic beads of the activated crosslinked BNP monoclonal antibody, cleaning, separating and storing by using a storage buffer solution.
In the above-mentioned BNP chemiluminescent detection kit, a solution of 3-mercaptopropionic acid and aminoguanidine hemisulfate is used in the blocking treatment.
In the above BNP chemiluminescence detection kit, the mass ratio of 3-mercaptopropionic acid to aminoguanidine hemisulfate is (1-3): 1.
after the magnetic beads are crosslinked with the antibodies, the magnetic beads are subjected to post-treatment by aminoguanidine hemisulfate and 3-thiopropionic acid, so that on one hand, binding sites of the magnetic beads, which are not crosslinked with the antibodies, can be blocked, and on the other hand, a polar monolayer can be formed and arranged on the surfaces of the magnetic beads. Therefore, the electrification condition of the magnetic beads can be changed, the phenomena of agglomeration and wall attachment are greatly reduced, and meanwhile, the nonspecific adsorption of serum can be weakened. After the carboxyl magnetic beads are activated and crosslinked, some sites can be not bound by the antibody (the antibody is Y-shaped and has steric hindrance), and the aminoguanidine hemisulfate and the 3-thiopropionic acid molecules have small spatial structures and can be blocked. On the other hand by-NH on aminoguanidine hemisulfate2And reacts with-COOH groups on the 3-thiopropionic acid to form a reticular polar monolayer, and in the preservation buffer solution, the magnetic beads have the same electrical property and have repulsion action with each other, so that the agglomeration and the wall attachment are not facilitated.
In the above BNP chemiluminescence detection kit, the storage buffer comprises: 10-50mM PBS, 0.3-1% protamine, 0.3-1% para-methylpropylamine, 0.3-1% sarcosine, 0.3-1% phenylalanine, 0.02-0.5% SDS, 1-10% chitosan, 0.05-0.15% NaN3At a pH of7.5。
In the above-mentioned BNP chemiluminescence detection kit, the preparation method of the acridinium ester labeled BNP monoclonal antibody solution comprises: and mixing the BNP monoclonal antibody and acridinium ester for incubation, and storing after ultrafiltration.
In the above BNP chemiluminescence detection kit, the crosslinking agent in the process of activating the crosslinked BNP monoclonal antibody is EDC.
In the above-mentioned BNP chemiluminescence detection kit, the cleaning solution comprises: 8-15mM/L PBS, 0.02-0.06% TW-20, 0.01-0.02% NaN3The pH was 7.4.
In the above chemical light emission detection kit for BNP, the excitation liquid is a and B, where a includes: 0.05-0.2M/L HNO3、0.05-0.2％H2O2(ii) a The component B comprises: 0.1-0.3M/L NaOH, 1-3% TritonX-100.
The application of the BNP chemical light-emitting detection kit comprises the steps of firstly adding acridinium ester labeled monoclonal antibody solution into a sample for incubation, then adding magnetic bead mixed solution coated with the BNP monoclonal antibody for incubation, separating and cleaning, finally adding excitation liquid for chemiluminescence reaction, and collecting signals.
Compared with the prior art, the invention has the following advantages:
according to the invention, the binding sites of the non-crosslinked antibodies on the magnetic beads are sealed by sealing treatment, so that not only is the stronger false positive of the magnetic beads avoided, but also the phenomena of agglomeration and wall attachment in the detection process are reduced, and therefore, the steps of marking the antibodies by the magnetic beads are optimized, and the purpose of improving the performance of the reagent is achieved; the invention also optimizes the components and conditions of the working solution stored by the magnetic beads and prolongs the stability and the storage time of the reagent.
Drawings
FIG. 1 shows the state of a magnetic bead mixture after 6 months of storage in the blocking treatment.
FIG. 2 shows the state of a magnetic bead mixture after 6 months of storage without blocking treatment.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.
For convenience of description, the buffers used in the examples are listed below (in liters below):
crosslinking buffer I: 100mM/L MES, pH 5.0;
crosslinking buffer II: 100mM/L CB, pH 9.0;
the crosslinking buffer III was TBS-T: 25mM Tris, 0.9% NaCl, 0.05% TW-20, pH 7.2;
crosslinking buffer IV: 10mM/L PBS, pH 7.4;
cleaning solution: 10mM/L PBS, 0.05% TW-20, 0.01% NaN3，PH 7.4；
Excitation liquid A: 0.1M/L HNO3、0.1％H2O2；
Excitation liquid B: 0.25M/L NaOH, 2% TritonX-100;
preservation buffer solution: 20mM PBS, 0.5% protamine, 0.5% p-methylpropylamine, 0.75% sarcosine, 0.3% phenylalanine, 0.05% SDS, 5% chitosan, 0.1% NaN3，pH7.5。
Example 1:
(1) labeling the antibody with magnetic beads:
centrifuging 20mg1.5um magnetic beads at 15000rpm for 10min → precipitating, adding 1ml of crosslinking buffer I, centrifuging at 15000rpm for 10min → dissolving and precipitating with 1ml of crosslinking buffer I, centrifuging at 15000rpm for 10min → taking precipitate 1 after 2min ultrasonic treatment;
② adding 1.8ml of crosslinking buffer solution I into the precipitate 1, carrying out ultrasonic treatment for 4min, adding 200ul EDC (10mg/ml), mixing uniformly, and shaking for 30min at room temperature;
thirdly, taking the activated substance, separating the magnetic beads for 4min by using a magnetic plate, and discarding the supernatant;
adding 200ug of BNP monoclonal antibody (firstly diluting the antibody, adding magnetic beads in the same volume), fixing the volume to 2ml, and incubating at room temperature for 3 h;
fifthly, taking the hatching fluid, centrifuging for 10min at 15000rpm, discarding the supernatant, adding 1ml of 2% 3-thiopropionic acid and 1ml of 1% aminoguanidine hemisulfate for resuspension, and shaking for 1h at 40 ℃.
Sixthly, separating the magnetic beads by using a magnetic plate for 1min, discarding the supernatant, adding 1ml of crosslinking buffer solution III, mixing and washing the magnetic beads, separating the magnetic beads by using the magnetic plate in the period, finally preserving the magnetic beads by using 4ml of preservation buffer solution with the concentration of 5mg/ml, and diluting the magnetic beads by using a washing solution for 10 times when in use.
(2) Acridinium ester labeled antibody
Mixing 200ug of monoclonal antibody with 20ug of acridinium ester (mixed in an equal volume ratio) in a shaking table, incubating at room temperature in the dark for 2h, ultrafiltering the mixed solution to obtain a crosslinking buffer solution II, and storing with 100ul of crosslinking buffer solution IV at a concentration of 2 mg/ml. When in use, the solution is diluted by 10000 times with a cleaning solution.
(3) Luminescence detection
Preparing BNP calibrators with the concentrations of 3100pg/ml, 516pg/ml, 86pg/ml, 14.3pg/ml, 2.39pg/ml and 0 pg/ml;
adding 100ul BNP sample into a reaction cup, adding 100ul acridinium ester labeled monoclonal antibody mixed solution, incubating at 37 ℃ for 6.3min, adding 200ul magnetic bead labeled monoclonal antibody mixed solution, and incubating at 37 ℃ for 3.0 min;
then separating and absorbing, cleaning the reaction cup with cleaning solution, adding 300ul of excitation liquid A and B, exciting the chemiluminescence reaction, and collecting signals.
The BNP calibrators with the concentrations of 3100pg/ml, 516pg/ml, 86pg/ml, 14.3pg/ml, 2.39pg/ml and 0pg/ml were respectively detected according to the preparation process.
Comparative example 1:
the difference from example 1 is only that the magnetic beads for activating the cross-linked BNP monoclonal antibody are not blocked in the process of labeling the antibody with the magnetic beads.
Comparative example 2:
the difference from example 1 is only that the treated magnetic beads which activate the crosslinked BNP monoclonal antibody are blocked, washed, separated and then stored in a common buffer. The buffer solution comprises the following components: 20mM PBS, 0.1% NaN3，pH7.5。
Table 1: example 1BNP calibration Curve data
| Calibrator | Signal value |
| pg/ml | RLU |
| 3100 | 3633770 |
| 516 | 615588 |
| 86 | 100221 |
| 14.3 | 16112 |
| 2.39 | 2994 |
| 0 | 354 |
Table 2: comparative example 1BNP calibration curve data
| Calibrator | Signal value |
| pg/ml | RLU |
| 3100 | 3400600 |
| 516 | 577206 |
| 86 | 104461 |
| 14.3 | 14572 |
| 2.39 | 5677 |
| 0 | 5024 |
As can be seen from the results in tables 1 and 2, the results in example 1 are more desirable in terms of distinguishing the low value of BNP standard, 2.39pg/ml, from the 0-value blank, and at 0, the particle background is lower after blocking with aminoguanidine hemisulfate and 3-thiopropionic acid.
Table 3: results of performance tests on the magnetic bead mixtures of example 1 and comparative example 1 after 3, 6, 9, and 12 months of storage
As can be seen from FIGS. 1 and 2, the phenomenon of wall hanging and agglomeration of the magnetic beads without the blocking treatment is obvious. The data from the magnetic bead test in conjunction with Table 3 shows that the stability of the beads was better after blocking with aminoguanidine hemisulfate and 3-thiopropionic acid.
Table 4: results of performance tests of the magnetic bead mixed solution of example 1 and comparative example 2 after 3, 6, 9 and 12 months of storage
As can be seen from the test results in Table 4, the optimized preservation buffer solution of the present invention still has good activity of the magnetic bead mixed solution after 12 months of preservation, and greatly improves the stability of the reagent, thereby improving the accuracy of the reagent detection result. Comparative example 2 the long-term preservation signal decreased all the time and the 0 value increased non-specifically.
Table 5: example 1 comparison of serum values with Yapei reagent assay
As can be seen from the results in table 5: the concentration of serum BNP is measured by the closed magnetic beads and is correlated with the concentration measured by yapei chemiluminescence, and the detection results of the BNP and the yapei chemiluminescence are found to have better consistency. Blocking the crosslinked particles with aminoguanidine hemisulfate and 3-thiopropionic acid gave results that are superior to conventional methods, whether the sample or serum sample was tested. The BNP standard curve is wider in linearity and better in background; compared with the detection result commonly used in the market, the serum detection has better accuracy.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (10)
1. A BNP chemiluminescence detection kit is characterized by comprising: the kit comprises a mixed magnetic bead solution coated with a BNP monoclonal antibody, a BNP calibrator solution, a BNP monoclonal antibody solution labeled with acridinium ester, a cleaning solution and an excitation solution.
2. The BNP chemiluminescence detection kit is characterized in that the preparation method of the magnetic bead mixed solution coated with the BNP monoclonal antibody comprises the following steps: activating the crosslinked BNP monoclonal antibody on the magnetic beads, sealing the magnetic beads of the activated crosslinked BNP monoclonal antibody, cleaning, separating and storing by using a storage buffer solution.
3. The BNP chemiluminescent detection kit according to claim 2, wherein 3-mercaptopropionic acid and aminoguanidine hemisulfate solution are used in the blocking treatment.
4. The BNP chemiluminescence detection kit according to claim 3, wherein the mass ratio of the 3-mercaptopropionic acid to the aminoguanidine hemisulfate is (1-3): 1.
5. the BNP chemiluminescence detection kit according to claim 2, wherein the storage buffer composition comprises: 10-50mM PBS, 0.3-1% protamine, 0.3-1% para-methylpropylamine, 0.3-1% sarcosine, 0.3-1% phenylalanine, 0.02-0.5% SDS, 1-10% chitosan, 0.05-0.15% NaN3The pH was 7.5.
6. The chemical light-emitting BNP detection kit according to claim 1, wherein the preparation method of the acridinium ester labeled monoclonal antibody solution comprises the following steps: and mixing the BNP monoclonal antibody and acridinium ester for incubation, and storing after ultrafiltration.
7. The BNP chemiluminiscence detection kit according to claim 2, wherein the cross-linking agent in the process of activating the cross-linked BNP monoclonal antibody is EDC.
8. The BNP chemiluminescence detection kit according to claim 1, wherein the cleaning solution comprises: 8-15mM/L PBS, 0.02-0.06% TW-20, 0.01-0.02% NaN3The pH was 7.4.
9. The BNP chemiluminescence detection kit according to claim 1, wherein the excitation liquid is A and B, wherein A comprises: 0.05-0.2M/L HNO3、0.05-0.2％H2O2(ii) a The component B comprises: 0.1-0.3M/L NaOH, 1-3% TritonX-100.
10. The application of the BNP chemiluminescence detection kit of claim 1, wherein a sample is added with a BNP monoclonal antibody solution labeled with acridinium ester for incubation, then a magnetic bead mixed solution coated with the BNP monoclonal antibody is added for incubation, separation and cleaning are carried out, and finally an excitation liquid is added for chemiluminescence reaction, and signals are collected.
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