CN111912838B - BNP chemiluminescence detection kit and application thereof - Google Patents
BNP chemiluminescence detection kit and application thereof Download PDFInfo
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- CN111912838B CN111912838B CN202010592531.XA CN202010592531A CN111912838B CN 111912838 B CN111912838 B CN 111912838B CN 202010592531 A CN202010592531 A CN 202010592531A CN 111912838 B CN111912838 B CN 111912838B
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Abstract
The invention relates to a BNP chemical light-emitting detection kit and application thereof, and belongs to the technical field of medical detection. According to the invention, the binding sites of the uncrosslinked antibodies on the magnetic beads are blocked through blocking treatment, so that not only is the strong false positive of the magnetic beads avoided, but also the blocking and wall attachment phenomena in the detection process are reduced, thereby achieving the purposes of optimizing the step of labeling the antibodies by the magnetic beads and improving the reagent performance; the invention optimizes the components and conditions of the magnetic bead preservation working solution, and prolongs the stability and preservation time of the reagent.
Description
Technical Field
The invention relates to a BNP chemiluminescence detection kit and application thereof, and belongs to the technical field of medical detection.
Background
BNP (brain natriuretic peptide) is a polypeptide neurohormone, has the functions of natriuretic, vasodilating and renin resisting, can be used as an effective marker of myocardial injury, predicts the ventricular pressure increase condition, and has important value in auxiliary diagnosis, treatment, evaluation and detection of congestive heart failure and dyspnea complications caused by the congestive heart failure, primary hypertension, evaluation of cardiovascular related diseases such as left ventricular dysfunction and the like.
After stimulation of the cardiomyocytes, pro-B-type natriuretic peptide precursors (Pre-proBNP) containing 134 amino acids are produced, followed by formation of BNP precursors (proBNP) containing 108 amino acids, which are cleaved by endonucleases into NT-proBNP containing 76 amino acids, which is biologically inactive, and B-type natriuretic peptide (BNP) containing 32 amino acids, which is active. 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 20min.
In the detection process of the BNP chemiluminescent detection reagent, the magnetic bead labeled antibody (particularly after the antibody is added) is easy to agglomerate and adhere to the wall, so that the operation time and the difficulty are increased, and when a blank sample is detected, non-specific adsorption can occur, so that the sensitivity is reduced. In addition, the magnetic bead labeled antibody can be subjected to caking, wall attachment and sedimentation after long-term storage, so that the stability of the reagent is greatly influenced.
Disclosure of Invention
Aiming at the existing problems of the detection method, the invention provides the BNP chemiluminescence detection kit and the application thereof, solves the problems that the magnetic bead labeled antibody is easy to agglomerate and adhere to the wall, and reduces the operation time and difficulty.
The aim of the invention is realized by the following technical scheme:
a BNP chemiluminescent detection kit, comprising: the BNP monoclonal antibody-coated magnetic bead mixed solution, BNP calibrator solution, acridinium ester marked BNP monoclonal antibody solution, cleaning solution and excitation solution.
The invention provides a BNP chemiluminescence detection kit, a preparation method and application thereof. Mixing biological sample (serum, plasma, whole blood), acridine ester marked BNP monoclonal antibody solution and magnetic bead marked BNP monoclonal antibody solution, fixing the mixture of double antibody sandwich under the action of magnetic field, cleaning and discarding the rest acridine ester marked antibody, adding light promoter, exciting chemical reaction, and collecting light signal.
In the BNP chemiluminescence detection kit, the preparation method of the magnetic bead mixed solution coated with the BNP monoclonal antibody comprises the following steps: firstly, activating and crosslinking BNP monoclonal antibody is carried out on the magnetic beads, then, sealing treatment is carried out on the magnetic beads of the activating and crosslinking BNP monoclonal antibody, and the magnetic beads are preserved by a preservation buffer solution after washing and separation.
In one of the above-mentioned BNP chemiluminescent detection kit, 3-mercaptopropionic acid and aminoguanidine hemisulfate solution are used in the blocking treatment.
In the BNP chemiluminescent detection kit, the mass ratio of 3-mercaptopropionic acid to aminoguanidine hemisulfate is (1-3): 1.
after the magnetic beads are crosslinked with antibodies, the magnetic beads are subjected to aftertreatment by aminoguanidine hemisulfate and 3-azulene propionic acid, on the one handThe binding sites of the uncrosslinked antibodies on the magnetic beads are 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 agglomeration and wall attachment phenomena are greatly reduced, and the serum nonspecific adsorption can be weakened. After the carboxyl magnetic beads are activated and crosslinked, some sites are possibly not bound by the antibody (the antibody is Y-shaped and has steric hindrance), and the amino guanidine hemisulfate and the 3-azulene propionic acid have small molecular space structures, so that the sites can be blocked. On the other hand by-NH on aminoguanidine hemisulfate 2 Reacts with-COOH groups on 3-azyl propionic acid to form a net-shaped polar monolayer, and in the preservation buffer solution, the magnetic bead bands have the same electrical property and mutually repulsive effect, so that agglomeration and wall attachment are not facilitated.
In the above-mentioned BNP chemiluminescent detection kit, the preservation buffer comprises the following components: 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 and 0.05-0.15% NaN 3 The pH was 7.5.
In the BNP chemiluminescent detection kit, the preparation method of the acridinium ester marked BNP monoclonal antibody solution comprises the following steps: BNP monoclonal antibody and acridinium ester are mixed and incubated, and the mixture is preserved after ultrafiltration.
In the BNP chemiluminescent detection kit, the cross-linking agent in the process of activating and cross-linking the BNP monoclonal antibody is EDC.
In one of the above-described chemiluminescent detection kit for BNP, the washing solution comprises: 8-15mM/L PBS, 0.02-0.06% TW-20, 0.01-0.02% NaN 3 The pH was 7.4.
In the above-mentioned kit for detecting BNP by chemiluminescence, the excitation solution is A and B, wherein the component A comprises: 0.05-0.2M/L HNO 3 、0.05-0.2％H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the The component B comprises the following components: 0.1-0.3M/L NaOH, 1-3% TritonX-100.
The BNP chemiluminescent detection kit is applied, wherein acridinium ester marked monoclonal antibody solution is added into a sample for incubation, then magnetic bead mixed solution coated with BNP monoclonal antibody is added for incubation, separation and cleaning are carried out, finally excitation solution is added for chemiluminescent reaction, and signals are collected.
Compared with the prior art, the invention has the following advantages:
according to the invention, the binding sites of the uncrosslinked antibodies on the magnetic beads are blocked through blocking treatment, so that not only is the strong false positive of the magnetic beads avoided, but also the blocking and wall attachment phenomena in the detection process are reduced, thereby achieving the purposes of optimizing the step of labeling the antibodies by the magnetic beads and improving the reagent performance; the invention optimizes the components and conditions of the magnetic bead preservation working solution, and prolongs the stability and preservation time of the reagent.
Drawings
FIG. 1 shows the state of the magnetic bead mixture after 6 months of storage in a closed state.
FIG. 2 shows the state of the bead mixture after 6 months of storage without sealing.
Detailed Description
The following are specific examples of the present invention, and the technical solutions of the present invention are further described, but the present invention is not limited to these examples.
For convenience of description, 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;
cross-linking buffer III is TBS-T:25mM Tris, 0.9% NaCl, 0.05% TW-20, pH 7.2;
crosslinking buffer IV:10mM/L PBS, pH7.4;
cleaning liquid: 10mM/L PBS, 0.05% TW-20, 0.01% NaN 3 ，PH 7.4；
Excitation liquid a:0.1M/L HNO 3 、0.1％H 2 O 2 ；
Excitation liquid B:0.25M/L NaOH, 2% TritonX-100;
preservation buffer: 20mM PBS,0.5% protamine, 0.5% para-methylpropylamine, 0.75% sarcosine, 0.3% phenylalanine, 0.05% SDS,5% chitosan, 0.1% NaN 3 ，pH7.5。
Example 1:
(1) Magnetic bead labeled antibodies:
(1) taking 20mg of 1.5um magnetic beads, centrifuging at 15000rpm for 10min, adding 1ml of crosslinking buffer solution I, centrifuging at 15000rpm for 10min, dissolving the precipitate with 1ml of crosslinking buffer solution I, centrifuging at 15000rpm for 10min after 2min of ultrasonic treatment, and taking precipitate 1;
(2) adding 1.8ml of crosslinking buffer solution I into the precipitate 1, performing ultrasound for 4min, adding 200ul EDC (10 mg/ml), mixing, and shaking at room temperature for 30min;
(3) separating the activated substance with magnetic plate for 4min, and discarding supernatant;
(4) adding 200ug BNP monoclonal antibody (diluting antibody first, adding magnetic beads in equal volume), fixing volume to 2ml, and incubating for 3h at room temperature;
(5) the hatching fluid was centrifuged at 15000rpm for 10min, the supernatant was discarded, 1ml of 2% 3-azulene and 1ml of 1% aminoguanidine hemisulphate were added for resuspension, and shaking was performed at 40℃for 1h.
(6) Separating magnetic beads with magnetic plate for 1min, discarding supernatant, adding 1ml crosslinking buffer solution III, mixing and cleaning magnetic beads, separating magnetic beads with magnetic plate during the period, and finally storing magnetic beads with 4ml preservation buffer solution with concentration of 5mg/ml, and diluting with cleaning solution for 10 times.
(2) Acridinium ester labeled antibody
200ug of monoclonal antibody and 20ug of acridinium ester are mixed (mixed in the proportion of equal volume) in a shaking table, incubated for 2 hours at room temperature and away from light, the mixed solution is ultrafiltered, the ultrafiltered cleaning solution is crosslinking buffer II, and finally 100ul of crosslinking buffer IV is used for storage, and the concentration is 2mg/ml. When in use, the cleaning solution is diluted 10000 times for use.
(3) Luminescence detection
BNP calibrator with concentration of 3100pg/ml, 516pg/ml, 86pg/ml, 14.3pg/ml, 2.39pg/ml, 0pg/ml was prepared;
adding 100ul BNP sample into a reaction cup, adding 100ul of acridinium ester marked monoclonal antibody mixed solution, incubating for 6.3min at 37 ℃, adding 200ul of magnetic bead marked monoclonal antibody mixed solution, and incubating for 3.0min at 37 ℃;
then separating and sucking, cleaning the reaction cup with cleaning liquid, adding 300ul of excitation liquid A and B, exciting chemiluminescence reaction, and collecting signals.
BNP calibrator concentrations of 3100pg/ml, 516pg/ml, 86pg/ml, 14.3pg/ml, 2.39pg/ml, 0pg/ml were tested according to the preparation procedure described above.
Comparative example 1:
the only difference from example 1 is that the magnetic beads of the activated cross-linked BNP monoclonal antibody were not subjected to the blocking treatment during the labeling of the antibodies by the magnetic beads.
Comparative example 2:
the only difference from example 1 is that the magnetic beads of the activated cross-linked BNP monoclonal antibody after blocking treatment are washed and separated and stored in a common buffer. The buffer composition comprises: 20mM PBS,0.1% NaN 3 ，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 |
From the results in tables 1 and 2, it can be seen that the results of example 1 are more optimal in terms of the low value 2.39pg/ml versus the 0 value blank discrimination for the BNP standard, and that the particle background after blocking treatment with aminoguanidine hemisulphate and 3-coloured base propionic acid is lower at the 0 value.
Table 3: example 1 and comparative example 1 results of Performance test of magnetic bead mixtures after 3, 6, 9, 12 months of storage, respectively
As can be seen from FIGS. 1 and 2, the wall built-up and agglomeration of the beads without the sealing treatment are evident. The bead test data in combination with Table 3 demonstrate better stability of the beads after blocking with aminoguanidine hemisulfate and 3-cysteic acid.
Table 4: example 1 and comparative example 2 magnetic bead mixtures after 3, 6, 9, 12 months of storage
As can be seen from the test results in Table 4, the optimized preservation buffer solution still has good activity after being preserved for 12 months, and greatly improves the stability of the reagent, thereby improving the accuracy of the reagent detection result. Comparative example 2 shows that the long-term preservation signal is always decreasing and the value 0 is nonspecifically increasing.
Table 5: example 1 serum value comparison results with the yabang reagent test
From the results in table 5, it can be seen that: the concentration of serum BNP is measured by using the closed magnetic beads, and the correlation is made between the concentration measured by the Atlantic chemiluminescence, so that the detection results of the BNP and the Atlantic chemiluminescence have better consistency. Whether the sample or serum sample is tested, the blocking of the crosslinked particles with aminoguanidine hemisulfate and 3-zemopropionic acid gives results that are advantageous over conventional methods. BNP standard curve is wider in linearity and better in background; serum detection has better accuracy than the detection results commonly used in the market.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the invention pertains or may be substituted in a similar manner without departing from the spirit of the invention or beyond the scope of 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 (8)
1. A BNP chemiluminescent detection kit, comprising: a magnetic bead mixed solution coated with BNP monoclonal antibody, BNP calibrator solution, acridinium ester marked BNP monoclonal antibody solution, cleaning solution and excitation solution;
the preparation method of the magnetic bead mixed solution for coating the BNP monoclonal antibody comprises the following steps: firstly, activating and crosslinking BNP monoclonal antibody is carried out on magnetic beads, then sealing treatment is carried out on the magnetic beads of the activated and crosslinked BNP monoclonal antibody, and the magnetic beads are preserved by a preservation buffer solution after washing and separation;
the blocking treatment uses 3-mercaptopropionic acid and aminoguanidine hemisulfate solution.
2. The BNP chemiluminescent detection kit of claim 1 wherein the mass ratio of 3-mercaptopropionic acid to aminoguanidine hemisulfate is (1-3): 1.
3. according to claimThe BNP chemiluminescent detection kit of claim 1 wherein the preservation 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% NaN 3 The pH was 7.5.
4. The BNP chemiluminescent detection kit of claim 1 wherein the acridinium ester-labeled monoclonal antibody solution is prepared by a method comprising: BNP monoclonal antibody and acridinium ester are mixed and incubated, and the mixture is preserved after ultrafiltration.
5. The BNP chemiluminescent assay kit of claim 1, wherein the cross-linking agent in the process of activating the cross-linked BNP monoclonal antibody is EDC.
6. The BNP chemiluminescent assay kit of claim 1, wherein the washing solution comprises: 8-15mM/L PBS, 0.02-0.06% TW-20, 0.01-0.02% NaN 3 The pH was 7.4.
7. The BNP chemiluminescent detection kit of claim 1, wherein the excitation fluid is a and B, wherein a comprises: 0.05-0.2M/L HNO 3 、0.05-0.2% H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the The component B comprises the following components: 0.1-0.3M/L NaOH, 1-3% TritonX-100.
8. Use of the BNP chemiluminescent detection kit of claim 1 wherein the solution of the acridinium ester-labeled BNP monoclonal antibody is added to the sample for incubation, then the mixed solution of magnetic beads coated with the BNP monoclonal antibody is added for incubation, separation and washing are performed, and finally the excitation solution is added for chemiluminescent reaction, and signals are collected.
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