CN118243936A - N-terminal brain natriuretic peptide precursor detection kit - Google Patents

N-terminal brain natriuretic peptide precursor detection kit Download PDF

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CN118243936A
CN118243936A CN202410659250.XA CN202410659250A CN118243936A CN 118243936 A CN118243936 A CN 118243936A CN 202410659250 A CN202410659250 A CN 202410659250A CN 118243936 A CN118243936 A CN 118243936A
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natriuretic peptide
probnp
streptavidin
brain natriuretic
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李有生
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Guangzhou Rhfay Biological Medical Technology Co ltd
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Guangzhou Rhfay Biological Medical Technology Co ltd
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Abstract

The invention discloses an N-terminal brain natriuretic peptide precursor detection kit and a preparation method thereof, belonging to the technical field of biological detection. The kit comprises a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, magnetic particles coupled with streptavidin, a calibrator, a substrate solution and a cleaning solution, wherein the biotin-labeled NT-proBNP antibody and the Alkaline Phosphatase (AP) -labeled paired NT-proBNP antibody are combined with NT-proBNP in a sample, a calibrator or a quality control product to form a sandwich complex. And then adding the magnetic particles connected with the streptavidin, and combining the antigen-antibody immune complex on the magnetic particles through the specific combination of the streptavidin and the biotin, so that the detection sensitivity is high, the linear range is wide, the accuracy is high, the stability is good, and the development of N-terminal brain natriuretic peptide precursor detection is facilitated.

Description

N-terminal brain natriuretic peptide precursor detection kit
Technical Field
The invention relates to the technical field of biological detection, in particular to an N-terminal brain natriuretic peptide precursor physical examination test kit.
Background
N-terminal brain natriuretic peptide precursor (NT-proBNP) is mainly derived from ventricular myocytes and is generated after digestion of B-type Brain Natriuretic Peptide (BNP), and can be divided and increased when the load of the cardiac myocytes is increased, so that the concentration of NT-proBNP in blood is closely related to the severity of Heart Failure (HF), and has important clinical significance for the prediction of congestive Heart failure risk stage, the treatment and the estimated evaluation of cardiac sudden death and the monitoring of Heart failure; meanwhile, the method has important clinical guidance significance for differential diagnosis of cardiac and non-cardiac acute dyspnea.
At present, domestic researches on the technology of NT-proBNP detection products are still in a starting stage, and methods for detecting NT-proBNP mainly comprise an enzyme-linked immunosorbent assay (ELISA), an electrochemiluminescence method, a radioimmunoassay, a colloidal gold method and the like, wherein the electrochemiluminescence method has strong specificity and high sensitivity, but expensive instruments and equipment are required during detection, and the requirements are met for operators; the radioimmunoassay has radioactive pollution, and the reagent has short validity period; the colloidal gold method is simple in detection, but only can be used for qualitative test, and has low sensitivity. For this reason, the development of NT-proBNP detection technology is favored if a product for accurately and rapidly quantitatively detecting NT-proBNP can be developed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention designs a kit which can accurately and quantitatively detect the N-terminal brain natriuretic peptide precursor.
In order to achieve the above purpose, the technical solution of the present invention is as follows:
In one aspect, the invention provides an N-terminal brain natriuretic peptide precursor detection kit, which comprises a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, streptavidin-coupled magnetic particles, a calibrator, a substrate solution and a cleaning solution;
wherein the magnetic particles are zinc iron oxide, and the particle size is 0.5-3 mu m.
Preferably, the preparation of the kit comprises: firstly preparing a diluent 1, then respectively preparing magnetic particles coupled with streptavidin and an alkaline phosphatase-marked NT-proBNP antibody solution by using the diluent 1, and then preparing a biotin-marked NT-proBNP antibody.
Preferably, the diluent 1 comprises 1-4% bovine serum albumin, 0.01-0.1% ProClin, 120-170 mmol/L NaCl, 0.2-0.7% Tween-20, 0.5-2% trehalose, 2-7% sucrose, 0.5-2% sodium citrate, 0.5-2% threonine, 0.5-2% ethanol, 5-15mmol/L tris, and pH 7.0-7.8.
Preferably, the diluent 1 comprises 3% bovine serum albumin, 0.05% ProClin, 150 mmol/L NaCl, 0.5% Tween-20, 1% trehalose, 5% sucrose, 1% sodium citrate, 1% threonine, 1% ethanol, 10 mmol/L tris, pH 7.5.
Preferably, the preparation of the streptavidin-coupled magnetic particles comprises: re-suspending the magnetic particles with a pH5.0 buffer solution, then adding 5-15mg/mL activator to activate for 0.5-2h, adding streptavidin to react for 1-3h at room temperature, washing after magnetically separating supernatant, adding a sealing agent to seal for 0.5-2h, and then adding a diluent 1 to perform magnetic attraction to prepare the magnetic particles coupled with the streptavidin;
The blocking agent is phosphate buffer solution with pH of 7.0-7.8 and containing 2-8% BSA and 0.1% ProClin300,300.
Preferably, the activator is one or more of carbodiimide hydrochloride, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide, suberic acid imine, N-hydroxy inositol, succinic anhydride and N, N' -dicyano-N, N- (dimethylformamide) guanidine.
Preferably, the alkaline phosphatase-labeled NT-proBNP antibody solution: preparing 10-15 mg/mL alkaline phosphatase solution by using 0.02-0.1M buffer solution, adding 100-150 mu L K 2Cr2O7 solution for reaction for 1-3h, adding 100-150 mu L N-methyl pyrrolidone solution for reaction for 1-3h, adding antibody, uniformly mixing, dialyzing for 20-28h, adding 8-15 mu L NaBH 4 solution for reaction for 1-3h, purifying, and finally diluting by using a diluent 1 for 1000 times to obtain the alkaline phosphatase marked NT-proBNP antibody solution.
Preferably, the biotin-labeled NT-proBNP antibody comprises: adding the antibody into p H to 7.5 to 0.02 to 0.1M buffer solution, adding 15 to 25mg/mL biotin solution, standing at room temperature for 4 to 8 hours, purifying, collecting the purified solution, adding 0.05 to 0.2M of 4.0 to 5.0 of citric acid buffer solution, and diluting to 0.2 to 1mg/mL.
On the other hand, the invention also provides application of the N-terminal brain natriuretic peptide precursor detection kit in detecting the N-terminal brain natriuretic peptide precursor.
Compared with the prior art, the invention has the following beneficial effects:
The invention prepares an N-terminal brain natriuretic peptide precursor detection kit, which comprises a biotin-marked NT-proBNP antibody, an alkaline phosphatase-marked NT-proBNP antibody, streptavidin-coupled magnetic particles, a calibrator, substrate liquid and a cleaning solution. The kit disclosed by the invention utilizes the specific combination of streptavidin and biotin, has the advantages of high detection sensitivity, wide linear range, high accuracy and good stability, and is favorable for the development of N-terminal brain natriuretic peptide precursor detection. And the sample does not need to be diluted when in use, can be automatically detected, has wide application prospect in the aspects of clinical examination, medication guidance and the like, and fills the blank of the detection field of N-terminal brain natriuretic peptide precursor by combining streptavidin and biotin with chemical immunofluorescence technology.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard graph of the N-terminal brain natriuretic peptide precursor detection kit of example 1.
FIG. 2 is a standard graph of the N-terminal brain natriuretic peptide precursor detection kit of example 2.
FIG. 3 is a standard graph of the N-terminal brain natriuretic peptide precursor detection kit of example 3.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The kit adopts the principle of a direct sandwich method, takes magnetic microparticles as a solid phase of immune reaction, and utilizes a chemiluminescent enzyme-linked immunoassay method to be matched with a chemiluminescent type measuring instrument for detecting the NT-proBNP content in human serum and plasma blood samples.
The technical principle is as follows: the biotin-labeled NT-proBNP antibody is combined with an Alkaline Phosphatase (AP) -labeled paired NT-proBNP antibody to form a "sandwich" complex with NT-proBNP in a sample, calibrator or quality control. Subsequently, magnetic microparticles to which streptavidin is attached are added, and antigen-antibody immune complexes are bound to the magnetic microparticles by specific binding of streptavidin to biotin. Under the action of an externally applied magnetic field, separating the compound formed by the immune reaction from other unbound substances, cleaning the compound, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, and photons are emitted when the excited state intermediate returns to the ground state to form a luminescence reaction, so that the luminescence intensity of the reaction can be detected by using a chemiluminescent instrument. The concentration of NT-proBNP in the sample can be calculated by fitting using a fitting equation in which the luminescence intensity is proportional to the content of NT-proBNP in the sample in the measurement wavelength (230-700 nm).
Example 1
An N-terminal brain natriuretic peptide precursor detection kit comprising a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, magnetic microparticles conjugated with streptavidin, a calibrator, a substrate solution (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy-ethylene disodium salt), a washing solution (Tris buffer, pH 8.0). The specific preparation is as follows:
1. Dilution 1:3% Bovine Serum Albumin (BSA), 0.05% ProClin% 300, 150 mmol/L NaCl, 0.5% Tween-20, 1% trehalose, 5% sucrose, 1% sodium citrate, 1% threonine, 1% ethanol, 10 mmol/L Tris (Tris), and adjusting pH to be stable at 7.5.
2. Magnetic microparticles coupled with streptavidin:
Taking 10mg of magnetic particles (1.0 mu m, zinc oxide iron) for magnetic separation for 1min, removing supernatant, re-suspending by 1mL of 0.1M MES buffer solution (pH is 5.0), adding 120 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the concentration of 10mg/mL for activation for 1h, adding 1mg of streptavidin for reaction for 2h at room temperature, washing 3 times by using a washing liquid after the supernatant is removed by magnetic separation for 1min, adding 1mL of phosphate buffer solution with the pH of 7.5 and containing 5% BSA and 0.1% ProClin for sealing for 1h at room temperature, absorbing magnetism for 10. 10 min in a magnetic frame, removing supernatant, repeatedly absorbing magnetism by using diluent 1, fixing the volume to 500 mL, and storing at 4 ℃ to obtain the magnetic particles coupled with the streptavidin.
3. Alkaline phosphatase-labeled NT-proBNP antibody solution:
(1) 2mg alkaline phosphatase was dissolved in 120. Mu.L of 0.05M carbonate buffer (p H =9.0) to a concentration of 12 mg/mL, then a solution of 120. Mu. L K 2Cr2O7 was added, vortexed and mixed, and reacted at 4℃in the absence of light for 2 h, then a solution of 120. Mu. L N-methylpyrrolidone was added, reacted at 4℃in the absence of light for 2 h, then 2mg of NT-proBNP antibody was added, vortexed and mixed, and transferred to a 3.5K Slide-A-Lyzer ™ microdialysis device, and the 0.05M carbonate buffer (p H =9.5) was dialyzed at 4℃in the absence of light for 24 h.
(2) After the dialysis was completed, 10. Mu.L of Na BH4 solution was added and the reaction was carried out at 4℃for 2 hours in the absence of light, and finally, unbound alkaline phosphatase and rabbit anti-pig Ig GNT-proBNP antibody were purified.
(3) Alkaline phosphatase-labeled NT-proBNP antibodies were subjected to 1000-fold dilution with the prepared dilution 1.
4. Biotin-labeled NT-proBNP antibodies:
5mg of NT-proBNP antibody was added to 100. Mu.L of 0.05M PBS buffer (p H =7.2), followed by 35mL of biotin solution (a mixed solution of 20mg/mL biotin in water N, N-dimethylformamide) and mixed well at room temperature, and left to stand for 6 hours, followed by purification by a protein purification instrument, and the purified solution was collected and diluted to 0.5mg/mL by adding 0.1M of 4.2 pH citrate buffer.
Example 2
An N-terminal brain natriuretic peptide precursor detection kit comprising a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, magnetic microparticles conjugated with streptavidin, a calibrator, a substrate solution (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy-ethylene disodium salt), a washing solution (Tris buffer, pH 8.0). The specific preparation is as follows:
1. Dilution 1:1% Bovine Serum Albumin (BSA), 0.1% ProClin% 300, 170mmol/L NaCl, 0.2% Tween-20, 0.5% trehalose, 7% sucrose, 0.5% sodium citrate, 0.5% threonine, 2% ethanol, 15 mmol/L Tris, and the pH was adjusted to be stable at 7.0.
2. Magnetic microparticles coupled with streptavidin:
Taking 10mg of magnetic particles (0.5 mu m, zinc oxide iron) for magnetic separation for 1min, removing supernatant, re-suspending by using 1mL of 0.1M MES buffer solution (pH is 5.0), adding 120 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the concentration of 5mg/mL for activation for 2h, adding 1mg of streptavidin for reaction for 3h at room temperature, washing 3 times by using a washing liquid after the supernatant is removed by magnetic separation for 1min, adding 1mL of phosphate buffer solution with the pH of 7.0 and the pH of 8% BSA and 0.1% ProClin for sealing for 2h at room temperature, absorbing magnetism for 10. 10 min in a magnetic frame, removing supernatant, repeatedly absorbing magnetism by using a diluent 1, fixing the volume to 500 mL, and storing at 4 ℃ to obtain the magnetic particles coupled with the streptavidin.
3. Alkaline phosphatase-labeled NT-proBNP antibody solution:
(1) The alkaline phosphatase 2mg was dissolved in 120. Mu.L of 0.1M carbonate buffer (p H =9.0) to a concentration of 10mg/mL, then 100. Mu. L K 2Cr2O7 solution was added and vortexed to react 1h at 4℃in the absence of light, then 150. Mu. L N-methylpyrrolidone solution was added and reacted 3h at 4℃in the absence of light, then 2mg of NT-proBNP antibody was added and vortexed to transfer to a 3.5K Slide-A-Lyzer ™ microdialysis unit, and the carbonate buffer 0.05M (p H =9.5) was dialyzed 20 h at 4℃in the absence of light.
(2) After the dialysis was completed, 8. Mu.L of NaBH 4 solution was added and reacted at 4℃in the absence of light for 1 hour, and finally unbound alkaline phosphatase and rabbit anti-pig Ig GNT-proBNP antibody were purified.
(3) Alkaline phosphatase-labeled NT-proBNP antibodies were subjected to 1000-fold dilution with the prepared dilution 1.
4. Biotin-labeled NT-proBNP antibodies:
5mg of NT-proBNP antibody was added to 100. Mu.L of 0.02M PBS buffer (p H =7.0), followed by 35mL of biotin solution (15 mg/mL of biotin mixed solution prepared from water N, N-dimethylformamide) and mixed well at room temperature, and left to stand for 8 hours, followed by purification by a protein purification instrument, and the purified solution was collected and diluted to 0.2mg/mL by adding 0.2M of 4.0-p-H in citric acid buffer.
Example 3
An N-terminal brain natriuretic peptide precursor detection kit comprising a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, magnetic microparticles conjugated with streptavidin, a calibrator, a substrate solution (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy-ethylene disodium salt), a washing solution (Tris buffer, pH 8.0). The specific preparation is as follows:
1. Dilution 1:4% Bovine Serum Albumin (BSA), 0.01% ProClin% 300, 120 mmol/L NaCl, 0.7% Tween-20, 2% trehalose, 2% sucrose, 2% sodium citrate, 2% threonine, 0.5% ethanol, 5 mmol/L Tris (Tris), and the pH was adjusted to be stable at 7.8.
2. Magnetic microparticles coupled with streptavidin:
Taking 10mg of magnetic particles (3.0 mu m, zinc oxide iron) for magnetic separation for 1min to remove supernatant, re-suspending by using 1mL of 0.1M MES buffer solution (pH is 5.0), adding 120 mu L of N-hydroxysuccinimide with the concentration of 15mg/mL for activating for 0.5h, adding 1mg of streptavidin for reaction for 1h at room temperature, washing 3 times by using a washing liquid after the supernatant is removed for magnetic separation for 1min, adding 1mL of phosphate buffer solution with the pH of 7.8 and the concentration of 2% BSA and 0.1% ProClin for sealing for 0.5h at room temperature, carrying out magnetic attraction on the supernatant by using a magnetic frame for 10 min, removing the supernatant, repeatedly carrying out magnetic attraction by using a diluent 1, and keeping the constant volume at 500 and mL ℃ to obtain the magnetic particles coupled with the streptavidin.
3. Alkaline phosphatase-labeled NT-proBNP antibody solution:
(1) The alkaline phosphatase 2mg was dissolved in 120. Mu.L of 0.02M carbonate buffer (p H =9.0) to a concentration of 15mg/mL, then 150. Mu. L K 2Cr2O7 solution was added and vortexed to react for 3h at 4℃in the absence of light, then 100. Mu. L N-methylpyrrolidone solution was added and reacted for 1h at 4℃in the absence of light, then 2mg of NT-proBNP antibody was added and vortexed to transfer to a 3.5K Slide-A-Lyzer ™ microdialysis unit, and the carbonate buffer 0.05M (p H =9.5) was dialyzed for 28h at 4℃in the absence of light.
(2) After the dialysis was completed, 15. Mu.L of NaBH 4 solution was added and reacted at 4℃in the dark for 3 hours, and finally the unbound alkaline phosphatase and rabbit anti-pig Ig GNT-proBNP antibody were purified.
(3) Alkaline phosphatase-labeled NT-proBNP antibodies were subjected to 1000-fold dilution with the prepared dilution 1.
4. Biotin-labeled NT-proBNP antibodies:
5mg of NT-proBNP antibody was added to 100. Mu.L of 0.1M PBS buffer (p H =7.5), followed by 35mL of biotin solution (a mixed solution of 25mg/mL biotin in water N, N-dimethylformamide) and mixed well at room temperature, and left to stand for 4 hours, followed by purification with a protein purifier, and the purified solution was collected and diluted to 1mg/mL by adding 0.05M of 5.0-P in citric acid buffer.
Comparative example 1
Comparative example 1 differs from example 1 in that the diluent 1 was replaced with pure water of the same quality, all the other things being equal.
Comparative example 2
Comparative example 2 differs from example 1 in that diluent 1 was formulated differently, all the other things being equal.
Dilution 1:4% Bovine Serum Albumin (BSA), 0.01% ProClin% 300, 120 mmol/L NaCl, 0.7% Tween-20, 2% trehalose, 2% sucrose, 0.5% ethanol, 5 mmol/L Tris (hydroxymethyl) aminomethane (Tris), and adjusting pH to be stable at 7.5.
Comparative example 3
Comparative example 3 differs from example 1 in that diluent 1 was formulated differently, all the other things being equal.
Dilution 1:4% Bovine Serum Albumin (BSA), 0.01% ProClin% 300, 120 mmol/L NaCl, 0.7% Tween-20, 2% trehalose, 2% sodium citrate, 2% threonine, 5 mmol/L Tris (hydroxymethyl) aminomethane (Tris), and the pH was adjusted to be stable at 7.5.
Test example 1
The kits of examples 1-3 and comparative examples 1-3 were operated according to the chemiluminescent assay protocol.
Before a new kit is started for the first time, the kit is carefully inverted for a plurality of times, so that the magnetic beads are uniformly mixed, and the opened kit is not inverted. The calibrator/quality control product needs to be balanced to room temperature before detection. And placing the reagent bottle into an instrument reagent bin, placing a calibrator in an instrument sample bin, and placing a sample to be detected in the instrument sample bin.
1. Linear experiments
A calibrator dilution was prepared and pH was adjusted to 7.2 using 20% BSA, 150 mmol/L sodium chloride, 0.5% 3ProClin, 0.5% Tween-20, 1% trehalose, 20 mmol/L Tris, 10% glycerol. NT-proBNP was diluted with calibrator dilutions to prepare calibrators at concentrations of 30, 500, 2,000, 5000, 10000, 20000, 30000 pg/mL.
Adding 50 mu L of a sample to be detected, 50 mu L of biotin-labeled NT-proBNP antibody, 50 mu L of alkaline phosphatase-labeled NT-proBNP antibody and 20 mu L of streptavidin-coupled magnetic particles into an instrument, incubating for 10min at 37 ℃ and performing magnetic separation and cleaning; 200. Mu.L of the luminescent substrate 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphorus oxy) -phenyl-1, 2-dioxy-ethylene disodium salt (AMPPD) was added in a dark environment; the total relative photon quantity is read. And drawing a calibration curve, detecting a sample, and calculating the concentration through the calibration curve according to the relative luminescence value measured by the sample to be detected. And setting 3 repetitions for each concentration, taking an average value, taking the concentration as an abscissa and the fluorescence signal intensity test value as an ordinate, and establishing a fitting standard curve.
As shown in FIGS. 1-3, the kits of examples 1-3 had a good linear relationship between 30-30000pg/mL, with R values of 0.9969, 0.9908, 0.9901, respectively.
2. Precision experiments
20 Test cards were prepared in examples 1-3 and comparative examples 1-3, and were tested with 500, 2000pg/mL of serum containing NT-proBNP standard, respectively, the experiment was repeated three times, the average value and the standard value were calculated from the test results, and the coefficient of variation CV was calculated from the average value and the standard value, as follows:
；
The results show that the precision of comparative examples 1-3 is 8-10% in batch and 15-20% in batch, while the kit of examples 1-3 is less than or equal to 8% in batch and less than or equal to 15% in batch, indicating that the kit of the invention has the advantage of good reproducibility.
3. Stability test
The kits of examples 1-3 and comparative examples 1-3 were stored at 4℃and tested for NT-proBNP quality control at a concentration of 500pg/mL for 3 months, 6 months, 12 months, 20 months, respectively, 5 replicates each time. The results show that the relative deviation of the test results of the examples 1-3 is not more than +/-10%, and the relative deviation of the test results of the comparative examples 1-3 is more than +/-10%, which indicates that the stability of the kit is good.
Furthermore, the kit of the invention is useful in the case of the crossover material ANP:3.1 μg/mL, CNP:2.2 μg/mL, angiotensin I:0.6 ng/mL, angiotensin II:0.6 ng/mL, angiotensin III: 1ng/mL, epinephrine: 1ng/mL, no cross-reaction. Bilirubin is less than or equal to 20mg/dL, hemoglobin is less than or equal to 500mg/dL, triglyceride is less than or equal to 1500mg/dL, biotin is less than or equal to 30pg/mL, rheumatoid factor is less than or equal to 1500IU/mL, and human anti-mouse antibodies (HAMAs) are less than or equal to 30ng/mL in the sample to be detected, and the detection is not affected. The detection is carried out by using anticoagulants (EDTA and heparin) without interference. HOOK effect: no hook effect occurs at 300000 pg/mL.
In summary, the invention provides a kit for detecting N-terminal brain natriuretic peptide precursors and a preparation method thereof, wherein the kit comprises a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, magnetic particles coupled with streptavidin, a calibrator, substrate solution and a cleaning solution, and the biotin-labeled NT-proBNP antibody and the Alkaline Phosphatase (AP) -labeled paired NT-proBNP antibody are combined to form a sandwich compound with NT-proBNP in a sample, a calibrator or a quality control product. And then adding magnetic particles connected with streptavidin, combining an antigen-antibody immune complex on the magnetic particles through the specific combination of the streptavidin and the biotin, and thus, the method has the advantages of high detection sensitivity, wide linear range, high accuracy and good stability, is favorable for the development of N-terminal brain natriuretic peptide precursor detection, and fills the blank of the detection field of the N-terminal brain natriuretic peptide precursor by the streptavidin and biotin combined chemical immunofluorescence technology.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (9)

1. An N-terminal brain natriuretic peptide precursor test kit, which is characterized by comprising a biotin-labeled NT-proBNP antibody, an alkaline phosphatase-labeled NT-proBNP antibody, streptavidin-coupled magnetic particles, a calibrator, a substrate solution and a cleaning solution;
wherein the magnetic particles are zinc iron oxide, and the particle size is 0.5-3 mu m.
2. The N-terminal brain natriuretic peptide precursor test kit of claim 1, wherein the preparation of the kit comprises: firstly preparing a diluent 1, then respectively preparing magnetic particles coupled with streptavidin and an alkaline phosphatase-marked NT-proBNP antibody solution by using the diluent 1, and then preparing a biotin-marked NT-proBNP antibody.
3. The kit of claim 2, wherein the diluent 1 comprises 1-4% bovine serum albumin, 0.01-0.1% ProClin, 120-170 mmol/L NaCl, 0.2-0.7% tween-20, 0.5-2% trehalose, 2-7% sucrose, 0.5-2% sodium citrate, 0.5-2% threonine, 0.5-2% ethanol, 5-15mmol/L tris, and a pH of 7.0-7.8.
4. The kit of claim 3, wherein the diluent 1 comprises 3% bovine serum albumin, 0.05% ProClin, 150 mmol/L NaCl, 0.5% tween-20, 1% trehalose, 5% sucrose, 1% sodium citrate, 1% threonine, 1% ethanol, 10 mmol/L tris, pH 7.5.
5. The N-terminal brain natriuretic peptide precursor test kit of claim 2 wherein the preparation of the streptavidin-coupled magnetic microparticles comprises: re-suspending the magnetic particles with a pH5.0 buffer solution, then adding 5-15mg/mL activator to activate for 0.5-2h, adding streptavidin to react for 1-3h at room temperature, washing after magnetically separating supernatant, adding a sealing agent to seal for 0.5-2h, and then adding a diluent 1 to perform magnetic attraction to prepare the magnetic particles coupled with the streptavidin;
The blocking agent is phosphate buffer solution with pH of 7.0-7.8 and containing 2-8% BSA and 0.1% ProClin300,300.
6. The kit of claim 5, wherein the activator is one or more of carbodiimide hydrochloride, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide, suberic acid imine, N-hydroxystearinositol, succinic anhydride, and N, N' -dicyano-N, N- (dimethylformamide) guanidine.
7. The N-terminal brain natriuretic peptide precursor test kit of claim 2 wherein the alkaline phosphatase-labeled NT-proBNP antibody solution: preparing 10-15 mg/mL alkaline phosphatase solution by using 0.02-0.1M buffer solution, adding 100-150 mu L K 2Cr2O7 solution for reaction for 1-3h, adding 100-150 mu L N-methyl pyrrolidone solution for reaction for 1-3h, adding antibody, uniformly mixing, dialyzing for 20-28h, adding 8-15 mu L NaBH 4 solution for reaction for 1-3h, purifying, and finally diluting by using a diluent 1 for 1000 times to obtain the alkaline phosphatase marked NT-proBNP antibody solution.
8. The N-terminal brain natriuretic peptide precursor test kit of claim 2, wherein the biotin-labeled NT-proBNP antibody comprises: adding the antibody into p H to 7.5 to 0.02 to 0.1M buffer solution, adding 15 to 25mg/mL biotin solution, standing at room temperature for 4 to 8 hours, purifying, collecting the purified solution, adding 0.05 to 0.2M of 4.0 to 5.0 of citric acid buffer solution, and diluting to 0.2 to 1mg/mL.
9. Use of an N-terminal brain natriuretic peptide precursor detection kit according to any one of claims 1-8 for detecting an N-terminal brain natriuretic peptide precursor.
CN202410659250.XA 2024-05-27 2024-05-27 N-terminal brain natriuretic peptide precursor detection kit Pending CN118243936A (en)

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