CN115494234A

CN115494234A - B-type natriuretic peptide detection kit and preparation method thereof

Info

Publication number: CN115494234A
Application number: CN202211233965.6A
Authority: CN
Inventors: 王胜岚; 彭永林; 李峰; 彭梅
Original assignee: Zhongshan Bio Tech Co ltd
Current assignee: Zhongshan Bio Tech Co ltd
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2022-12-20

Abstract

The invention provides a B-type natriuretic peptide detection kit, which is used for detecting the content of B-type natriuretic peptide in human serum, plasma and whole blood by adopting a fluorescence immunochromatography principle, is mainly used for clinically assisting diagnosis of heart failure, and has the advantages of low background, long luminous life, strong fluorescence signal, high signal to noise ratio and the like by adopting a rare earth nano fluorescent microsphere to mark an antibody, wherein a rare earth complex is Eu (hfpd) 3phen, and the kit has the characteristics of a detection range frame, high sensitivity, high accuracy, quick and simple detection and the like, can be stably stored by reacting with free amino on the antibody after the surface of the fluorescent microsphere is activated, and can be used for quick detection.

Description

B-type natriuretic peptide detection kit and preparation method thereof

Technical Field

The invention belongs to the technical field of detection kits, and particularly relates to a B-type natriuretic peptide detection kit and a preparation method thereof.

Background

Type B Natriuretic Peptide (BNP) is a peptide hormone synthesized and secreted by cardiac myocytes, and is an important biological marker reflecting cardiac function. BNP is secreted mainly according to the cardiac function of the patient, when the cardiac function of the patient is abnormal, the level value of the BNP can be obviously improved, and the content of the BNP is in direct proportion to the pressure of the ventricles, the degree of dyspnea and the condition of a neurohormonal regulation system. In the middle of the twenty-first century, the american college of cardiology and the american college of cardiology take significant steps to improve the long-standing clinical testing standards, issue the latest guidelines for grouping and treating heart failure, and definitely use BNP as one of the most important markers. The dynamic observation of the change of the BNP content in vivo can provide reference for the development of clinical diagnosis and treatment work.

At present, the detection method aiming at the BNP mainly comprises an enzyme-linked immunosorbent assay, an electrochemiluminescence method, a radioimmunoassay, a colloidal gold immunochromatography and the like, and the enzyme-linked immunosorbent assay has poor quantitative accuracy, long operation time and low automation degree and is mainly used for qualitative detection; the sensitivity of the radioimmunoassay can reach 4pg/ml, the operation is simple, the measurement is accurate, and the defects are that the required time is long, and radioactive pollution and radiation danger exist; the electrochemical luminescence method has strong specificity, high sensitivity and high accuracy, but needs expensive instruments and equipment and operators with rich experience, and is generally used in specific medical institutions; although the colloidal gold immunochromatographic assay has the advantages of small sample dosage, simplicity, rapidness and low cost, the colloidal gold immunochromatographic assay has low sensitivity, generally can only be used for qualitative detection and cannot be used for quantitative detection, and particularly, the defect of poor repeatability limits the clinical application of the colloidal gold immunochromatographic assay, and is not particularly suitable for the quantitative detection of the humoral marker protein which needs to be used for assisting in the diagnosis of diseases through accurate quantification.

Therefore, a BNP measurement product with higher sensitivity, rapidness and convenience is needed.

Disclosure of Invention

The invention provides a B-type natriuretic peptide detection kit which is high in sensitivity, rapid, simple and convenient to realize by using the sensitivity of fluorescence immunochromatography and combining a fluorescence immunochromatography analyzer and can accurately quantify, aiming at overcoming the defects and shortcomings in the prior art;

a B-type natriuretic peptide detection kit comprises a PVC base plate, and a sample pad, a combination pad, a detection pad and an absorption pad which are sequentially arranged on the PVC base plate, wherein the detection pad comprises a detection area and a quality control area, and the detection area is coated with a BNP monoclonal antibody;

the quality control area is coated with a chicken IgY monoclonal antibody;

the binding pad is coated with a rare earth fluorescent microsphere labeled anti-BNP monoclonal antibody;

the rare earth fluorescent microsphere material comprises polystyrene-methyl methacrylate copolymer and rare earth complex, wherein the rare earth complex is Eu (hfpd) ₃ phen, since photoluminescence in rare earth complexes is usually quenched in the presence of high energy oscillators (C-H, O-H and N-H bonds), due to efficient vibrational coupling to the ground state, whereas substitution with deuterated or fluorinated ligands can suppress deactivation of excited states, fluorine-containing β -diketone ligands are extremely effective for synthesis of highly efficient Ln complexes. By adopting (hfpd =1, 5-hexafluoropentane-2, 4-dione anion form) as a first ligand and phenanthroline phen as a second ligand, water molecules in an internal coordination sphere can be replaced, so that the non-radiative quenching process is disabled, the generated ternary lanthanide complex relatively emits light, the fluorescent microsphere is used as a rare earth nano fluorescent microsphere labeled antibody, and has the advantages of low background, long luminescent life, strong fluorescent signal, high signal-to-noise ratio and the like, after being activated, the surface of the fluorescent microsphere reacts with free amino groups on the antibody to form covalent bonds, can be stably stored, has the characteristics of wide detection range, high sensitivity, high accuracy, quick and simple detection and the like, and can be used for quick detection, wherein Eu (hfpd) ₃ The molecular formula of phen is:

a preparation method of a B-type natriuretic peptide detection kit comprises the following steps:

s1, preparing a BNP monoclonal antibody labeled by a rare earth fluorescent microsphere;

s2, preparing a BNP detection kit:

the method for preparing the envelope membrane by using the BNP monoclonal antibody and the chicken IgY antibody comprises the following steps:

adopting Tris buffer solution with pH8.0 to prepare chicken IgY antibody into solution with concentration of 1mg/ml, respectively preparing BNP monoclonal antibody into solution with concentration of 1mg/ml, selecting XYZ3050 film spraying system of Biodot to spray the chicken IgY antibody to the position of a quality control line of a coating film (a nitrocellulose film), spraying the BNP antigen coating antibody to the position of a detection line, dehumidifying for 4 hours in a drying workshop with relative humidity of less than 10%, and drying for later use to obtain the coating film with the detection line and the quality control line.

Soaking the glass fiber paper in the Tris buffer solution for half an hour at the soaking temperature of 37 ℃, dehumidifying for 4 hours under the same dehumidifying condition, diluting the fluorescent microsphere labeled BNP antibody liquid obtained in the step S1 with the membrane processing buffer solution respectively until the content of the fluorescent microsphere labeled BNP antibody is 0.05mg/ml, spraying the fluorescent microsphere labeled BNP antibody liquid onto the processed glass fiber membrane by using an XYZ3050 membrane spraying system of Biodot to prepare a binding pad, and drying under the same dehumidifying condition. After the dried envelope film with the detection line and the quality control line, the sample pad, the water absorption pad and the back plate are matched and assembled in a 10 ten thousand grade clean and dry workshop, a CM4000 cutting system of Biodot is adopted to cut the pasted paper board into 4 mm/strip width, and a clamping piece for detection is arranged for standby.

The preparation method of the B-type natriuretic peptide detection kit comprises the following steps of:

(1) Adding the rare earth fluorescent microspheres into MES buffer solution, dissolving, and fixing the volume to the concentration of 30-100 ul/ml to obtain a rare earth fluorescent microsphere storage solution, mixing the rare earth fluorescent microsphere storage solution with EDC/NHS, taking the MES buffer solution as a reaction medium, and culturing for 30-120 min at 25 ℃ to obtain an activated rare earth fluorescent microsphere storage solution;

(2) Centrifuging and resuspending the activated rare earth fluorescent microsphere storage solution in the step (1) to remove a supernatant, and adding the same volume of MES buffer solution;

(3) Adding BNP monoclonal antibody into the rare earth fluorescent microsphere storage solution subjected to the first resuspension in the step (2), reacting for 60min at 20-25 ℃, adding 500ul of 3% casein, and sealing for 30min;

(4) Centrifuging after sealing is finished, wherein the centrifuging condition is 15000rpm,20min and 25 ℃; washing the centrifuged marker solution by using Tris-HCl buffer solution, wherein the centrifugation conditions are 12000rpm,15min and 25 ℃, repeating for 2-3 times, the last centrifugation conditions are 15000rpm,20min and 25 ℃, and removing supernatant to obtain the BNP monoclonal antibody marked by the rare earth fluorescent microspheres.

The preparation method of the rare earth fluorescent microsphere comprises the following steps:

adding 3v/v% rare earth complex Eu (hfpd) into polystyrene-methyl methacrylate copolymer ₃ phen, mixing uniformly by ultrasonic to obtain solution A;

dissolving 0.1v/v% hydroxymethyl cellulose and 0.1v/v% sodium bicarbonate in water to obtain solution B;

and adding the solution A into the solution B, performing ultrasonic treatment for 10 minutes, introducing nitrogen into the obtained mixed solution for 30 minutes, uniformly stirring at the speed of 100r/min, heating to 60 ℃, adding 0.01-0.1 v/v% of potassium persulfate into the mixed solution, and reacting for 12 hours to obtain the rare earth fluorescent microsphere.

The particle size of the rare earth fluorescent microsphere is 130-200nm, and the rare earth complex Eu (hfpd) is promoted by ultrasonic waves ₃ phen is loaded in a pore channel of the polystyrene-methyl methacrylate copolymer, has higher light stability and photoluminescence performance, and the mesoporous structure can also promote energy transfer between the emission ions and the ligands, so that the excitation spectrum and the emission spectrum are not overlapped, and the mutual interference phenomenon does not exist.

According to the B-type natriuretic peptide detection kit, the rare earth fluorescent microspheres are stable under the ground state, and emit fluorescence with the wavelength range of 580-700nm under the action of an excitation light source of 360 nm.

The detection kit for B-type natriuretic peptide, wherein the detection pad is a nitrocellulose membrane.

The B-type natriuretic peptide detection kit is characterized in that a test sample of the kit is human serum, plasma or whole blood.

Compared with the prior art, the invention has the following advantages:

1. the invention provides a B-type natriuretic peptide detection kit, which adopts the fluorescence immunochromatography principle to detect the content of B-type natriuretic peptide in human serum, plasma and whole blood, is mainly used for the auxiliary diagnosis of heart failure clinically, adopts rare earth nano fluorescent microspheres to mark antibodies, and adopts a rare earth complex Eu (hfpd) ₃ phen has the advantages of low background, long luminescent life, strong fluorescence signal, high signal-to-noise ratio and the like, the surface of the fluorescent microsphere is activated and then reacts with free amino on the antibody to form a covalent bond, and the fluorescent microsphere can be stably stored, has the characteristics of a detection range frame, high sensitivity, high accuracy, rapidness, convenience and the like, and can be used for rapid detection.

2. The preparation method of the rare earth fluorescent microsphere is promoted by ultrasonic waves, and the rare earth complex Eu (hfpd) ₃ phen is loaded in a pore channel of the polystyrene-methyl methacrylate copolymer, has higher light stability and photoluminescence performance, and the mesoporous structure can also promote energy transfer between the emission ions and the ligands, so that the excitation spectrum and the emission spectrum are not overlapped, and the mutual interference phenomenon does not exist.

Detailed Description

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. In addition, the term "coating" in the present invention is a term in the field of immunization, and includes adsorption and immobilization.

The following describes a specific technical scheme of the present invention with reference to specific examples:

(1) Preparation of fluorescent microsphere labeled probe

(2) Coating of antibodies at test zone T line and quality control line

Adopting a film spraying instrument, spraying BNP monoclonal antibodies on the detection lines of the film coating test areas, and spraying chicken IgY antibodies on the quality control lines.

(3) Coating of labeled probes at the conjugate pad

And spraying a BNP monoclonal antibody marked by fluorescent microspheres at a specific position of the sample pad by using a spraying instrument.

(4) Kit assembly molding

A coating film serving as a test area is adhered to the middle of the PVC bottom plate, a combination pad is adhered to the detection end of the coating film, and a water absorption pad is adhered to the quality control end. A test paper splitting machine is adopted to split the paper into paper strips with certain width, clamping pieces are arranged in the paper strips, and aluminum foil bags filled with drying agents are used for packaging.

(5) Formation of antigen-antibody fluorescent immune complexes

Adding a sample to be detected at the sample adding end of the assembled reaction plate, combining a BNP antigen in the sample with a BNP monoclonal antibody marked by a fluorescent microsphere, then carrying out chromatography to a detection line, capturing the BNP antigen by the sprayed BNP monoclonal antibody, forming an antibody-antigen-fluorescent microsphere marked antibody immune complex at the detection line, and forming a fluorescent marked immune complex by the redundant BNP monoclonal antibody marked by the fluorescent microsphere with goat anti-chicken IgY and chicken IgY at a quality control line.

(6) Fluorescence intensity detection of fluorescence-labeled immune complexes

And (3) measuring the fluorescence intensity at the detection line and the quality control line by using a fluorescence detector, calculating by using a curve equation of the fluorescence intensity and the standard concentration to obtain a quantitative result, and using the measurement result of the quality control line as a quality control internal standard of the measurement method.

The fluorescence intensity of the fluorescence labeling immune complex is a numerical value obtained after the number of the combined fluorescent microspheres respectively retained at the detection line and the quality control line is measured by a fluorescence detector. And (3) drawing a standard concentration curve by measuring a large number of samples with different standard concentrations under the condition of double-antibody sandwich immunoreaction, obtaining a standard concentration curve equation, and calculating the concentration of the detected sample according to the standard concentration curve equation. And the quality control line measurement result is used as the quality control internal standard of the measurement method.

Example 1

1. Preparation of BNP detection kit

Preparation of BNP labeled antibody and goat anti-chicken IgY labeled antibody respectively labeled at different concentrations

Adding the rare earth fluorescent microspheres into MES buffer solution, dissolving, and fixing the volume to the concentration of 30-100 ul/ml to obtain a rare earth fluorescent microsphere storage solution, mixing the rare earth fluorescent microsphere storage solution with EDC/NHS, taking the MES buffer solution as a reaction medium, and culturing for 30-120 min at 25 ℃ to obtain an activated rare earth fluorescent microsphere storage solution;

centrifuging and resuspending the activated rare earth fluorescent microsphere storage solution to remove the supernatant, and adding MES buffer solution with the same volume; respectively adding BNP monoclonal antibodies and goat anti-chicken IgY with different concentrations into the rare earth fluorescent microsphere storage solution subjected to primary resuspension, reacting for 60min at 20-25 ℃ under the feeding combination condition shown in Table 1, adding 500ul of 3% casein, and sealing for 30min;

centrifuging after sealing is finished, wherein the centrifuging condition is 15000rpm,20min and 25 ℃; washing the centrifuged labeling solution with Tris-HCl buffer solution, repeating the centrifugation conditions of 12000rpm,15min and 25 ℃ for 2-3 times, removing the supernatant under the last centrifugation conditions of 15000rpm,20min and 25 ℃, uniformly mixing the labeled BNP marker and the labeled goat anti-chicken IgY marker according to a ratio of 1 (V: V) to 1 (V: V), and fully performing ultrasonic treatment to obtain the rare earth fluorescent microsphere labeled BNP monoclonal antibody and goat anti-chicken IgY.

Table 1: feeding combination condition of T line and C line

(II) preparing the bonding pad

Loading a BNP monoclonal antibody marked by rare earth fluorescent microspheres and goat anti-chicken IgY into a gold spraying and scribing machine, and spraying the BNP monoclonal antibody and goat anti-chicken IgY onto the cut combination pad; drying overnight (16-24 h).

The prepared test materials were assembled into reagent cards, and then evaluated with the enterprise reference materials as shown in table 2.

Table 2: T/C precision results of different T line and C line labeled antibody feeding ratios

As can be seen from the above table, the combination 5T/C is less than 6% in precision, which is relatively optimal.

As can be seen from Table 2, the concentration gradient ratio of the reference substances of each enterprise in the combination 5 is relatively best, the sensitivity is higher, the linearity is better, and the precision is less than 6%, 10 mu g of BNP labeled antibody is selected for feeding, and 16 mu g of goat anti-chicken IgY labeled antibody is selected for feeding in a combination, namely the fluorescent microspheres: antibody =20, 1 is the T-line dosage ratio; fluorescent microspheres: antibody =25, C-line dosage ratio.

(III) determination of sample application amount

The preparation of the reagent card is carried out according to the determined production process, the prepared detection card detects B-type natriuretic peptide (BNP) reference substances with the concentrations of 5000pg/mL, 3000pg/mL, 1000pg/mL, 100pg/mL and 5pg/mL according to the set sample loading amounts (80 μ L, 90 μ L, 100 μ L, 110 μ L and 120 μ L), and the sample loading chromatography conditions are shown in Table 3.

Table 3: effect of different sample addition amounts on the results

As can be seen from Table 3, the reaction takes longer to complete with the sample addition amount of less than 100. Mu.L, and the T/C is stable later; the reaction was completed at 10min with a sample addition of 100. Mu.L or more, and T/C stabilized earlier.

As can be seen from Table 3, the chromatography effect was good at a loading of 100. Mu.L, and T/C was stable earlier, and 100. Mu.L was selected as the loading.

(IV) determination of stock solution

4 different marking storage solutions are prepared for marking and storing 20mL of each marker (the formula is calculated according to 1000 mL)

Table 4: stock solutions of different formulations

Loading the rare earth fluorescent microsphere labeled antibody solution into a gold spraying and scribing machine, and spraying the rare earth fluorescent microsphere labeled antibody solution onto the cut bonding pad according to the spraying amount of 4 mu L/cm; drying overnight for 24h; the test materials prepared from the various raw materials were assembled into reagent cards and evaluated with corporate reference products, the results of which are shown in table 5.

Table 5 different formulations marking stock solution markers observations

As can be seen from the above table, the Tris buffer is more stable than the PB buffer, and the rare earth fluorescent microspheres with pH8.0 have good release effect.

Preparation of BNP detection kit

adopting Tris buffer solution with pH8.0 to prepare chicken IgY antibody into solution with concentration of 1mg/ml, respectively preparing BNP monoclonal antibody into solution with concentration of 1mg/ml, selecting XYZ3050 film spraying system of Biodot to spray goat anti-chicken IgY antibody to the position of a quality control line of a coating film (nitrocellulose film), spraying BNP antigen coating antibody to the position of a detection line, dehumidifying for 4 hours in a drying workshop with relative humidity of less than 10%, and drying for later use to obtain the coating film with the detection line and the quality control line.

Soaking the glass fiber paper in the Tris buffer solution for half an hour at the soaking temperature of 37 ℃, dehumidifying for 4 hours under the same dehumidifying condition, diluting the rare earth fluorescent microsphere labeled BNP antibody liquid with the membrane processing buffer solution respectively until the content of the fluorescent microsphere labeled BNP antibody is 0.05mg/ml, spraying the rare earth fluorescent microsphere labeled BNP antibody liquid onto the treated glass fiber membrane by adopting an XYZ3050 membrane spraying system of BioDot to prepare a binding pad, and drying under the same dehumidifying condition. After the dried envelope film with the detection line and the quality control line, the sample pad, the water absorption pad and the back plate are matched and assembled in a 10 ten thousand grade clean and dry workshop, a CM4000 cutting system of Biodot is adopted to cut the pasted paper board into 4 mm/strip width, and a clamping piece for detection is arranged for standby.

Example 2 evaluation of a kit for detecting BNP

(I) detection sensitivity

The sensitivity of the lateral chromatography detection reagent for detecting BNP in example 1 is determined by taking the BNP antigen as a sample to be detected.

Meanwhile, BNP antigen is respectively prepared into a series of concentrations (0, 0.5, 1,5, 10, 50, 100 and 500 ng/mL) by using a Tris buffer solution containing 3% lamb serum and having the pH value of 8.0, the concentrations are respectively added into the sample adding end of the BNP detection kit obtained in the example 1, and a fluorescence detector is adopted to detect the fluorescence intensity. A detection step: before detection, the sample to be detected is restored to room temperature, 100 mu l of the sample to be detected is taken by a precise pipette and vertically and slowly dripped into the sample adding end of the lateral chromatographic detection reagent for detecting BNP obtained in example 1, and a fluorescence detector is used for testing after 10 minutes.

The results are shown in Table 6 below. From the results of the measurement, it was found that the sensitivity for BNP antigen of example 1 was 0.5ng/mL. Detecting the correlation coefficient R of the BNP antigen according to a calibration curve formed by the detection value and the concentration ² =0.9994, linear range 0-500ng/mL.

TABLE 6 kit detection values of different sample concentrations of BNP

Performance analysis evaluation summary

1. Detection limit: 0.5ng/mL.

2. Linearity: within the range of [5,5000] ng/mL, the linear correlation coefficient r should be not less than 0.9900.

3. Precision: the variation Coefficient (CV) in batch is less than or equal to 15%.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. The utility model provides a B type natriuretic peptide detect reagent box, includes the PVC bottom plate, and set gradually in sample pad, combination pad, detection pad and absorption pad on the PVC bottom plate, it includes detection zone and matter accuse district, its characterized in that to detect the pad: the detection area is coated with BNP monoclonal antibody;

the quality control area is coated with a chicken IgY monoclonal antibody;

the rare earth fluorescent microsphere material comprises polystyrene-methyl methacrylate copolymer and rare earth complex, wherein the rare earth complex is Eu (hfpd) ₃ phen, the molecular formula is:

2. the B-type natriuretic peptide detection kit of claim 1, wherein: the preparation method of the rare earth fluorescent microsphere comprises the following steps:

3. The detection kit for B-type natriuretic peptide according to claim 1, characterized in that: the particle size of the rare earth fluorescent microsphere is 130-200nm.

4. The detection kit for B-type natriuretic peptide according to claim 1, characterized in that: the rare earth fluorescent microspheres are stable under a ground state, and emit fluorescence with the wavelength range of 580-700nm under the action of an excitation light source of 360 nm.

5. The detection kit for B-type natriuretic peptide according to claim 1, characterized in that: the detection pad is a nitrocellulose membrane.

6. The B-type natriuretic peptide detection kit of claim 1, wherein: the test sample of the kit is human serum, plasma or whole blood.

7. A preparation method of a B-type natriuretic peptide detection kit is characterized by comprising the following steps:

s2, preparing a BNP detection kit:

the BNP monoclonal antibody and the goat anti-chicken IgY antibody are used for preparing the coating film, and the specific method is as follows:

preparing a goat anti-chicken IgY antibody into a solution with the concentration of 1mg/ml by adopting a Tris buffer solution with the pH of 8.0, respectively preparing a BNP monoclonal antibody into a solution with the concentration of 1mg/ml, spraying the goat anti-chicken IgY antibody to the position of a quality control line of a coating film by adopting an XYZ3050 film spraying system of Biodot, spraying the BNP antigen coating antibody to the position of a detection line, dehumidifying for 4 hours in a drying workshop with the relative humidity of less than 10%, and drying for later use to obtain the coating film with the detection line and the quality control line;

soaking the glass fiber paper in the Tris buffer solution for half an hour at the soaking temperature of 37 ℃, dehumidifying for 4 hours under the same dehumidifying condition, diluting the fluorescent microsphere labeled BNP antibody liquid obtained in the step S1 with the membrane processing buffer solution respectively until the content of the fluorescent microsphere labeled BNP antibody is 0.05mg/ml, spraying the fluorescent microsphere labeled BNP antibody liquid on the treated glass fiber membrane respectively by using a Biodot XYZ3050 membrane spraying system to form a binding pad, drying under the same dehumidifying condition, matching and assembling the dried coating membrane with the detection line and the quality control line, the sample pad, the water absorption pad and the back plate in a 10 ten thousand clean and dry workshop, cutting the pasted paperboard into 4 mm/strip width by using a Biodot CM4000 cutting system, and filling the paperboard into a detection clamping piece for standby.

8. The method for preparing a B-type natriuretic peptide detection kit according to claim 7, characterized in that: the preparation method of the BNP monoclonal antibody marked by the rare earth fluorescent microspheres on the bonding pad comprises the following steps:

(1) Adding the rare earth fluorescent microspheres into MES buffer solution, dissolving, and fixing the volume to 30-100 ul/ml to obtain a rare earth fluorescent microsphere storage solution, mixing the rare earth fluorescent microsphere storage solution with EDC/NHS, taking the MES buffer solution as a reaction medium, and culturing at 25 ℃ for 30-120 min to obtain an activated rare earth fluorescent microsphere storage solution;

(2) Centrifuging and resuspending the activated rare earth fluorescent microsphere storage solution in the step (1) to remove supernatant, and adding MES buffer solution with the same volume;

(3) Adding BNP monoclonal antibody into the rare earth fluorescent microsphere storage solution subjected to first resuspension in the step (2), reacting for 60min at 20-25 ℃, adding 500ul of 3% casein, and sealing for 30min;

(4) Centrifuging after sealing is finished, wherein the centrifuging condition is 15000rpm,20min and 25 ℃; washing the centrifuged labeling solution with Tris-HCl buffer solution, repeating the centrifugation conditions of 12000rpm,15min and 25 ℃ for 2-3 times, removing the supernatant under the last centrifugation conditions of 15000rpm,20min and 25 ℃, adding a redissolution with the same volume as the MES buffer solution in the step (1), and carrying out ultrasonic resuspension to obtain the BNP monoclonal antibody labeled by the rare earth fluorescent microspheres.