CN112986586A

CN112986586A - Amino-terminal brain natriuretic peptide detection method and kit

Info

Publication number: CN112986586A
Application number: CN202110192561.6A
Authority: CN
Inventors: 刘春艳; 崔琪瑶; 邓盛花
Original assignee: Guangdong Fapon Biotech Co Ltd
Current assignee: Guangdong Fapon Biotech Co Ltd
Priority date: 2021-02-20
Filing date: 2021-02-20
Publication date: 2021-06-18
Also published as: WO2022174570A1

Abstract

The invention provides a method and a kit for detecting amino-terminal brain natriuretic peptide, which adopt a double-antibody sandwich method to detect the content of the amino-terminal brain natriuretic peptide; by adopting the amino-terminal brain natriuretic peptide detection method and the kit, the specificity and/or the detection rate of detection can be effectively improved.

Description

Amino-terminal brain natriuretic peptide detection method and kit

Technical Field

The present invention relates to the field of protein detection. In particular, the invention relates to a detection method and a kit of amino-terminal brain natriuretic peptide.

Background

Heart failure (heart failure) is the severe and terminal stage of various heart diseases, and is clinically manifested as dyspnea, fatigue, fluid retention, and the like. According to statistics, the current heart failure disease estimation of China is about 1.3%, the number of patients with the current disease is about 1000 thousands, and China has become the world with the largest heart failure disease population. Early diagnosis is critical due to the preventive control of cardiovascular disease.

The detection of serum markers is one of the important means for clinical early diagnosis and prognosis of heart failure. The serum marker comprises Brain Natriuretic Peptide (BNP) and amino terminal brain natriuretic peptide (NT-proBNP), and is a preferred serum marker for heart failure recommended by the heart failure guidelines at home and abroad (ECS/ACC/AHA/HFSA/CSC).

Compared with BNP, NT-proBNP has longer half-life and better stability, so that NT-proBNP has higher sensitivity in the detection of early or mild heart failure and is more suitable for clinical application.

Disclosure of Invention

The inventor obtains the amino-terminal brain natriuretic peptide antigen region combination which can be used for detecting the amino-terminal brain natriuretic peptide by analyzing and researching various NT-proBNP antigen intervals to be detected and detection antibodies by fully considering the structural characteristics of the amino-terminal brain natriuretic peptide through a large amount of theoretical research and experimental groping.

In some embodiments, the invention may include one or more of the following:

1. an amino terminal brain natriuretic peptide detection kit, which comprises a first antibody and a second antibody for detecting the amino terminal brain natriuretic peptide, wherein the first antibody is combined with the 27 th-31 th amino acid segment of the amino terminal brain natriuretic peptide; the second fragment binds to amino acid fragment 42-46 of the amino terminal brain natriuretic peptide.

2. The kit according to item 1, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the second antibody is a labeled antibody and the first antibody is a coated antibody.

3. The kit of item 1, further comprising a third antibody that detects an amino-terminal brain natriuretic peptide, wherein the third antibody binds to amino acid fragment 13-27 of the amino-terminal brain natriuretic peptide;

for example, antibodies for pairing are divided into a first set of antibodies and a second set of antibodies, wherein the first set of antibodies and the second set of antibodies are selected from the group consisting of:

1) the first set of antibodies comprises a first antibody and the second set of antibodies comprises a second antibody and a third antibody;

2) the first set of antibodies comprises a second antibody and the second set of antibodies comprises a first antibody and a third antibody;

3) the first set of antibodies includes a third antibody and the second set of antibodies includes a first antibody and a second antibody.

4. The kit of any one of items 1 to 3, wherein the reactivity of the antibody is OD evaluation by ELISA₄₀₅≥0.5。

5. The kit of any of items 1-4, wherein the labeled antibody is labeled with a detectable label such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label, and/or a binding partner; for example, the enzyme can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the labeled antibody is labeled with a binding partner and a detectable label.

6. The kit of any of items 1-5, wherein the coated antibody is attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate, a nitrocellulose membrane, or a microfluidic chip and/or a binding partner; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to a solid phase via a binding partner.

7. An antibody combination for detecting an amino-terminal brain natriuretic peptide, comprising a first antibody and a second antibody; optionally, a third antibody is also included; wherein the first antibody, the second antibody, the third antibody are defined according to any one of items 1-6.

8. Use of the antibody combination of item 7 in the preparation of a kit for detecting an amino-terminal brain natriuretic peptide.

9. The use of item 8, wherein the kit is used for 1) detection of amino terminal brain natriuretic peptide content, and/or 2) prediction, diagnosis, prognostic assessment and therapy guidance of heart failure, including for example acute heart failure.

10. A method of making the antibody combination of item 7, the method comprising:

1) immunizing an animal with an antigen or hapten comprising a fragment 1-fragment 3 selected from the group consisting of:

fragment 1: amino-terminal brain natriuretic peptide amino acid fragment 13-27;

fragment 2: amino-terminal brain natriuretic peptide amino acid fragment 27-31;

fragment 3: amino-terminal brain natriuretic peptide amino acid fragments 42-46; and

2) obtaining antibodies from said animal that bind to said fragments 1-3, respectively.

Drawings

In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a linear range for detection of 1+1 pairings with recombinant antigens on a chemiluminescent platform;

FIG. 2 is a linear range for detection of 2+1 pairing using fluorescent microsphere detection.

Detailed Description

In some embodiments, the kits of the invention comprise a first antibody and a second antibody for detecting an amino-terminal brain natriuretic peptide, wherein the first antibody binds to amino acid fragment 27-31 of the amino-terminal brain natriuretic peptide; the second antibody binds to an amino acid fragment 42-46 of the amino-terminal brain natriuretic peptide; the detection of the amino-terminal brain natriuretic peptide by combining the antibody pairs of the amino acid fragments can improve the detection specificity. For example, the second antibody is a coated antibody when the first antibody is a labeled antibody; or the second antibody is a labeled antibody and the first antibody is a coated antibody.

The kit comprises an amino-terminal brain natriuretic peptide detection reagent card (test strip). In some embodiments, NT-proBNP in a sample may be detected using the principle of the double antibody sandwich method by labeling the antibody with a detectable label, such as a fluorescent microsphere, using immunofluorescence techniques. In some embodiments, the methods and kits of the invention can improve specificity and/or reduce missed detection.

In some embodiments, the invention may be performed by or include reagents that perform fluorescence immunochromatography. In some embodiments, immunochromatography can be achieved by a test strip by a double antibody sandwich method. In some embodiments, the reagents and apparatus for performing the double antibody sandwich method are not particularly limited, and any suitable reagents and/or apparatus may be employed. For example, the fluorescence immunochromatographic test strip may include a sample pad, a conjugate pad, an NC membrane, and a water absorbent pad. In some embodiments, at least one fluorophore-labeled antibody is immobilized on the conjugate pad; in some embodiments, the NC film may be coated with two lines, for example T and C lines, respectively; the T line can be coated with at least one antibody; can capture the fluorescent group labeled antibody from the binding pad to form a complex with the antigen in the sample, thereby forming a double antibody sandwich. In some embodiments, a sample to be detected is added into a sample adding port of the detection reagent card, and under the lateral capillary action, the sample to be detected firstly passes through the combination pad to be specifically and immunologically combined with a fluorescent group labeled antibody on the combination pad, and the antigen-antibody fluorescent complex is formed by combination of the combination pad and the fluorescent group labeled antibody, so that the sample to be detected is fixed in the T line. The C line is coated with a substance that reacts with the free fluorophore-labeled antibody, and when the free fluorophore-labeled antibody passes through the C line, it is immobilized in the C line by specific immunological binding to the substance on the C line. The control band (C) is inversely proportional to the fluorescence intensity of the test band (T), so that the individual differences of the samples can be corrected. The fluorescence intensity of the two bands detected by the fluorescence immunoassay analyzer is represented by peak areas, a T/C value (T peak area/C peak area) is calculated by calculation software of the fluorescence immunoassay analyzer, the T/C value is fitted to a set standard curve, and the fluorescence immunoassay analyzer automatically converts the concentration value of NT-proBNP in a corresponding sample to be detected.

In some embodiments, the invention can utilize antibodies that bind different amino acid fragments to recognize multiple positions on an antigen, reduce the risk of missed detection, and increase the detection rate.

In some embodiments, further comprising a third antibody that detects an amino-terminal brain natriuretic peptide, wherein the third antibody binds to amino acid fragment 13-27 of the amino-terminal brain natriuretic peptide. In some embodiments, antibodies that bind the 13-27aa fragment of the amino terminal brain natriuretic peptide are used as a first set of antibodies, and antibodies that bind the 27-31aa fragment of the amino terminal brain natriuretic peptide and antibodies that bind the 42-46aa fragment of the amino terminal brain natriuretic peptide are used as a second set of antibodies; in some embodiments, antibodies that bind the 27-31aa fragment of the amino terminal brain natriuretic peptide are used as a first set of antibodies, and antibodies that bind the 13-27aa fragment of the amino terminal brain natriuretic peptide and antibodies that bind the 42-46aa fragment of the amino terminal brain natriuretic peptide are used as a second set of antibodies; in some embodiments, antibodies that bind the 42-46aa fragment of the amino terminal brain natriuretic peptide are used as a first set of antibodies, and antibodies that bind the 13-27aa fragment of the amino terminal brain natriuretic peptide and antibodies that bind the 27-31aa fragment of the amino terminal brain natriuretic peptide are used as a second set of antibodies.

The term "antibody" is used herein in the broadest sense. In some embodiments, the antibodies of the invention may be monoclonal or polyclonal antibodies. In some embodiments, the antibodies of the invention can be prepared using methods known in the art. In some embodiments, an antibody of the invention can be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) may be coupled to immunoreactive substances (e.g., epitope peptides). The carrier protein may comprise a protein or polypeptide, which may function as a carrier for the immunogen. These types of polypeptides include albumin, serum proteins, globulins, lens proteins, lipoproteins, and/or fragments thereof. In some embodiments, immunoreactive substances (e.g., but not limited to, amino-terminal brain natriuretic peptide amino acid fragments) can be used to generate antibodies having affinity for NT-proBNP.

In some embodiments, binding of an antibody to an amino acid fragment corresponding to an amino-terminal brain natriuretic peptide can refer to the ability of the antibody to bind to the amino acid fragment, but the amino acid fragment need not be the smallest binding fragment.

In some embodiments, the effect of the antibodies of the invention, such as binding activity and/or cross-reactivity, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, and the like. In some embodiments, the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibodies of the invention to antigen (antigenic peptide) binding is characterized, for example, in an ELISA, for better reactivity, for example, as determined by a peroxidase-labeled ELISA reading a response value of 0.5 or greater at 405nm, and can be used in immunoassays.

In some embodiments, a first antibody, a second antibody; in some embodiments, a third antibody is also included in the kit; in some embodiments, other antibodies may also be used as coating antibodies or labeled antibodies.

In some embodiments, the coated antibody is bound to a solid phase. In some embodiments, the manner in which the coated antibody is bound to the solid phase may be direct or indirect. In some embodiments, the coated antibody can be used to coat a solid support. In some embodiments, the solid support is not particularly limited and can be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips. In some embodiments, the coated antibody binds to a binding partner, such as biotin/avidin, biotin/streptavidin; in some embodiments, the coated antibody is attached to a solid phase via a binding partner.

In some embodiments, the labeled antibody is labeled with a detectable label. In some embodiments, the labeled antibody and detectable label may be bound directly or indirectly. In some embodiments, a detectable label such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label; in some embodiments, the detectable label can be, for example, a rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase label. In some embodiments, the labeled antibody is linked to a binding partner, such as biotin/avidin, biotin/streptavidin. In some embodiments, the labeled antibody is labeled with a binding partner and a detectable label.

In some embodiments, the kits of the invention comprise reagents suitable for performing an immunoassay. In some embodiments, the kits of the invention can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assays IFA, radioimmunoassays RIA, and other non-enzyme-linked antibody binding assays or methods.

In some embodiments, the present invention provides an antibody combination for the detection of an amino-terminal brain natriuretic peptide, comprising a first antibody and a second antibody as defined above; in some embodiments, a third antibody as defined above is also included.

In some embodiments, the invention provides the use of an antibody combination in the manufacture of a kit for detecting an amino-terminal brain natriuretic peptide. Wherein the kit is used for 1) detection of amino-terminal brain natriuretic peptide content, and/or 2) prediction, diagnosis, prognostic assessment and treatment guidance of heart failure, including for example acute heart failure.

The term "diagnosis" is used herein in the broadest sense. In some embodiments, the diagnosis of the invention may be the identification of a health state. In some embodiments, the diagnosis of the invention may be the exclusion of negative samples. In some embodiments, the diagnosis of the invention may be a secondary diagnosis. In some embodiments, the diagnosis of the present invention may be risk stratification.

In some embodiments, the present invention provides a method for producing an antibody for detecting an amino-terminal brain natriuretic peptide, the method comprising immunizing an animal with an immunoreactive polypeptide comprising an amino acid fragment described herein as an antigen or a hapten, respectively, thereby producing an antibody, such as a monoclonal antibody or a polyclonal antibody, for detecting an amino-terminal brain natriuretic peptide. Monoclonal or polyclonal antibodies can be made by methods known in the art.

In some embodiments, the invention provides the use of an immunoreactive polypeptide comprising an amino acid fragment as described herein in the preparation of a reagent or kit for detecting an amino-terminal brain natriuretic peptide.

In this context, the sample to be tested for NT-proBNP may comprise biological tissues, cells or body fluids of a healthy or pathological state, such as blood samples, e.g. plasma, serum, blood products.

The present invention will be described in further detail with reference to specific examples. The following examples are provided to illustrate embodiments of the present invention and are not intended to limit the invention. The invention may optionally include embodiments that are not illustrated by the examples.

Preparation of amino-terminal brain natriuretic peptide monoclonal antibody

1. Immunizing animals: taking a BALB/c mouse which is 8-12 weeks old and has the same line with myeloma cells, fully and uniformly mixing NT-proBNP antigen containing 100 mu g/mouse protein and an equal amount of Freund's complete adjuvant, injecting the mixture into the abdominal cavity of the mouse, fully and uniformly mixing NT-proBNP antigen containing 100 mu g/mouse protein and an equal amount of Freund's incomplete adjuvant every 2 weeks, and injecting the mixture into the abdominal cavity of the mouse for multiple times to strengthen immunity. By detecting mouse serum (indirect ELISA method), the mice with titer of more than 1:2000 can be used for fusion, and 3 days before fusion, the mice are boosted again in the abdominal cavity, and the dosage is 50 mug/mouse.

2. Preparation of feeder cells

BALB/c mouse peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mouse neck-pulled to be killed, 75% alcohol soaked in whole body, in super clean bench, using scissors to cut off abdominal skin under aseptic operation, exposing peritoneum, using syringe to inject 5mL of RPMI 1640 basic culture solution into abdominal cavity, repeatedly washing, recovering washing solution, 1000rpm, centrifuging for 5min, leaving precipitate, using RPMI 1640 to screen culture solution (in complete culture solution of RPMI 1640 containing HAT) to resuspend, regulating cell concentration to 1 × 10⁵Add to 96-well plate at 150. mu.L/well, 37 ℃ with 5% CO₂The culture was carried out overnight.

3. Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleen is taken out under the aseptic condition, placed in a plate, washed once by RPMI 1640 basic culture solution, placed on a nylon net of a small beaker, ground and filtered to prepare cell suspension. Centrifuging, discarding supernatant, resuspending RPMI 1640 basic culture solution, repeating the steps three times, and counting.

4. Cell fusion

(1) Respectively putting 40mL HAT culture solution, 15mL DMEM serum-free culture solution and 1mL 50% PEG (M12000) in a water bath at 37 ℃ for pre-warming;

(2) separately, mouse myeloma cells Sp2/0 (stored by Roc Bio Inc.) (2-5X 10)⁷) The above immune spleen cell (10)⁸) The suspension is added into a 50mL centrifuge tube for even mixing, and DMEM serum-free culture solution is added to the centrifuge tube until the volume is 40 mL. Centrifuging for 10min, pouring out the supernatant, and mixing;

(3) the centrifuge tube was placed in pre-warmed water at 37 ℃ and 0.7mL of pre-warmed 50% PEG solution was allowed to stand for 90 seconds. Immediately dropwise adding 15mL of prewarmed serum-free culture solution at 37 ℃;

(4) DMEM serum-free culture solution is supplemented to 40mL, centrifugation is carried out for 10 minutes, and the supernatant is poured out. Adding 40mL HAT culture solution containing 15% -20% fetal calf serum. Mixing with a pipette, dropping 2 drops into small holes of 4 pieces of 96-well cell culture plate containing feeder cells, placing at 37 deg.C and 7% CO₂Cultured in an incubator.

5. Selective culture of hybridoma cells

The immune mouse spleen cells and mouse myeloma cells are treated by PEG to form a mixture of various cell components, including unfused myeloma cells and immune spleen cells, a corebody of myeloma cells and a corebody of immune spleen cells, and a corebody of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. For this purpose, it is necessary to remove unfused cells and homologously fused coenckaryons from the various cell mixtures and to select for true hybrid cells. Therefore, the cells were cultured with the HAT medium on days 1, 3, 5, and 7 after cell fusion.

6. Detection of antibodies and hybridoma cell cloning

The supernatant of each culture well was aspirated, and culture wells containing antibodies to NT-proBNP in the culture were detected by indirect ELISA. Hybridoma cells were cloned by limiting dilution. After being cultured, the single cell can be proliferated into homologous cell clone; cell strains which have better reactivity and stably secrete the anti-NT-proBNP monoclonal antibody are obtained through reactivity screening.

Method for reactivity screening: at room temperature, the microtiter plates (Nunc, Maxisorb) were coated with 100. mu.L/well of coating buffer containing 2.5. mu.g/mL human NT-proBNP (as antigen) for 1 hour while stirring. Post-coating treatments were incubated in PBS buffer and 1% monoclonal antibody for 30 min. Subsequently, washing was performed with a washing buffer. 100 μ L/well antibody samples were incubated for 1 hour while stirring at room temperature. Then washed 2 more times with the washing solution. Then, the goat anti-mouse IgG conjugated with the detection antibody peroxidase diluted with PBS buffer at 1:40000 was incubated with 100. mu.L/well for another 1 hour at room temperature while stirring. After washing again with the washing buffer, the peroxidase activity is measured in a conventional manner (for example, by reading the reaction value at 405nm with an ELISA reader) to obtain the OD₄₀₅More than or equal to 0.5 (with better reactivity).

Identification of antibody binding fragments

Different NT-proBNP antigen peptides are adopted to respectively coat micropores, PBS + 20% NBS is used as diluent, monoclonal antibodies are diluted to be 1 mu g/mL of primary antibody concentration, goat anti-mouse IgG-HRP is used as secondary antibody, and the binding fragments of the monoclonal antibodies are determined according to the reaction condition of each monoclonal antibody to different antigens. The results of identifying binding fragments of the cell lines used in the examples are given in the following table:

binding fragments	Cell line
		1-12aa	1NT-9；7NT-3
13-27aa	3NT-12-1；5NT-6；5NT-8
		27-31aa	6NT-1；8NT-7
31-39aa	2NT-17；3NT-16
		38-44aa	2NT-9-1；8NT-5
42-46aa	1NT-3-14；3NT-22
		62-76aa	7NT-1-5；32NT-1

Third, screening by pairing

Selecting antibodies of different binding fragments for coating and marking respectively, and carrying out cross-pairing experiments, wherein the screening process is as follows:

1. marking

(1) Taking 100uL of fluorescent microspheres, centrifuging at 15000rpm/8min/4 ℃, removing supernatant after centrifugation, resuspending to 100uL, and performing ultrasonic treatment for 2-3 times;

(2) preparing 10mg/mL EDC and NHS;

(3) and (3) activation: activating fluorescent microspheres to a final volume of 200uL by using EDC and NHS, uniformly mixing for 18min in a dark place, centrifuging at 15000rpm/10min/4 ℃, and removing supernatant;

(4) washing: resuspending to 100uL, sonicating for 2-3 times at 15000rpm/8min/4 deg.C, removing supernatant, and repeating for 2 times;

(5) coupling: resuspending to 200uL, performing ultrasonic treatment for 2-3 times, adding the antibody into the fluorescent microspheres to a final volume of 300uL, uniformly mixing in a dark place, and reacting at 37 ℃ for 2 h;

(6) and (3) sealing: adding 100uL of sealant, uniformly mixing in dark, and reacting for 1h at 37 ℃;

(7) washing: after blocking, the cells were centrifuged at 15000rpm/12min/4 ℃ to remove the supernatant. Resuspend to 100uL, sonicate 2-3 times. Centrifuging at 15000rpm/8min/4 deg.C, and removing supernatant;

(8) and (3) storage: resuspending to 100uL, sonicating for 2-3 times, and storing at 4 ℃ for later use.

2. Coating quilt

(1) Assembling the nitrocellulose membrane and the fluorescent PVC base plate for later use;

(2) diluting the antibody to 1.0-2.0mg/mL, uniformly drawing a line on an NC film by using a gold spraying film drawing instrument, and then drying in a 37 ℃ thermostat for at least over 45 min. Assembling the cutting strips, and detecting the sample adding.

3. Detection of

(1) Quality control product: NT-proBNP antigen, diluted to 35000, 10000, 1000, 500pg/mL with PBS;

(2) the detection method comprises the following steps: detection was performed using a fluorometer. The reading value T/C of the instrument represents the ratio of the area of the T peak to the area of the C peak, the reaction pairing activity is high, and the higher the T/C is, the higher the activity is, under the condition of quality control and positive samples.

TABLE 1 first round of screening

Cross-pairing the above pairings in the first round of screening were all active, and 138 randomized clinical sera were immediately tested for specificity evaluation, with the results shown in Table 1: the NT-proBNP antibody combined with 27-31aa and the NT-proBNP antibody combined with 42-46aa have better pairing specificity, and are obviously better than the antibody pairing of other binding fragments.

Fourth, chemical luminous platform verification

The preferred pairs (6NT-1 and 1NT-3-14) are continuously applied to a chemiluminescence platform, and indexes such as a linear range, a minimum detection limit and the like are tested. The operation steps are as follows:

label AE: (1) 0.5mg of NT-proBNP antibody is dialyzed into 20mM pH7.4 PB buffer and dialyzed at 4 ℃ for 4 hours; (2) adding dissolved 10mM acridinium ester into the protein, and carrying out rotary reaction for 15min at room temperature; (3) adding 0.1M glycine in 10 times excess of AE, and rotating at room temperature for 30 min; (4) the marker is dialyzed again into 20mM pH7.2PB buffer and dialyzed overnight at 4 ℃; (5) taking out the marker, adding glycerol and storing in dark.

Labeling magnetic beads: (1) 10mg of carboxyl magnetic beads are washed 4 times with 50mM MES, each time is 1mL, then 0.8mL of activation buffer solution is added, ultrasonic dispersion is carried out, 0.1mL of NHS and 0.1mL of EDC are added, the mixture is fully mixed, and the reaction is carried out for 10 minutes at room temperature (30 rpm); (2) magnetic separation, discarding supernatant, adding 50mM MES and NT-proBNP antibody, and rotating at room temperature (30rpm) for 4 hours; (3) washing for 2 times, 1mL each time, adding 1mL of confining liquid, and rotating (30rpm) at room temperature to react for 4 hours; (4) resuspend to 10 mg/mL.

And (3) detection process: (1) adding 50 mu L of magnetic bead working solution, 100 mu L of sample to be detected and 50 mu L of marked AE working solution into each hole, and incubating for 15min at 37 ℃; (2) adding washing solution into each hole, and washing for 4 times; (3) adding chemiluminescence substrate solution into each hole, and measuring the luminescence value of the quality control product by using a chemiluminescence automatic instrument.

The detection results are as follows:

as shown in FIG. 1, the linear range of pairing is wide, hook effect does not occur at 50000pg/mL, and the lowest detection limit is 4.3 pg/mL; other pairs, such as 3NT-12-1 as labeled antibody and 6NT-1 as coated antibody, have a minimum detection limit of 7.9pg/mL in parallel experiments.

Fifth and second round of paired screening

To reduce the risk of missed detection, the next step is to test for antibodies that incorporate additional binding fragments. The missing of each pair was examined with 96 positive samples. The results of introducing the third antibody at the labeled end and the coated end, respectively, are shown in tables 2 and 3: the NT-proBNP antibody combined with 13-27aa is introduced into the advantageous combination obtained in the 1+1 mode, namely the NT-proBNP antibody combined with 27-31aa and the NT-proBNP antibody combined with 42-46aa, so that a plurality of binding fragments of the NT-proBNP antigen can be recognized, the defects of respective detection are supplemented, and the pairing performance is better than that of antibodies combined with other binding fragments and different strains of the same fragment; on the basis of good specificity (Table 1), omission is further reduced by supplementing the binding fragments, and the accuracy of detection results is improved.

TABLE 2, 2+1 Pattern evaluation

TABLE 31 +2 Pattern evaluation

To further verify that the combination of binding fragments has high specificity and low omission factor not brought by specific antibodies, different antibodies of the same binding fragment were replaced, wherein 5NT-6 and 3NT-12-1 are antibodies binding to the 13-27aa fragment, 8NT-7 and 6NT-1 are antibodies binding to the 27-31aa fragment, and 3NT-22 and 1NT-3-14 are antibodies binding to 42-46aa, and the results are shown in Table 4.

TABLE 4 replacement verification

Marking	Coating quilt	Rate of missed examination
			5NT-6+6NT-1	1NT-3-14	0/96
6NT-1	5NT-6+1NT-3-14	0/96
			3NT-12-1+3NP-22	6NT-1	0/96
1NT-3-14	3NT-12-1+8NT-7	0/96
			3NT-12-1	6NT-1+1NT-3-14	0/96
6NT-1+1NT-3-14	3NT-12-1	1/96

To further verify the superior performance of the combination of binding fragments, the advantages obtained in the 2+1 mode were combined, namely the NT-proBNP antibody binding to 13-27aa, the NT-proBNP antibody binding to 27-31aa and the NT-proBNP antibody binding to 42-46 aa.

Sixth, fluorescent microsphere platform verification

Indexes such as linear range, lowest detection limit and the like are tested by taking 5NT-6 and 6NT-1 as labeled antibodies for mixed labeling and 1NT-3-14 as a coating antibody as an example.

The detection results are as follows:

as shown in FIG. 2, the linear range of pairing is broad, hook effect does not occur at 50000pg/mL, and the minimum detection limit is 3.5 pg/mL.

The invention also provides an antibody which is prepared by taking the NT-proBNP amino acid fragment 27-31 coupled carrier protein as immunogen immunization through the antibody preparation scheme, for example, the antibody which is combined with the 27-31aa fragment is evaluated by ELISA through a reactivity screening reaction₄₀₅NT30C-3 antibody of ≥ 0.5, and the antibody conjugated with 42-46aa fragment prepared by immunizing with NT-proBNP amino acid fragment 42-46 coupled carrier protein as immunogen, for example, evaluating OD by ELISA₄₀₅The pairing specificity of the antibody NT25D-7 and the like of not less than 0.5 is higher than 99.2 percent (the evaluation is carried out by the fluorescent microsphere detection method); coupling by NT-proBNP amino acid fragment 13-27Immunization with the carrier protein as an immunogen to produce antibodies that bind to the 13-27aa fragment, e.g., by assessing OD in ELISA using a reactive screening reaction₄₀₅The false positive rate of the antibodies NT32A-9 and the like which are more than or equal to 0.5 and the former two antibodies are not more than 1.0% (evaluated by the fluorescent microsphere detection method).

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An amino terminal brain natriuretic peptide detection kit, which is characterized by comprising a first antibody and a second antibody for detecting the amino terminal brain natriuretic peptide, wherein the first antibody is combined with the amino acid fragment at the 27 th-31 th positions of the amino terminal brain natriuretic peptide; the second antibody binds to amino acid fragment 42-46 of the amino terminal brain natriuretic peptide.

2. The kit of claim 1, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the second antibody is a labeled antibody and the first antibody is a coated antibody.

3. The kit of claim 1, further comprising a third antibody that detects an amino-terminal brain natriuretic peptide, wherein the third antibody binds to amino acid fragment 13-27 of the amino-terminal brain natriuretic peptide;

4. The kit of any one of claims 1 to 3, wherein the reactivity of the antibody is an ELISA-assessed OD₄₀₅≥0.5。

5. The kit of any one of claims 1 to 4, wherein the labeled antibody is labeled with a detectable label such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label, and/or a binding partner; for example, the enzyme can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the labeled antibody is labeled with a binding partner and a detectable label.

6. The kit of any one of claims 1 to 5, wherein the coated antibody is attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate, a nitrocellulose membrane or a microfluidic chip and/or a binding partner; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to a solid phase via a binding partner.

7. An antibody combination for detecting an amino-terminal brain natriuretic peptide, comprising a first antibody and a second antibody; optionally, a third antibody is also included; wherein the first antibody, the second antibody, the third antibody are as defined in any one of claims 1 to 6.

8. Use of the antibody combination of claim 7 in the preparation of a kit for detecting amino-terminal brain natriuretic peptide.

9. Use according to claim 8, wherein the kit is for 1) detection of the amino terminal brain natriuretic peptide content and/or 2) prediction, diagnosis, prognostic assessment and therapy guidance of heart failure, including for example acute heart failure.

10. A method of making the antibody combination of claim 7, comprising:

fragment 1: amino-terminal brain natriuretic peptide amino acid fragment 13-27;

fragment 2: amino-terminal brain natriuretic peptide amino acid fragment 27-31;