WO2022174570A1 - Method and kit for detecting amino-terminal pro-brain natriuretic peptide - Google Patents

Abstract

A kit and a method for detecting amino-terminal pro-brain natriuretic peptide. The kit comprises a first antibody and a second antibody, the first antibody binds to amino acid fragments at positions 27 to 31 of the amino-terminal pro-brain natriuretic peptide, and the second antibody binds to amino acid fragments at positions 42 to 46 of the amino-terminal pro-brain natriuretic peptide. The detection method comprises: contacting antibodies and a sample to generate a binding reaction, and detecting immune complexes generated from the binding reaction.

Description

A kind of amino terminal brain natriuretic peptide detection method and kit

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims the priority of the Chinese patent application with the application number "202110192561.6" and the title "A method and kit for detecting amino-terminal brain natriuretic peptides" filed with the China Patent Office on February 20, 2021, the entire contents of which are approved by References are incorporated in this disclosure.

technical field

The present disclosure relates to the field of protein detection. Specifically, the present disclosure relates to detection methods and kits for amino-terminal brain natriuretic peptides.

Background technique

Heart failure (HF) is a severe and terminal stage of various heart diseases, and clinically mainly manifests as dyspnea, fatigue, and fluid retention. According to statistics, the current prevalence of heart failure in my country is estimated to have reached about 1.3%, and there are about 10 million patients with the current disease. China has become the country with the largest group of heart failure patients in the world. Due to the preventable and controllable nature of cardiovascular disease, early diagnosis is crucial.

Serum marker detection is one of the important means for the early diagnosis and prognosis of heart failure in clinical practice. These include brain natriuretic peptide (BNP) and amino-terminal brain natriuretic peptide (NT-proBNP), which are the preferred serum markers for heart failure recommended by both domestic and foreign (ECS/ACC/AHA/HFSA/CSC) heart failure guidelines.

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

SUMMARY OF THE INVENTION

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

The present disclosure provides an amino-terminal brain natriuretic peptide detection kit, which includes a first antibody and a second antibody for detecting amino-terminal brain natriuretic peptide, wherein the first antibody binds to the 27th N-terminal brain natriuretic peptide -31 amino acid fragment; the second fragment binds the 42-46 amino acid fragment of the N-terminal brain natriuretic peptide.

In some embodiments, the second antibody is a coating antibody when the first antibody is a labeled antibody; or the first antibody is a coating antibody when the second antibody is a labeled antibody.

In some embodiments, it further comprises a third antibody that detects amino-terminal brain natriuretic peptide, the third antibody binds to the amino acid fragment of amino-terminal brain natriuretic peptide at positions 13-27;

For example, the antibodies used for pairing are divided into a first group of antibodies and a second group of antibodies, wherein the first group of antibodies and the second group of antibodies are selected from the following group:

1) the first group of antibodies includes a first antibody, and the second group of antibodies includes a second antibody and a third antibody;

2) the first group of antibodies includes the second antibody, and the second group of antibodies includes the first antibody and the third antibody;

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

The present disclosure provides any one of the above-mentioned kits, wherein the reactivity of the antibody is OD ₄₀₅ ≥ 0.5 as assessed by ELISA.

The present disclosure provides the kit of any of the above, wherein the labeled antibody is labeled with a detectable label and/or a binding partner, such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, Chemiluminescent, radioactive, or enzymatic labels; for example, may be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridine esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-half Lactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; binding partners are eg biotin/avidin, biotin /Streptavidin; eg, a labeled antibody is labeled with a detectable label by a binding partner.

The present disclosure provides the kit of any of the above, wherein the coated antibody is attached to a solid phase and/or a binding partner, such as magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidics Control chip; the binding partner is eg biotin/avidin, biotin/streptavidin; eg the coated antibody is linked to the solid phase through the binding partner.

The present disclosure provides antibody combinations for the detection of amino-terminal brain natriuretic peptides, including a first antibody and a second antibody; optionally, further including a third antibody; wherein the first antibody, the second antibody, and the third antibody are according to any one of the above defined.

The present disclosure provides use of any of the above-described kits or the antibody combination in an immunoassay.

The present disclosure provides the use of any one of the above-mentioned kits or the antibody combination in the detection of amino-terminal brain natriuretic peptides.

The present disclosure provides the use of any one of the above-mentioned kits or the antibody combination for detecting amino-terminal brain natriuretic peptides.

The present disclosure provides a method for detecting amino-terminal brain natriuretic peptide, comprising:

A) contacting the antibody combination with a sample under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure provides a method for diagnosing an N-terminal brain natriuretic peptide-related disease in a subject, comprising:

A) contacting the antibody combination with a sample from the subject under conditions sufficient for a binding reaction to occur to effect a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

In some embodiments, the disease associated with N-terminal brain natriuretic peptide comprises heart failure;

Preferably, the heart failure is selected from the group consisting of congestive heart failure, systolic heart failure, diastolic heart failure, heart failure with reduced ejection fraction (HFREF), heart failure with preserved ejection fraction (HFPEF), intermediate ejection fraction Extensive chronic heart failure (HFmEF), chronic heart failure, acute heart failure, post-acute heart failure, chronic heart failure of ischemic and non-ischemic origin, advanced heart failure, compensated heart failure, decompensated heart failure, right heart failure, left heart failure, total heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart failure associated with heart valve defects, heart failure associated with combined heart valves Defect-related heart failure, diabetic heart failure, heart failure associated with cardiac storage disease.

The present disclosure provides use of any of the above-described kits or the antibody combination in at least one of prediction, diagnosis, prognostic assessment, or treatment guidance of heart failure, including, eg, acute heart failure.

The present disclosure provides methods for any of the above-described kits or the antibody combination in at least one of the prediction, diagnosis, prognostic assessment or treatment guidance of heart failure, comprising:

A) contacting the antibody combination with a sample under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure provides use of the antibody combination in the preparation of a kit for detecting amino-terminal brain natriuretic peptide.

In some embodiments, the kit is used for 1) the detection of amino-terminal brain natriuretic peptide content, and/or 2) the prediction, diagnosis, prognosis assessment and treatment guidance of heart failure, including, for example, acute heart failure exhaustion.

The present disclosure provides a method for preparing the above-mentioned antibody combination, the method comprising:

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

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 natriuretic peptide amino acid fragment 42-46; and

2) Obtaining antibodies that bind the fragment 1 to fragment 3, respectively, from the animal.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. The drawings are some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

Figure 1 shows the linear range of 1+1 pairing detection with recombinant antigen on a chemiluminescence platform;

Figure 2 shows the linear range of 2+1 pairing detected by fluorescent microsphere detection method.

Detailed ways

Definition of Terms

As used herein, "aa" refers to the amino acid at position ... of the peptide.

After a lot of theoretical research and experimental exploration, the authors fully considered the structural properties of the amino-terminal brain natriuretic peptide, and analyzed and studied various NT-proBNP antigen ranges and detection antibodies to be tested. Detected combination of amino-terminal brain natriuretic peptide antigen regions.

In some embodiments, the kits of the present disclosure include a first antibody and a second antibody for detecting an amino-terminal brain natriuretic peptide, wherein the first antibody binds to the amino acid fragment at positions 27-31 of the amino-terminal brain natriuretic peptide ; The second antibody binds the amino-terminal natriuretic peptide at the 42-46th position; the antibody paired with the above amino acid fragments can improve the detection specificity for detection of the amino-terminal natriuretic peptide. For example, when the first antibody is a labeled antibody, the second antibody is a coated antibody; or when the second antibody is a labeled antibody, the first antibody is a coated antibody.

The kit of the present disclosure includes an amino-terminal brain natriuretic peptide detection reagent card (test strip). In some embodiments, immunofluorescence techniques can be used to label antibodies on detectable labels such as fluorescent microspheres, and the principle of double-antibody sandwich method can be used to detect NT-proBNP in samples. In some embodiments, the methods and kits of the present disclosure can improve specificity and/or reduce missed detection.

In some embodiments, the present disclosure may be performed by or include reagents for performing fluorescent immunochromatography. In some embodiments, immunochromatography can be accomplished by a double-antibody sandwich method with test strips. In some embodiments, the reagents and devices for performing the double-antibody sandwich method are not particularly limited, and any suitable reagents and/or devices can be used. For example, fluorescent immunochromatography strips can include, but are not limited to, sample pads, binding pads, NC membranes, and absorbent pads. In some embodiments, at least one fluorophore-labeled antibody is immobilized on the binding pad; in some embodiments, the NC membrane can be coated with two lines, such as T line and C line, respectively; the T line can be coated At least one antibody; capable of capturing the fluorophore-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, the sample to be tested is added to the injection port of the detection reagent card, and under the action of the lateral capillary, the sample to be tested first passes through the binding pad, and undergoes specific immunological binding with the fluorophore-labeled antibody on the binding pad. Binds to form antigen-antibody fluorescent complexes, which are immobilized in the T-line. The C line is coated with a substance that reacts with the free fluorophore-labeled antibody. When the free fluorophore-labeled antibody passes through the C line, it can specifically immunocombine with the substance on the C line, thereby being immobilized on the C line. in line. The fluorescence intensity of the control band (C) is inversely proportional to the test band (T), so that individual differences between samples can be corrected. The fluorescence intensity of the two bands detected by the fluorescence immunoassay analyzer is reflected in the peak area, and the T/C value (T peak area/C peak area) is calculated by the instrument's own calculation software and fitted to the set standard curve. The fluorescence immunoassay analyzer automatically converts the concentration value of NT-proBNP in the corresponding sample to be tested.

In some embodiments, the present disclosure enables the use of antibodies that bind different amino acid fragments to recognize multiple positions on an antigen, reducing the risk of missed detection and improving the detection rate.

In some embodiments, a third antibody for detecting amino-terminal brain natriuretic peptide is further included, and the third antibody binds to the amino acid fragment at positions 13-27 of the amino-terminal brain natriuretic peptide. In some embodiments, an antibody that binds the 13-27aa fragment of the amino-terminal brain natriuretic peptide is used as the first set of antibodies, and an antibody that binds the 27-31aa fragment of the amino-terminal brain natriuretic peptide and the antibody that binds the amino-terminal brain natriuretic peptide are used as the first set of antibodies. Antibodies to the 42-46aa fragment of the N-terminal natriuretic peptide are used as the second group of antibodies; in some embodiments, the antibody that binds the 27-31aa fragment of the amino-terminal brain natriuretic peptide is used as the first group of antibodies, and the amino-terminal brain natriuretic peptide is used. - 27aa fragment antibodies and antibodies that bind to the 42-46aa fragment of the amino-terminal brain natriuretic peptide are used as the second set of antibodies; in some embodiments, antibodies that bind the 42-46aa fragment of the amino-terminal brain natriuretic peptide are used as the first set of antibodies As the second group of antibodies, an antibody binding to the 13-27aa fragment of the amino-terminal brain natriuretic peptide and an antibody binding to the 27-31aa fragment of the amino-terminal brain natriuretic peptide were used.

The term "antibody" is used herein in the broadest sense. In some embodiments, the antibodies of the present disclosure may be monoclonal or polyclonal antibodies. In some embodiments, the antibodies of the present disclosure can be prepared using methods known in the art. In some embodiments, antibodies of the present disclosure can be prepared by immunizing animals with antigens comprising the amino acid fragments described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA (bovine serum albumin), ovalbumin, KLH (keyhole limpet hemocyanin), etc.) can be conjugated to immunoreactive substances (eg, epitope peptides) . Carrier proteins can include proteins or polypeptides that can function as immunogenic carriers. These types of polypeptides include, but are not limited to, albumin, serum albumin, globulin, crystallin, lipoprotein, and/or fragments thereof. In some embodiments, immunoreactive substances (eg, but not limited to, amino-terminal brain natriuretic peptide amino acid fragments) can be used to generate antibodies with affinity for NT-proBNP.

In some embodiments, binding of an antibody to an amino acid fragment corresponding to an amino-terminal brain natriuretic peptide may mean that the antibody is capable of binding to the amino acid fragment, but the amino acid fragment is not necessarily the smallest binding fragment.

In some embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc., can be used to detect the effects of the disclosed antibodies, such as binding activity and/or cross-reactivity. In some embodiments, the assay may include, but is not limited to, eg, ELISA (enzyme-linked immunosorbent assay), FACS (flow cytometry) binding assay, Biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibody of the present disclosure to antigen (antigenic peptide) binding is characterized, for example, in ELISA, for example, a reaction value ≥ 0.5 at 405 nm read by a peroxidase-labeled ELISA method is determined to be good Reactivity, can be used in immunoassays.

In some embodiments, the kit includes a first antibody and a second antibody; in some embodiments, the kit also includes a third antibody; in some embodiments, other antibodies can also be used as coating antibodies or labels Antibody.

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 it can be, for example, including but not limited to 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 passes The binding partner is attached to the solid phase.

In some embodiments, the labeled antibody is labeled with a detectable label. In some embodiments, the labeled antibody can be bound directly or indirectly to a detectable label. 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 enzymatic label; in some embodiments, the detectable label, for example, can be Rhodamine, Luciferin, Fluorescent Microspheres, Colloidal Gold, Acridine Esters, Luciferase, Horseradish Peroxidase, Alkaline Phosphatase, Beta-Galactosidase, Glucoamylase, Lysozyme, Sugar Oxidation Enzyme, 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 detectable label by a binding partner.

In some embodiments, the kits of the present disclosure include reagents suitable for performing immunoassays. In some embodiments, the kits of the present disclosure can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay IFA (indirect immunofluorescence assay), radioimmunoassay RIA (radioimmunoassay), and other non-enzyme-linked antibody binding assays or method.

In some embodiments, the present disclosure provides antibody combinations for the detection of amino-terminal brain natriuretic peptides, comprising a first antibody and a second antibody as defined above; in some embodiments, a third antibody as defined above.

In some embodiments, the present disclosure provides the use of antibody combinations in the manufacture of kits for the detection of amino-terminal brain natriuretic peptides. Wherein the kit is used for 1) detecting the content of amino-terminal brain natriuretic peptide, and/or 2) predicting, diagnosing, evaluating and treating 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 present disclosure may be the identification of a health state. In some embodiments, the diagnosis of the present disclosure may be the exclusion of negative samples. In some embodiments, the diagnosis of the present disclosure may be an auxiliary diagnosis. In some embodiments, the diagnosis of the present disclosure may be risk stratification.

In some embodiments, the present disclosure provides a method of preparing an antibody that detects an amino-terminal brain natriuretic peptide, the method comprising immunizing an animal as an antigen or hapten with an immunoreactive polypeptide comprising an amino acid fragment described herein, respectively, thereby preparing the assay Antibodies to amino-terminal brain natriuretic peptides such as monoclonal or polyclonal antibodies. Monoclonal or polyclonal antibodies can be produced by methods known in the art.

In some embodiments, the present disclosure provides the use of an immunoreactive polypeptide comprising an amino acid fragment described herein in the manufacture of a reagent or kit for the detection of amino-terminal brain natriuretic peptide.

Herein, a sample to be tested for NT-proBNP may include, but is not limited to, biological tissues, cells or body fluids in healthy or pathological states, such as blood samples such as plasma, serum, blood products.

The present disclosure will be described in further detail below mainly in conjunction with the embodiments. The following examples are provided to illustrate embodiments of the present disclosure and are not intended to limit the present disclosure. The present disclosure may optionally include embodiments not shown by way of example.

1. Preparation of monoclonal antibody against amino-terminal brain natriuretic peptide

1. Immune animals: Take 8-12-week-old BALB/c (albino laboratory mice) mice of the same strain as myeloma cells, and immunize them with NT-proBNP antigen containing 100 μg/mouse of protein and the same amount of complete Freund's adjuvant. The agent was thoroughly mixed and injected into the abdominal cavity of mice, and 100 μg/mouse of NT-proBNP antigen was fully mixed with the same amount of incomplete Freund's adjuvant every 2 weeks, and was injected into the abdominal cavity of mice for multiple times for boosting immunization. After testing mouse serum (indirect ELISA method), those with a titer above 1:2000 can be used for fusion. Three days before fusion, the mice were boosted by intraperitoneal immunization again at a dose of 50 μg/mouse.

2. Preparation of Feeder Cells

BALB/c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB/c mice were sacrificed by pulling their necks, immersed in 75% alcohol, and in a clean bench, the abdominal skin was cut open with scissors under aseptic operation, the peritoneum was exposed, and 5 mL of RPMI 1640 basal medium was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000rpm for 5 minutes, leave the precipitate, resuspend with RPMI 1640 screening medium (complete RPMI 1640 medium containing HAT), adjust the cell concentration to 1×10 ⁵ cells/mL, add to 96 wells Plate, 150 μL/well, incubated overnight at 37°C, 5% CO ₂ .

3. Preparation of Immune Splenocytes

Three days after the last immunization of the mice, the spleen was taken out under sterile conditions, placed in a petri dish, washed once with RPMI 1640 basal medium, and placed on a nylon mesh in a small beaker to grind and filter to prepare a cell suspension. Centrifuge, discard the supernatant, resuspend in RPMI 1640 basal medium, repeat this three times, and count.

4. Cell Fusion

(1) Take 40 mL of HAT medium, 15 mL of DMEM serum-free medium and 1 mL of 50% PEG (M12000) and place them in a 37°C water bath to pre-warm;

(2) Take mouse myeloma cell Sp2/0 (preserved by Philips Biotech Co., Ltd.) (2-5×10 ⁷ ), and the immune splenocyte (10 ⁸ ) suspension obtained in the above step 3, respectively, and add it to a 50 mL centrifuge tube Mix well, and add DMEM serum-free medium to 40 mL. Centrifuge for 10 minutes, pour off the supernatant, and mix.

(3) Place the centrifuge tube in pre-warmed water at 37°C, take 0.7 mL of the pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of pre-warmed serum-free medium at 37°C dropwise;

(4) Supplement DMEM serum-free medium to 40 mL, centrifuge for 10 minutes, and pour off the supernatant. Add 40 mL of HAT medium containing 15% to 20% fetal bovine serum. Mix well with a pipette, add dropwise to the small wells of 4 96-well cell culture plates containing feeder cells, 2 drops per well, and culture in an incubator at 37°C and 7% CO ₂ .

5. Selective Culture of Hybridoma Cells

The immune mouse spleen cells and mouse myeloma cells obtained in step 4-(4) are treated with PEG to form a mixture of various cellular components, including unfused myeloma cells and immune spleen cells, myeloma cells The synkaryon of cells and the synkaryon of immune spleen cells, and the conductor of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. For this purpose, unfused cells and homofused synkaryons must be removed from this multi-cell mixture and true hybrid cells selected. Therefore, on the 1st, 3rd, 5th, and 7th days after cell fusion, the culture medium was exchanged with the aforementioned HAT medium.

6. Detection of antibodies and cloning of hybridoma cells

The supernatant of each culture well was aspirated, and the culture wells containing the antibody of NT-proBNP in the culture medium were detected by indirect ELISA method. Hybridoma cells were cloned by limiting dilution. After culturing, a single cell can be proliferated into a homologous cell clone; a cell line with better reactivity and stable secretion of anti-NT-proBNP monoclonal antibody is obtained through reactivity screening.

Method for reactivity screening: Microtiter plates (Nunc, Maxisorb) were coated with 100 μL/well of coating buffer containing 2.5 μg/mL human NT-proBNP (as antigen) for 1 hour at room temperature with stirring. Post-coating treatments were incubated in PBS buffer and 1% monoclonal antibody for 30 minutes. Then, wash with wash buffer. 100 μL/well of antibody samples were incubated with agitation for 1 hour at room temperature. Then wash 2 more times with washing solution. It was then incubated with 100 μL/well of the detection antibody peroxidase-conjugated goat anti-mouse IgG diluted 1:40000 in PBS buffer for 1 hour at room temperature with stirring. After washing with washing buffer again, the peroxidase activity is measured by conventional methods (eg, the reaction value at 405 nm is read with an ELISA plate reader), and an antibody with OD ₄₀₅ ≥ 0.5 (with good reactivity) is obtained.

2. Identification of Antibody Binding Fragments

Different NT-proBNP antigen peptides were used to coat the microwells respectively, PBS+20% NBS was used as the diluent, the primary antibody was diluted to a concentration of 1 μg/mL, and the goat anti-mouse IgG-HRP was used as the secondary antibody. The response to different antigens is used to determine the binding fragment of the monoclonal antibody. The identification results of the binding fragments of the cell lines used in the embodiments are as follows:

binding fragment	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

3. Paired screening

Antibodies with different binding fragments were selected for coating and labeling, and cross-pairing experiments were performed. The screening process was as follows:

1. Mark

(1) Take 100uL of fluorescent microspheres, centrifuge at 15000rpm/8min/4°C, remove the supernatant after centrifugation, resuspend to 100uL, and sonicate for 2-3 times;

(2) Prepare 10 mg/mL of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide);

(3) Activation: use EDC and NHS to activate the fluorescent microspheres to a final volume of 200uL, mix well in the dark for 18 minutes, centrifuge at 15000rpm/10min/4°C, and remove the supernatant;

(4) Washing: resuspend to 100uL, ultrasonicate 2-3 times, 15000rpm/8min/4℃, remove supernatant, repeat twice;

(5) Coupling: resuspend to 200uL, sonicate for 2-3 times, add the antibody to the fluorescent microspheres to a final volume of 300uL, mix well in the dark, and react at 37°C for 2h;

(6) Blocking: Add 100uL of blocking agent, mix well in the dark, and react at 37°C for 1h;

(7) Washing: After blocking, centrifuge at 15000rpm/12min/4°C and remove the supernatant. Resuspend to 100uL and sonicate 2-3 times. Centrifuge again at 15000rpm/8min/4℃, and remove the supernatant;

(8) Storage: resuspend to 100uL, ultrasonicate 2-3 times, and store at 4°C for later use.

2. Coat

(1) Assemble the nitrocellulose membrane and the fluorescent PVC bottom plate for subsequent use;

(2) Dilute the antibody to 1.0-2.0 mg/mL, draw a line on the NC film evenly with a gold spray film machine, and then put it into a 37°C incubator for drying for at least 45 minutes. Assemble and cut strips, add samples for testing.

3. Detection

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

(2) Detection method: use a fluorometer to detect. The instrument reading T/C represents the ratio of the T peak area to the C peak area, and the activity of the reaction pairing. The higher the T/C in the quality control and positive samples, the higher the activity.

Table 1. The first round of screening

mark	Coated	specificity
1NT-9	3NT-12-1	12/138
1NT-9	6NT-1	11/138
1NT-9	2NT-17	9/138
1NT-9	2NT-9-1	7/138
1NT-9	1NT-3-14	7/138

1NT-9	7NT-1-5	10/138
3NT-12-1	6NT-1	3/138
3NT-12-1	2NT-17	8/138
3NT-12-1	2NT-9-1	4/138
3NT-12-1	1NT-3-14	4/138
3NT-12-1	7NT-1-5	5/138
6NT-1	2NT-17	14/138
6NT-1	2NT-9-1	5/138
6NT-1	1NT-3-14	0/138
6NT-1	7NT-1-5	4/138
2NT-17	2NT-9-1	10/138
2NT-17	1NT-3-14	12/138
2NT-17	7NT-1-5	9/138
2NT-9-1	1NT-3-14	8/138
2NT-9-1	7NT-1-5	6/138
1NT-3-14	7NT-1-5	9/138

The above pairings in the first round of cross-pairing screening were all active, and then 138 random clinical sera were tested to evaluate specificity. The results are shown in Table 1: NT-proBNP antibody binding 27-31aa and NT-proBNP binding 42-46aa The specificity of the antibody pairing is better, significantly better than the antibody pairing of other binding fragments.

4. Chemiluminescence platform validation

The preferred pairings (6NT-1 and 1NT-3-14) were continued to be applied to the chemiluminescence platform, and their linear range, minimum detection limit and other indicators were tested. The operation steps are as follows:

Labeling AE: (1) dialyze 0.5 mg of NT-proBNP antibody into 20 mM pH7.4 PB buffer, and dialyze at 4°C for 4 hours; (2) add dissolved 10 mM acridine ester to the protein, and rotate at room temperature for 15 min; (3) Add 10 times excess 0.1M glycine to AE, and rotate at room temperature for 30min; (4) Re-dialyze the marker to 20mM pH7.2 PB buffer, dialyze overnight at 4°C; (5) Take out the marker, Add glycerol to protect from light.

Labeled magnetic beads: (1) Take 10 mg of carboxyl magnetic beads and wash 4 times with 50 mM MES, 1 mL each time, then add 0.8 mL of activation buffer, ultrasonically disperse, add 0.1 mL of NHS and 0.1 mL of EDC, mix well, rotate at room temperature ( 30rpm) for 10 minutes; (2) Magnetic separation, discard the supernatant, add 50mM MES and NT-proBNP antibody, rotate at room temperature (30rpm) and react for 4 hours; (3) Wash 2 times, 1mL each time and add 1mL blocking solution , Rotate (30rpm) at room temperature for 4 hours; (4) Resuspend to 10mg/mL.

Detection process: (1) Add 50 μL magnetic bead working solution, 100 μL sample to be tested and 50 μL labeled AE working solution to each well, incubate at 37°C for 15 min; (2) Add washing solution to each well and wash 4 times; (3) Each well The chemiluminescence substrate solution was added, and the luminescence value of the quality control substance was measured with a chemiluminescence automatic instrument.

The test results are as follows:

As shown in Figure 1, the pairing has a wide linear range, no hook (hook) effect occurs at 50,000 pg/mL, and the minimum detection limit is 4.3 pg/mL; other pairings such as 3NT-12-1 as the labeled antibody, Taking 6NT-1 as an example of a coated antibody, the minimum detection limit in parallel experiments was 7.9 pg/mL.

5. The second round of matching screening

In order to reduce the risk of missed detection, the next step is to consider the introduction of antibodies with other binding fragments for testing. 96 positive samples were used to investigate the missed detection of each pair. The third antibody was introduced at the labeled end and the coated end, respectively, and the results are shown in Table 2 and Table 3: the advantageous combination obtained in the 1+1 mode, that is, the NT-proBNP antibody that binds 27-31aa and the antibody that binds 42-46aa. The introduction of NT-proBNP antibody binding to 13-27aa in the NT-proBNP antibody combination can recognize multiple binding fragments of NT-proBNP antigen, supplement the shortcomings of each detection, and have better pairing performance than introducing other binding fragments and binding the same fragment. Excellent; On the basis of good specificity (Table 1), the missed detection was further reduced by supplementing the binding fragment, and the accuracy of the detection result was improved.

Table 2. Evaluation of 2+1 Mode

Table 3. 1+2 mode evaluation

In order to further verify that the high specificity and low missed detection rate of the combination of binding fragments are not caused by specific antibodies, different strain antibodies of the same binding fragment were replaced, wherein 5NT-6 and 3NT-12-1 were combined with 13- 8NT-7 and 6NT-1 are antibodies that bind to the 27aa fragment, and 3NT-22 and 1NT-3-14 are antibodies that bind the 42-46aa fragment. The results are shown in Table 4.

Table 4. Replacement verification

mark	Coated	Missing detection rate
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

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

6. Validation of fluorescent microsphere platform

Taking 5NT-6 and 6NT-1 as labeled antibodies for mixed standard, and 1NT-3-14 as coating antibody as an example, the linear range, minimum detection limit and other indicators were tested.

The test results are as follows:

As shown in Figure 2, the paired linear range is wide, no hook effect occurs at 50,000 pg/mL, and the minimum detection limit is 3.5 pg/mL.

The present disclosure also uses the above-mentioned antibody preparation scheme, using NT-proBNP amino acid fragment 27-31 conjugated carrier protein as an immunogen to prepare an antibody that binds to the 27-31aa fragment, for example, OD ₄₀₅ is evaluated by ELISA through the reactive screening reaction Antibody NT30C-3 with ≥0.5, etc., is immunized with NT-proBNP amino acid fragment 42-46 conjugated carrier protein as immunogen to obtain an antibody that binds to 42-46aa fragment, for example, OD ₄₀₅ ≥0.5 is evaluated by ELISA after reactivity screening reaction The pairing specificity of antibodies such as NT25D-7 was higher than 99.2% (evaluated by the aforementioned fluorescent microsphere detection method); the NT-proBNP amino acid fragment 13-27 conjugated carrier protein was used as an immunogen to prepare an antibody that binds to the 13-27aa fragment. For example, the antibody NT32A-9 with OD ₄₀₅ ≥ 0.5 is evaluated by ELISA after reactivity screening reaction, and the missed detection rate of pairing with the former two is not more than 1.0% (evaluated by the aforementioned fluorescent microsphere detection method).

The above-mentioned embodiments further describe the purpose, technical solutions and beneficial effects of the present disclosure in detail. It should be understood that the above-mentioned embodiments are only examples of the present disclosure, and are not intended to limit the present disclosure. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the disclosure should be included within the protection scope of the present disclosure.

Industrial Applicability

The present disclosure provides an amino-terminal brain natriuretic peptide detection method and a kit. The antibody combination provided by the present disclosure has high specificity and can be widely used in the detection of the amino-terminal brain natriuretic peptide content and the detection of cardiac Prediction, diagnosis, prognostic assessment and treatment guidance of failure. The prepared kit also has the same technical effect as the antibody, and has high application value and broad market application prospect.

Claims (18)

1. An amino-terminal brain natriuretic peptide detection kit, characterized in that it comprises a first antibody and a second antibody for detecting amino-terminal brain natriuretic peptide, wherein the first antibody binds to the first antibody of the amino-terminal brain natriuretic peptide Amino acid fragment 27-31; the second antibody binds amino-terminal natriuretic peptide 42-46 amino acid fragment.
2. The kit according to claim 1, wherein when the first antibody is a labeled antibody, the second antibody is a coated antibody; or when the second antibody is a labeled antibody, the first antibody is a coated antibody.
3. The kit according to claim 1 or 2, wherein the kit further comprises a third antibody for detecting amino-terminal brain natriuretic peptide, the third antibody binds to the 13th- 27 amino acid fragment;

   For example, the antibodies used for pairing are divided into a first group of antibodies and a second group of antibodies, wherein the first group of antibodies and the second group of antibodies are selected from the following group:

   1) the first group of antibodies includes a first antibody, and the second group of antibodies includes a second antibody and a third antibody;

   2) the first group of antibodies includes the second antibody, and the second group of antibodies includes the first antibody and the third antibody;

   3) The first set of antibodies includes the third antibody, and the second set of antibodies includes the first antibody and the second antibody.
4. The kit according to any one of claims 1-3, wherein the reactivity of the antibody is OD ₄₀₅ ≥ 0.5 evaluated by ELISA.
5. The kit according to any one of claims 1-4, wherein the labeled antibody is labeled with a detectable label and/or a binding partner, such as a metal particle, a fluorescent label, a chromophore Group label, electron dense label, chemiluminescence label, radiolabel, or enzyme label; for example, can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridine ester, luciferase, horseradish peroxidase, alkali Sex phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase label; binding partners such as biotin/ Avidin, biotin/streptavidin; eg, labeled antibodies are labeled with a detectable label by a binding partner.
6. The kit of any one of claims 1-5, wherein the coated antibody is linked to a solid phase and/or a binding partner, such as a solid phase such as magnetic particles, latex particles, microtiter plates, Nitrocellulose membranes or microfluidic chips; binding partners are eg biotin/avidin, biotin/streptavidin; eg coated antibodies are attached to the solid phase via binding partners.
7. An antibody combination for detecting amino-terminal brain natriuretic peptide, characterized in that it includes a first antibody and a second antibody; optionally, it also includes a third antibody; wherein the first antibody, the second antibody, and the third antibody are according to the claims As defined in any one of 1-6.
8. Use of the kit according to any one of claims 1-6 or the antibody combination according to claim 7 in an immunoassay.
9. Use of the kit according to any one of claims 1-6 or the antibody combination according to claim 7 in the detection of amino-terminal brain natriuretic peptide.
10. Use of the kit according to any one of claims 1-6 or the antibody combination according to claim 7 for the detection of amino-terminal brain natriuretic peptide.
11. A method of detecting amino-terminal brain natriuretic peptide, comprising:

    A) contacting the antibody combination of claim 7 with a sample to effect a binding reaction under conditions sufficient for the binding reaction to occur; and

    B) Detection of immune complexes produced by the binding reaction.
12. Use of the kit of any one of claims 1-6 or the antibody combination of claim 7 in at least one of prediction, diagnosis, prognostic assessment or treatment guidance of heart failure including, for example, acute heart failure.
13. The method of the kit of any one of claims 1-6 or the antibody combination of claim 7 in at least one of prediction, diagnosis, prognosis assessment or treatment guidance of heart failure, comprising:

    A) contacting the antibody combination of claim 7 with a sample to effect a binding reaction under conditions sufficient for the binding reaction to occur; and

    B) Detection of immune complexes produced by the binding reaction.
14. A method of diagnosing an N-terminal brain natriuretic peptide-related disorder in a subject, comprising:

    A) contacting the antibody combination of claim 7 with a sample from the subject under conditions sufficient for a binding reaction to occur to effect a binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.
15. The method of claim 14, wherein the disease associated with N-terminal brain natriuretic peptide comprises heart failure;

    Preferably, the heart failure is selected from the group consisting of congestive heart failure, systolic heart failure, diastolic heart failure, heart failure with reduced ejection fraction (HFREF), heart failure with preserved ejection fraction (HFPEF), intermediate ejection fraction Extensive chronic heart failure (HFmEF), chronic heart failure, acute heart failure, post-acute heart failure, chronic heart failure of ischemic and non-ischemic origin, advanced heart failure, compensated heart failure, decompensated heart failure, right heart failure, left heart failure, total heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart failure associated with heart valve defects, heart failure associated with combined heart valves Defect-related heart failure, diabetic heart failure, heart failure associated with cardiac storage disease.
16. Use of the antibody combination according to claim 7 in the preparation of a kit for detecting amino-terminal brain natriuretic peptide.
17. The use according to claim 16, wherein the kit is used for 1) detection of N-terminal brain natriuretic peptide content, and/or 2) prediction, diagnosis, prognosis evaluation and treatment of heart failure Guidance, heart failure includes, for example, acute heart failure.
18. A method of preparing the antibody combination of claim 7, wherein the method comprises:

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

    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 natriuretic peptide amino acid fragment 42-46; and

    2) Obtaining antibodies that bind the fragment 1 to fragment 3, respectively, from the animal.

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