CN112759639A

CN112759639A - N-terminal brain natriuretic peptide precursor polypeptide, antibody and kit

Info

Publication number: CN112759639A
Application number: CN202011644512.3A
Authority: CN
Inventors: 易维京; 姬成东; 吴春容; 温雅雯; 张鹤铭; 汤定斌
Original assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-07

Abstract

The invention relates to the field of biotechnology, in particular to an N-terminal brain natriuretic peptide precursor polypeptide, an antibody and a kit, which can specifically identify the N-terminal brain natriuretic peptide precursor.

Description

N-terminal brain natriuretic peptide precursor polypeptide, antibody and kit

Technical Field

The invention relates to the field of biotechnology, in particular to an N-terminal brain natriuretic peptide precursor polypeptide, an antibody and a kit

Background

N-terminal brain natriuretic peptide (N-terminal fragment of the BNP precorsor, NT-proBNP) is a heart failure detection marker widely used at present, and has been widely applied to early diagnosis of heart failure, risk grade division of heart failure, identification of pulmonary dyspnea and cardiogenic dyspnea, high risk group screening of heart failure, guidance of treatment and prognosis of heart failure and the like. NT-proBNP in blood is derived from brain natriuretic peptide precursor (proBNP) and is formed by enzyme digestion. Currently, NT-proBNP content in the blood of patients is mainly detected by an immune-based method. Namely, the combination of antibodies specifically recognizing NT-proBNP is adopted to realize the capture and quantitative reading of NT-proBNP. Therefore, good antibodies are the key to the development of good quality NT-proBNP diagnostic reagents. At present, many manufacturers in China develop diagnostic kits of NT-proBNP, but detection reagents with excellent performance mostly depend on imported antibody raw materials and are high in price. Therefore, it is necessary to develop a low-cost NT-proBNP antibody material having excellent clinical manifestations.

At present, some reports of attempts to develop NT-proBNP antibodies exist in China, but the properties of detection sensitivity, clinical goodness of fit and the like are still inferior to those of imported antibodies when the antibodies are used for clinical detection. Studies have shown that there are heterogeneous glycosylation modifications of proBNP and NT-proBNP proteins in human blood circulation. The current knowledge of the biological function and physiological significance of glycosylation modification on proBNP and NT-proBNP is also very limited. However, these glycosylation modifications clearly have a significant influence on the selection and detection of antibodies in clinical tests. On the other hand, in the development of related antibodies, synthetic linear polypeptides are mostly used for animal immunization, so that the developed antibodies can recognize related epitopes which are not modified by glycosylation, but O-glycosylation modification on NT-proBNP in human blood may cause failure of antibody recognition, thereby affecting detection quality. On the other hand, NT-proBNP is derived from the enzymatic processing of proBNP, and glycosylation modification also has an influence on the processing process. Thus, differences in antibody recognition sites lead to differences in the ability of the antibody to discriminate between NT-proBNP and proBNP, and thus to differences in the final assay results.

Disclosure of Invention

Based on the above recognition, the present invention has developed four antibody combinations: ab21, Ab22, Ab23 or Ab25 can realize the specific detection of NT-proBNP and improve the detection sensitivity and affinity.

In one aspect, the invention provides an amino-terminal pro-brain natriuretic peptide (NT-proBNP) polypeptide, which is characterized in that the amino acid sequence is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

In another aspect, the present invention provides an anti-NT-proBNP monoclonal antibody or an antigen-binding portion thereof, wherein the NT-proBNP monoclonal antibody or the antigen-binding portion thereof is formed by immunizing an animal with a recombinant protein formed by coupling the polypeptide with a carrier protein.

In another aspect, the invention discloses an anti-NT-proBNP monoclonal antibody or an antigen-binding portion thereof that specifically binds to amino acid regions 13-27, 27-31, or 42-46 of NT-proBNP. The binding of the antibody bound in said amino acid region to NT-proBNP or proBNP is not affected by the level of modification of the protein glycosylation.

Preferably, said anti-NT-ProBNP monoclonal antibody or antigen binding portion thereof comprises:

a) a heavy chain variable region CDR1 as set forth in SEQ ID No. 5;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 6;

c) heavy chain variable region CDR3 as set forth in SEQ ID No. 7;

d) light chain variable region CDR1 as set forth in SEQ ID No. 8;

e) light chain variable region CDR2 as set forth in SEQ ID No. 9;

f) light chain variable region CDR3 as set forth in SEQ ID No. 10;

the anti-NT-proBNP monoclonal antibody or the antigen-binding portion thereof is named Ab21

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 11;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 12;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 13;

d) light chain variable region CDR1 as set forth in SEQ ID No. 14;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 15;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 16;

the anti-NT-proBNP monoclonal antibody or the antigen-binding portion thereof is named Ab22

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 17;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 18;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 19;

d) light chain variable region CDR1 as set forth in SEQ ID No. 20;

e) light chain variable region CDR2 as set forth in SEQ ID No. 21;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 22;

the anti-NT-proBNP monoclonal antibody or the antigen-binding portion thereof is named Ab23

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 23;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 24;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 25;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 26;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 27;

f) light chain variable region CDR3 as set forth in SEQ ID No. 28;

the anti-NT-proBNP monoclonal antibody or the antigen-binding portion thereof is named Ab25

The present invention also relates to a NT-proBNP detection kit comprising the monoclonal antibody or the antigen binding portion thereof.

The invention also relates to a conjugate comprising a chemical label or a biomarker covalently linked to any of the monoclonal antibodies described above.

The invention also relates to a conjugate formed by coupling the monoclonal antibody and/or the conjugate with a solid medium or a semi-solid medium

The invention also relates to the application of the monoclonal antibody and/or the conjugate in the preparation of products for detecting Ab21, Ab22, Ab23 or Ab25 expression.

Drawings

FIG. 1: ab21 stability test gel pattern;

FIG. 2: ab25 stability test gel pattern;

FIG. 3: ab22 stability test gel pattern;

FIG. 4: ab23 stability test gel pattern;

FIG. 5: ab21 and Ab25 were applied to a standard curve in the detection of colloidal gold immunochromatography;

FIG. 6: ab21 and Ab25 applied to the accuracy curve of the colloidal gold immunochromatography detection;

FIG. 7: ab23 and Ab25 were applied to a standard curve in fluorescence immunochromatographic assay;

FIG. 8: ab21 and Ab22 were applied to a standard curve in chemiluminescent kit detection;

FIG. 9: ab21 and Ab22 are applied to the clinical recombination rate in the chemiluminescence kit detection;

FIG. 10: ab21 and Ab22 were applied to detection correlation in chemiluminescence kit detection.

Detailed Description

The embodiments of the present invention are illustrated below by specific examples, and the scope of the present invention is not limited to the specific embodiments described below. The examples are merely illustrative of the principles of the present invention and its efficacy, and are not to be construed as limiting the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Example 1: preparation of immunogens

In order to screen out antibodies aiming at specific epitopes, peptide fragments aiming at different epitopes are specifically synthesized, and the sequences are shown as follows:

(1)ETS GLQEQ RNHLQ GK；

(2)KLSE LQVEQ TSLEP LQ；

(3)TSLEP LQESP RPTGV W；

(4)KSRE VATEG IRGHR KMVLY T；

coupling of small peptides to BSA protein carriers by carbodiimide (EDC): respectively weighing 15mg of small peptide, 25mg of EDC-HCl and 5mg of N-hydroxysuccinimide (NHS), respectively dissolving in 0.25ml of ultrapure water to fully dissolve, mixing and stirring the small peptide and the EDC-HCl at 4 ℃ in dark place for reacting overnight, slowly dropwise adding about 0.5ml of ultrapure water containing 5mg of BSA into the mixed solution, adjusting the pH value to 7.4, continuing to stir for reacting for 6h, fully dialyzing the reaction mixture with 10mM PBS (phosphate buffer solution) with pH7.4, changing the dialysate about every 5h, changing the dialysate for 6 times, and finally subpackaging at-20 ℃ for storage for later use.

Example 2: preparation of anti-NT-proBNP monoclonal antibody

Preparation process of mouse antibody

The recombinant proteins obtained in example 1 were used as immunogens to immunize mice, respectively, to prepare monoclonal antibodies.

The preparation method comprises the following steps:

a) mouse immunization and antibody detection: the recombinant protein of 2mg/ml and Freund complete adjuvant are mixed in equal volume and emulsified, and then injected into 3 SPF female Balb/c mice of 6-8 weeks old, 40 mu g of each mouse, the recombinant protein and incomplete adjuvant are mixed, emulsified and injected into 40 mu g of the mice two weeks after the initial immunization, blood is collected through tail veins after two weeks, supernatant is collected by centrifugation and serum titer is detected by ELISA, and the recombinant protein and incomplete adjuvant are immunized once every two weeks and serum titer is detected. Screening serum titer 10⁶The above mice were immunized, and spleen of the immunized mice was used to isolate lymphocytes for cell fusion.

b) Cell fusion:

lymphocytes from the immunized mice were isolated and either PEG 1500-mediated or electrofused with cultured SP2/0 myeloma cells. Fusion cell culture was screened in HAT-1640 medium containing 20% FBS serum. After one week, the medium was changed and after another 4 days of culture, culture supernatants were taken for positive clone screening.

Preparation process of sheep antibody

(1) Chemically synthesized human recombinant peptide is used for immunizing the Boer sheep after being coupled with BSA; separating sheep spleen cells, extracting total RNA, and preparing cDNA by reverse transcription technology;

(2) using cDNA as template to respectively synthesize sheep IgG heavy chain coding sequence and light chain coding sequence, and connecting the heavy chain coding sequence and the light chain coding sequence by using overlap extension PCR technology;

(3) inserting the coding sequence obtained after the connection in the step (2) into a phagemid vector to construct an immune antibody library; screening to obtain a single-chain antibody specifically combined with human NT-proBNP through phage display and antigen specificity panning;

(4) the DNA fragment of the single-chain antibody for coding the human NT-proBNP is connected with the coding fragment of the human IgG Fc segment and then is subcloned into a eukaryotic expression vector to construct recombinant plasmids, the recombinant plasmids are transfected into host cells to be expressed, and the cell culture supernatant is subjected to affinity purification to obtain the specific recombinant goat monoclonal antibody of the human NT-proBNP.

c) And (3) cell screening:

hybridoma cells were cultured on 96-well cell culture plates using HAT selection medium with a total fusion rate > 95% as detected under a microscope. Selecting a hole with a monoclonal cell from a 96-well plate, detecting the supernatant of the monoclonal cell hole by adopting a coating natural NT-proBNP protein, selecting the cell in the hole with OD450 more than 0.8 for subcloning, and finally obtaining a cell clone with the reaction positive rate of more than 99 percent to the natural NT-proBNP as a positive hybridoma cell strain secreting the anti-NT-proBNP monoclonal antibody.

d) Cloning and determination of antibody Gene sequences

The subtypes of the heavy and light chains of the monoclonal antibody were identified by the SBA cloning System-HRP kit from southern Biotech according to the protocol of the instruction. The specific operation is as follows:

1. the capture antibody was diluted to 1 μ g/mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), added to the microplate at 100 μ L/well, and coated overnight at 4 ℃. The plate was washed 3 times with PBS buffer (wash solution) containing 0.05% Tween-20.

2. The culture supernatant of the hybridoma to be tested was diluted 1: 1 with a diluent (1% BSA, 0.1% PBST), 100. mu.L/well of the diluted culture supernatant was applied to an ELISA plate, and the plate was incubated at 37 ℃ for 30 minutes. The corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) were diluted 1: 3000 with the diluent.

3. After washing the plate with the plate washing solution 3 times, 100. mu.L of diluted enzyme-labeled antibody was added to each well, and the mixture was incubated at 37 ℃ for 30 minutes. The plate was washed 3 times again, then the developing solution was added, and after about 5 minutes (depending on the intensity of the reaction), 2M sulfuric acid was added to terminate the reaction, and the OD450 absorbance was read.

The heavy chains of the four antibodies are identified as IgG2b, and the light chains are Kappa. According to antibody subtype results, a mature technical route is adopted to clone an antibody gene sequence. The hybridoma cells with good growth state were collected, total RNA of the hybridoma cells was extracted by Trizol of Thermo, mRNA was Reverse-transcribed into cDNA according to the protocol of PrimeScript II Reverse Transcriptase Transcriptase of Takara, and the cDNA was frozen at-20 ℃ for use.

The specific operation flow of reverse transcription is as follows:

first, 10. mu.L of a total volume of a premixed solution of template RNA/primer DNA was prepared, which contained 1. mu.L of 10. mu.M concentration of specific primers (sequences atgggtgccagtgtctcttagga and gaagcctccaagaccttagaagggaa), 4. mu.L of a mixture of dNTPs at 2.5mM, and 5. mu.L of RNA (total amount less than 5. mu.g). After mixing, the mixture was treated at 65 ℃ for 5 minutes and immediately placed on ice.

mu.L of 5 XPrimeScript II buffer, 20 units of RNase inhibitor and 1. mu.L of PrimeScript II RTase (200 units) were added to the premix, mixed well, reacted at 42 ℃ for 60 minutes, treated at 70 ℃ for 15 minutes, and then cooled on ice until needed.

And (3) respectively and independently carrying out amplification attempts by using cDNA as a template and antibody gene amplification primers reported in the literature, and screening out the primers capable of efficiently amplifying the antibody genes. PCR was performed according to the protocol of Phanta Max Super-Fidelity DNA Polymerase of Nanjing Nodezak

The PCR reaction system is as follows:

25μL 2×Phanta

1μL dNTP

4 uL of 10uM primer pair

4 μ L hybridoma cell cDNA

1μL DNA polymerase

15μL dd H2O

The total reaction volume was 50. mu.L. The amplification conditions were: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 30 s; annealing at 56 deg.C for 30 s; extension was 72 ℃ for 2 min. The PCR product was Gel recovered according to the OMEGA Gel Extraction Kit instructions of OMEGA, and the antibody gene sequence was obtained by plasmid sequencing after the amplified product was linked to the T vector.

e) Antibody titer determination

NT-proBNP full-length prokaryotic antigen is diluted to 1ug/ml, added to a 96-well plate according to 100ul per well, incubated overnight at 4 ℃, and washed. The antibody is diluted to a proper concentration, diluted according to a 3-fold ratio, 100ul is put in each hole, incubated at 37 ℃ for 1h, and plates are washed. Diluting goat anti-mouse HRP enzyme-labeled antibody at a ratio of 1: 3000, incubating at 37 ℃ for 30min, developing for 3min, and detecting absorbance by A450 nm.

The control was the antibody itself incubated at 4 ℃.

TABLE 1 Ab21 Indirect potency assay

TABLE 2 Ab22 Indirect potency assay

TABLE 3 Ab23 Indirect potency assay

TABLE 4 Ab25 Indirect potency assay

The results show that: under lower concentration, the detection signal has larger gradient, which indicates that the sensitivity of the four antibodies is higher, and the four antibodies are suitable for subsequent tests.

f) Determination of antibody stability:

the antibodies were thermally accelerated at 37 ℃ for 7 days in a defined buffer, and the accelerated antibodies were evaluated by SDS-PAGE and ELISA indirect methods, and the long-term stability of the antibodies was identified in comparison with 4 ℃. The freeze-thaw stability of the antibody was evaluated by repeating 5 times of freeze-thawing at-20 ℃ according to the same standard.

TABLE 5 Ab21 stability data

The results are shown in table 5 and fig. 1: wherein the rightmost side is Marker, the antibody reagents added in the lane from right to left are Ab 2137 ℃ accelerated 31d non-reduction, Ab 2137 ℃ accelerated 31d reduction, Ab 2137 ℃ accelerated 24d non-reduction, Ab 2137 ℃ accelerated 24d reduction, Ab 2137 ℃ accelerated 10d non-reduction, Ab 2137 ℃ accelerated 10d reduction, Ab 214 ℃ non-reduction, Ab 214 ℃ reduction, Ab21 freeze-thaw for 5 times non-reduction, and Ab21 freeze-thaw for 5 times reduction.

The experimental result shows that the Ab21 can be stably stored at 4 ℃, the property is still stable after being accelerated for one month at 37 ℃, and the performance of using the reagent when the bottle is opened is ensured, so that the accuracy and stability of the detection result are ensured.

Table 6 AB25 stability data

The results are shown in table 6 and fig. 2: the rightmost side is Marker, and the antibody reagents added in the lane from right to left are Ab25 freeze-thawing for 5 times of non-reduction and Ab25 freeze-thawing for 5 times of reduction. Ab 2537 ℃ accelerated 31d non-reduction, Ab 2537 ℃ accelerated 31d reduction, Ab 2537 ℃ accelerated 14d non-reduction, Ab 2537 ℃ accelerated 14d reduction, Ab 2537 ℃ accelerated 7d non-reduction, Ab 2537 ℃ accelerated 7d reduction, Ab 254 ℃ non-reduction, Ab 254 ℃ reduction.

The experimental result shows that the Ab25 can be stably stored at 4 ℃, the property is still stable after being accelerated for one month at 37 ℃, and the performance of using the reagent when the bottle is opened is ensured, so that the accuracy and stability of the detection result are ensured.

TABLE 7 Ab22 stability evaluation data

The results are shown in table 7 and fig. 3: the rightmost side is Marker, and the antibody reagents added in the lane from right to left are Ab22 freeze-thawing for 3 times and Ab22 freeze-thawing for 5 times. Ab 2237 ℃ accelerated 31d non-reduction, Ab 2237 ℃ accelerated 14d non-reduction, Ab 2237 ℃ accelerated 7d reduction.

The experimental result shows that the Ab22 can be accelerated at 37 ℃ for one month, the property is still stable, the performance of using the reagent when the bottle is opened is ensured, and therefore the accuracy and the stability of the detection result are ensured.

Ab23 stability assessment data

The results are shown in table 8 and fig. 4: where from left to right, lane 1 is: ab 234 ℃ reduction, lane 2 Ab 234 ℃ non-reduction, lane 3 Ab 2337 ℃ accelerated 7d reduction, lane 4 Ab 2337 ℃ accelerated 7d non-reduction, lane 5 Ab 2337 ℃ accelerated 14d reduction, lane 6 Ab 2337 ℃ accelerated 14d non-reduction, lane 7 Ab 2337 ℃ accelerated 31d reduction, lane 8 Ab 2337 ℃ accelerated 31d non-reduction, lane 9 Ab23 freeze-thaw 5 times reduction, lane 10 freeze-thaw 5 times non-reduction.

The experimental result shows that the Ab23 can be accelerated at 37 ℃ for one month, the property is still stable, the performance of using the reagent when the bottle is opened is ensured, and therefore the accuracy and the stability of the detection result are ensured.

In conclusion, the four antibody strains of the invention are similar in detection, the gradient of measurement can be pulled up at a low value, and the sensitivity is high.

In addition, can freeze thawing repeatedly and use, and can be at 37 ℃ stable preservation, the performance is very stable, can be applicable to actual operating environment, because the reagent can not be used up once, and many times freezes the storage, then dissolves the demand of use, and stable preservation in 37 ℃ service environment simultaneously is very important to the accuracy of the result of detection.

Example 3: ab21 and Ab25 are applied to colloidal gold immunochromatography detection

1) Preparation of the detection device

The detection device comprises detection test paper, a card shell and a matched immunochromatography result reading recorder. The detection test paper comprises a colloidal gold pad, an NC membrane, a sample pad, a sample sucking pad and a plastic bottom plate. Wherein the colloidal gold pad contains a colloidal gold labeled Ab25 antibody, and the antibody can be combined with NT-proBNP protein in a sample to form an antigen-antibody-colloidal gold complex. The NC membrane was provided with a T-line (coated with Ab21 antibody for capturing antigen-antibody-colloidal gold complex) and a C-line (coated with anti-mouse IgG antibody). During detection, a sample is added into a sample hole, NT-proBNP in blood is combined with Ab25 marked by colloidal gold, then an antigen-antibody-colloidal gold compound is chromatographed to a detection area along a nitrocellulose membrane and is combined with Ab21 antibody coated by the detection area, so that a color band is formed in the detection area, the intensity of a signal of the color band is analyzed through a middle-element Zybio-Q7 immunochromatography detector, and the concentration of NT-proBNP in the sample is calculated according to a preset analysis module.

2) Creation of standard music

5 test paper prepared above are taken, 5 concentrations (10ng/ml, 100ng/ml, 300ng/ml, 500ng/ml and 1000ng/ml) of calibrator are respectively added on the sample pad of the test paper, each concentration point is tested repeatedly for three times, the sample adding amount is 70 mu l, and after 3 minutes of reaction, the calibrator concentration is used as an X axis for detection on Zhongyuan Hui Ji Q7. The mean value of the signal values of 3 from each point test is the Y-axis, and a standard curve is plotted, as shown in FIG. 5.

The results of the Roche sample detection based on the standard curve are shown in FIG. 6.

The experimental result shows that when the Ab21 and Ab25 antibodies are applied to immunochromatography colloidal gold detection, the coincidence rate with 38 Roche samples reaches 0.99, low-value and high-value samples can be accurately detected, samples in the range of 200-400ng/mL which are most easy to generate false positive experimental results can be also accurately detected, and false positive or false negative results cannot occur. The two antibodies have excellent effect in the immunochromatography colloidal gold chromatography card detection and high clinical coincidence rate, and can be applied to the methodology.

Example 4: ab23 and Ab25 are applied to fluorescence immunochromatography detection

1) Preparation of the detection device

The detection device comprises detection test paper, a card shell and a matched immunochromatography result reading recorder. The detection test paper comprises an NC film, a sample pad, a sample sucking pad and a plastic bottom plate. Wherein, the NC membrane comprises the biotin mark line, the fluorescent microsphere mark line, the streptavidin detection line (T line) and the quality control line (C line), the biotin mark line comprises a biotin-marked Ab25 which can be combined with NT-proBNP protein in a sample to form an antigen-Ab 25 antibody-biotin compound, the fluorescent microsphere mark line comprises a fluorescent-marked Ab23 antibody, and the Ab23 antibody is combined with an antigen to form: fluorescent microsphere-Ab 23 antibody-antigen-Ab 25 antibody-biotin. The streptavidin detection line was used for capture: fluorescent microsphere-Ab 23 antibody-antigen-Ab 25 antibody-biotin complex) and C-line (antibody coated anti-mouse IgG). After the sample is added, the signal intensity is detected by a middle-element Zybio-Q8 Pro immunofluorescence analyzer, and the concentration of NT-proBNP in the sample is calculated according to a preset analysis module.

2) Creation of standard music

10 test strips prepared above are taken, 10 calibrators with the concentrations (5pg/mL, 142pg/mL, 314pg/mL, 545pg/mL, 936pg/mL, 1884pg/mL, 3122pg/mL, 5793pg/mL, 11692pg/mL, 22766 pg/mL) are respectively added on a sample pad of the test strip, each concentration point is subjected to three-time repeated tests, the sample adding amount is 120 mu l, and after 10 seconds of reaction, the machine is used for detection, and the concentration of the calibrators is taken as an X axis. The mean value of the signal values for each point test 3 is the Y-axis and a standard curve is plotted, as shown in figure 7.

3) False positive experiment of NT-proBNP content detection kit

TABLE 9 false positive test of NT-proBNP content detection kit

Sample(s)	Negative of	Positive for	Total of
				Negative of	50	0	50
Positive for	0	50	50

Example 5: ab21 and Ab22 are applied to chemiluminescence kit detection

1) Preparation of detection kit

The kit comprises R1, R2, magnetic particles, a calibrator and a quality control product.

Wherein R1 comprises biotin-labeled NT-proBNP, R2 comprises alkaline phosphatase-labeled Ab21, and Ab22 is labeled on magnetic particles

Specific invention R1: 50mM PBS, 100mM NaCl, 1% BSA, 0.1% Tween20, 0.1% PC 300;

R2：50mM PBS、100mMNaCl、1％BSA、1mM MgCl2、1mM ZnCl2

2) creation of standard music

Preparation of NT-proBNP calibrator: the prepared NT-proBNP solutions were formulated at concentrations (0ng/ml, 116.5ng/ml, 436.9ng/ml, 1103.9ng/ml, 2446.6ng/ml, 16662.5ng/ml and 45510.0 ng/ml) with PBS buffer at pH 7.4.

The results are shown in FIG. 8.

3) False positive experiment of NT-proBNP content detection kit

TABLE 10 false positive test of NT-proBNP content detection kit

4) Evaluation of clinical examination

The clinical compliance rate is shown in fig. 9 and 10, and the results show that the detection results are all in the range of 20% deviation, most values fall in the confidence interval, and the detection results are accurate and effective.

The evaluation result shows that after Ab21 and Ab22 are applied to chemiluminescence clinical evaluation and concentration calculation is carried out by means of a standard, the coincidence rate of 187 clinical samples reaches 0.99, and the two antibodies are suitable for chemiluminescence detection.

In conclusion, the antibody of the invention is suitable for being applied to a detection kit of NT-proBNP, can be applied to various detection methodologies such as colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence and the like, can be matched with various detection kits due to stable and excellent performance, presents excellent detection performance, and realizes high-sensitivity, high-specificity and accurate detection of NT-proBNP.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Sequence listing

<110> Yuanhui Biotechnology Ltd in Chongqing

<120> N-terminal brain natriuretic peptide precursor polypeptide, antibody and kit

<130> 2020.12.30

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<210> 20

<211> 16

<212> PRT

<213> mouse

<400> 20

Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 21

<211> 7

<212> PRT

<213> mouse

<400> 21

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 22

<211> 9

<212> PRT

<213> mouse

<400> 22

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 23

<211> 5

<212> PRT

<213> mouse

<400> 23

Asn Tyr Asp Met Ser

1 5

<210> 24

<211> 17

<212> PRT

<213> mouse

<400> 24

Thr Ile Ser Gly Ala Asp Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 9

<212> PRT

<213> mouse

<400> 25

Pro Ser Thr Gly Asp Trp Phe Ala Tyr

1 5

<210> 26

<211> 16

<212> PRT

<213> mouse

<400> 26

Lys Ser Ser Gln Ser Ile Val Phe Ser Asn Gly Ile Thr Tyr Leu Glu

1 5 10 15

<210> 27

<211> 7

<212> PRT

<213> mouse

<400> 27

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> mouse

<400> 28

Phe Gln Gly Ser His Leu Pro Trp Thr

1 5

Claims

1. An amino-terminal pro-brain natriuretic peptide (NT-proBNP) polypeptide, wherein the amino acid sequence is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No. 4.

2. An anti-NT-proBNP monoclonal antibody or an antigen-binding portion thereof, wherein the NT-proBNP monoclonal antibody or the antigen-binding portion thereof is formed by immunizing an animal with a recombinant protein formed by coupling the polypeptide of claim 1 to a carrier protein.

3. An anti-NT-proBNP monoclonal antibody or an antigen-binding portion thereof, characterized in that said monoclonal antibody or antigen-binding portion thereof specifically binds to amino acid region 13-27, 27-31 or 42-46 of NT-proBNP.

4. The anti-NT-proBNP monoclonal antibody or antigen-binding portion thereof according to claim 2 or 3, comprising:

a) a heavy chain variable region CDR1 as set forth in SEQ ID No. 5;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 6;

c) heavy chain variable region CDR3 as set forth in SEQ ID No. 7;

d) light chain variable region CDR1 as set forth in SEQ ID No. 8;

e) light chain variable region CDR2 as set forth in SEQ ID No. 9;

f) light chain variable region CDR3 as set forth in SEQ ID No. 10;

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 11;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 12;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 13;

d) light chain variable region CDR1 as set forth in SEQ ID No. 14;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 15;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 16;

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 17;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 18;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 19;

d) light chain variable region CDR1 as set forth in SEQ ID No. 20;

e) light chain variable region CDR2 as set forth in SEQ ID No. 21;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 22;

Or

a) A heavy chain variable region CDR1 as set forth in SEQ ID No. 23;

b) a heavy chain variable region CDR2 as set forth in SEQ ID No. 24;

c) a heavy chain variable region CDR3 as set forth in SEQ ID No. 25;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 26;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 27;

f) light chain variable region CDR3 as set forth in SEQ ID No. 28;

the anti-NT-proBNP monoclonal antibody or the antigen-binding portion thereof is named Ab 25.

5. An NT-proBNP assay kit comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 2-4.

6. A conjugate comprising a chemical label or a biomarker covalently linked to the monoclonal antibody of any one of claims 2-4.

7. A conjugate formed by coupling the monoclonal antibody of any one of claims 2 to 4, and/or the conjugate of claim 6 to a solid or semi-solid medium

8. Use of a monoclonal antibody according to any one of claims 2 to 4 and/or a conjugate according to claim 6 and/or a conjugate according to claim 7 for the preparation of a product for detecting the expression of Ab21, Ab22, Ab23 or Ab 25.