CN111333728B

CN111333728B - Preparation of anti-DNP monoclonal antibody and hybridoma producing the same

Info

Publication number: CN111333728B
Application number: CN202010026953.0A
Authority: CN
Inventors: 张茂祥; 海那尔·乌拉孜巴依; 裴毓; 章文
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2022-01-07
Anticipated expiration: 2040-01-10
Also published as: CN111333728A

Abstract

Preparation of monoclonal antibody against DNP and hybridoma producing the same. The invention relates to the technical field of detection, and particularly relates to a hybridoma cell strain, a D-type natriuretic peptide monoclonal antibody based on the hybridoma cell strain and a detection kit. The preservation number of the hybridoma cell strain is CCTCC NO: C2019291. the invention has the technical effects that: the D-type natriuretic peptide monoclonal antibody produced by the hybridoma cell strain has high titer, can be used for detecting D-type natriuretic peptide by an enzyme-linked immunosorbent assay (ELISA), and has the characteristics of convenient sample operation, low cost, quick reaction, high sensitivity, strong specificity and the like.

Description

Preparation of anti-DNP monoclonal antibody and hybridoma producing the same

Technical Field

The invention relates to the technical field of small molecule immunochemistry analysis and detection, in particular to preparation of a DNP (deoxyribose nucleic acid) resisting monoclonal antibody and a hybridoma cell strain thereof.

Background

The natriuretic peptide family includes, as endocrine hormones, Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), C-type natriuretic peptide (C-type natriuretic peptide) and D-type natriuretic peptide (DNP), which play important roles in regulating blood volume, vascular elasticity, blood pressure, and the like through the pathways of natriuretic, diuretic, vasodilation, and anti-renin-angiotensin-aldosterone system. Other natriuretic peptides include dendroaspin natriuretic peptide, urodilatin, intestinal epithelium derived peptide, guanylin, and uroguanylin, etc., which have similar cardiovascular effects and are involved in water balance and blood pressure regulation. Research on natriuretic peptides (particularly brain natriuretic peptides) shows that the plasma natriuretic peptide level of heart disease patients such as heart failure is increased, the increased natriuretic peptide indicates high risk of cardiovascular events, and the monitoring of the plasma natriuretic peptide is helpful for diagnosis, treatment guidance and prognosis evaluation of heart failure. Besides the natriuretic effect, natriuretic peptides have many other important roles.

The natriuretic peptide family are neuroendocrine hormones released during heart failure and play a role in the regulation of the kidney, blood vessels, and endocrine system by activating second messenger guanylate cyclase (cGMP). The plasma has good stability, simple and quick operation and high sensitivity, and is a good biochemical index for diagnosing heart failure, guiding treatment and judging curative effect at present.

Compared with other natriuretic peptides, DNP has a longer amino terminus (C-terminus), is more resistant to degradation by neutral endonucleases (NEP natriuretic peptide lyase), and has good plasma stability. Has effects of activating second messenger, promoting cGMP activity, increasing cGMP in blood plasma and urinary system, and inhibiting renin activity, thereby inducing natriuresis and diuresis, and blocking adverse effects caused by excessive activation of endocrine system during heart failure. The D-type natriuretic peptide (DNP), a new member of the family of natriuretic peptides discovered in the last decade, appears in human plasma and atrial muscularis and increases in the plasma of heart failure patients, and its biological activity in Congestive Heart Failure (CHF) is under investigation, but current studies demonstrate that DNP may play an important role in the diagnosis and treatment of CHF.

In the prior art, the accuracy of a DNP detection result is determined by the performance of an antibody to a great extent, and the antibody with strong specificity and high binding speed is helpful for accurately and quickly detecting the DNP level.

Disclosure of Invention

The invention aims to prepare a DNP monoclonal antibody and a monoclonal antibody cell strain by using a completely new BNP natural analogue D-type natriuretic peptide (DNP) (DNP:38aa, amino acid sequence: EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA) which is derived from the venom of a narrow-head eye of an eastern African tree and is prepared previously as an antigen.

In order to achieve the purpose, the invention adopts the technical scheme that:

a D-type natriuretic peptide monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: and C2019291.

In the invention, the preparation method of the D-type natriuretic peptide monoclonal antibody comprises the following steps: a D-type natriuretic peptide is used as an immunogen to immunize a mouse for multiple times, spleen cells and myeloma cells of the immunized mouse are fused, a limited dilution method and an indirect ELISA screening technology are adopted to obtain a positive monoclonal hybridoma cell strain, ascites is induced, and the D-type natriuretic peptide monoclonal antibody is obtained after purification.

Preferably, the immunogen is prepared by coupling a D-type natriuretic peptide to a hemocyanin carrier protein.

Preferably, the myeloma cell is an SP2/0 myeloma cell.

Preferably, the mouse is a female C57BL/6 mouse.

The invention also provides application of the D-type natriuretic peptide monoclonal antibody in preparation of a D-type natriuretic peptide detection kit.

The invention also provides a D-type natriuretic peptide detection kit, which comprises the D-type natriuretic peptide monoclonal antibody provided by the invention.

Preferably, the D-type natriuretic peptide detection kit further comprises a coating antigen.

Preferably, the coating antigen is a conjugate of hemocyanin and D-type natriuretic peptide.

Preferably, the kit further comprises a D-type natriuretic peptide standard solution, a coating solution, a washing solution, a confining solution, an enzyme-labeled secondary antibody, a substrate developing solution and a stop solution.

Preferably, the enzyme-labeled secondary antibody is a goat anti-mouse enzyme-labeled antibody.

Preferably, the D-type natriuretic peptide detection kit further comprises a solid phase carrier.

In the embodiments provided herein, the solid support is an elisa plate.

The invention also provides an indirect ELISA detection method of the D-type natriuretic peptide, which comprises the following steps:

coating the conjugate of hemocyanin and D-type natriuretic peptide on a solid phase carrier, and sealing to obtain the solid phase carrier coated with the coating antigen; incubating the D-type natriuretic peptide standard product, the D-type natriuretic peptide monoclonal antibody and a solid phase carrier coated with a coating antigen together, adding an enzyme-labeled secondary antibody for color development, and obtaining a standard curve; incubating a sample to be detected, the D-type natriuretic peptide monoclonal antibody and a solid phase carrier coated with a coating antigen together, adding an enzyme-labeled secondary antibody for color development, and obtaining the concentration of glycocholic acid in the sample to be detected according to a standard curve.

The invention has the beneficial effects that: the invention provides a D-type natriuretic peptide monoclonal antibody, a hybridoma cell secreting the antibody and a kit for detecting D-type natriuretic peptide. The D-type natriuretic peptide monoclonal antibody is prepared from the following components in percentage by weight with the preservation number of CCTCC NO: the hybridoma cell line is obtained by secretion of the hybridoma cell of C2019291, the D-type natriuretic peptide monoclonal antibody produced by the hybridoma cell line has high titer, can be used for qualitative or quantitative detection of D-type natriuretic peptide content by an enzyme-linked immunosorbent assay (ELISA), and has the characteristics of convenient sample operation, low cost, rapid reaction, high sensitivity, strong specificity and the like.

Biological preservation description: and (3) classification and naming: the hybridoma cell strain 1A1-2-4 is preserved in China center for type culture Collection in 2019, 12 months and 22 days, the address of the preservation center is the first attached small face of Wuhan university in eight paths 299 # Wuhan university in Wuhan district in Wuhan city, Hubei province, and the preservation number is CCTCC NO: C2019291.

drawings

FIG. 1 is a graph of a BSA standard curve;

FIG. 2 shows the detection of DNP coupling by EDC method by KLH SDS-PAGE (M: Marker; 1: KLH-10. mu.g; 2: DNP-KLH-6. mu.g; 3: DNP-KLH-9. mu.g);

FIG. 3 is a graph of the UV absorption spectrum of KLH-coupled DNP protein at 250nm to 400 nm;

FIG. 4 shows the results of SDS-PAGE of anti-KLH-DNP antibody (M: Marker; 1: 1. mu.g anti-KLH-DNP antibody; 2: 4. mu.g anti-KLH-DNP antibody; 3: 6. mu.g anti-KLH-DNP antibody; 4: 8. mu.g anti-KLH-DNP antibody);

FIG. 5 is a graph of anti-DNP monoclonal antibody titers after ELISA detection and purification;

FIG. 6 is a graph showing the results of Western blot analysis of anti-KLH-DNP antibody (1: Marker; 2: 4. mu.g of anti-KLH-DNP antibody, primary antibody; DNP antibody; secondary antibody against goat anti-rabbit antibody).

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

Example 1 preparation of hybridoma cell lines, comprising the steps of:

immunization

1. Preparation of immunogens

(1) Coupling D-type natriuretic peptide (DNP) to the carrier protein KLH using carbodiimide EDC:

(a) 5 mg/1250. mu.L of DNP polypeptide (equimolar) was added to an EP tube containing 5 mg/500. mu.L of KLH solution and shaken slowly in a shaker to form a mixture of polypeptide and carrier.

(b) Dissolving the ready-prepared EDC solution, quickly adding 250 mu L into DNP-KLH mixed solution, placing in a shaking table, and shaking at low speed of 100rpm for 2 h.

(c) Desalting column (10kD) pretreatment: 1000rcf, 2min, centrifuge, discard bottom solution, wash 3-4 times with 1 × PBS. Note that: the column indicates the level of the centrifuged liquid in the tube.

(d) Desalting the column: PBS was washed 3-4 times.

(e) The DNP-KLH after coupling for 2h was added to 1 desalting column, 1000rcf, 2min, centrifuged and the centrifuged solution collected in a 5ml EP tube. The EP tube was labeled with DNP-KLH conjugate.

(f) The BCA method quantitatively detects the concentration of DNP-KLH coupled protein.

Measurement of BSA protein Standard at 0. mu.g/. mu.L, 0.025. mu.g/. mu.L, 0.05. mu.g/. mu.L, 0.1. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.3. mu.g/. mu.L, 0.4. mu.g/. mu.L, 0.5. mu.g/. mu.L, absorbance at 562nm, and a calibration curve was prepared. And obtaining an equation according to the standard curve. Diluting the sample 2 times, 5 times, 10 times, 15 times, and 20 times, measuring absorbance at 562nm, substituting into standard curve equation to calculate sample dilution concentration, and BCA quantitative BSA protein standard curve is shown in FIG. 1, where linear equation y is 1.2094x +0.0105, R is²BCA quantified DNP-KLH-coupled protein concentration of 1.58 μ g/μ L ═ 0.9982.

(g) SDS-PAGE detection: based on the BCA protein quantification, samples were prepared in the amounts of KLH, DNP, DNP-KLH-coupled protein (FIG. 2).

Ultraviolet spectroscopic scanning: detection of coupling efficiency

Taking 200 mu L of KLH, DNP and DNP-KLH coupled protein and each sample in a 96-well plate, and measuring the absorbance of the absorbed light with the total wavelength of 250nm-400nm to obtain a spectrogram. Compared with the absorbance of the DNP small molecule protein, the absorbance peak of the coupled protein sample is obviously different at 280 nm-290 nm (as shown in figure 3).

2. Animal immunization

The DNP-KLH coupled protein antigen is extracted, the concentration of the primary antigen is 1mg/mL, and the immunization dose of the mouse is 0.1 mL/mouse. The concentration of the antigen from the second immunity to the fourth immunity is 0.5mg/mL, and the immunization dose of the mouse is 0.1 mL/mouse. After DNP-KLH coupled protein and an adjuvant are uniformly mixed in a ratio of 1:1 (Freund complete adjuvant is adopted for the first immunization, Freund incomplete adjuvant is adopted for the second immunization and the fourth immunization), a 7-week-old (7-10-week-old) female C57BL/6 mouse is immunized by subcutaneous point injection at the back, and the operation time is as follows: second immunization, third immunization and fourth immunization are carried out 21 days after the first immunization, and the interval time between the third immunization and the fourth immunization is 14 days. And (5) carrying out orbital blood collection detection on the 7 th day after the four-time immunization of the mice.

Serum centrifugation: and (4) performing orbital blood collection detection at 4000rpm for 15min on the 7 th day after the four-time immunization of the mouse, and taking the supernatant.

Second, potency assay

1. ELISA for detecting serum titer

Coating: the coated DNP was diluted to 1. mu.g/mL with coating buffer, 50. mu.L/well was added to the microplate and coated overnight at 4 ℃.

Washing: the microplate was washed 3 times by adding a washing solution (1 XTSST) to the microplate in an amount of 180. mu.L/well.

And (3) sealing: and (3) drying the liquid in the holes, adding 1% BSA (bovine serum albumin) into the ELISA plate in an amount of 100 mu L/hole, and incubating for 1h in an electrothermal constant-temperature incubator at 37 ℃.

Adding a primary antibody: and (6) drying the liquid in the holes. The sera of immunized mice were diluted with PBS in a gradient of 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:2900, 1: 218700. 50 mu L/hole is added into the enzyme label plate according to the dilution ratio from small to large, and BSA is used as a negative control. Incubation was carried out at 37 ℃ for 30 min.

Adding a secondary antibody: the microplate was washed 3 times by adding a washing solution (1 XTSST) to the microplate in an amount of 180. mu.L/well. GAM-HRP was diluted to working concentration (1:3000) with 1% BSA, 50. mu.L/well was added to the microplate, and incubation was carried out at 37 ℃ for 30 min.

Color development, stop and read: discarding liquid in the hole, adding a washing solution into the ELISA plate according to the amount of 180 mu L/hole, and washing the ELISA plate for 3 times; adding 100 mu L of newly prepared TMB chromogenic substrate into each reaction hole, and incubating for 5min at constant temperature of 37 ℃; the reaction was then stopped by the addition of 90. mu.L/well of stop solution and the OD at 450nm was measured on a microplate reader.

2. The result of the detection

After three times of immunization, the serum of the mouse is diluted by 1 multiplied by PBS, and simultaneously, the nonimmune serum negative control and the PBS blank control are set for ELISA detection, and the maximum dilution times with similar positive percentages are taken as antibody titer, and the result shows that: the mouse serum antibody titer after the third immunization was 1:218700, as shown in table 1.

TABLE 1 serum titre test results

Cell fusion

1. Culture of mouse myeloma cells

Reviving the mouse myeloma cell line sp/20 cell strain frozen in liquid nitrogen two weeks before fusion, changing the liquid at proper time, and performing expanded culture 2 days before fusion so that the cells reach the number of cells required by fusion and are in a logarithmic growth phase. The cells in good growth state are round and transparent, have uniform size, few particles in the cells and have no vacuole.

2. Spleen cell preparation

(1) And (3) taking the mice on the fourth day after the fourth immunization, grasping the tail of the mice, tightening the neck skin of the mice to keep the heads of the mice unable to rotate freely, and enabling the eyeball of the mice to protrude outwards. The protruding eyeball was removed with an elbow forceps and the blood left from the orbit was collected. Treating blood at 37 deg.C for 30min, ice-cooling at 4 deg.C for 1h, centrifuging at 10000rpm for 10min, and collecting supernatant as positive serum;

(2) after the blood flow was completed, the mouse was sacrificed by cutting the neck, and placed in a beaker containing 75% alcohol for sterilization, and the beaker was brought into a sterile room together with the mouse and alcohol.

(3) The mouse is taken out of the alcohol, drained and placed in a kidney-shaped disc on a super clean bench, the abdominal cortex of the mouse is lifted up by big tweezers, an inverted triangle incision is cut by aseptic eye scissors, and the abdominal cortex is torn by hemostatic forceps to expose the abdominal cavity. The mouse peritoneal membrane was lifted with a sterile pair of tweezers and cut open with another sterile ophthalmic scissors until the entire abdominal cavity was exposed. The spleen was carefully removed and placed in a glass plate containing pre-heated 1640 medium, three plates were changed and washed three times.

(4) A small hole was made in one end of the spleen with forceps, then the spleen cells were squeezed out with two forceps, blown up with a 1mL pipette tip, collected into a 50mL centrifuge tube, and centrifuged at 1500rpm for 3 min.

(5) The supernatant was discarded, and after the precipitate was knocked out, 30mL of 1640 culture medium was added, followed by centrifugation at 1500rpm for 3 min. Repeating the steps once, and uniformly mixing and counting for later use.

3. Cell fusion

(1) SP2/0 cells with good growth state are blown down, centrifuged at 1500rpm for 3min, the supernatant is discarded, 30mL of preheated 1640 culture solution is used for resuspending the cells, and centrifuged at 1500rpm for 3 min. Repeating the steps once.

(2) The supernatant was discarded and the cells were resuspended by adding an appropriate amount of pre-warmed 1640 medium.

(3) After 50. mu.L of the cell suspension was mixed with 150. mu.L of trypan blue, the cell suspension was counted under a microscope. Corresponding cell amounts were taken according to the ratio of mouse splenocytes to SP2/0 cells 10:1, mixed well in an imported 50mL centrifuge tube, and centrifuged at 1500rpm for 3 min.

(4) The supernatant was discarded, and 1mL of preheated PEG (1500) was added slowly along the bottom wall of the centrifuge tube and placed in a 37 ℃ water bath for 1 min.

(5) 5mL of preheated 1640 culture solution is slowly dropped along the tube wall, then the speed is gradually increased, 15mL of preheated 1640 culture solution is added, the volume is finally increased to 40mL, and the tube is centrifuged at 1200rpm for 5 min.

(6) The supernatant was discarded, 25mL of pre-warmed SP2/0 myeloma cell culture medium was added, and the cells were carefully resuspended using a 10mL pipette.

(7) The hybridoma cell suspension after fusion was plated on a 96-well cell culture plate in an amount of 100. mu.L/well, and cultured in a 5% carbon dioxide incubator at 37 ℃.

4. Selective culture of hybridoma cells

After cell fusion, wait 4-6 h, add 100. mu.L of 2 XHAT medium per well.

Fourth, hybridoma cell screening and subcloning

1. And (3) cell screening process:

and detecting the positive cells after the cells grow for 7 days after fusion, selecting the positive cells, uniformly blowing the cells in positive cell holes, paving 200 cells in a 96-well plate for subcloning screening, selecting a monoclonal antibody, detecting the supernatant after the subcloning plate cells grow for 8-10 days generally, and selecting positive cell strains. If the positive cell strain is not selected, subcloning is carried out again until the monoclonal positive cell strain is selected. After selecting the monoclonal cell strain, the cell strain is enlarged and cultured, and cell supernatant is collected and frozen to store the cell.

2. The method for positive screening after subcloning comprises the following steps: indirect ELISA

(1) Wrapping a plate: antigen was diluted to 1. mu.g/mL with coating buffer, 50. mu.L was added to a 96-well plate of ELSIA plate, overnight at 4 ℃ and 1 × TBST wash buffer was used twice at 180. mu.L per well for 1 wash.

(2) And (3) sealing: blocking was performed by adding 60. mu.L/well of 1% BSA, incubating at 37 ℃ for 1h, and then removing the blocking solution.

(3) Sample adding: cell supernatant was added to 50. mu.L of the solution in the above-mentioned well. Positive (1: 500 dilution of mouse serum after immunization) and negative control wells (1% BSA added) were also set. Incubate at 37 ℃ for 30min, wash 2 times with 180. mu.L of 1 XTSST wash buffer per well.

(4) Adding an enzyme-labeled antibody: diluted goat anti-mouse secondary antibody-HRP (1:3000 diluted with 1% BSA) was added to wells of an ELISA plate at 50. mu.L/well, incubated at 37 ℃ for 30min, and washed 3 times with 180. mu.L/well of 1 XTSST wash buffer.

(5) Adding a substrate solution for color development: mu.L of the prepared TMB substrate solution (solution A and solution B in a ratio of 1:1, ready for use) was added to each reaction well, and the mixture was allowed to react at 37 ℃ for 5 min.

(6) And (3) terminating the reaction: the reaction was terminated by adding 100. mu.L of 2M sulfuric acid to each reaction well.

(7) Reading a plate: and (4) placing the ELISA plate in a preheated ELISA reader for reading and analyzing.

3. Positive cell monoclonal cloning assay procedure:

(1) the 1640 medium was previously incubated at 37 ℃.

(2) Positive hybridoma cells were counted.

(3) Plate paving: adding 6.5mL of subclone culture medium into the sample adding groove, and adding positive hybridoma cells into the culture medium to enable the cell concentration to be 125 cells/200 mu L; cells were added to a 96-well cell culture plate at 200. mu.L/well, and 24 wells in 3 rows were added; then 5mL of fresh subcloning culture medium is added, and 3 rows of plates are paved according to the same sample adding amount for 24 holes; the operation is then repeated in sequence. Four cell gradients were finally formed, namely 125/well, 25/well, 5/well and 1/well. Placing at 37 ℃ and 5% CO₂Culturing in medium.

(4) Observing on an inverted microscope after culturing for 4 days (4-5 days), wherein small cell clones can be seen, and at the 10 th day, white punctate cell clone clusters can be seen by naked eyes, and detecting the antibody of the culture supernatant;

(5) selecting monoclonal cells with strong detection positive and good state, transferring the monoclonal cells into a 24-well plate for culture and retesting, and carrying out subcloning on the positive wells for 2 times and 3 times according to the steps until monoclonal cell strains 1A1-2-4, 1H11-3-4-6, 1A11-3-7-4-8, 1A2-3-5-7-3 and 2A6-3-6) with basically stable secreted antibodies are obtained, wherein the monoclonal cells are shown in the table 2.

(6) Freezing and preserving the cell strains (1A1-2-4, 1H11-3-4-6, 1A11-3-7-4-8, 1A2-3-5-7-3 and 2A6-3-6), preserving 1A1-2-4 cell strains in the preservation unit: china center for type culture Collection, collection address: the preservation center of Wuhan university in Wuhan university, eight paths 299 # Wuhan university in Wuhan city, Hubei province has the preservation date: 12 and 22 months in 2019.

TABLE 2DNP monoclonal cell supernatant competition ELISA validation

Example 2 preparation of DNP monoclonal antibody (mouse peritoneal cavity induction method) comprising the following steps:

1. preparation of ascites

(1) One week ahead of time, the liquid paraffin is injected into the abdominal cavity of the mouse after being filtered and sterilized, and 0.5mL of the liquid paraffin is injected into each mouse for later use;

(2) the cells were gently tapped off and then transferred to a 50mL sterile centrifuge tube; centrifuging at 1500rpm for 3min, and discarding the supernatant; adding 10mL 1640 medium for counting; centrifuging at 1500rpm for 3min, and discarding the supernatant; cell pellet knock-off according to 1X10⁶1640 medium was added per mL.

(3)7 days later, hybridoma cells, 1X10 were injected intraperitoneally⁶One/only.

(4) Forming ascites of the mouse after 7 days generally, puncturing the ascites into the abdominal cavity of the mouse by using a 20G sterile needle, and collecting the ascites into a centrifugal tube;

(5) centrifuging ascites at 5000rpm/min for 3min, collecting oily supernatant, packaging, and storing at-80 deg.C.

2. Protein G affinity chromatography for purifying antibodies

(1) Preparation of Protein G affinity chromatography column after packing (1ml) was packed into the column, 3-5 bed volumes of ultrapure water was washed to remove ethanol, and 5-10 bed volume of equilibrated column was washed with equilibration buffer.

(2) Loading: the sample was applied to the column, diluted 1:2 before application and filtered through 0.45 μm. Bind to column, 1 h.

(3) Washing: the column was removed and washed 5-10 column volumes with PBS.

(4) And (3) elution: elution was performed with 0.1M glycine buffer for recovery.

(5) Concentration: the eluted sample was concentrated and washed with PBS.

(6) Measurement of concentration: the concentration of the purified ascites is 3.68mg/mL by ultraviolet spectroscopy.

(8) And (3) storage: the purified samples were stored at-20 ℃.

3. Purity of monoclonal antibody was verified by SDS-PAGE

The purity of the mAbs was analyzed by 10% SDS-PAGE combined with Coomassie blue staining and the results are shown in FIG. 4, where M is Marker, 1 is 1. mu.g, 2 is 4. mu.g, 3 is 6. mu.g, and 4 is 8. mu.g. As can be seen from FIG. 4, there are mainly two bands of heavy chain and light chain of the antibody, indicating that the purity of the antibody is significantly improved after purification.

EXAMPLE 3 method for identifying monoclonal antibody specificity

1. The titer of the purified DNP monoclonal antibody was measured by ELISA, and the result is shown in FIG. 5, wherein the titer of the purified DNP monoclonal antibody by ELISA was 1:50 ten thousand.

2. The purified DNP monoclonal antibody specificity is detected by Western blot, and two clear bands appear between 35kDa and 45kDa in a lane 2 as can be seen from figure 6. The upper is the heavy chain and the lower is the light chain.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A D-type natriuretic peptide monoclonal antibody is characterized in that the D-type natriuretic peptide monoclonal antibody is prepared by the following components in percentage by weight with a preservation number of CCTCC NO: and C2019291.

2. The use of the D-type natriuretic peptide monoclonal antibody of claim 1 for the preparation of a D-type natriuretic peptide detection kit.

3. A D-type natriuretic peptide detection kit comprising the D-type natriuretic peptide monoclonal antibody of claim 1.

4. The D-type natriuretic peptide detection kit of claim 3 further comprising a coating antigen.

5. The D-type natriuretic peptide detection kit of claim 4 wherein the coating antigen is a conjugate of hemocyanin and D-type natriuretic peptide.

6. The D-type natriuretic peptide detection kit of claim 5, characterized in that, the kit further comprises a D-type natriuretic peptide standard solution, a coating solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, a substrate developing solution and a stop solution.