Rat brain natriuretic peptide bnp detection kit and application thereof

Abstract

The application provides a rat brain natriuretic peptide (BNP) detection kit and application thereof. A rat BNP monoclonal antibody specific to rat BNP is prepared and obtained by taking rat BNP as an immunogen, the rat BNP monoclonal antibody has good specificity and affinity, and after the rat BNP monoclonal antibody is prepared into an ELISA detection kit, the rat BNP monoclonal antibody can be effectively used for content determination of rat BNP, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a rat brain natriuretic peptide (BNP) detection kit and application thereof. BACKGROUND

[0002] Brain natriuretic peptide (BNP) is a member of the natriuretic peptide family secreted by the heart, and has species specificity. Rat BNP is composed of 45 amino acids, and has important pathophysiological significance. BNP can dilate blood vessels, promote sodium and urine excretion, etc., and plays a vasoconstrictor role in the renin-angiotensin-aldosterone system (RAAS). Heart dysfunction can greatly activate the natriuretic peptide system, and increased ventricular load leads to BNP release. Therefore, BNP is generally used as a screening index for heart failure.

[0003] The main function of brain natriuretic peptide (BNP) is to inhibit sodium reabsorption, and to promote sodium and water excretion, while inhibiting aldosterone production in the RAAS. BNP is widely distributed in tissues such as the brain, spinal cord, heart and lung, and the content in the heart is the highest. BNP in the heart mainly exists in the left and right atria, and the content in the right atrium is more than 3 times that in the left atrium. The content of BNP in the ventricle is about 1 / 20 of that in the atrium. The reason for the low content of BNP in the ventricle is that the BNP precursor is not stored in the ventricle, and only when the ventricular wall tension increases can the BNP gene be rapidly expressed, a large amount of BNP is synthesized and secreted into the blood. BNP is involved in the regulation of blood pressure, blood volume and water-salt balance, increases the glomerular filtration rate, promotes natriuresis and diuresis, dilates blood vessels, reduces systemic vascular resistance and plasma volume, and all of these play a role in maintaining heart function. BNP is currently the only recommended cardiac function marker for patients suspected of heart failure. BNP negative can exclude heart failure.

[0004] Currently, brain natriuretic peptide (BNP) is an important indicator for the detection of heart failure. Common methods for detecting target proteins include gene chips, gene editing, proteomics, high-throughput screening, molecular imaging, and ELISA. ELISA, or enzyme-linked immunosorbent assay, is a widely used and relatively sensitive method for detecting and measuring protein content in samples. Over time and with rapid technological development, ELISA technology has made important breakthroughs in many areas. First, it can more accurately and specifically identify different proteins. By using specific antibodies to recognize the structure or amino acid sequence of different proteins, the ability to identify specific proteins has been greatly enhanced. This advancement has broadened the scope of ELISA applications, enabling it to play an important role in disease diagnosis, new drug development, and biomarker discovery. Second, enhancing the sensitivity and specificity of ELISA detection is another key development direction. By optimizing the ratio of antibodies and enzymes, improving signal amplification mechanisms, and developing more standardized detection processes, researchers have effectively improved the sensitivity and specificity of ELISA detection. This means that ELISA can accurately detect low concentrations of proteins while reducing background interference signals to ensure more accurate results. In addition, innovative ELISA technologies are constantly emerging, such as the combination of microfluidic technology and digital PCR technology, which has created a highly sensitive digital ELISA technology capable of detecting single molecule-level proteins. This new technology, with its ultra-high sensitivity and accuracy, opens up new possibilities for biomarker research and clinical diagnosis. In summary, ELISA technology has made remarkable achievements in the specific identification of proteins, the improvement of detection sensitivity and specificity, and the development of new technologies, continuing to consolidate its core position in life science and medical research.

[0005] However, there are not enough studies on the core monoclonal antibodies for ELISA detection of BNP, and the available options are not diverse enough, especially for monoclonal antibodies with strong binding capacity. Further technological improvements are needed. SUMMARY

[0006] In view of this, the present application provides a rat brain natriuretic peptide (BNP) detection kit and its application. The rat BNP monoclonal antibody is prepared using rat BNP as an immunogen and has good specificity and affinity. The rat BNP monoclonal antibody can be effectively used for the determination of the content of rat BNP after being prepared into an ELISA detection kit, and has good application prospects.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a rat brain natriuretic peptide (BNP) detection kit, comprising a monoclonal antibody BNP-6C5 specific to rat BNP.

[0009] Preferably, the kit further comprises a washing solution, a substrate reaction solution and a reaction termination solution.

[0010] Preferably, the washing solution comprises a PBST washing solution; and / or,

[0011] the substrate reaction solution comprises 3,3,5,5-tetramethylbenzidine, H2O2 and sodium acetate solution; and / or,

[0012] the termination solution comprises H2SO4 and water.

[0013] In a second aspect, the present application provides an antibody for a rat brain natriuretic peptide (BNP) detection kit, comprising a monoclonal antibody BNP-6C5 specific to rat BNP.

[0014] Preferably, the light chain variable region amino acid sequence of the monoclonal antibody BNP-6C5 is shown in SEQ ID NO. 1; and the heavy chain variable region amino acid sequence of the monoclonal antibody BNP-6C5 is shown in SEQ ID NO. 2.

[0015] Specifically, SEQ ID NO. 1 is

[0016] EVKLVESGGGLVKPGGSLKLSCAASGFIFSTYYMSWVRQTPEKRLE

[0017] LVATIHGNGNSIYYLDSVKGRFAISRDNAKNTLYLQMSSLKSEDTALYYCVRHDGYYVDHAMDCWGQGTSVTVSS;

[0018] SEQ ID NO. 2 is

[0019] DVLMTQTPLSLPVSLGDQASISCRSSQRIVHVNGNTYLDWYLQKPG

[0020] QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVAAEDLGVYYC FQGSHVPRTFGGGTRLEMK.

[0021] In a third aspect, the present application provides use of the antibody in the preparation of a kit for detecting BNP.

[0022] Finally, the present application provides a preparation method of a rat brain natriuretic peptide (BNP) detection kit, comprising the following steps:

[0023] S1, mixing PBS buffer and monoclonal antibody BNP-6C5, coating the enzyme label plate and standing overnight;

[0024] S2, washing the coated enzyme label plate;

[0025] S3, adding blocking solution to the washed enzyme label plate;

[0026] S4, after washing the enzyme label plate containing the blocking solution, drying and vacuum storage.

[0027] Preferably, in step S1, after mixing PBS buffer and monoclonal antibody BNP-6C5, the concentration of monoclonal antibody BNP-6C5 is 0.5 μg / mL.

[0028] Preferably, in step S1, the PBS buffer and the monoclonal antibody BNP-6C5 are mixed to obtain a mixed solution, and the mixed solution contained in each well of the enzyme label plate is 100 μL.

[0029] Preferably, the blocking solution comprises a PBS solution and BSA.

[0030] Compared with the prior art, the beneficial effects of the present application are:

[0031] (1) The present application uses rat BNP as an immunogen to prepare and obtain a monoclonal antibody specific to rat BNP, which has good specificity and affinity. After being prepared into an ELISA detection kit, it can be effectively used for the determination of the content of rat BNP, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Gel electrophoresis map of the recombinant BNP protein provided in Example 2 of the present application;

[0033] Figure 2 Western blotting detection map of the monoclonal antibody prepared in Example 4 and the recombinant BNP protein provided in Example 2 of the present application;

[0034] Figure 3 Competitive ELISA test standard curve map of the antibody provided in Example 8 of the present application. DETAILED DESCRIPTION

[0035] The present application will be further described in detail below in conjunction with specific examples, so that those skilled in the art can more clearly understand the present application.

[0036] Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that the embodiments described below or the technical features can be combined to form new embodiments. The methods and applications of the present application have been described by preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application to realize and apply the present application technology. The methods, devices and materials in the following examples are conventional methods, devices and materials in the art, which can be purchased from the market.

[0037] It is also pointed out herein that, in order to avoid obscuring the present application due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present application are shown in the drawings, and other details not closely related to the present application are omitted.

[0038] Example 1 Construction of rat BNP prokaryotic expression plasmid pET-28a-RBNP

[0039] According to the nucleotide sequence of rat BNP in the NCBI database and with the aid of computer-aided analysis, primers were designed on both sides of the gene sequence encoding the 77-121 amino acids of rat BNP protein. The primer sequences are shown in Table 1 (the underlined italicized part is the vector part).

[0040] Table 1 Primer sequences

[0041] Primer Primer sequence (5'-3') Upstream TAAGAAGGAGATATACCATGTCTCA AGACAGCGCCTTC Downstream GTGGTGGTGGTGGTGGTGAAACAACCTCAGCCCGTCA

[0042] Using the above primers, the rat BNP gene was amplified by PCR amplification technology, and the PCR conditions were as follows: 95°C pre-denaturation for 3 min, 95°C for 15 s, 58°C for 15 s, 72°C for 15 s, 35 cycles, and 72°C for 5 min. After PCR, centrifuge for a while, then take 5 μL of PCR product for 2% agarose gel electrophoresis detection. The PCR product and the pET-28a vector digested by EcoR I and Nco I were purified by ethanol precipitation method, and the purified rat BNP gene was homologously recombined with the pET-28a fragment. The recombinant product was transformed into E. coli DH5α competent cells, and the transformation product was spread on LB agar plates containing kanamycin and cultured at 37°C for 10 h. Single colonies were picked and cultured, and plasmids were extracted. The extracted plasmids were identified by PCR and sequencing, and the correct plasmid was named pET-28a-RBNP.

[0043] Example 2 Expression and purification of rat BNP protein

[0044] The pET-28a-RBNP recombinant plasmid 50 ng was transformed into E. coli BL21 (DE3) competent cells, and then coated on LB agar plates containing kanamycin. A single colony was inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking.

[0045] The cultured bacteria were inoculated into 10 mL LB liquid medium containing kanamycin at a ratio of 1:100, and cultured at 37°C with shaking at 200 r / min in an incubator for about 3 h, until the OD 600nm value was between 0.6 and 0.8. IPTG was added to a final concentration of 0.1 mmol / L, and induced at 37°C with shaking at 200 r / min for 6 h. After induction, the bacteria were collected by centrifugation at 4°C and 8000 r / min for 10 min, resuspended in 1 mL sterile PBS, and transferred to a 1.5 mL centrifuge tube. 200 μL was taken as a whole bacteria sample, and the remaining bacteria were sonicated. The 1.5 mL centrifuge tube containing the bacteria was placed in an ice-water mixture and placed in a sonicator. The sonication conditions were: power 25 w, working for 4 s, stopping for 6 s, and sonication for 2 min. After sonication, the sample was centrifuged at 4°C and 12000 r / min for 15 min. 200 μL supernatant was taken and added to 50 μL 5x loading buffer, and the remaining supernatant was discarded. The precipitate was resuspended with 200 μL 1x loading buffer. The whole bacteria sample was also added to 50 μL 5x loading buffer. The whole bacteria, supernatant, and precipitate samples were boiled in a 95°C metal bath for 10 min, and then centrifuged at 4°C and 12000 r / min for 1 min. SDS-PAGE was performed to identify the expression of the target protein, which was expressed in the form of inclusion bodies.

[0046] The expression bacteria pET-28a-RBNP were cultured and induced to express in large quantities. After the bacteria were broken, affinity chromatography rProteinA column was used for purification. The purified target protein sample was identified by SDS-PAGE. The results are shown in Figure 1 M represents marker, and R represents recombinant BNP protein. Lane 2 showed a clear band at about 5 KDa, indicating that a relatively pure BNP recombinant protein was prepared and obtained. The protein was quantified using a protein quantification kit after dialysis, refolding, and concentration, and the protein concentration was adjusted to 1 mg / mL for standby.

[0047] Example 3 Preparation of rat BNP monoclonal antibody

[0048] The purified BNP protein of Example 2 was mixed with an equal volume of adjuvant and emulsified, and 6-week-old female Balb / c mice (100 μg / mouse) were immunized by subcutaneous injection in the nape of the neck in multiple sites. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations, with a 3-week interval between each immunization, and a total of 4 immunizations. Blood was collected from the tail of the mice 21 days after the fourth immunization. The purified BNP protein was used to coat the enzyme-labeled plate (10 mg / L), and the serum titer of the immunized mice was determined by indirect enzyme-linked immunosorbent assay (iELISA). The results are shown in Table 2.

[0049] Table 2 iELISA titers of the serum of the immunized mice

[0050] Mouse number 1:4000 1:8000 1:16000 1:32000 1:64000 1:128000 1 2.242 1.681 0.945 0.566 0.239 0.221 2 2.799 2.012 1.501 0.742 0.363 0.245 Negative control 0.166 0.154 0.123 0.108 0.121 0.109

[0051] Note: The first column in the table is the volume ratio between the antibody and the diluent. The negative control refers to the serum titer of mice that were not injected with BNP protein and adjuvant.

[0052] The results show that the titer of No. 2 mouse was the highest. The mouse was boosted by intraperitoneal injection of antigen at a dose twice that of the immunization dose and without adjuvant, and the spleen of the mouse was aseptically removed 7 days later for cell fusion. The method of cell fusion was as follows. The mouse was sacrificed, the spleen was removed, and a cell suspension was prepared by grinding. The spleen cell suspension (1 x 10 8 cells) of the immunized mouse was mixed with the Sp2 / 0 myeloma cell suspension (1 x 10 7 cells), and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, and the centrifuge tube was gently shaken to loosen the cells. In a 37°C water bath environment, 1 mL of preheated fusion-promoting agent PEG was added while stirring. The preheated RPMI-1640 / HAT / 10% FBS culture solution was added, and the cells were inoculated into a 96-well plate with prepared feeder cells at 100 μL per well. The plate was placed in a 37°C, 5% CO2 cell incubator for culture.

[0053] On the 10th day of culture, the supernatant of the hybridoma cells was detected by iELISA, and the hybridoma cells with positive results were transferred to a 24-well cell plate for expansion culture for 4 days. The iELISA was repeatedly tested for strong positive wells, which were subjected to limited dilution for 3 times. Finally, the well-grown monoclonal hybridoma cell BNP-6C5 was selected, expanded, and centrifuged to collect the cells, which were resuspended in serum-free cell freezing solution and frozen in liquid nitrogen for storage.

[0054] The monoclonal antibody was produced by in vivo ascites induction. One 10-week-old female Balb / c mouse was used for sensitization pretreatment by intraperitoneal injection of incomplete Freund's adjuvant (0.5 mL / mouse). Three days later, the expanded hybridoma cell strain 6C5 was collected, washed twice with sterile PBS, and finally resuspended in 0.5 mL of sterile PBS for intraperitoneal injection into the mouse (10 5 -106 cells / only). After about 14 days of observation, when the abdomen of the mouse was obviously swollen, ascites was collected aseptically and centrifuged at 5000 r / min for 15 min. The supernatant was collected and the monoclonal antibody was purified by Protein A affinity chromatography and stored at -20°C for later use.

[0055] Example 4 Specificity identification of the monoclonal antibody BNP-6C5

[0056] SDS-PAGE electrophoresis was performed on the rat BNP protein, and then the proteins separated by gel electrophoresis were transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 h. The membrane was washed with PBST for 3 times. The monoclonal antibody BNP-6C5 to be detected was added and incubated overnight in a refrigerator at 4°C. The membrane was washed with PBST with sufficient shaking. HRP-labeled goat anti-mouse IgG diluted with 1% skimmed milk powder (the mass ratio of goat anti-mouse IgG to 1% skimmed milk powder was 1:1000) was added and incubated at 37°C for 1 h. The membrane was washed with PBST with sufficient shaking. Developing solution A and B (1:1) were mixed and dropped on the membrane for incubation for 3 min. The results were observed in a gel imaging instrument. As shown in Figure 2 Figure 2, M represents marker, and R represents the recombinant BNP protein incubated with the 6C5 monoclonal antibody. A clear band appeared at about 5 KDa in lane 2, indicating that the monoclonal antibody BNP-6C5 specifically recognized the rat BNP protein.

[0057] Example 5 Affinity identification of the monoclonal antibody BNP-6C5

[0058] The BNP recombinant protein obtained in Example 2 was coated with a coating buffer, blocked with 5% skimmed milk powder, and washed with a washing buffer PBST. 100 μL of the monoclonal antibody was added to the wells of an enzyme-labeled plate, and incubated at 37°C for 1 h. After washing the plate with PBST, different concentrations of sodium thiocyanate were used for elution. 60 μL of a solution of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mol / L was added in sequence, and the plate was left to stand at room temperature for 15 min. The plate was washed with PBST. 100 μL of diluted HRP-labeled goat anti-mouse IgG (1:1000) was added to each well, and the plate was incubated at room temperature for 45 min. 100 μL of TMB developing solution was added to each well, and 50 μL of a stop solution was added to each well after 10 min. The OD value was measured at a wavelength of 450 nm by using an enzyme-labeled instrument. The relative affinity constant of the antibody was the concentration of sodium thiocyanate corresponding to 50% of the OD value after elution. The results showed that the affinity of the monoclonal antibody BNP-6C5 could reach 4.6 mol / L. 450

[0059] Example 6 Preparation of a BNP antigen capture ELISA kit

[0060] ​Dilute BNP monoclonal antibody (e.g. BNP-6C5 prepared in Example 3) to a concentration of 0.5 μg / mL with PBS buffer to coat 96-well enzyme-coated plates, 100 μL / well, 4°C overnight. After removal, wash the plate with PBST for 3 times, and spin dry. Dissolve 3% BSA in PBS solution as blocking solution, 200 μL / well to the enzyme-coated plate, 37°C blocking for 1 h. After removal, wash the plate with PBST for 3 times, 3 min each time, spin dry, and store in vacuum after drying.

[0061] Example 7 Preparation of BNP antigen capture ELISA kit

[0062] Dilute BNP antigen (e.g. prepared in Example 2) to a concentration of 0.5 μg / mL with PBS buffer to coat 96-well enzyme-coated plates, 100 μL / well, 4°C overnight. After removal, wash the plate with PBST for 3 times, and spin dry. Dissolve 3% BSA in PBS solution as blocking solution, 200 μL / well to the enzyme-coated plate, 37°C blocking for 1 h. After removal, wash the plate with PBST for 3 times, 3 min each time, spin dry, and store in vacuum after drying.

[0063] Bioinylate BNP monoclonal antibody. Bring the reagents and the antibody to be labeled to room temperature (about 20-26°C), weigh a certain amount of biotin, dissolve and dilute the biotin to a final concentration of 0.1 mg / mL with PBS. The mass ratio of biotin to antibody is 3.4:100, mix the calculated mass of biotin and the antibody to be tested, and the total volume should be greater than 100 μL. Rotate the reaction at room temperature for 1 h in the dark. After the reaction is completed, dialyze with 1xPBS solution at 4°C overnight. The next day, continue dialysis with fresh 1xPBS solution for 5 h. After dialysis, collect and aliquot, and store at -20°C in the dark. Add glycerol to a final ratio of 60% for long-term storage.

[0064] Example 8 Use of BNP antigen capture ELISA kit

[0065] Set up standard wells, blank wells, and sample wells. Add 50 μL of BNP antigen diluted by a certain factor to the standard wells as standard, and add 50 μL of the sample to be tested to the remaining wells. The sample can be rat serum, rat plasma, rat tissue grinding supernatant, or other biological samples. (It is recommended to set up duplicate wells for all samples to be tested and standards; the dilution factor of the sample to be tested can be determined through pre-experiments or consultation with technical support.) Immediately add 50 μL of the prepared biotinylated antibody (prepared in Example 7) working solution to each well. Cover the enzyme-coated plate with film, and incubate at 37°C for 45 min. Note: When adding the sample, add the sample to the bottom of the enzyme-coated plate, try not to touch the wall of the well, and gently shake to mix, avoiding the formation of bubbles. The addition time should be controlled within 10 min.

[0066] Remove the liquid in the hole, and dry it on a clean absorbent paper. Add 350 μL of washing solution to each hole, soak for 1 minute, and remove or shake off the liquid in the enzyme-labeled plate. Dry it. Repeat this washing step 3 times. Note: This and other washing steps can use a plate washer (reference Beijing Tuopu DEM-3 plate washer parameter settings: 2-point suction, add 350 μL of washing solution to each hole, shake the plate for 5 seconds, and suck for 0.5 seconds). After washing the plate, please immediately proceed to the next step, do not let the microplate dry.

[0067] Add 100 μL of HRP enzyme conjugate working solution to each hole, and cover the enzyme-labeled plate with a film. Incubate at 37°C for 30 minutes. Shake off the liquid in the hole, and wash the plate 5 times as described above. Add 90 μL of substrate solution (TMB) to each hole, cover the enzyme-labeled plate with a film, and incubate at 37°C in the dark for about 15 minutes. Note: The actual color development time can be shortened or lengthened as appropriate, but not more than 30 minutes. When the standard holes show a clear gradient (the first 4 color development holes show a clear blue gradient), the reaction can be terminated. Turn on the enzyme-labeled instrument 15 minutes in advance for preheating. Add 50 μL of stop solution to each hole to terminate the reaction. Note: The order of adding the stop solution should be as close as possible to the order of adding the substrate solution. Immediately measure the optical density (OD value) of each hole at 450 nm wavelength using the enzyme-labeled instrument.

[0068] It should be noted that in the above method, 3,3,5,5-tetramethylbenzidine TMB (SIGMA) is used as the substrate of horseradish peroxidase. Add 5 mL of 30% H2O2, 1 mL of TMB, and 4 mL of sodium acetate solution (pH = 4.3) to make 10 mL of substrate reaction solution (freshly prepared). The washing solution is PBST washing solution, and the reaction termination solution is 2M H2SO4 solution prepared with deionized water.

[0069] Results:

[0070] Determination of the optimal working concentration of the capture antibody and the detection antibody:

[0071] The clone number of the detection antibody is 10E12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1, which is specifically:

[0072] EVKLVESGGGLVKPGGSLKLSCAASGFIFSTYYMSWVRQTPEKRLE

[0073] LVATIHGNGNSIYYLDSVKGRFAISRDNAKNTLYLQMSSLKSEDTA

[0074] LYYCVRHDGYYVDHAMDCWGQGTSVTVSS

[0075] The corresponding nucleotide sequence is shown in SEQ ID NO. 3, which is specifically:

[0076] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTACTTATTACATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTTGGTCGCAACCATTCATGGTAATGGTAATAGCATCTACTATCTAGACAGTGTGAAGGGTCGATTCGCCATCTCCAGAGACAATGCCAAGAACACTCTGTACCTGCAGATGAGCAGTCTGAAGTCTGAGGACACAGCCCTGTATTACTGTGTAAGACATGATGGTTATTACGTGGACCATGCTATGGACTGCTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0077] The amino acid sequence of the light chain variable region of the detection antibody is shown in SEQ ID NO. 2, specifically: DVLMTQTPLSLPVSLGDQASISCRSSQRIVHVNGNTYLDWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVAAEDLGVYYCFQGSHVPRTFGGGTRLEMK

[0078] The corresponding nucleotide sequence is shown in SEQ ID NO. 4, specifically: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGGATTGTACATGTTAATGGAAACACCTATTTAGATTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTAATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGCGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCGGACGTTCGGTGGAGGCACCAGGCTGGAAATGAAA

[0079] The standard concentration and the OD value of the enzyme label are shown in Table 3. The standard curve is drawn by fitting software Figure 3 .

[0080] Table 3: The OD value of the optimal working concentration of the antibody

[0081] Standard concentration pg / ml OD (Optical Density) 2000 0.318 1000 0.427 500 0.568 250 0.746 125 1.053 62.5 1.460 31.25 1.776 0 2.303

[0082] Tables 4-6 are other data information of the monoclonal antibody for the competitive ELISA detection

[0083] Table 4: The minimum detection limit and the linear range of the monoclonal antibody for the competitive ELISA

[0084]

[0085] Table 5: The dilution recovery rate of the monoclonal antibody for the competitive ELISA detection of samples

[0086]

[0087] Table 6: The in-plate and inter-plate difference range of the monoclonal antibody for the competitive ELISA

[0088]

[0089] In the present application, the materials not specifically described are all existing substances, which can be directly purchased from the market.

[0090] The above are only the preferred embodiments of the present application, and are not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A rat brain natriuretic peptide (BNP) detection kit, characterized in that, The invention includes a monoclonal antibody BNP-6C5 specifically targeting rat BNP, the amino acid sequence of the light chain variable region of the monoclonal antibody BNP-6C5 being shown in SEQ ID NO.1; and the amino acid sequence of the heavy chain variable region of the monoclonal antibody BNP-6C5 being shown in SEQ ID NO.

2.

2. The detection kit according to claim 1, characterized in that, It also includes washing solution, substrate reaction solution, and reaction termination solution.

3. The detection kit according to claim 2, characterized in that, The washing solution includes PBST washing solution; and / or, The substrate reaction solution comprises 3,3,5,5-tetramethylbenzidine, H₂O₂, and sodium acetate solution; and / or, The terminating solution comprises H2SO4 and water.

4. An antibody for use in a rat brain natriuretic peptide (BNP) detection kit, characterized in that, The invention includes a monoclonal antibody BNP-6C5 specifically targeting rat BNP, the amino acid sequence of the light chain variable region of the monoclonal antibody BNP-6C5 being shown in SEQ ID NO.1; and the amino acid sequence of the heavy chain variable region of the monoclonal antibody BNP-6C5 being shown in SEQ ID NO.

2.

5. Use of the antibody according to claim 4 in the preparation of a kit for detecting BNP.

6. A method for preparing a rat brain natriuretic peptide (BNP) detection kit, characterized in that, Includes the following steps: S1. Mix PBS buffer and monoclonal antibody BNP-6C5, coat the microplate with the mixture, and let it stand overnight. The amino acid sequence of the light chain variable region of the monoclonal antibody BNP-6C5 is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the monoclonal antibody BNP-6C5 is shown in SEQ ID NO.

2. S2. Clean the coated ELISA plate; S3. Add blocking solution to the cleaned ELISA plate; S4. After cleaning the ELISA plate containing the blocking solution, dry and vacuum store it.

7. The preparation method according to claim 6, characterized in that, In step S1, after mixing the PBS buffer and the monoclonal antibody BNP-6C5, the concentration of the monoclonal antibody BNP-6C5 is 0.5 μg / mL.

8. The preparation method according to claim 6, characterized in that, In step S1, PBS buffer and monoclonal antibody BNP-6C5 are mixed to obtain a mixture, and each well of the ELISA plate contains 100 μL of the mixture.

9. The preparation method according to claim 6, characterized in that, The blocking solution includes PBS solution and BSA.

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