Rat brain natriuretic peptide BNP detection kit and application thereof

Abstract

According to the rat brain natriuretic peptide BNP detection kit and the application thereof, the rat BNP is used as an immunogen to prepare and obtain a specific monoclonal antibody aiming at the rat BNP, the rat BNP monoclonal antibody has good specificity and affinity, after the rat BNP monoclonal antibody is prepared into the ELISA detection kit, the ELISA detection kit can be effectively used for measuring the content of the rat BNP, and the detection sensitivity is high. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a rat brain natriuretic peptide BNP detection kit and application thereof. Background Art

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

[0003] The main function of brain natriuretic peptide BNP is to inhibit sodium reabsorption, excrete sodium and water, and inhibit aldosterone production in RAAS. It is widely distributed in tissues such as the brain, spinal cord, heart and lungs, among which the heart has the highest content. In the heart, BNP is mainly present in the left and right atria, among which the content in the right atrium is more than 3 times that in the left atrium, and the BNP content in the ventricle is less than 1 / 20 of that in the atrium. The low BNP content in the ventricle is because the BNP precursor is not stored in the ventricle. Only when the ventricular wall tension increases will it quickly stimulate the high expression of the BNP gene, and a large amount of synthesized BNP will be secreted into the blood. BNP is involved in the regulation of blood pressure, blood volume and water-salt balance, increases glomerular filtration rate, promotes natriuresis and diuresis, dilates blood vessels, reduces systemic vascular resistance and plasma volume, all of which 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 rule out heart failure.

[0004] At present, brain natriuretic peptide BNP is an important test indicator for effective detection of heart failure. Common methods for detecting target proteins include gene chips, gene editing, proteomics, high-throughput screening and molecular imaging, and ELISA. ELISA, or enzyme-linked immunosorbent assay, is a widely used and relatively sensitive method for detecting and measuring the protein content in samples. With the passage of time and the rapid development of technology, ELISA technology has achieved important breakthroughs in many aspects. First, it can more accurately and specifically identify different proteins. Through specific antibodies, the structure or amino acid sequence of different proteins is recognized, thereby greatly enhancing the ability to identify specific proteins. This progress has broadened the scope of application of ELISA, making it play an important role in disease diagnosis, new drug development, and the search for biomarkers. Secondly, enhancing the sensitivity and specificity of ELISA detection is another key development direction. By optimizing the ratio of antibodies and enzymes, improving the mechanism of signal amplification, and formulating a more standardized detection process, researchers have effectively improved the sensitivity and specificity of ELISA detection. This means that ELISA can accurately detect proteins with lower concentrations while reducing background interference signals to ensure more accurate results. In addition, innovative ELISA technologies are emerging one after another. For example, the combination of microfluidics and digital PCR technology has created a highly sensitive digital ELISA technology that can detect proteins at the single molecule level. This new technology has opened up new possibilities for biomarker research and clinical diagnosis due to its ultra-high sensitivity and accuracy. In short, ELISA technology has made remarkable achievements in the specific identification of proteins, improving the sensitivity and specificity of detection, and promoting the development of new technologies, and continues to consolidate its core position in life science and medical research.

[0005] However, there is not enough research on the core monoclonal antibodies for ELISA detection of BNP, and the available options are not rich enough, especially monoclonal antibodies with strong binding ability, which requires further improvement of technology. Summary of the invention

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

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0009] Preferably, it also includes a washing solution, a substrate reaction solution and a reaction termination solution.

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

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

[0012] The stop solution includes H2SO4 and water.

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

[0014] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody BNP-6C5 is shown as SEQ ID NO.1; and the amino acid sequence of the heavy chain variable region of the monoclonal antibody BNP-6C5 is shown as 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 invention provides a use of the antibody in preparing a kit for detecting BNP.

[0022] Finally, the present invention provides a method for preparing a rat brain natriuretic peptide (BNP) detection kit, comprising the following steps:

[0023] S1. Mix PBS buffer and monoclonal antibody BNP-6C5, coat the ELISA plate and let stand overnight;

[0024] S2, cleaning the coated ELISA plate;

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

[0026] S4. After washing the ELISA plate containing the blocking solution, dry it and store it in a vacuum.

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

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

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

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention uses rat BNP as an immunogen to prepare and obtain a monoclonal antibody specific for rat BNP. The rat BNP monoclonal antibody 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 prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the gel electrophoresis diagram of the recombinant BNP protein provided in Example 2 of the present invention;

[0033] Figure 2 This is a Western blotting test of the monoclonal antibody prepared in Example 4 of the present invention and the recombinant BNP protein provided in Example 2;

[0034] Figure 3 This is a standard curve diagram of the competitive ELISA test performed on the antibody provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0036] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that, under the premise of not conflicting, the embodiments described below or the technical features can be arbitrarily combined to form a new embodiment. The method and application of the present invention have been described by preferred embodiments, and 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 invention to realize and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art and can be purchased from the market.

[0037] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present invention are shown in the drawings, while other details that are not closely related to the present invention 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-assisted analysis, primers were designed on both sides of the gene sequence encoding the 77th to 121st amino acids of the rat BNP protein. The primer sequences are shown in Table 1 (the underlined italics are the vector parts).

[0040] Table 1 Primer sequences

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

[0042] The above primers were used to amplify the rat BNP gene by PCR amplification technology. The PCR conditions were: 95℃ pre-denaturation for 3min, 95℃ for 15s, 58℃ for 15s, 72℃ for 15s, 35 cycles, and 72℃ extension for 5min. After the PCR was completed, centrifuged for a while, and then 5μL of the PCR product was taken for 2% agarose gel electrophoresis detection. The PCR product and the pET-28a vector double-digested by EcoRⅠ and NcoⅠ were purified by ethanol precipitation. The purified rat BNP gene was homologously recombined with the pET-28a fragment, and the recombinant product was transformed into E.coil DH5α competent cells, and the transformation product was coated on LB agar plates containing kanamycin and cultured at 37℃ for 10h. A single colony was picked, amplified and cultured, and the plasmid was extracted. The extracted plasmid was 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] Take 50 ng of the pET-28a-RBNP recombinant plasmid and transform it into E. coli BL21 (DE3) competent cells, and spread it on LB agar plates containing kanamycin. Pick a single colony and inoculate it into LB liquid culture medium containing kanamycin, and culture it at 37°C with shaking.

[0045] The cultured bacterial solution was inoculated into 10 mL of LB liquid medium containing kanamycin at a ratio of 1:100, and cultured in a shaking incubator at 37°C and 200 rpm for about 3 h until the OD 600nm The value is between 0.6 and 0.8, IPTG is added to a final concentration of 0.1mmol / L, and induced at 37℃, 200r / min for 6h. After the induction, the bacterial solution was collected by centrifugation at 4℃, 8000r / min for 10min, and the bacteria were resuspended in 1mL sterile PBS and transferred to a 1.5mL centrifuge tube. 200μL was taken out as the whole bacterial sample, and the remaining bacterial solution was ultrasonically broken. The 1.5mL centrifuge tube containing the bacterial solution was placed in an ice-water mixture and put into the ultrasonic crusher. The crushing conditions were: power 25w, working 4s, stopping 6s, and crushing 2min. After the crushing was completed, centrifuged at 4℃, 12000r / min for 15min, 200μL of supernatant was taken out and added to 50μL 5×loading Buffer, the rest of the supernatant was discarded, and the precipitate was resuspended with 200μL 1×loading Buffer. 50μL 5×loading Buffer was also added to the previous whole bacterial sample. The whole bacteria, supernatant and precipitate samples were boiled in a 95°C metal bath for 10 min, centrifuged at 4°C, 12000 r / min for 1 min, and then subjected to SDS-PAGE identification to determine whether the target protein was expressed in the form of inclusion bodies.

[0046] The expression bacteria pET-28a-RBNP was expanded and induced to express in large quantities. After the bacteria were broken, they were purified using affinity chromatography rProteinA column. The purified target protein samples were identified by SDS-PAGE. The results are shown in Figure 1 As shown, M represents marker, R represents recombinant BNP protein, and a clear band appears at about 5KDa in lane 2, indicating that a relatively pure BNP recombinant protein was prepared and obtained. The protein was deformed and renatured by dialysis, and then quantified using a protein quantification kit after concentration, and the protein concentration was adjusted to 1 mg / mL for later use.

[0047] Example 3 Preparation of rat BNP monoclonal antibody

[0048] The purified BNP protein expressed in Example 2 was mixed with an equal volume of adjuvant and emulsified, and 2 female Balb / c mice aged 6 weeks were immunized (100 μg / mouse) by subcutaneous injection at the back of the neck, wherein Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the subsequent booster immunization, each immunization was 3 weeks apart, and a total of 4 immunizations were performed, and blood was collected by tail cutting on the 21st day after the 4 immunizations. The purified BNP protein was coated on an ELISA plate (10 mg / L), and the serum titer of the immunized mice was detected by indirect enzyme-linked immunosorbent assay (iELISA). The results are shown in Table 2.

[0049] Table 2 iELISA titer of sera from 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 showed that the titer of mouse No. 2 was the highest. The mice were given a 2-fold immunization dose of antigen without adjuvant for booster immunization. After 7 days, the spleen of the mice was removed aseptically for cell fusion. The cell fusion method was to kill the mice, dissect and remove the spleen, grind and prepare a cell suspension, and then add the spleen cell suspension of the immunized mice (1×10 8 cells) and Sp2 / 0 myeloma cell suspension (1×10 7 cells) and centrifuged at 1000r / min for 10min; discard the supernatant and gently shake the centrifuge tube to loosen the cells; add 1mL of preheated fusion promoter PEG in a 37℃ water bath environment and stir while adding. Add preheated RPMI-1640 / HAT / 10% FBS culture medium and inoculate the cells into a 96-well plate prepared with feeder cells, 100μL per well, and culture in a 37℃, 5% CO2 cell culture incubator.

[0053] On the 10th day of culture, the hybridoma cell supernatant was tested according to the iELISA method, and the hybridoma cells with positive results were transferred to a 24-well cell plate for expansion culture for 4 days. The strongly positive wells in the iELISA retest were selected for limiting dilution 3 times, and finally the monoclonal hybridoma cell BNP-6C5 in good condition was selected. After expansion culture, the cells were collected by centrifugation, resuspended in serum-free cell freezing solution, and frozen in liquid nitrogen for later use.

[0054] Monoclonal antibodies were produced by in vivo ascites induction method. A 10-week-old female Balb / c mouse was injected intraperitoneally with incomplete Freund's adjuvant for sensitization pretreatment (0.5 mL / mouse); 3 days later, the hybridoma cell line 6C5 was collected and cultured, washed twice with sterile PBS, and finally resuspended in 0.5 mL sterile PBS and injected intraperitoneally into mice (10 5 -106 After 14 days of observation, when the abdomen of the mice was obviously swollen, the ascites was collected aseptically, centrifuged at 5000 r / min for 15 min, and the supernatant was collected. The monoclonal antibody was purified by Protein A affinity chromatography and stored at -20°C for later use.

[0055] Example 4 Specificity Identification of Monoclonal Antibody BNP-6C5

[0056] The rat BNP protein was subjected to SDS-PAGE electrophoresis, and then the protein separated by gel electrophoresis was transferred to a PVDF membrane, and the PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 hours. The membrane was washed 3 times with PBST; the monoclonal antibody BNP-6C5 to be tested was added, incubated in a 4°C refrigerator overnight, and washed with PBST by shaking thoroughly; HRP-labeled goat anti-mouse IgG diluted with 1% skimmed milk powder (the mass ratio of goat anti-mouse IgG: 1% skimmed milk powder is 1:1000), incubated at 37°C for 1 hour, and washed with PBST by shaking thoroughly. The developer A and B (1:1) were mixed and dropped on the membrane for incubation for 3 minutes, and the results were observed in a gel imager. The results are shown in Figure 2 As shown, M represents marker, R represents recombinant BNP protein incubated with 6C5 monoclonal antibody, and a clear band appears at about 5 KDa in lane 2, indicating that the monoclonal antibody BNP-6C5 specifically recognizes rat BNP protein.

[0057] Example 5 Affinity Identification of Monoclonal Antibody BNP-6C5

[0058] The BNP recombinant protein obtained in Example 2 was coated with coating buffer, blocked with 5% skimmed milk powder, and washed with washing buffer PBST. 100 μL of monoclonal antibody was added to the wells of the ELISA plate, incubated at 37°C for 1 hour, and eluted with sodium thiocyanate of different concentrations after washing with PBST. 60 μL / well 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 solutions were added in sequence, allowed to stand at room temperature for 15 minutes, and the plate was washed with PBST. 100 μL of diluted HRP-labeled goat anti-mouse IgG (1:1000) was added to each well and incubated at room temperature for 45 minutes. 100 μL / well of TMB colorimetric solution was added, and 50 μL / well of stop solution was added after 10 minutes. The OD value was measured at a wavelength of 450 nm using an ELISA reader. After elution, the OD 450 The sodium thiocyanate concentration corresponding to the 50% of the uneluted concentration is the relative affinity constant of the antibody. The results show that the affinity of the monoclonal antibody BNP-6C5 can reach 4.6 mol / L.

[0059] Example 6 Preparation of BNP Antigen Capture ELISA Kit

[0060] Dilute the BNP monoclonal antibody (BNP-6C5 prepared as in Example 3) to a concentration of 0.5 μg / mL with PBS buffer, coat the 96-well ELISA plate, 100 μL / well, and leave overnight at 4°C. After taking out, wash the plate 3 times with PBST and spin dry. Dissolve 3% BSA in PBS solution as a blocking solution, add 200 μL / well to the ELISA plate, and block at 37°C for 1h. After taking out, wash the plate 3 times with PBST, 3 minutes each time, spin dry, and store in a vacuum seal after drying.

[0061] Example 7 Preparation of BNP Antigen Capture ELISA Kit

[0062] Dilute the BNP antigen (prepared as in Example 2) with PBS buffer to a concentration of 0.5 μg / mL, coat a 96-well ELISA plate, 100 μL / well, and leave overnight at 4°C. After taking out, wash the plate 3 times with PBST and spin dry. 3% BSA dissolved in PBS solution as a blocking solution, 200 μL / well added to the ELISA plate, and block at 37°C for 1h. After taking out, wash the plate 3 times with PBST, 3 minutes each time, spin dry, and vacuum seal and store after drying.

[0063] Label the BNP monoclonal antibody with biotin. Return the reagents and the antibody to be labeled to room temperature (about 20-26°C), weigh a certain amount of biotin, use PBS to dissolve and dilute the biotin to a final concentration of 0.1 mg / mL. The mass ratio of biotin to antibody is 3.4:100. Mix the calculated corresponding mass of biotin and the antibody to be tested, and the total volume must be greater than 100 μL. Rotate and react for 1 hour at room temperature in the dark. After the reaction is completed, dialyze overnight on a shaker at 4°C with 1×PBS solution. The next day, replace with a new 1×PBS solution and continue dialysis for 5 hours. After the dialysis is completed, collect and package, store at -20°C in the dark, and add 60% glycerol for long-term storage.

[0064] Example 8 Use of BNP Antigen Capture ELISA Kit

[0065] Set up standard wells, blank wells and sample wells respectively. Add 50 μL of diluted BNP antigen as a standard to the standard wells, and add 50 μL of the sample to be tested to the remaining wells. The samples are biological samples such as rat serum, rat plasma, and rat tissue grinding supernatant. (It is recommended to set up duplicate wells for all samples and standards to be tested in the test; it is recommended to determine the dilution multiple of the sample to be tested through preliminary experiments or consulting technical support). Immediately add 50 μL of the prepared biotinylated antibody (prepared in Example 7) working solution to each well. Cover the ELISA plate with a film and incubate at 37°C for 45 minutes. Tip: When adding the sample, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, and shake gently to mix and avoid bubbles. The sample addition time should be controlled within 10 minutes.

[0066] Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, suck or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Tip: Here and other washing steps can use a plate washer (refer to the parameter settings of Beijing Top DEM-3 plate washer: 2-point aspiration, add 350μL of washing solution to each well, vibrate the plate for 5 seconds, and aspirate for 0.5s). Please proceed to the next step immediately after washing the plate, and do not let the microplate dry.

[0067] Add 100μL of HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 30 minutes. Shake off the liquid in the wells and wash the plate 5 times, using the same method as the above-mentioned plate washing steps. Add 90μL of substrate solution (TMB) to each well, cover the ELISA plate with a film, and incubate at 37℃ in the dark for about 15 minutes. Tip: Shorten or extend the time as appropriate according to the actual color development situation, but do not exceed 30 minutes. When a clear gradient appears in the standard well (a clear blue gradient appears in the first 4 color-developing wells), it can be terminated. Turn on the ELISA reader 15 minutes in advance to preheat. Add 50μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. Immediately measure the optical density (OD value) of each well at a wavelength of 450nm using an ELISA reader.

[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. 4 mL of sodium acetate solution (pH = 4.3), 5 mL of 30% H2O2, and 1 mL of TMB are added to form 10 mL of substrate reaction solution (prepared immediately before use). The washing solution is PBST washing solution, and the reaction termination solution is 2M H2SO4 solution prepared with deionized water.

[0069] result:

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

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

[0072] EVKLVESGGGLVKPGGSLKLSCAASGFIFSTYYMSWVRQTPEKRLE

[0073] LVATIHGNGNSIYYLDSVKGRFAISRDNAKNTLYLQMSSLKSEDTA

[0074] LYYCVRHDGYYVDHAMDCWGQGTSVTVSS

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

[0076] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTACTTATTACATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTTGGTCGCAACCATTCATGGTAATGGTAATAGCATCTACTATCTAGACAGTGTGAAGGGTCGATTCGCCATCTCCAGAGACAATGCCAAGAACACTCTGTACCTGCAGATGAGCAGTCTGAAGTCTGAGGACACAGCCCTGTATTACTGTGTAAGACATGATGGTTATTACGTGGACCATGCTATGGACTGCTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0077] The amino acid sequence of the light chain variable region of the detected 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 concentration of the standard and the reading of the microplate reader are shown in Table 3. The standard curve was drawn by fitting software. Figure 3 .

[0080] Table 3 OD values ​​of optimal working concentrations of antibodies

[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] Table 4-6 shows other data information of the monoclonal antibody used in competitive ELISA detection

[0083] Table 4 Minimum detection limit and linear range of the monoclonal antibody used in competitive ELISA:

[0084]

[0085] Table 5 Dilution recovery rate of the monoclonal antibody used in competitive ELISA test samples:

[0086]

[0087] Table 6 The intra-plate and inter-plate differences of the monoclonal antibody used in competitive ELISA:

[0088]

[0089] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0090] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A rat brain natriuretic peptide (BNP) detection kit, characterized in that: Includes BNP-6C5, a monoclonal antibody specific for rat BNP.

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

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

4. An antibody for a rat brain natriuretic peptide (BNP) detection kit, characterized in that: Includes BNP-6C5, a monoclonal antibody specific for rat BNP.

5. The antibody according to claim 4, characterized in that 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.

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

7. A method for preparing a rat brain natriuretic peptide (BNP) detection kit, characterized in that: The following steps are involved: S1. Mix PBS buffer and monoclonal antibody BNP-6C5, coat the ELISA plate and let stand overnight; S2, cleaning the coated ELISA plate; S3, adding blocking solution to the washed ELISA plate; S4. After washing the ELISA plate containing the blocking solution, dry it and store it in a vacuum.

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

9. The preparation method according to claim 7, characterized in that: In step S1, PBS buffer and monoclonal antibody BNP-6C5 are mixed to obtain a mixed solution, and each well of the ELISA plate contains 100 μL of the mixed solution.

10. The preparation method according to claim 7, characterized in that: The blocking solution includes PBS solution and BSA.

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