A monoclonal antibody combination for detecting cat NT-proBNP protein and its application

Abstract

This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody combination for detecting feline NT-proBNP protein and its applications. The monoclonal antibody combination of the present invention comprises monoclonal antibodies 3A6 and 5H5. These two antibodies possess unique heavy and light chain variable region complementarity-determining regions, respectively, and their amino acid sequences have been fully defined (SEQ ID NOs. 1-12). This antibody combination can specifically recognize and efficiently bind to feline NT-proBNP protein, making it suitable for the development of a highly sensitive and specific diagnostic tool for feline cardiomyopathy. By constructing a double-antibody sandwich ELISA assay system, the minimum detection limit can reach 100 pg / mL, and it exhibits excellent specificity and stability.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody combination for detecting cat NT-proBNP protein and an application thereof. Background Art

[0002] With the rapid development of the pet economy, cats, as one of the most popular companion animals, are loved by more and more families. However, because feline cardiomyopathy is a multifactorial disease with complex etiology and an incompletely understood pathogenesis, early identification and diagnosis of feline heart disease in clinical practice still face many challenges.

[0003] Currently, clinical diagnostic methods for feline cardiomyopathy primarily include X-rays, electrocardiograms, echocardiograms, and biomarker testing. X-rays may appear normal in many cats even when they have progressed to congestive heart failure, making them of limited diagnostic value. Conventional and dynamic electrocardiograms (ACDs) can help assess heart rhythm changes but cannot directly reflect structural changes in the heart. Echocardiography, considered the gold standard for diagnosing feline cardiomyopathy, requires the cat to be in a resting state, often requiring the use of sedatives. Furthermore, it places high demands on equipment and technicians, making large-scale screening and rapid diagnosis difficult.

[0004] Brain natriuretic peptide (BNP) and its precursor (NT-proBNP) have been extensively studied and clinically applied. Studies have shown that NT-proBNP concentrations above 46 pmol / L can be used to distinguish latent cardiomyopathy in cats with high specificity and sensitivity. This marker not only effectively assists in distinguishing cardiac from non-cardiac diseases (such as asthma and pneumonia), but also can be used for disease monitoring, efficacy assessment, and prognosis assessment, making it a crucial tool in the management of feline cardiomyopathy.

[0005] Although NT-proBNP is widely used in clinical practice, there is a lack of highly specific and sensitive antibodies and test kits for feline NT-proBNP. Existing detection methods often rely on commercially available antibodies, which suffer from strong cross-reactivity and insufficient sensitivity, making them inadequate for the precise diagnosis of feline cardiomyopathy. Therefore, there is an urgent need to develop a monoclonal antibody that can efficiently and accurately detect feline NT-proBNP levels, as well as its application method, to provide new technical support and tools for the early screening, diagnosis, and prognostic assessment of feline cardiomyopathy. Summary of the Invention

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a monoclonal antibody combination for detecting feline NT-proBNP protein and its application, which solves the technical problems in existing detection methods such as poor antibody specificity, low sensitivity, and easy cross-reaction, which make it difficult to meet the clinical needs for accurate diagnosis of feline cardiomyopathy.

[0007] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a monoclonal antibody combination for detecting feline NT-proBNP protein, the monoclonal antibody combination comprising monoclonal antibody 3A6 and monoclonal antibody 5H5;

[0009] The heavy chain variable region of monoclonal antibody 3A6 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0010] The light chain variable region of monoclonal antibody 3A6 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0011] The heavy chain variable region of monoclonal antibody 5H5 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 9, respectively;

[0012] The light chain variable region of monoclonal antibody 5H5 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 10 to SEQ ID NO. 12, respectively.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.14.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.16.

[0015] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3A6 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3A6 is shown as SEQ ID NO.18.

[0016] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5H5 is shown as SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5H5 is shown as SEQ ID NO. 20.

[0017] In some embodiments, the monoclonal antibody combination specifically recognizes feline NT-proBNP recombinant protein and feline NT-proBNP protein.

[0018] In a second aspect, the present invention provides use of the combination of the above-mentioned monoclonal antibodies in preparing a tool for detecting feline NT-proBNP protein.

[0019] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0020] In some embodiments, the kit comprises a double antibody sandwich ELISA kit.

[0021] In some embodiments, the ELISA kit uses monoclonal antibody 3A6 as a coating antibody and monoclonal antibody 5H5 as a labeling antibody.

[0022] Beneficial effects:

[0023] The present invention provides a monoclonal antibody combination for detecting feline NT-proBNP protein. This combination consists of monoclonal antibodies 3A6 and 5H5, each with distinct and well-defined amino acid sequences (SEQ ID NOs. 1-12) of the complementarity-determining regions (CDRs) of the heavy and light chain variable regions, respectively. These antibodies are capable of efficiently and specifically recognizing feline NT-proBNP protein. These two monoclonal antibodies significantly improve the sensitivity and specificity of a double-antibody sandwich ELISA assay, achieving a minimum detection limit of 100 pg / mL and exhibiting no cross-reactivity with unrelated antigens. This high-affinity and highly specific antibody pairing not only enhances the accuracy of the detection signal but also provides a reliable foundation for the development of a stable and reproducible diagnostic kit. Therefore, this invention provides strong technical support for the early screening, accurate diagnosis, and monitoring of feline cardiomyopathy, and possesses significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 The SDS-PAGE identification results of protein purification;

[0026] Figure 2 This is the ELISA identification result of feline NT-proBNP recombinant protein;

[0027] Figure 3 It is a double antibody sandwich ELISA sensitivity and specificity test;

[0028] Figure 4 To identify the binding activity of paired monoclonal antibodies. DETAILED DESCRIPTION

[0029] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0030] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0031] Example 1

[0032] 1. Recombinant protein gene synthesis

[0033] The feline NT-proBNP protein gene (downloaded from the NCBI database) was synthesized by Qingke Biotechnology after codon optimization and cloned into the pET32a vector. The nucleotide sequence corresponding to the feline NT-proBNP protein gene is shown in SEQ ID NO. 21:

[0034] CATCCGCTGGGTGGTCCAGGTCCAGCGTCTGAGGCTTCTGCGATCCAAGAACTGCTGGACGGTCTGCGTGACACTGTTTCTGAACTGCAAGAAGCGCAGATGGCACTGGGTCCGCTGCAGCAGGGTCACTCTCCGGCTGAAAGCTGGGAAGCTCAGGAAGAACCGCCAGCGCGTGTTCTGGCTCCGCATGACAACGTTCTGCGTGCTCTGCGT.

[0035] The amino acid sequence corresponding to the cat NT-proBNP protein gene is shown in SEQ ID NO.22:

[0036] HPLGGPGPASEASAIQELLDGLRDTVSELQEAQMALGPLQQGHSPAESWEAQEEPPARVLAPHDNVLRALR.

[0037] The synthetic plasmid pET32a-feline NT-proBNP was transformed into BL21(DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) using conventional methods. Transformants were plated on LB agar plates (containing 100 μg / mL ampicillin) and cultured overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured with shaking at 37°C, 220 rpm, and 1% of the total volume of the culture was inoculated into LB medium (containing 100 μg / mL ampicillin). The culture was shaken at 37°C, 220 rpm, and cultured for approximately 3 hours to an OD600 of 0.5. The culture was then cooled at 16°C for 1.5 hours. After induction with a final concentration of 0.1 mM IPTG at 180 rpm at 16°C for 16 hours, the cells were harvested and purified.

[0038] Feline NT-proBNP (also known as fNT-proBNP) recombinant protein and feline NT-proBNP protein are essentially the same protein, but they differ in their sources. Feline NT-proBNP protein is a naturally occurring cardiac protein secreted by cats. Its concentration increases significantly in cats with cardiomyopathy or increased cardiac workload, making it considered an important biomarker for heart disease. Recombinant feline NT-proBNP protein, on the other hand, is artificially synthesized through genetic engineering techniques. Typically, the gene encoding feline NT-proBNP is cloned into an expression vector, induced for expression, and purified.

[0039] 2. Purification of recombinant protein

[0040] Because the expressed feline NT-proBNP recombinant proteins all carry histidine tags, GE's AKTA Start, HisTrap TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The collected bacterial pellet expressing feline NT-proBNP recombinant protein was then resuspended in buffer A and disrupted in ice water by sonication for 30 minutes, followed by 5-second intervals and 5-second intervals. The supernatant was centrifuged at 12,000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm filter and loaded onto the column. The column was washed with buffer A and then eluted with a gradient of buffer B. Purification was assessed by SDS-PAGE. The target protein peak was selected and dialyzed into buffer A. The purified protein was collected and its concentration was determined using a Nanodrop. The protein was aliquoted into 1 mL tubes and stored at -20°C. The pET32a-feline NT-proBNP recombinant protein was soluble after low-temperature induction at 16°C, and the main elution peak was collected at 500 mM imidazole during Ni affinity chromatography purification.

[0041] 3. Identification of recombinant protein

[0042] 3.1. Identify protein purity by SDS-PAGE electrophoresis

[0043] Protein sample pretreatment: Add an equal volume of 2x SDS loading buffer to each sample, boil in a boiling water bath for 10 minutes, and centrifuge at 12,000 rpm for 3 minutes. Dilute 5x glycine buffer to the working concentration and add it to the electrophoresis tank until the liquid level is appropriate. Gently remove the comb from the solidified gel. Add a protein marker and 10 μl of the treated protein sample to the sample wells. Connect the power supply and adjust the voltage to 80V for constant voltage electrophoresis until the resolving gel is reached, then adjust it to 120V until the bromophenol blue reaches the bottom of the gel. Cut the gel from the glass plate and stain with Coomassie Brilliant Blue solution. Stain with shaking for 4 hours, then destain with destaining solution until the bands are clear.

[0044] See also Figure 1 , M: protein marker, No. 1: before induction, No. 2: precipitation, No. 3: supernatant, No. 4: flow-through, No. 5: 100mM elution, No. 6: 500mM elution, SDS-PAGE results showed a clear main band around 25-33kDa, consistent with the estimated size of the fusion protein (26.3kDa), and the purity was above 85%.

[0045] 3.2. ELISA to identify the activity of recombinant protein

[0046] Purified feline NT-proBNP recombinant protein was coated (1 μg / mL), and its reaction with the commercially available feline NT-proBNP monoclonal antibody M101706M (Nanjing Fuxiao Biotechnology Co., Ltd.) was identified by indirect ELISA. An irrelevant fCys-C monoclonal antibody M102202M (Nanjing Fuxiao Biotechnology Co., Ltd.) was used as a control. First, microplates were coated with feline NT-proBNP recombinant protein (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a coating concentration of 1 μg / mL, 50 μl / well, and incubated at 4°C overnight. The plates were blocked with 1% BSA (100 μl per well) at 37°C for 2 hours, washed once with washing buffer (PBST, PBS containing 0.05% Tween-20) and patted dry. The monoclonal antibody was diluted in PBS at a gradient of 10 μg / mL, 1 μg / mL, 100 ng / mL, and 10 ng / mL, and 50 μL was added to the antigen-coated microplate. An equal concentration of fCys-C monoclonal antibody M102202M was also added as a negative control, and the plates were reacted at 37°C for 30 minutes. The liquid in the wells was discarded, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again, and 50 μL / well of TMB color development solution was added to develop at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added to terminate the reaction, and the OD450 nm value was measured with a microplate reader.

[0047] The reaction of recombinantly expressed feline NT-proBNP antigen with the commercially available monoclonal antibody M101706M was identified by indirect ELISA, coated with feline NT-proBNP (1 μg / mL) recombinant protein. Figure 2 , Figure 2 M101706M represents the commercially available feline NT-proBNP monoclonal antibody M101706M, and Ctrl represents the commercially available fCys-C monoclonal antibody M102202M. The results showed that the antigen still had a positive reaction when the purchased monoclonal antibody was diluted to 100 ng / mL-10 ng / mL, indicating that the recombinant protein has good biological activity and the protein was successfully expressed.

[0048] 4. Mouse immunization

[0049] Six-week-old female BALB / c mice were immunized intramuscularly with a mixture of feline NT-proBNP recombinant protein and an equal volume of MF59 adjuvant (200 μl). Immunizations were repeated at two and four weeks using the same protocol. At five weeks, sera were collected from the mice to measure antibody titers using the expressed feline NT-proBNP recombinant protein. Mice with the highest titers were boosted with 20 μg of feline NT-proBNP recombinant protein via the tail vein. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0050] 5. Screening, preparation of hybridoma cell lines and antibody purification

[0051] 5.1. Screening of Hybridoma Cells

[0052] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, clones positive for feline NT-proBNP protein were screened by indirect ELISA. Positive cells were then cloned to monoclonal status by limiting dilution, and the cell line was expanded and cryopreserved.

[0053] Positive clones were screened by indirect ELISA. Microplates were coated with feline NT-proBNP recombinant protein (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a coating concentration of 1:100 (presumably 0.000 ng / well) at 4°C overnight. Plates were then blocked with 1% gelatin (150 μL per well) at 37°C for 2 hours. The plates were washed once with detergent and patted dry. 50 μL of cell culture supernatant was added and incubated at 37°C for 30 minutes. The wells were then discarded, washed four times with PBST, and patted dry. HRP-conjugated goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added at 50 μL / well and incubated at 37°C for 30 minutes. The plates were then washed four times, patted dry, and TMB color development solution (50 μL / well) was added for development at room temperature for 10 minutes. The reaction was terminated by the addition of 0.5 M sulfuric acid, and the OD 450 nm was measured using a microplate reader. Positive cell lines that reacted only with feline NT-proBNP recombinant protein but not with the control antigen fcTnI were selected for subsequent experiments.

[0054] The screening process is shown in Table 1.

[0055] Table 1: Indirect ELISA reaction results of hybridoma cell line culture supernatant to feline NT-proBNP recombinant protein and control antigen.

[0056]

[0057] In addition, the test results of several groups of monoclonal antibodies were not ideal and were not suitable for subsequent testing, so they were not shown in the table.

[0058] 5.2 Preparation of Monoclonal Antibody Ascites

[0059] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0060] 5.3. Affinity chromatography purification of monoclonal antibodies (Protein G)

[0061] The ascites was centrifuged at 12,000 r / min for 5 minutes, and the supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4). The supernatant was then filtered through a 0.22 μm filter. The filtered sample was pumped at a low speed through a peristaltic pump into a Protein G (Cytiva) purification column equilibrated with binding buffer. The column was connected to an AKTA purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak was flattened. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected and the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, placed in a dialysis bag (MW: 8,000-14,000), and dialyzed against 0.01 M PBS, pH 7.4, at 2-8°C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration was measured using an ultramicro spectrophotometer and the supernatant was stored in aliquots.

[0062] 6. Establishment and Condition Optimization of Double Antibody Sandwich ELISA

[0063] 6.1. HRP-labeled monoclonal antibodies

[0064] The screened antibodies were labeled with HRP according to the instructions of the G-Biosciences HOOK™ HRP PLUS Labeling Kit (Cat#: 786-313). The labeled antibodies were dialyzed overnight in 0.01M PBS, pH 7.4 buffer, and glycerol was added at a 1:1 volume ratio. The antibodies were stored in aliquots at -20°C.

[0065] Specifically, dilute the antibody to be labeled with coupling buffer (provided with the kit) to a final concentration of 2 mg / mL. Add the diluted antibody solution to the tube containing HRP (provided with the kit) and mix thoroughly by pipetting. Incubate at room temperature for 1 hour, mixing regularly during the incubation period. Add 50 μL of stop solution and mix for 15 minutes to terminate the labeling reaction. Dialyze the solution overnight against PBS buffer and add an equal volume of glycerol for storage.

[0066] 6.2. Establishment of the double antibody sandwich method:

[0067] The purified monoclonal antibodies were diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 1% gelatin at 150 μL / well. The plates were incubated at 37°C for 2 h, the blocking solution was discarded, and the plates were patted dry. The test antigen feline NT-proBNP and the control antigen fcTnI were diluted to 100 ng / mL in PBS and added to an ELISA plate at 50 μL / well. The plates were incubated at 37°C for 35 minutes. The plates were washed four times with PBST and patted dry. An enzyme-labeled monoclonal antibody diluted 4000-fold in PBS was added at 50 μL / well. The plates were incubated at 37°C for 35 minutes. The plates were washed four times and patted dry. TMB color development solution (50 μL / well) was added and color was developed at room temperature for 10 minutes. Finally, 0.5 M sulfuric acid (50 μL / well) was added to terminate the reaction. The OD450 nm value was measured using a microplate reader. The paired monoclonal antibody with the highest P / N value was selected. The results showed that the coating antibody (3A6) and the labeled antibody (5H5) had the highest P / N value, and this paired monoclonal antibody was used for further testing.

[0068] The screening process is shown in Table 2.

[0069] Table 2: Comparison of the reactivity of different monoclonal antibody combinations against feline NT-proBNP recombinant protein and control antigen in double antibody sandwich ELISA.

[0070]

[0071] “*” in the table indicates the dilution multiple.

[0072] 6.3 Optimization of the Double Antibody Sandwich ELISA Method

[0073] The purified monoclonal antibody 4D5 was coated at a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6) 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the plate was blocked with 1% BSA at 150 μL / well and incubated at 37°C for 2 h. The blocking solution was discarded and the antigen to be tested and the control antigen were diluted with PBS at 100 ng / mL and added to the ELISA plate at 50 μL / well and incubated at 37°C for 35 min. The plate was washed 4 times with PBST and added with PBS. Add 50 μL / well of HRP-labeled monoclonal antibody at 2000-, 4000-, and 6000-fold dilutions and incubate at 37°C for 35 minutes. Wash the plate four times, pat dry, and add 50 μL / well of TMB colorimetric solution. Develop at room temperature for 10 minutes. Finally, add 50 μL / well of 0.5 M sulfuric acid to terminate the reaction. Measure the OD450 nm value using a microplate reader. Select the pairing condition with the highest P / N value for the next step.

[0074] The screening process is shown in Table 3.

[0075] Table 3: Optimization of optimal coating and labeling conditions for sandwich ELISA.

[0076]

[0077] The optimal reaction conditions for the double-antibody sandwich ELISA were as follows: monoclonal antibody 3A6 was used as the coating antibody at a coating concentration of 1 μg / mL; HRP-labeled monoclonal antibody 5H5 was used as the labeling antibody and diluted 4000 times before use.

[0078] 6.4. Double Antibody Sandwich ELISA Sensitivity and Specificity Test

[0079] Referring to the above optimal detection conditions and steps, the feline NT-proBNP protein was first diluted in a gradient manner with PBS buffer solution to concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. At the same time, two recombinant proteins, fcTnI and fSAA, were added to each well at the same concentration for detection, and the sensitivity and specificity of the detection system for the recombinant proteins were determined.

[0080] according to Figure 3 Available, Figure 3fNT-proBNP represents recombinant fNT-proBNP protein, while fcTnI and fSAA represent recombinant fcTnI and fSAA proteins, respectively. The genes for the recombinant fcTnI and fSAA proteins are synthesized and expressed by Qingke Biotechnology. This double-antibody sandwich ELISA, using monoclonal antibody 3A6 as the coating antibody and monoclonal antibody 5H5 as the labeling antibody, shows a positive reaction at a dilution of 100 pg / mL for feline NT-proBNP protein and is unreactive with unrelated antigens. It demonstrates excellent sensitivity and specificity, providing a new tool for the diagnosis of feline cardiomyopathy.

[0081] 7. Identification of binding activity of paired monoclonal antibodies

[0082] Referring to the aforementioned indirect ELISA method, the two monoclonal antibodies were serially diluted at 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL. The binding activity of the feline NT-proBNP monoclonal antibody was determined using another unrelated mouse monoclonal antibody, fCys-C monoclonal antibody M102202M, as a negative control.

[0083] See also Figure 4 , Figure 4 In the table, 3A6 represents monoclonal antibody 3A6, 5H5 represents monoclonal antibody 5H5, and Ctrl represents fCys-C monoclonal antibody M102202M. Both monoclonal antibodies 3A6 and 5H5 specifically reacted with feline NT-proBNP recombinant protein (antigen) at concentrations of 1-10 ng / mL and did not react with the control monoclonal antibody, demonstrating that this paired monoclonal antibody pair has high binding activity.

[0084] 8. Antibody variable region gene cloning and sequencing

[0085] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0086] Mouse monoclonal antibody light and heavy chain gene sequences:

[0087] Monoclonal antibody 3A6:

[0088] Light chain:

[0089] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.18:

[0090] GACATCGAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATCACCTGCAGCGCCAGCAGCAGCAGGAGCTACATGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCCAGAGCTTCATCCACAG CGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTGCCAGCAGAAGAGGAGCTACCCCATCACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0091] The amino acid sequence of the light chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.14:

[0092] DIEMTQSPAIMSASPGEKVTITCSASSSRSYMHWFQQKPGTSPKLWIYSQSFIHSGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQKRSYPITFGAGTKLEIKRTV.

[0093] CDR region annotation:

[0094] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 3A6 are shown in SEQ ID NO. 4: SASSSRSYMH;

[0095] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of monoclonal antibody 3A6 are shown in SEQ ID NO. 5: SQSFIHS;

[0096] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO. 6: QQKRSYPIT.

[0097] Heavy chain:

[0098] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.17:

[0099] CAGGGTGCAGCTGAAGCAGAGCGGCAGCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGGTACAGCATGCACTGGGTGAAGCAGAGGCACGGCCAGGGCCTGGAGTGGATCGGCAACATCTACCCCGGCAGCGGCAGGACC AACTACGACGAGAAGTTCAAGAGCAAGGGCACCCTGACCGCCGACACCAGCAGCAACACCGCCTACATGCACCTTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCATGATCGAGGACTACAACGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC.

[0100] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO.13:

[0101] QVQLKQSGSELVRPGASVKLSCKASGYTFTRYSMHWVKQRHGQGLEWIGNIYPGSGRTNYDEKFKSKGTLTADTSSNTAYMHLSSLTSEDSAVYYCTMIEDYNDYWGQGTTLTVSA.

[0102] CDR region annotation;

[0103] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 3A6 are shown in SEQ ID NO. 1: RYSMH;

[0104] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 3A6 are shown in SEQ ID NO. 2: NIYPGSGRTNYDEKFKS;

[0105] The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 3A6 is shown in SEQ ID NO. 3: IEDYNDY.

[0106] Monoclonal antibody 5H5:

[0107] Light chain:

[0108] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.20:

[0109] GACATCCAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATCACCTGCAGCGCCAGCAGGAGGAGCAACTACATGAAGTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCACCAGCAACACCAACAG CGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGGGCAGCGCCGAGGACGCCGCCACCTACTACTGCGGCAGGAGGAGCAGCAACCCCCTGGCCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0110] The amino acid sequence of the light chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.16:

[0111] DIQMTQSPAIMSASPGEKVTITCSASRRSNYMKWFQQKPGTSPKLWIYSTSNTNSGVPARFSGSGSGTSYSLTISRGSAEDAATYYCGRRSSNPLAFGAGTKLELKRTV.

[0112] CDR region annotation:

[0113] The amino acid sequences of the complementarity determining regions (CDR-L1) of the light chain variable region of the monoclonal antibody 5H5 are shown in SEQ ID NO. 10: SASRRSNYMK;

[0114] The amino acid sequences of the complementarity determining regions CDR-L2 of the light chain variable region of the monoclonal antibody 5H5 are shown in SEQ ID NO. 11: STSNTNS;

[0115] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO. 12: GRRSSNPLA.

[0116] Heavy chain:

[0117] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.19:

[0118] CAGATGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCGAGAAGACCAGCGGCTACACCTTCACCTTCGGCACCAGGAGGTGGGTGAACCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCTGGATCAAGAGGCAGGACGGCAGGTTCAAGTACAA CAGCGAGAAGAGGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGAACAGCGCCGTGTACTTCTGCGCCAGGGGCGGCAGCAGCGGCTACAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0119] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5H5 is shown in SEQ ID NO.15:

[0120] QMQLQQSGPELVKPGASMKISEKTSGYTFTFGTRRWVNQRPGQGLEWIGWIKRQDGRFKYNSEKRGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGGSSGYSYAMDYWGQGTSVTVSS.

[0121] CDR region annotation:

[0122] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 5H5 are shown in SEQ ID NO. 7: FGTRR;

[0123] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of the monoclonal antibody 5H5 are shown in SEQ ID NO. 8: WIKRQDGRFKYNSEKRG;

[0124] The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO. 9: GGSSGYSYAMDY.

Claims

1. A monoclonal antibody combination for detecting feline NT-proBNP protein, characterized in that: The monoclonal antibody combination includes monoclonal antibody 3A6 and monoclonal antibody 5H5; The heavy chain variable region of the monoclonal antibody 3A6 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of the monoclonal antibody 3A6 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; The heavy chain variable region of the monoclonal antibody 5H5 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively; The light chain variable region of the monoclonal antibody 5H5 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting feline NT-proBNP protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3A6 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3A6 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting feline NT-proBNP protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting feline NT-proBNP protein according to claim 3, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3A6 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3A6 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting feline NT-proBNP protein according to claim 4, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5H5 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting feline NT-proBNP protein according to claim 5, characterized in that: The monoclonal antibody combination specifically recognizes feline NT-proBNP recombinant protein and feline NT-proBNP protein.

7. Use of the combination of monoclonal antibodies according to claim 1 in preparing a tool for detecting feline NT-proBNP protein.

8. The use according to claim 7, characterized in that The tools include reagents, test kits, test strips and antibody chips.

9. The use according to claim 8, characterized in that The kit includes a double antibody sandwich ELISA kit.

10. The use according to claim 9, characterized in that The ELISA kit uses the monoclonal antibody 3A6 as the coating antibody and the monoclonal antibody 5H5 as the labeling antibody.

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