Monoclonal antibody combination for detecting cat ngal protein and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting cat NGAL protein and application thereof. The monoclonal antibody combination of the application comprises monoclonal antibodies 1E2 and 2D6. The two antibodies have unique heavy chain and light chain variable region complementarity determining regions, and the amino acid sequences thereof have been defined in detail (SEQ ID NO. 1-12). The antibody combination can specifically recognize and efficiently bind to cat NGAL protein, and is suitable for developing a diagnostic tool with high sensitivity and high specificity. By constructing a double-antibody sandwich ELISA detection system, the minimum detection limit can reach 1 ng / mL, and good specificity and stability are shown.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for detecting feline NGAL protein and its application. Background Technology

[0002] Chronic kidney disease (CKD) in cats is a common and irreversible disease. Due to its slow and progressive development, early diagnosis and accurate assessment of kidney function are crucial for the management and treatment of the disease.

[0003] Currently, clinical assessment of feline kidney function primarily relies on indicators such as glomerular filtration rate (GFR), serum urea nitrogen, and creatinine concentration. GFR is considered the "gold standard" for evaluating kidney function, but its measurement requires specialized equipment and strict sampling conditions, making its practical clinical application challenging. While serum urea nitrogen and creatinine are commonly used indicators, they have poor sensitivity in reflecting early kidney damage and are easily affected by non-renal factors (such as diet and muscle mass), making it difficult to accurately reflect the true state of kidney function.

[0004] In recent years, with the development of molecular biology and biomarker research, neutrophil gelatinase-associated lipocarboxin (NGAL) has been found to be closely related to various types of kidney injury. NGAL is a glycoprotein with a molecular weight of approximately 25 kDa, which is upregulated not only during inflammatory responses but also significantly elevated when epithelial cells are damaged. Studies have shown that the concentration of NGAL in blood and urine can effectively reflect the degree of kidney injury. Although NGAL shows promising application prospects in the diagnosis of feline chronic kidney disease, a highly efficient, sensitive, and specific detection method has not yet been established. Existing immunological detection methods generally suffer from insufficient sensitivity and severe cross-reactivity, limiting their widespread clinical application. Summary of the Invention

[0005] Given the lack of efficient, sensitive and specific methods for detecting feline NGAL protein in existing technologies, this invention provides a combination of monoclonal antibodies for detecting feline NGAL protein and its application, solving the problems of low sensitivity and severe cross-reactivity in existing immunoassay methods.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides a monoclonal antibody combination for detecting feline NGAL protein, the monoclonal antibody combination comprising monoclonal antibody 1E2 and monoclonal antibody 2D6.

[0008] The heavy chain variable region of monoclonal antibody 1E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0009] The light chain variable region of monoclonal antibody 1E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0010] The heavy chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0011] The light chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.14.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.16.

[0014] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO. 18.

[0015] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO. 20.

[0016] In some embodiments, the monoclonal antibody combination specifically recognizes the recombinant fNGAL protein and the feline NGAL protein.

[0017] Secondly, the present invention provides the application of the above-mentioned combination of monoclonal antibodies in the preparation of a tool for detecting feline NGAL protein.

[0018] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0019] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0020] In some embodiments, the ELISA kit uses monoclonal antibody 1E2 as the coating antibody and monoclonal antibody 2D6 as the labeling antibody.

[0021] In addition, the double-antibody sandwich ELISA detection method is not used for disease diagnosis and treatment.

[0022] Beneficial effects:

[0023] The monoclonal antibody ensemble (including monoclonal antibodies 1E2 and 2D6) provided by this invention for detecting feline NGAL protein exhibits high specificity and sensitivity. This antibody ensemble, designed and screened with clearly defined complementarity-determining region (CDR) sequences, can efficiently recognize feline NGAL protein (i.e., feline NGAL protein) and avoid cross-reactivity with other feline proteins. Specifically, monoclonal antibodies 1E2 and 2D6 target different antigenic epitopes of the NGAL recombinant protein, making them suitable for constructing detection systems such as double-antibody sandwich ELISA, significantly improving the stability and accuracy of the detection signal.

[0024] The detection method established based on this antibody combination has good sensitivity, with a detection limit as low as 1 ng / mL. It can be widely used for the detection of various sample types such as serum, urine, and tissue fluid. It is expected to provide a reliable tool for the early diagnosis and disease monitoring of feline chronic kidney disease through the development and application of various platforms such as diagnostic kits, test strips, and antibody chips. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the SDS-PAGE identification results of the purified protein.

[0027] Figure 2 The graph shows the sensitivity and specificity of the double-antibody sandwich ELISA method.

[0028] Figure 3 This is a diagram illustrating the binding activity of paired monoclonal antibodies. Detailed Implementation

[0029] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0030] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated 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 NGAL protein gene (downloaded from the NCBI database) was synthesized by Qingke Biotechnology and cloned into the pET32a vector. The nucleotide sequence corresponding to the feline NGAL protein gene is shown in SEQ ID NO.21.

[0034] caggattccaccccaaacctgatcccagccccgcctctgctcttggtccctgtggagcctgacttccagaatgagcagttccaggggaaatggtacttcttaggattggcagggaacggattcaataaagaaaagcacaggaggatgaagatgtacattgccaactacgagctgaacgaagacaacagctacaatgtcacctctactgtggcctggaaccagacctgtcatccctcgaccaaaattttcctcccaaatttgcatctaggccaattcaacctgggcaacattgagcgttacactggaatccagaactacacttcaaaagtggtgaccacagactacaaccagtttgccatactgtacttcaagaaagttcatgacaaccaggagtacatcaaggtcatcctctatgggaggaccaaggaggtgccttctgtaccgaaggcaatcttcatcagcttcatcaaatccctgggcctcaccgacgaccacatcatcttccctatccccaatgatgagtgcatggataag。

[0035] The amino acid sequence corresponding to the feline NGAL protein gene is shown in SEQ ID NO.22:

[0036] QDSTPNLIPAPPLLLVPVEPDFQNEQFQGKWYFLGLAGNGFNKEKHRRMKMYIANYELNEDNSYNVTSTVAWNQTCHPSTKIFLPNLHLGQFNLGNIERYTGIQNYTSKVVTTDYNQFAILYFKKVHDNQEYIKVILYGRTKEVPSVPKAIFISFIKSLGLTDDHIIFPIPNDECMDK。

[0037] Seamless cloning primers for the pCold-TF vector fNGAL were designed. Using the synthetic plasmid pET32a-fNGAL as a template, the fNGAL fragment was amplified. The fragment was ligated to the vector according to the kit instructions (In-Fusion HD Cloning kits, TaKaRa), and transformed into BL21(DE3) competent cells using standard methods (Molecular Cloning, 3rd edition, Science Press). Transformed cells were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours, until OD (dose elapsed). 600 The concentration was 0.5, and the temperature was lowered to 16℃ for 1.5 hours. Then, IPTG was added to a final concentration of 0.1 mM, and the cells were collected after induction at 16℃ and 180 rpm for 16 hours.

[0038] Feline NGAL protein generally refers to a protein naturally occurring in cats. Its expression level increases significantly under pathological conditions such as inflammation, infection, and tissue damage. In felines, NGAL is often used as a biomarker for kidney injury or inflammatory responses. Recombinant feline NGAL protein (fNGAL recombinant protein) is an NGAL protein synthesized in an in vitro expression system using genetic engineering techniques. It is obtained by cloning the feline NGAL gene into a suitable expression vector and expressing it in a host system. The recombinant protein has the same amino acid sequence as the feline NGAL protein and, after purification, can be used for scientific research, diagnostic reagent development, or functional studies.

[0039] 2. Purification and concentration identification of recombinant proteins

[0040] Since the expressed fNGAL recombinant proteins all carry histidine tags, purification was performed using GE's AKTA Start and HisTrap™ HP affinity chromatography columns. 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 chromatography columns were equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 20 min, the precipitate was resuspended in buffer A, and the mixture was sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter, loaded onto the column, washed with buffer A, and finally eluted with a gradient of buffer B. The purification status was observed by SDS-PAGE protein gel electrophoresis. Collect the target protein peak and dialyze it into a buffer solution of 50 mM PB, 300 mM NaCl, pH 8.0. Determine the concentration of fNGAL recombinant protein using Nanodrop, aliquot into 1 ml tubes, and store at -20°C.

[0041] 3. Identification of recombinant proteins

[0042] This invention uses SDS-PAGE electrophoresis to identify protein purity.

[0043] Protein sample pretreatment: For each sample, add an equal volume of 2x SDS loading buffer, boil in a water bath for 10 min, and centrifuge at 12000 rpm for 3 min. Dilute 5x glycine buffer to the working concentration, add to the electrophoresis tank to the appropriate liquid level, and gently remove the comb from the solidified gel. Add protein marker and 10 μl of the pretreated protein sample to each well. Turn on the power and adjust the voltage to a constant 80V for electrophoresis until the separating gel is reached, then change to 120V until bromophenol blue reaches the bottom of the gel. Cut the gel from the glass plate, place it in Coomassie Brilliant Blue staining solution, shake and stain for 4 h, then destain with destaining solution until the bands are clear.

[0044] See results Figure 1 , Figure 1 In the diagram, M represents the protein marker; sequence 1: pre-induction; sequence 2: precipitation; sequence 3: supernatant; sequence 4: flow-through; sequence 5: 100 mM elution-1; sequence 6: 100 mM elution-2; sequence 7: 100 mM elution-3; sequence 8: 500 mM elution-1; and sequence 9: 500 mM elution-2. A distinct main band is visible between 70 and 95 kDa, indicating a protein purity of approximately 85% or higher, consistent with the estimated antigen size (74.4 kDa). The purified fNGAL recombinant protein can be used for further downstream experiments.

[0045] 4. Mouse immunization

[0046] Female BALB / c mice were immunized intramuscularly with a mixture of recombinant fNGAL protein and an equal volume of MF59 adjuvant (200 μL) for 6 weeks. Repeat immunizations were administered at 2 and 4 weeks. At week 5, mouse serum was collected to detect antibody titers using the recombinant fNGAL protein. Mice with the highest titers were selected for a booster immunization of 20 μg of recombinant fNGAL protein via tail vein. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0047] 5. Screening, preparation, and antibody purification of hybridoma cell lines

[0048] 5.1 Screening of hybridoma cells

[0049] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for fNGAL recombinant protein were selected by indirect ELISA. The positive cells were then cloned into monoclonal states by limiting dilution, and the cell lines were expanded and cryopreserved.

[0050] Indirect ELISA screening of positive clones: Hybridoma cell lines that secrete recombinant fNGAL protein specifically recognized by the cell fusion were selected by indirect ELISA.

[0051] fNGAL recombinant protein and control antigen fCys-C were coated separately in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water) at a concentration of 1 μg / mL and incubated overnight at 4°C. The plates were then blocked with 1% gelatin (150 μL per well) at 37°C for 2 hours. After washing once with washing buffer and patting dry, 50 μL of cell culture supernatant was added and incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After patting dry, 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) was added per well and incubated at 37°C for 30 min. After washing four more times and patting dry, 50 μL of TMB chromogenic buffer was added per well and incubated at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added to stop the reaction, and the OD was measured using a microplate reader. 450 nm value. Positive cell lines that react only with the fNGAL recombinant protein and not with the control antigen fCys-C were selected for subsequent experiments.

[0052] The screening process is shown in Table 1.

[0053] Table 1: Results of indirect ELISA reaction of hybridoma cell line supernatant to fNGAL recombinant protein and control antigen.

[0054]

[0055] As can be seen from the indirect ELISA screening results shown in the table above, monoclonal antibodies 1E2, 1A8, and 2D6 exhibited strong reactivity in recognizing recombinant fNGAL protein, with OD450nm values ​​of 1.738, 1.276, and 1.696, respectively, which were significantly higher than those of other monoclonal antibodies. Furthermore, none of them showed cross-reactivity with the control antigen fCys-C, demonstrating good specificity.

[0056] Given that the test results of several monoclonal antibodies are not ideal (not shown), considering both reaction strength and specificity, clones 1E2, 1A8, and 2D6 are selected for subsequent experiments.

[0057] 5.2 Preparation of Monoclonal Antibody Ascites

[0058] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the mice's abdomens were significantly swollen, ascites fluid was collected using a sterile syringe needle. The collected ascites fluid was centrifuged at 3000 rpm for 10 minutes, and the intermediate layer was collected.

[0059] 5.3 Affinity chromatography purification of monoclonal antibodies (Protein G)

[0060] Centrifuge the ascites fluid at 12000 rpm for 5 minutes. Take the supernatant and dilute it 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4). Filter the supernatant through a 0.22 μm filter. Pump the filtered sample slowly into a Protein G (Cytiva) purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to an AKTA purification instrument and wash with binding buffer for 5-10 column volumes until the UV absorption peak is leveled off. Then elute with elution buffer (0.1 M glycine, pH 2.7). Collect the elution peak. Adjust the pH of the collected sample to neutral with 1 M Tris-HCl (pH 9) and place it in a dialysis bag (MW: 8000~14000). Dialyze the sample in 0.01 M PBS (pH 7.4) at 2~8℃ for 14 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 r / min for 5 minutes. The supernatant is the purified monoclonal antibody. Determine the concentration using an ultra-micro spectrophotometer and aliquot for storage.

[0061] 6. Establishment and optimization of conditions for double-antibody sandwich ELISA

[0062] 6.1 HRP-labeled monoclonal antibodies

[0063] The monoclonal antibodies obtained from screening were labeled according to the instructions of G-Biosciences' HOOK™ HRP PLUS Labeling Kit (Cat#: 786-313). Finally, 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 aliquots were stored at -20°C.

[0064] Specifically: Dilute the antibody to be labeled to a final concentration of 2 mg / mL with conjugation buffer (as provided with the kit). Then, add the diluted antibody solution to a tube containing HRP (as provided with the kit), mix well by pipetting, and incubate at room temperature for 1 hour, mixing periodically during incubation. Add 50 μL of stop solution, mix for 15 minutes to stop the labeling reaction, dialyze overnight in PBS buffer, and then add an equal volume of glycerol for freezing.

[0065] 6.2 Establishment of the double-antibody sandwich method

[0066] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was patted dry and blocked with 1% BSA at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. The test antigen (fNGAL recombinant protein) and control antigen (fCys-C recombinant protein) were diluted to 100 ng / ml with PBS and added to the microplate at 50 μL / well. The plate was incubated at 37°C for 35 min. The plate was washed four times with PBST wash buffer, blotted dry, and then 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS was added. The plate was incubated at 37°C for 35 min, washed four more times, blotted dry, and then 50 μL / well of TMB chromogenic buffer was added. The plate was incubated at room temperature for 10 min. Finally, 50 μL / well of 0.5 M sulfuric acid was added to terminate the reaction. The OD was measured using a microplate reader. 450 nm value. The paired monoclonal antibodies with the highest P / N value were selected. Results showed that monoclonal antibody 1E2, used as the coating antibody, and monoclonal antibody 2D6, used as the labeling antibody, had the highest P / N value. These paired monoclonal antibodies were then used for further testing. The screening process is shown in Table 2.

[0067] Table 2: Comparison of the reactivity of different monoclonal antibody combinations to fNGAL recombinant protein and control antigen in double antibody sandwich ELISA.

[0068]

[0069] "*" indicates the dilution factor.

[0070] 6.3 Optimization of the Double Antibody Sandwich ELISA Method

[0071] The purified monoclonal antibody 1E2 was diluted with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, and incubated overnight at 4°C. The coating buffer was discarded the next day, and the plates were blocked with 1% BSA at 150 μL / well. The plates were incubated at 37°C for 2 h, and the blocking buffer was discarded. The test antigen (fNGAL recombinant protein) and the control antigen (fCys-C recombinant protein) were diluted with PBS at 100 ng / mL and added to the microplates at 50 μL / well. The plates were incubated at 37°C for 35 min, washed four times with PBST, and then coated with PBS. HRP-labeled monoclonal antibody 2D6 diluted 1000, 2000, and 3000 times, 50 μL / well, was incubated at 37°C for 35 min. After washing the plate four times and patting it dry, 50 μL / well of TMB chromogenic buffer was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL / well of 0.5 M sulfuric acid was added to stop the reaction, and the OD was measured using a microplate reader. 450 nm value. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The screening process is shown in Table 3.

[0072] Table 3: Optimization of optimal coating and labeling conditions for sandwich ELISA.

[0073]

[0074] The optimal reaction conditions are as follows: using monoclonal antibody 1E2 as the coating antibody at a concentration of 2 μg / mL; using HRP-labeled monoclonal antibody 2D6 as the labeling antibody, and diluting it 2000 times before use.

[0075] 6.4 Sensitivity and Specificity Tests of Double-Antibody Sandwich ELISA

[0076] The optimal coating concentration of 2 μg / mL and the HRP-labeled monoclonal antibody dilution of 2000-fold were determined. Following the above detection steps, the fNGAL recombinant protein was first serially diluted with PBS buffer to concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL. Simultaneously, two recombinant proteins, fCys-C and fNT-proBNP, were added to each well at the same concentration (50 μL) for detection. The sensitivity and specificity of this detection system for recombinant proteins were then determined. Figure 2 We can obtain, Figure 2In this study, fCys-C, fNT-proBNP, and fNGAL represent the recombinant proteins fCys-C, fNT-proBNP, and fNGAL, respectively. The double-antibody sandwich ELISA assay using these paired antibodies showed a positive reaction for the fNGAL recombinant protein at a 1 ng / ml dilution, and exhibited no reaction with irrelevant antigens, demonstrating good sensitivity and specificity. This is of significant importance for diagnosing chronic kidney injury in cats. Both fCys-C and fNT-proBNP recombinant protein genes were synthesized and expressed by Qingke Biotechnology.

[0077] 7. Identification of binding activity of paired monoclonal antibodies

[0078] Following the aforementioned indirect ELISA method, serial dilutions of the two monoclonal antibodies (10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 0.1 ng / ml) were performed. Another murine-derived unrelated monoclonal antibody, fNT-proBNP monoclonal antibody M101706M (Nanjing Fuxiao Biotechnology), was used as a negative control. The binding activity of the fNGAL monoclonal antibody was measured. Results are as follows... Figure 3 The diagram shows that Ctrl represents the fNT-proBNP monoclonal antibody, 2D6 represents monoclonal antibody 2D6, and 1E2 represents monoclonal antibody 1E2. Monoclonal antibodies 2D6 and 1E2 showed specific reactions with the recombinant fNGAL antigen at concentrations between 1-10 ng / ml, but did not react with the control monoclonal antibody, indicating that the paired monoclonal antibodies possess high binding activity.

[0079] 8. Cloning and sequencing of antibody variable region genes

[0080] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after incubation at 37°C for 14 h. The sequenced sequences are shown below:

[0081] Monoclonal antibody 2D6:

[0082] Heavy chain:

[0083] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.19:

[0084] GAGGTGAAGCTGGAGGAGAGCGGCAGCGTGCTGGTGAGGCCCGGCGGCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCAGGTGGATGCACTGGGCCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGGTGCTGCCCACCAGCGGCACCGACAA CTACAACGAGAAGTTCAGGGGCAAGGCCACCCTGACCGTGGACACCTTCAGCAGCACCGCCTACGTGGACCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAACGACGTGAGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0085] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.15: EVKLEESGSVLVRPGGSVKLSCKASGYTFTSRWMHWAKQRPGQGLEWIGEVLPTSGTDNYNEKFRGKATLTVDTFSSTAYVDLSSLTSEDSAVYYCARNDVSAMDYWGQGTSVTVSS.

[0086] CDR area annotation:

[0087] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.7: SRWMH;

[0088] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.8: EVLPTSGTDNYNEKFRG;

[0089] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.9: NDVSAMDY.

[0090] Light chain:

[0091] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.20:

[0092] GACATCCAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATCACCTGCAGCAGCGGCGGCAGCCTGAACTACATCCTGTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCGACAGCAACTACTGGAG CGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCTACGGCACCAGCTACAGCCTGACCATCGGCACCATGGAGGCCGAGGACGTGGCCACCTACTACTGCCAGCACGGCAGCAGGTTCCAGAGGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0093] The amino acid sequence of the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.16: DIQMTQSPAIMSASPGEKVTITCSSGGSLNYILWFQQKPGTSPKLWIYSDSNYWSGVPARFSGSGYGTSYSLTIGTMEAEDVATYYCQHGSRFQRTFGAGTKLELKRTV.

[0094] CDR area annotation:

[0095] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain of monoclonal antibody 2D6 is shown in SEQ ID NO.10: SSGGSLNYIL;

[0096] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain of monoclonal antibody 2D6 is shown in SEQ ID NO.11: SDSNYWS;

[0097] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain of monoclonal antibody 2D6 is shown in SEQ ID NO.12: QHGSRFQRT.

[0098] Monoclonal antibody 1E2:

[0099] Heavy chain:

[0100] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.17: GAGATGCAGCTGGAGGAGAGCGGCCCCGTGCTGGTGAGGCCCGCCGGCAGCGTGAAGCTGAGCGAGAAGGCCAGCGGCTACACCTTCACCAGCAGGTGGAACTACTGGGCCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGGTGCTGAAGGTGAGCAGCACCGACAACTACAACGAGAAGTTCAGCGGCAAGGCCACCCTGACCGTGGACACCTTCAGCAGCACCGCCTACGTGGACCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGTGGGACGTGAGCGCCAGCCTGTACTGGGGACGTGAGCGCCAGCCTGTACTGGGCCAGGGCACCAGCGTGACCGTGAGCGCC.

[0101] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.13: EMQLEESGPVLVRPAGSVKLSEKASGYTFTSRWNYWAKQRPGQGLEWIGEVLKVSSTDNYNEKFSGKATLTVDTFSSTAYVDLSSLTSEDSAVYYCARWDVSASLYWGQGTSVTVSA.

[0102] CDR area annotation:

[0103] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.1: SRWNY;

[0104] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.2: EVLKVSSTDNYNEKFSG;

[0105] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.3: WDVSASLY.

[0106] Light chain:

[0107] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.18: AACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGGGCGGCCAGACCCTGGTGATCAACAACAGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGGTACAGCACCAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCCCAGCACCGAGGTGCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0108] The amino acid sequence of the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.14: NIVMTQSPLSLPVSLGDQASISCRGGQTLVINNSNTYLHWYLQKPGQSPKLLIYRYSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSPSTEVPFTFGSGTKLEIKRTV.

[0109] CDR area labeling;

[0110] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain of monoclonal antibody 1E2 is shown in SEQ ID NO.4: RGGQTLVINNSNTYLH;

[0111] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain of monoclonal antibody 1E2 is shown in SEQ ID NO.5: RYSTRFS;

[0112] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain of monoclonal antibody 1E2 is shown in SEQ ID NO.6: SPSTEVPFT.

[0113] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0114] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination for detecting feline NGAL protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 1E2 and monoclonal antibody 2D6; The heavy chain variable region of the monoclonal antibody 1E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 1E2 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 2D6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 2D6 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting feline NGAL protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1E2 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting feline NGAL protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2D6 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting feline NGAL protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1E2 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1E2 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting feline NGAL protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D6 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D6 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting feline NGAL protein according to claim 5, characterized in that, The monoclonal antibody combination specifically recognizes recombinant fNGAL protein and feline NGAL protein.

7. The use of a combination of monoclonal antibodies based on claim 1 in the preparation of a tool for detecting feline NGAL protein.

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

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

10. The application according to claim 9, characterized in that, The ELISA kit uses monoclonal antibody 1E2 as the coating antibody and monoclonal antibody 2D6 as the labeling antibody.

Images