Monoclonal antibody combination for detecting cat NGAL protein and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a monoclonal antibody combination for detecting feline NGAL protein and its application. Background Art

[0002] Feline Chronic Kidney Disease (CKD) is a common and irreversible disease in the feline population. Due to its slow and progressive development, early diagnosis and accurate assessment of renal function status are crucial for disease management and treatment.

[0003] Currently, the clinical assessment of feline renal function mainly relies on indicators such as Glomerular Filtration Rate (GFR), serum urea nitrogen, and creatinine concentration. Among them, GFR is considered the "gold standard" for evaluating renal function, but due to the special equipment and strict sampling conditions required in its measurement process, there are significant difficulties in actual clinical applications. Although serum urea nitrogen and creatinine are commonly used detection indicators, they are less sensitive in reflecting early kidney damage and are easily affected by non-renal factors (such as diet, muscle mass, etc.), making it difficult to accurately reflect the true renal function status.

[0004] In recent years, with the development of molecular biology and biomarker research, Neutrophil Gelatinase-Associated Lipocalin (NGAL) has been found to be closely related to various kidney injuries. NGAL is a glycoprotein with a molecular weight of approximately 25 kDa, which is not only upregulated during the inflammatory response but also significantly increased when epithelial cells are damaged. Existing studies have shown that the concentrations of NGAL in blood and urine can effectively reflect the degree of kidney injury. Although NGAL shows good application prospects in the diagnosis of feline chronic kidney disease, there is currently no efficient, sensitive, and highly specific detection method. Existing immunological detection methods generally suffer from problems such as insufficient sensitivity and severe cross-reactivity, which limit their wide application in clinical practice. Summary of the Invention

[0005] In view of the lack of an efficient, sensitive, and highly specific detection method for feline NGAL protein in the prior art, the present invention provides a monoclonal antibody combination for detecting feline NGAL protein and its application, which solves the problems of low sensitivity and severe cross-reactivity of existing immunological detection methods.

[0006] To achieve the above object, the main technical solutions adopted by the present invention include: In a first aspect, the present invention provides a monoclonal antibody combination for detecting feline NGAL protein, and the monoclonal antibody combination includes monoclonal antibody 1E2 and monoclonal antibody 2D6; The heavy chain variable region of monoclonal antibody 1E2 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively as shown in SEQ ID NO.1-SEQ ID NO.3; The light chain variable region of monoclonal antibody 1E2 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively as shown in SEQ ID NO.4-SEQ ID NO.6; The heavy chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively as shown in SEQ ID NO.7-SEQ ID NO.9; The light chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively as shown in SEQ ID NO.10-SEQ ID NO.12.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1E2 is as shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 1E2 is as shown in SEQ ID NO.14.

[0008] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 2D6 is as shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 2D6 is as shown in SEQ ID NO.16.

[0009] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E2 is as shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E2 is as shown in SEQ ID NO.18.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2D6 is as shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2D6 is as shown in SEQ ID NO.20.

[0011] In some embodiments, the monoclonal antibody combination specifically recognizes fNGAL recombinant protein and feline NGAL protein.

[0012] In a second aspect, the present invention provides the application of the above monoclonal antibody combination in the preparation of a tool for detecting feline NGAL protein.

[0013] In some embodiments, the tool includes reagents, reagent kits, test strips and antibody chips.

[0014] In some embodiments, the kit includes a double antibody sandwich ELISA kit.

[0015] In some embodiments, the ELISA kit uses monoclonal antibody 1E2 as the coating antibody and monoclonal antibody 2D6 as the labeled antibody.

[0016] In addition, the double antibody sandwich ELISA detection method is not used for disease diagnosis and treatment.

[0017] Beneficial effects: The monoclonal antibody combination for detecting feline NGAL protein provided by the present invention (including monoclonal antibodies 1E2 and 2D6) has high specificity and sensitivity. This antibody combination is designed and screened through clear complementarity determining region (CDR) sequences, and can efficiently recognize feline-derived NGAL protein (i.e., feline NGAL protein), avoiding cross-reaction with other feline-derived proteins. Among them, monoclonal antibody 1E2 and monoclonal antibody 2D6 target different antigenic epitopes of the NGAL recombinant protein respectively, and are suitable for constructing detection systems such as double antibody sandwich ELISA, significantly improving the stability and accuracy of the detection signal.

[0018] The detection method established based on this antibody combination has good sensitivity, with a minimum detection limit of up to 1 ng / mL, and can be widely applied to the detection of various sample types such as serum, urine, and tissue fluid. It is expected to be developed and applied on various platforms such as diagnostic kits, test strips, and antibody chips, providing a reliable tool for the early diagnosis and disease monitoring of feline chronic kidney disease. Brief description of the drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is a SDS-PAGE identification result diagram for protein purification; Figure 2 It is a sensitivity and specificity detection diagram for the double antibody sandwich ELISA method; Figure 3 It is a binding activity identification diagram for paired monoclonal antibodies. Detailed implementation manners

[0021] The following further describes in detail the implementation manners of the present application in conjunction with the accompanying drawings and embodiments. The detailed descriptions and drawings of the following embodiments are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application. The present application can be implemented in many different forms, not limited to the specific embodiments disclosed in the text, but including all technical solutions falling within the scope of the claims.

[0022] These embodiments of the present application are provided to make the present application thorough and complete, and to fully convey the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, the compositions of materials, numerical expressions and values described in these embodiments should be interpreted as merely exemplary, rather than as limitations.

[0023] Example 1 1. Recombinant protein gene synthesis The gene of feline NGAL protein (downloaded from the NCBI database) was synthesized by Tsingke Biotechnology and cloned into the pET32a vector. The corresponding nucleotide sequence of the feline NGAL protein gene is shown in SEQ ID NO.21: caggattccaccccaaacctgatcccagccccgcctctgctcttggtccctgtggagcctgacttccagaatgagcagttccaggggaaatggtacttcttaggattggcagggaacggattcaataaagaaaagcacaggaggatgaagatgtacattgccaactacgagctgaacgaagacaacagctacaatgtcacctctactgtggcctggaaccagacctgtcatccctcgaccaaaattttcctcccaaatttgcatctaggccaattcaacctgggcaacattgagcgttacactggaatccagaactacacttcaaaagtggtgaccacagactacaaccagtttgccatactgtacttcaagaaagttcatgacaaccaggagtacatcaaggtcatcctctatgggaggaccaaggaggtgccttctgtaccgaaggcaatcttcatcagcttcatcaaatccctgggcctcaccgacgaccacatcatcttccctatccccaatgatgagtgcatggataag.

[0024] The amino acid sequence corresponding to the cat NGAL protein gene is shown in SEQ ID NO.22: QDSTPNLIPAPPLLLVPVEPDFQNEQFQGKWYFLGLAGNGFNKEKHRRMKMYIANYELNEDNSYNVTSTVAWNQTCHPSTKIFLPNLHLGQFNLGNIERYTGIQNYTSKVVTTDYNQFAILYFKKVHDNQEYIKVILYGRTKEVPSVPKAIFISFIKSLGLTDDHIIFPIPNDECMDK。

[0025] Design seamless cloning primers for the fNGAL of the pCold-TF vector. Using the synthetic plasmid pET32a-fNGAL as a template, amplify the fNGAL fragment. Connect the fragment and the vector according to the kit instructions (In-Fusion HD Cloning kits, TaKaRa), and transform the BL21(DE3) competent cells by the conventional method (Molecular Cloning, Third Edition, Science Press). Spread the transformed bacteria on an LB agar plate (containing 100 μg / mL ampicillin) and culture overnight at 37°C. Pick a single colony and inoculate it into 5 mL of LB medium (containing 100 μg / mL ampicillin), and culture it with shaking at 37°C and 220 rpm overnight. Inoculate it into LB medium (containing 100 μg / mL ampicillin) at a volume of 1% of the total volume of the medium, and culture it with shaking at 37°C and 220 rpm for about 3 hours until the OD 600 reaches 0.5. Cool it down to 16°C for 1.5 hours, add IPTG with a final concentration of 0.1 mM, and induce it at 16°C and 180 rpm for 16 hours, then collect the bacteria.

[0026] Cat NGAL protein generally refers to the protein naturally present in cats. The expression level of this protein will increase 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 response. The cat NGAL recombinant protein (fNGAL recombinant protein) is a NGAL protein synthesized in an in vitro expression system through genetic engineering technology. It is a recombinant protein obtained by cloning the gene of cat NGAL into an appropriate expression vector and expressing it in a host system. The recombinant protein has the same amino acid sequence as the cat NGAL protein, and after purification, it can be used for scientific research experiments, diagnostic reagent development, or functional studies.

[0027] 2. Purification and concentration identification of the recombinant protein Since the expressed recombinant fNGAL protein all carried a histidine tag, it was purified using an AKTA Start and HisTrapTM HP affinity chromatography column from GE Healthcare. Buffer A was 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B was 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with buffer A. Then, the fermented bacterial solution was centrifuged at 8000 rpm for 20 min, the precipitate was resuspended with buffer A, ultrasonically disrupted in ice water for 30 min (ultrasonic for 5 s every 5 s interval), centrifuged at 12000 rpm for 30 min, the supernatant was filtered through a 0.22-μm filter, loaded onto the column, the column was washed with buffer A, and finally eluted with a gradient of buffer B. The purification was observed by SDS-PAGE protein gel electrophoresis. The target protein peak was collected and dialyzed into a buffer of 50 mM PB, 300 mM NaCl, pH 8.0. The concentration of the recombinant fNGAL protein was measured by Nanodrop, aliquoted into 1 ml / tube, and stored at -20 °C.

[0028] 3. Identification of the recombinant protein In the present invention, SDS-PAGE electrophoresis was used to identify the protein purity.

[0029] Protein sample pretreatment: Samples were taken and an equal volume of 2x SDS loading buffer was added, then placed in a boiling water bath for 10 min and centrifuged at 12000 rpm for 3 min. The 5x glycine buffer was diluted to the working concentration, added to the electrophoresis tank to the appropriate liquid level, and the comb was gently removed from the solidified gel. Protein marker and 10 μl of the pretreated protein sample were added to the sample wells. The power supply was turned on, the voltage was adjusted to 80 V for constant voltage electrophoresis until the separating gel, and then changed to 120 V until the bromophenol blue reached the bottom of the gel. The gel was cut from the glass plate, placed in Coomassie Brilliant Blue staining solution, and stained with shaking for 4 h, and then decolorized with decolorizing solution until the bands were clear.

[0030] The results are shown in Figure 1 , Figure 1 where M: protein marker, No. 1: before induction, No. 2: precipitate, No. 3: supernatant, No. 4: flow-through, No. 5: 100 mM elution -1, No. 6: 100 mM elution -2, No. 7: 100 mM elution -3, No. 8: 500 mM elution -1, No. 9: 500 mM elution -2. An obvious main band was visible between 70 - 95 kDa, and the protein purity was approximately above 85%, which was consistent with the predicted antigen size (74.4 kDa). The purified recombinant fNGAL protein could be used for further downstream experiments.

[0031] 4. Mouse immunization The fNGAL recombinant protein was mixed with an equal volume of MF59 adjuvant (200 μl) and then intramuscularly injected into 6-week-old female BALB / c mice. The immunization was repeated once at 2 weeks and 4 weeks according to the above method. At the 5th week, mouse serum was taken to detect the antibody titer using the recombinantly expressed fNGAL protein. The mice with the highest titers were boosted by intravenous injection of 20 μg of the fNGAL recombinant protein through the tail vein. Three days later, the spleens of the mice were taken for the preparation of hybridoma cells.

[0032] 5. Screening, preparation and antibody purification of hybridoma cell lines 5.1 Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then placed in HAT medium for screening culture. When the fused cells grew to half of the well bottom, positive clones for the fNGAL recombinant protein were screened by the indirect ELISA method. Then, the positive cells were cloned to the monoclonal state by the limiting dilution method, and the cell lines were further expanded and cryopreserved.

[0033] Screening of positive clones by the indirect ELISA method: By the indirect ELISA method, hybridoma cell lines that could secrete antibodies specifically recognizing the fNGAL recombinant protein were selected after cell fusion.

[0034] The fNGAL recombinant protein and the control antigen fCys-C were respectively coated in microtiter plates (coating buffer: carbonate buffer, 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentration was 1 μg / mL. Incubate overnight at 4°C; block with 1% gelatin, 150 μl per well, block at 37°C for 2 hours, wash the plate once with the washing solution, and pat dry; add 50 μL of cell culture supernatant, react at 37°C for 30 min. Discard the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, and then add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS), react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB chromogenic solution, develop color at room temperature for 10 min, and finally add 0.5 M sulfuric acid to terminate the reaction. Measure the OD 450 nm value. Positive cell lines that reacted only with the fNGAL recombinant protein and not with the control antigen fCys-C were selected for subsequent experiments.

[0035] The screening process is shown in Table 1.

[0036] Table 1: Indirect ELISA reaction results of hybridoma cell line supernatants against the fNGAL recombinant protein and the control antigen.

[0037]

[0038] As can be seen from the indirect ELISA screening results shown in the above table, monoclonal antibodies 1E2, 1A8, and 2D6 showed strong reactivity in recognizing the fNGAL recombinant protein, with OD450nm values of 1.738, 1.276, and 1.696 respectively, significantly higher than those of other monoclonal antibodies, and none of them cross-reacted with the control antigen fCys-C, showing good specificity.

[0039] Given that the detection results of multiple groups of monoclonal antibodies are not ideal at present (not shown), considering the reaction intensity and specificity comprehensively, three clones, 1E2, 1A8, and 2D6, were preferentially selected for subsequent experiments.

[0040] 5.2 Preparation of Monoclonal Antibody Ascites After the screened monoclonal cell lines were expanded in culture, 0.2 ml (containing 2.5×10 6 cells) of female BALB / c mice pretreated with Freund's incomplete adjuvant were injected intraperitoneally. After about 10 days, when the abdomen of the mice was significantly swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 r / min for 10 minutes, and the middle layer was collected.

[0041] 5.3 Affinity Chromatography Purification of Monoclonal Antibodies (Protein G) The ascites was centrifuged at 12000 r / min for 5 minutes, and the supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4), filtered through a 0.22-μm filter, and the filtered sample was slowly pumped into a Protein G (Cytiva) purification column equilibrated with binding buffer using a peristaltic pump. The AKTA purifier was connected, and the column was washed with binding buffer for 5 - 10 column volumes until the UV absorption peak was washed flat, and 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, loaded into a dialysis bag (MW: 8000 - 14000), and dialyzed in 0.01 M PBS, pH 7.4 solution 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, and the concentration was measured using an ultra-micro spectrophotometer and stored in aliquots.

[0042] 6 Establishment and Condition Optimization of Double Antibody Sandwich ELISA 6.1 HRP-Labeled Monoclonal Antibodies The obtained monoclonal antibodies 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 a 0.01M PBS buffer at pH 7.4, and glycerol was added in a 1:1 volume ratio. They were aliquoted and stored at -20°C.

[0043] Specifically: Dilute the antibody to be labeled with the coupling buffer (provided in the kit) to a final concentration of 2 mg / mL. Subsequently, add the diluted antibody solution to the tube containing HRP (provided in the kit), and pipette to mix well. Incubate at room temperature for 1 h, and mix regularly during the incubation. Add 50 μL of the termination solution, and mix for 15 min to terminate the labeling reaction. Dialyze overnight in PBS buffer and add an equal volume of glycerol for storage.

[0044] 6.2 Establishment of the double-antibody sandwich method Dilute the purified monoclonal antibodies to a concentration of 1 μg / mL with the coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L with pure water, pH 9.6), and add 50 μL per well to the ELISA plate. Incubate overnight at 4°C. The next day, discard the coating solution, wash the plate once with the washing solution (PBST, PBS containing 0.05% Tween-20), pat dry, and block with 1% BSA, 150 μL per well. Incubate at 37°C for 2 h, discard the blocking solution, and pat dry. Dilute the antigen to be detected (fNGAL recombinant protein) and the control antigen (fCys-C recombinant protein) to 100 ng / ml with PBS and add 50 μL per well to the ELISA plate. Incubate at 37°C for 35 min, wash the plate 4 times with PBST washing solution, pat dry, add the enzyme-labeled monoclonal antibody diluted 1000-fold with PBS, 50 μL per well. Incubate at 37°C for 35 min, wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution per well, and develop color at room temperature for 10 min. Finally, add 50 μL of 0.5M sulfuric acid per well to terminate the reaction, and measure the OD 450 nm value. Select the paired monoclonal antibodies with the largest P / N value. The results showed that monoclonal antibody 1E2 as the coating antibody and monoclonal antibody 2D6 as the labeled antibody had the largest P / N value, and this paired monoclonal antibody was further tested. The screening process is shown in Table 2.

[0045] Table 2: Comparison of the reactivity of different monoclonal antibody combinations to fNGAL recombinant protein and control antigen in double-antibody sandwich ELISA.

[0046]

[0047] "*" indicates the dilution factor.

[0048] 6.3 Optimization of the double-antibody sandwich ELISA method The purified monoclonal antibody 1E2 was diluted with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L with pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL per well, and coated overnight at 4°C. The next day, the coating solution was discarded, and blocked with 1% BSA, 150 μL per well, incubated at 37°C for 2 h. The blocking solution was discarded. The antigen to be detected (fNGAL recombinant protein) and the control antigen (fCys-C recombinant protein) were diluted with PBS at 100 ng / ml and added to the enzyme-linked immunosorbent assay (ELISA) plate, 50 μL per well, incubated at 37°C for 35 min. The plate was washed 4 times with PBST washing solution, and HRP-labeled monoclonal antibody 2D6 diluted 1000, 2000, and 3000 times with PBS was added, 50 μL per well, incubated at 37°C for 35 min. Then the plate was washed 4 times again, patted dry, and 50 μL of TMB chromogenic solution was added per well, developed at room temperature for 10 min. Finally, 50 μL of 0.5 M sulfuric acid was added per well to terminate the reaction, and the optical density (OD) 450 nm value was measured with an ELISA reader. The pairing conditions with the largest P / N value were selected for sensitivity and specificity tests. The screening process is shown in Table 3.

[0049] Table 3: Optimization of the best coating and labeling conditions for sandwich ELISA.

[0050]

[0051] The optimal reaction conditions were as follows: monoclonal antibody 1E2 was used as the coating antibody at a coating concentration of 2 μg / mL; HRP-labeled monoclonal antibody 2D6 was used as the labeling antibody and diluted 2000 times before use.

[0052] 6.4 Sensitivity and specificity tests of the double-antibody sandwich ELISA The optimal coating concentration of 2 μg / ml, the dilution factor of 2000 times for HRP-labeled monoclonal antibody, and other reaction conditions were determined. Referring to the above detection steps, the fNGAL recombinant protein was first serially diluted with PBS buffer solution, and the diluted concentrations were 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL. At the same time, two recombinant proteins, fCys-C and fNT-proBNP, were taken and added at the same concentration of 50 μL per well for detection to determine the detection sensitivity and specificity of the detection system for recombinant proteins. According to Figure 2 It can be obtained that Figure 2fCys-C, fNT-proBNP, and fNGAL represent recombinant fCys-C, fNT-proBNP, and fNGAL proteins, respectively. This double-antibody sandwich ELISA, comprised of paired antibodies, demonstrates a positive reaction at a dilution of 1 ng / ml for recombinant fNGAL protein and is unreactive with unrelated antigens. This assay demonstrates excellent sensitivity and specificity, making it a valuable biomarker for the diagnosis of chronic kidney injury in cats. Both the fCys-C and fNT-proBNP recombinant protein genes are synthesized and expressed by Qingke Biotechnology.

[0053] 7. Identification of binding activity of paired monoclonal antibodies Referring to the aforementioned indirect ELISA method, the two monoclonal antibodies were serially diluted at 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml, 1ng / ml, and 0.1ng / ml, and the binding activity of the fNGAL monoclonal antibody was determined using another unrelated mouse monoclonal antibody, fNT-proBNP monoclonal antibody M101706M (Nanjing Fuxiao Biotechnology), as a negative control. The results are shown in Figure 2. Figure 3 The following table shows the control panel (Ctrl), which represents the fNT-proBNP monoclonal antibody, 2D6 (2D6), and 1E2 (1E2). Both monoclonal antibodies 2D6 and 1E2 specifically reacted with the fNGAL recombinant protein at concentrations between 1 and 10 ng / ml and did not react with the control monoclonal antibody, demonstrating that this monoclonal antibody pairing has high binding activity.

[0054] 8. Antibody variable region gene cloning and sequencing Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104) and reverse transcribed using Random Primers to synthesize cDNA. 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 product was gel-cleaved and ligated into the pUC19 vector. The TOP10 strain was transformed and cultured at 37°C for 14 hours. Single colonies were picked and sequenced. The sequenced sequences are shown below: Monoclonal antibody 2D6: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.19: GAGGTGAAGCTGGAGGAGAGCGGCAGCGTGCTGGTGAGGCCCGGCGGCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCAGGTGGATGCACTGGGCCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGGTGCTGCCCACCAGCGGCACCGACAACTACAACGAGAAGTTCAGGGGCAAGGCCACCCTGACCGTGGACACCTTCAGCAGCACCGCCTACGTGGACCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAACGACGTGAGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC。

[0055] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.15: EVKLEESGSVLVRPGGSVKLSCKASGYTFTSRWMHWAKQRPGQGLEWIGEVLPTSGTDNYNEKFRGKATLTVDTFSSTAYVDLSSLTSEDSAVYYCARNDVSAMDYWGQGTSVTVSS。

[0056] CDR region annotation: 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; 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; 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.

[0057] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.20: GACATCCAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATCACCTGCAGCAGCGGCGGCAGCCTGAACTACATCCTGTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCGACAGCAACTACTGGAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCTACGGCACCAGCTACAGCCTGACCATCGGCACCATGGAGGCCGAGGACGTGGCCACCTACTACTGCCAGCACGGCAGCAGGTTCCAGAGGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0058] The amino acid sequence of the light chain variable region of monoclonal antibody 2D6 is shown in SEQ ID NO.16: DIQMTQSPAIMSASPGEKVTITCSSGGSLNYILWFQQKPGTSPKLWIYSDSNYWSGVPARFSGSGYGTSYSLTIGTMEAEDVATYYCQHGSRFQRTFGAGTKLELKRTV。

[0059] CDR region annotation: 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; 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; 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。

[0060] Monoclonal antibody 1E2: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.17: GAGATGCAGCTGGAGGAGAGCGGCCCCGTGCTGGTGAGGCCCGCCGGCAGCGTGAAGCTGAGCGAGAAGGCCAGCGGCTACACCTTCACCAGCAGGTGGAACTACTGGGCCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGGTGCTGAAGGTGAGCAGCACCGACAACTACAACGAGAAGTTCAGCGGCAAGGCCACCCTGACCGTGGACACCTTCAGCAGCACCGCCTACGTGGACCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGTGGGACGTGAGCGCCAGCCTGTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCGCC。

[0061] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.13: EMQLEESGPVLVRPAGSVKLSEKASGYTFTSRWNYWAKQRPGQGLEWIGEVLKVSSTDNYNEKFSGKATLTVDTFSSTAYVDLSSLTSEDSAVYYCARWDVSASLYWGQGTSVTVSA。

[0062] CDR region annotation: 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; 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; 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.

[0063] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.18: AACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGGGCGGCCAGACCCTGGTGATCAACAACAGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGGTACAGCACCAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCCCAGCACCGAGGTGCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0064] The amino acid sequence of the light chain variable region of monoclonal antibody 1E2 is shown in SEQ ID NO.14: NIVMTQSPLSLPVSLGDQASISCRGGQTLVINNSNTYLHWYLQKPGQSPKLLIYRYSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSPSTEVPFTFGSGTKLEIKRTV。

[0065] CDR region annotation; 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; 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; 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.

[0066] So far, the embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well 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.

[0067] Although some specific embodiments of the present 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 not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or some technical features can be equivalently replaced without departing from the scope and spirit of the present 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 monoclonal antibody 1E2 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown as SEQ ID NO.1-SEQ ID NO.3 respectively; The light chain variable region of monoclonal antibody 1E2 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown as SEQ ID NO.4-SEQ ID NO.6 respectively; The heavy chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown as SEQ ID NO.7-SEQ ID NO.9 respectively; The light chain variable region of monoclonal antibody 2D6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown as SEQ ID NO.10-SEQ ID NO.12 respectively.

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

14.

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

16.

4. 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 monoclonal antibody 1E2 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1E2 is shown as SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting feline NGAL protein according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2D6 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2D6 is shown as SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting feline NGAL protein according to claim 5, wherein The monoclonal antibody combination specifically recognizes the fNGAL recombinant protein and the cat NGAL protein.

7. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting cat NGAL protein.

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

9. The application according to claim 8, wherein 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 labeled antibody.

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