Monoclonal antibody combination for detecting cat Cys-C protein and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting cat Cys-C protein and application. The monoclonal antibody combination provided by the invention comprises monoclonal antibodies 4D5 and 3F3, and amino acid sequences of complementary determining regions (CDR) of variable regions of a heavy chain and a light chain of the monoclonal antibodies 4D5 and 3F3 are respectively shown as SEQ ID NO.1 to SEQ ID NO.12. The antibody combination has high specificity and sensitivity, and can effectively recognize cat Cys-C recombinant protein and natural cat Cys-C protein. According to the double-antibody sandwich ELISA detection method constructed based on the antibody combination, positive signals can still be generated under the low concentration of 1ng / mL, no obvious cross reaction exists, and the detection accuracy is remarkably improved. The antibody combination can be used for preparing a detection kit, a test strip, an antibody chip and other tools, and is suitable for cat kidney function evaluation and early auxiliary diagnosis of kidney diseases.

Description

Technical Field

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

[0002] In recent years, with the continuous increase in the number of dogs and cats raised, the incidence of kidney diseases in pet clinics has also shown an upward trend, often accompanied by a relatively high mortality rate. Research has shown that the occurrence of chronic renal failure in dogs and cats is closely related to various factors such as age, genetics, underlying diseases, diet, and living environment, and is usually the result of the combined action of multiple pathogenic factors. Many diseased animals, if not promptly diagnosed and treated effectively after acute nephritis, are extremely likely to cause irreversible kidney damage, and then develop into chronic renal failure, ultimately endangering life.

[0003] Early and accurate assessment of the renal function status of cats is of great significance for disease prevention and intervention. Cystatin C is a non-glycosylated basic protein belonging to the cysteine protease inhibitor family, with a molecular weight of approximately 13 kDa. Its structure is formed by multiple amino acid residues connected by peptide bonds to form a specific three-dimensional conformation, enabling it to specifically inhibit the activity of cysteine proteases. These proteases are involved in various physiological and pathological processes including apoptosis and protein degradation, and cystatin C regulates their activity by binding to them, thereby maintaining the stability of the internal environment.

[0004] Especially in felines, feline cystatin C (also known as fCys-C or cat Cys-C), as an important biomarker reflecting glomerular filtration function, has received extensive attention due to its good stability in plasma. Its concentration change is not affected by factors such as inflammation, infection, tumor, or liver function, and can reflect renal function abnormalities earlier and more accurately. Therefore, cat Cys-C is widely used in the auxiliary diagnosis of kidney-related diseases such as acute kidney injury and chronic kidney disease in cats, and can also be used to evaluate the recovery of renal function after kidney transplantation, and has important application value in clinical practice.

[0005] However, currently, the clinical diagnosis of feline renal failure still relies on comprehensive assessment methods, including biochemical tests, urine analysis, imaging examinations, and pathological detections, etc. These methods are not only complex in process but also costly, bringing a relatively large economic pressure to pet owners. In addition, traditional detection methods still have deficiencies in terms of sensitivity and specificity, and it is difficult to meet the needs of early and rapid diagnosis. Therefore, developing a highly sensitive and specific detection method for feline cystatin C, especially an immunoassay technology based on monoclonal antibodies, will help improve the diagnostic efficiency and accuracy of feline kidney diseases, and has significant clinical application prospects. Summary of the Invention

[0006] Given the lack of efficient, sensitive and specific feline Cys-C protein detection methods in the prior art, the present invention provides a monoclonal antibody combination for detecting feline Cys-C protein and its application, which solves the problems of low sensitivity and severe cross-reaction of existing immunoassay methods.

[0007] In order to achieve the above objectives, 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 Cys-C protein, the monoclonal antibody combination comprising monoclonal antibody 4D5 and monoclonal antibody 3F3; The heavy chain variable region of monoclonal antibody 4D5 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of monoclonal antibody 4D5 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively; The heavy chain variable region of monoclonal antibody 3F3 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 9, respectively; The light chain variable region of monoclonal antibody 3F3 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.

[0008] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.14.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.16.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4D5 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4D5 is shown as SEQ ID NO.18.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3F3 is shown as SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3F3 is shown as SEQ ID NO. 20.

[0012] In some embodiments, the monoclonal antibody combination specifically recognizes feline Cys-C recombinant protein and feline Cys-C protein.

[0013] In a second aspect, the present invention provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting feline Cys-C protein.

[0014] In some embodiments, the tool includes reagents, kits, test strips, and antibody chips.

[0015] In some embodiments, the kit includes a double antibody sandwich ELISA kit.

[0016] In some embodiments, the ELISA kit uses monoclonal antibody 4D5 as the coating antibody and monoclonal antibody 3F3 as the labeled antibody.

[0017] In addition, the double antibody sandwich ELISA detection method is not used for disease diagnosis and treatment.

[0018] Beneficial effects: The present invention provides a monoclonal antibody combination with high specificity and strong sensitivity for feline Cys-C protein, including monoclonal antibodies 4D5 and 3F3. Through the design and optimization of the complementary determining regions (CDRs) of their heavy and light chain variable regions, the CDR amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.12 respectively. This antibody combination can efficiently recognize feline Cys-C recombinant protein and native feline Cys-C protein, and has excellent binding activity and detection specificity.

[0019] Based on this antibody combination, the double antibody sandwich ELISA detection method has high sensitivity, can still produce a positive reaction at a low concentration of 1 ng / mL, and has no cross-reaction with other irrelevant antigens, significantly improving the accuracy of the detection results. The present invention also provides the amino acid sequences of the relevant antibody variable regions and their encoding nucleotide sequences, laying a foundation for the genetic engineering expression and large-scale production of subsequent antibodies.

[0020] This antibody combination can not only be used to prepare tools such as kits, test strips, and antibody chips for detecting feline Cys-C protein, but also be widely applied to the evaluation of feline renal function, early auxiliary diagnosis of acute kidney injury and chronic kidney disease, and has good clinical application prospects. Compared with traditional detection methods, the immunoassay method provided by the present invention is simple to operate, has controllable costs, and rapid response, overcomes the problems of insufficient sensitivity and serious cross-reaction in the prior art, and provides an efficient diagnostic solution for veterinary clinics. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 SDS-PAGE identification result diagram for protein purification; Figure 2 Activity identification diagram of feline Cys-C recombinant protein; Figure 3 Double-antibody sandwich ELISA sensitivity and specificity test diagram; Figure 4 Binding activity identification diagram of paired monoclonal antibodies. Detailed implementation manners

[0023] The following further describes the implementation manners of the present application in detail in conjunction with the accompanying drawings and embodiments. The detailed description and the accompanying drawings of the following embodiments are used to exemplarily illustrate the principle 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.

[0024] 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 components of materials, numerical expressions and values described in these embodiments should be construed as merely exemplary, rather than as limitations.

[0025] Example 1 1. Recombinant protein gene synthesis The feline cystatin C protein gene (downloaded from the NCBI database) was codon-optimized and synthesized by Tsingke Biotechnology, and cloned into the pET32a vector. The nucleotide sequence corresponding to the feline cystatin C protein gene is shown in SEQ ID NO.21: ATGTGCGCACTGCCGATCACCTCTTACGGTAACCTGTGCGCTGTTGATCGTGAACACACTATGGCGGGTTCTCTGCGTACTCCATTGTTGCTGCTGGCTGCGGTTGCTCTGACTCTGGCACTGGCTATGAGTCCGGGTACTGGTCGTCGTAACAACAAATCTGCTCTGGTTGGTGCACCGCTGGATGCGGACGTGAACGAAGAAGGTGTACAGCAGGCTCTGAACTTCGCGCTGTCTGAATACAACAAGGCTTCTAACGACGCGTACCACTCTCGTGCGATGCGTGTTGTTCGTGCTCGTAAACAGGTGGTTGCTGGTATGAACTACTTTCTGGACGTGGAAATCGGTCGTACTCGTTGCACCAAATCTCAGCCGAACCTGGACACCTGTCCGTTCCATGACCAGCCGCACCTGATGCGTAAAACTCTGTGCTCTTTCCAAATCTACACCGTGCCGTGGATGGGTAAGACCTCTCTGGTTAAATCTTCTTGCCAAGATGCA。

[0026] The amino acid sequence corresponding to the cystatin C protein gene is shown in SEQ ID NO. 22: MCALPITSYGNLCAVDREHTMAGSLRTPLLLLAAVALTLALAMSPGTGRRNNKSALVGAPLDADVNEEGVQQALNFALSEYNKASNDAYHSRAMRVVRARKQVVAGMNYFLDVEIGRTRCTKSQPNLDTCPFHDQPHLMRKTLCSFQIYTVPWMGKTSLVKSSCQDA。

[0027] Design seamless cloning primers for the feline Cys-C gene of the pCold-TF vector. Using the synthetic plasmid pET32a-feline Cys-C as a template, amplify the feline Cys-C fragment. Connect the fragment with the vector according to the kit instructions (Seamless Cloning Kit In-Fusion HD Cloning kits, TaKaRa), and transform 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 with shaking at 37°C and 220 rpm overnight. Inoculate into LB medium (containing 100 μg / mL ampicillin) at 1% of the total volume of the medium, and culture with shaking at 37°C and 220 rpm for about 3 hours until OD 600 reaches 0.5. Cool down to 16°C for 1.5 hours, add IPTG with a final concentration of 0.1 mM, and induce at 16°C and 180 rpm for 16 hours, then collect the bacterial cells to obtain the recombinant feline Cys-C protein.

[0028] 2. Purification of the recombinant protein Since the expressed recombinant feline Cys-C protein all carries a histidine tag, use the AKTA purifier and HisTrap TM HP affinity chromatography column of GE Company for purification. Buffer A is 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B is 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. Equilibrate the chromatography column with buffer A. Then centrifuge the fermented bacterial solution at 8000 rpm for 20 min, resuspend the precipitate with buffer A, ultrasonically disrupt it in ice water for 30 min, ultrasonicate for 5 s every 5 s, centrifuge at 12000 rpm for 30 min, filter the supernatant through a 0.22-μm filter and load the sample. Wash the chromatography column with buffer A, and finally elute with a gradient of buffer B. Observe the purification situation by SDS-PAGE protein gel electrophoresis. Select the collection peak of the target protein, dialyze and change the buffer to buffer A and concentrate and collect it. Measure the protein concentration with Nanodrop, aliquot into 1 ml / tube, and store at -20°C.

[0029] 3. Identification of the recombinant protein Use SDS-PAGE electrophoresis to identify the protein purity.

[0030] 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. After shaking and staining for 4 hours, destain with destaining solution until the bands are clear.

[0031] See also Figure 1 , Figure 1 M in the middle: protein marker. Number 1: before induction, Number 2: precipitate, Number 3: supernatant, Number 4: 100 mM elution, Number 5: 500 mM elution. Soluble expression of the feline Cys-C recombinant protein was achieved after low-temperature induction at 16°C. Purification by Ni affinity chromatography with the elution peak collected at 500 mM imidazole was performed. SDS-PAGE revealed a single band around 70 kDa, consistent with the estimated size of the fusion protein (estimated molecular weight, 72.1 kDa), and a purity exceeding 85%.

[0032] 4. ELISA to identify the activity of recombinant protein The plate was coated with purified feline Cys-C recombinant protein (1 μg / ml), and its reaction with the commercially available feline Cys-C monoclonal antibody M102202M (Nanjing Fuxiao Biotechnology) was identified by indirect ELISA. An irrelevant monoclonal antibody, fNT-proBNP monoclonal antibody M101706M, was used as a control. First, the recombinant protein was coated in a microplate (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 plate was blocked with 1% BSA, 100 μl per well, at 37°C for 2 hours. The plate was 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 unrelated monoclonal antibody, fNT-proBNP monoclonal antibody M101706M, was used as a negative control. The reaction was carried out at 37°C for 30 minutes. The liquid in the wells was shaken off, 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. After patting dry, 50 μL / well of TMB color development solution was added and color was developed at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added to stop the reaction. The OD was measured with a microplate reader. 450nm value.

[0033] See Figure 2 , M102202M represents the commercially available monoclonal antibody M102202M against feline Cys-C, and Ctrl represents the monoclonal antibody M101706M against fNT-proBNP.

[0034] The indirect ELISA method was used to identify the reaction of the expressed recombinant feline Cys-C protein with the commercially available monoclonal antibody M102202M. The recombinant feline Cys-C protein (1 μg / ml) was coated. The results showed that the commercially available monoclonal antibody still had a weak positive reaction with the recombinant feline Cys-C protein at a dilution of 10 ng / ml, indicating that the recombinant feline Cys-C protein had good biological activity and could be used for the next experiment.

[0035] 5. Mouse immunization The recombinant feline Cys-C protein (20 μg) was mixed with an equal volume of MF59 adjuvant (total volume 200 μl) and intramuscularly injected into 6-week-old female BALB / c mice. Immunization was repeated once more at 2 weeks and 4 weeks according to the above method. At the 5th week, mouse serum was taken to detect the antibody titer with the expressed recombinant feline Cys-C protein. The mouse with the highest titer was boosted by intravenous injection of 20 μg of recombinant feline Cys-C protein through the tail vein. Three days later, the mouse spleen was taken for the preparation of hybridoma cells.

[0036] 6. Screening of hybridoma cell lines All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then cultured in HAT medium for screening. When the fused cells grew to 1 / 2 of the well bottom, positive clones against the recombinant feline Cys-C 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.

[0037] 7. Screening of positive clones by indirect ELISA The recombinant feline Cys-C protein (expressed by the pCold-TF vector and containing His tag) was coated in a microplate (coating buffer: carbonate buffer, 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), coating concentration 1 μg / mL, overnight at 4°C; blocked with 1% gelatin, 150 μL per well, blocked at 37°C for 2 hours, washed the plate once with the washing solution, patted dry; added 50 μL of cell culture supernatant, reacted at 37°C for 30 min. Discarded the liquid in the wells, washed the plate 4 times with PBST washing solution, patted dry and then added 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) per well, reacted at 37°C for 30 min, washed the plate 4 times again, patted dry and then added 50 μL of TMB chromogenic solution per well for color development at room temperature for 10 min, finally added 0.5 M sulfuric acid to terminate the reaction, and measured OD with an enzyme-linked immunosorbent assay instrument 450nm value. Select positive cell lines that react only with the recombinant feline Cys-C protein and not with other control antigens (recombinant fNGAL protein, synthesized by Tsingke Biotechnology) for subsequent experiments.

[0038] The screening process is shown in Table 1.

[0039] Table 1: Results of indirect ELISA of hybridoma cell lines against recombinant feline Cys-C protein and control antigens.

[0040]

[0041] Only the results with better data readings among the screened monoclonal antibodies are shown in Table 1. The data of the remaining groups are not shown because they are not ideal enough.

[0042] 8. 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.

[0043] 9. Affinity chromatography purification (Protein G) of monoclonal antibodies 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 5-10 column volumes of binding buffer 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 a micro-spectrophotometer and stored in aliquots.

[0044] 10. Pairing of double antibody sandwich ELISA 10.1. HRP-labeled monoclonal antibody The selected antibodies were labeled with HRP 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, pH 7.4 buffer, and then glycerol was added at a 1:1 volume ratio, and they were stored in aliquots at -20°C. Specifically: Dilute the antibody to be labeled with the coupling buffer (provided in the kit) to a final concentration of 2 mg / mL. Then 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, mix for 15 min to terminate the labeling reaction, dialyze overnight in PBS buffer, and add an equal volume of glycerol for storage.

[0045] 10.2 Establishment of the double-antibody sandwich method: Respectively dilute the purified monoclonal antibodies 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) to a concentration of 1 μg / mL, and add 50 μL / well to the enzyme-linked immunosorbent assay (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 / well. Incubate at 37°C for 2 h, discard the blocking solution, and pat dry. Dilute the antigen to be tested and the control antigen (fNGAL) with PBS to 50 ng / ml and then add 50 μL / well to the ELISA plate. Incubate at 37°C for 35 min, wash the plate 4 times with the PBST washing solution, pat dry, add the enzyme-labeled monoclonal antibody diluted 1000-fold with PBS, 50 μL / well. Incubate at 37°C for 35 min, wash the plate 4 times again, pat dry, then add 50 μL / well of the TMB chromogenic solution, develop color at room temperature for 10 min, and finally add 50 μL / well of 0.5M sulfuric acid to terminate the reaction. Measure the OD 450 nm value. Select the pair of monoclonal antibodies with the largest P / N value. The results showed that the coating antibody (4D5) and the labeled antibody (3F3) had the largest P / N value, and this pair of monoclonal antibodies was further tested.

[0046] The screening process is shown in Table 2.

[0047] Table 2: Comparison of the reactivity of different monoclonal antibody combinations to the fCys-C (feline Cys-C) recombinant protein and the control antigen in a double-antibody sandwich ELISA.

[0048]

[0049] In Table 2, 1000* indicates a 1000-fold dilution, fCys-C represents the feline Cys-C recombinant protein, and fNGAL represents the feline NGAL recombinant protein.

[0050] The antibodies were separately coated and labeled for one-to-one pairing to initially screen out the paired antibodies. Monoclonal antibody 4D5 was used as the coating antibody, and monoclonal antibody 3F3 was used as the HRP-labeled antibody.

[0051] 11. Optimization of the double-antibody sandwich method Purified monoclonal antibody 4D5 was added to the microwells at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL in 50 μL of 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) per well, and coated overnight at 4°C. The next day, the coating solution was discarded, and the wells were blocked with 1% BSA, 150 μL per well, incubated at 37°C for 2 h, and then the blocking solution was discarded. The antigen to be tested and the control antigen were diluted with PBS to 50 ng / ml and added to the enzyme-linked immunosorbent assay (ELISA) plate, 50 μL per well, incubated at 37°C for 35 min, washed 4 times with PBST washing solution, and then HRP-labeled monoclonal antibody diluted 1000-fold, 2000-fold, and 3000-fold with PBS was added, 50 μL per well, incubated at 37°C for 35 min, washed 4 times again, patted dry, and then 50 μL of TMB chromogenic solution was added per well, developed at room temperature for 10 min, and finally 50 μL of 0.5 M sulfuric acid was added per well to terminate the reaction. The optical density (OD) 450 nm value was measured using an enzyme-linked immunosorbent assay reader. The pairing conditions with the highest P / N value were selected for sensitivity and specificity testing. The screening process is shown in Table 3.

[0052] Table 3: Optimization of the best coating and labeling conditions for the double-antibody sandwich ELISA.

[0053]

[0054] The optimal reaction conditions were as follows: the coating antibody concentration and the dilution factor of the labeled antibody were determined according to the pairing conditions with the highest P / N value. The detection results of the double-antibody sandwich ELISA were optimal when the coating antibody concentration was 1 μg / ml and the HRP-labeled antibody was diluted 2000-fold.

[0055] 12. Sensitivity and specificity testing of the double-antibody sandwich After determining the optimal reaction conditions such as a coating concentration of 1 μg / ml and a 2000-fold dilution of the HRP-labeled monoclonal antibody, referring to the above detection steps, the recombinant feline Cys-C protein was 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, fNGAL and fNT-pro BNP, 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 the recombinant proteins.

[0056] According toFigure 3 It can be seen that the double-antibody sandwich ELISA composed of this group of paired antibodies still has a positive reaction when the recombinant cat Cys-C protein is diluted at 1 ng / ml, and does not react with irrelevant antigens, showing good sensitivity and specificity. It is of great significance for the early diagnosis of the renal function status of cats, the auxiliary diagnosis of acute kidney injury, chronic kidney disease and other kidney-related diseases in cats. Figure 3 Among them, fNGAL, fCys-C, and fNT-pro BNP respectively represent the recombinant cat NGAL protein, the recombinant cat Cys-C protein, and the recombinant cat NT-proBNP protein. The recombinant fNGAL protein and the recombinant fNT-pro BNP protein were both synthesized and expressed by Tsingke Biological.

[0057] Specifically: The cat NT-proBNP protein gene (downloaded from the NCBI database) was synthesized by Tsingke Biological and cloned into the pET32a vector. The nucleotide sequence corresponding to the cat NT-proBNP protein gene is shown in SEQ ID NO.23: CATCCGCTGGGTGGTCCAGGTCCAGCGTCTGAGGCTTCTGCGATCCAAGAACTGCTGGACGGTCTGCGTGACACTGTTTCTGAACTGCAAGAAGCGCAGATGGCACTGGGTCCGCTGCAGCAGGGTCACTCTCCGGCTGAAAGCTGGGAAGCTCAGGAAGAACCGCCAGCGCGTGTTCTGGCTCCGCATGACAACGTTCTGCGTGCTCTGCGT.

[0058] The amino acid sequence corresponding to the cat NT-proBNP protein gene is shown in SEQ ID NO.24: HPLGGPGPASEASAIQELLDGLRDTVSELQEAQMALGPLQQGHSPAESWEAQEEPPARVLAPHDNVLRALR.

[0059] The synthetic plasmid pET32a-cat NT-proBNP was transformed into BL21 (DE3) competent cells by the conventional method (Molecular Cloning, Third Edition, Science Press). The transformed bacteria were spread on an LB agar plate (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 overnight with shaking at 37°C and 220 rpm. It was inoculated into LB medium (containing 100 μg / mL ampicillin) at a volume of 1% of the total volume of the medium, and cultured with shaking at 37°C and 220 rpm for about 3 hours until OD 600It was 0.5, cooled at 16 °C for 1.5 hours, added IPTG with a final concentration of 0.1 mM, induced at 16 °C and 180 rpm for 16 hours, then the bacterial cells were collected for the next purification experiment.

[0060] The feline NGAL protein gene (downloaded from the NCBI database) was synthesized by Tsingke Biological and cloned into the pET32a vector. The nucleotide sequence corresponding to the feline NGAL protein gene is shown in SEQ ID NO.25: caggattccaccccaaacctgatcccagccccgcctctgctcttggtccctgtggagcctgacttccagaatgagcagttccaggggaaatggtacttcttaggattggcagggaacggattcaataaagaaaagcacaggaggatgaagatgtacattgccaactacgagctgaacgaagacaacagctacaatgtcacctctactgtggcctggaaccagacctgtcatccctcgaccaaaattttcctcccaaatttgcatctaggccaattcaacctgggcaacattgagcgttacactggaatccagaactacacttcaaaagtggtgaccacagactacaaccagtttgccatactgtacttcaagaaagttcatgacaaccaggagtacatcaaggtcatcctctatgggaggaccaaggaggtgccttctgtaccgaaggcaatcttcatcagcttcatcaaatccctgggcctcaccgacgaccacatcatcttccctatccccaatgatgagtgcatggataag。

[0061] The amino acid sequence corresponding to the feline NGAL protein gene is shown in SEQ ID NO.26: QDSTPNLIPAPPLLLVPVEPDFQNEQFQGKWYFLGLAGNGFNKEKHRRMKMYIANYELNEDNSYNVTSTVAWNQTCHPSTKIFLPNLHLGQFNLGNIERYTGIQNYTSKVVTTDYNQFAILYFKKVHDNQEYIKVILYGRTKEVPSVPKAIFISFIKSLGLTDDHIIFPIPNDECMDK。

[0062] Design seamless cloning primers for the pCold-TF vector fNGAL. Using the synthetic plasmid pET32a-fNGAL as a template, amplify the fNGAL fragment. Connect the fragment with the vector according to the kit instructions (Seamless Cloning Kit In-Fusion HD Cloning kits, TaKaRa), and transform 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 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, culture with shaking at 37°C and 220 rpm for about 3 hours until the OD600 is 0.5, cool down at 16°C for 1.5 hours, add IPTG with a final concentration of 0.1 mM, and induce at 16°C and 180 rpm for 16 hours, then collect the bacteria.

[0063] 13. Identification of the binding activity of paired monoclonal antibodies Refer to the aforementioned indirect ELISA method, serially dilute the two monoclonal antibodies 4D5 and 3F3 at concentrations of 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Use other murine monoclonal antibody fNT-proBNP monoclonal antibody M101706M as a negative control to determine the binding activity of the feline Cys-C monoclonal antibody.

[0064] The results are shown in Figure 4 , monoclonal antibodies 4D5 and 3F3 still had a binding reaction with the antigen feline Cys-C recombinant protein at a concentration of 10 ng / ml, and the control monoclonal antibody did not react with the target antigen feline Cys-C recombinant protein, indicating that this paired monoclonal antibody has a high binding activity. Figure 4 In [reference], 3F3 is monoclonal antibody 3F3, 4D5 is monoclonal antibody 4D5, and Ctrl is fNT-proBNP monoclonal antibody M101706M.

[0065] 14. Cloning and sequencing of antibody variable region genes Total RNA of hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription with RandomPrimers; universal primers for the variable regions of mouse antibodies were designed, and the VH and VL genes were amplified by 2 rounds of PCR. Restriction enzyme cleavage sites of Age1 and Bsiw1 were introduced into the primers of the 3rd round of PCR. After gel purification of the PCR products, they were ligated to the pUC19 vector, transformed into TOP10 strains, and single colonies were picked for sequencing after culturing at 37°C for 14 h to obtain the gene sequences of the heavy and light chains of the monoclonal antibody.

[0066] Variable region sequence of monoclonal antibody Monoclonal antibody 3F3 Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.19: CAGATGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGACCAGCGGCTACACCTTCACCAACGTGGACCTGAACTGGGTGAACCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCAGCATCAGCCCCGGCGCCGCCAGCAGGAAGTACAACGAGACCTTCAGGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGAACAGCGCCGTGTACTTCTGCGCCAGGGGCAGCGACTACGGCTACAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0067] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.15: QMQLQQSGPELVKPGASMKISCKTSGYTFTNVDLNWVNQRPGQGLEWIGSISPGAASRKYNETFRGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGSDYGYSYAMDYWGQGTSVTVSS.

[0068] CDR region annotation: The amino acid sequence of the complementary determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ IDNO.7: NVDLN; The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.8 respectively: SISPGAASRKYNETFRG; The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.9 respectively: GSDYGYSYAMDY.

[0069] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.20: GACATCAAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGAGCACCATCACCTGCGAGGCCAGCAGCAGGCTGAACTACAAGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCACCATCAACCTGGACAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTTCAGCAGCGTGCCCCACACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0070] The amino acid sequence of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.16: DIKMTQSPAIMSASPGEKSTITCEASSRLNYKHWFQQKPGTSPKLWIYSTINLDSGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQFSSVPHTFGAGTKLEIKRTV.

[0071] CDR region annotation: The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.10 respectively: EASSRLNYKH; The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.11 respectively: STINLDS; The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.12 respectively: QQFSSVPHT.

[0072] Monoclonal antibody 4D5: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.17: GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGACCAGCGGCTACACCTTCACCAGGGTGTACGACAACTGGGTGAACCAGAGGCCCGGCCAGGGCCTGGAGTGGGGCGTGAGCATCAGCCCCAGGGTGGTGGACAGCAGGAAGTACAAGACCTTCAGGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGAACAGCGCCGTGTACTTCTGCGCCAGGGGCAGCGACTACAGCGACGAGAGCGCCATGGACGTGTGGGGCCAGGGCACCAGCGTGACCGTGAGCGCC。

[0073] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.13: EVQLQQSGPELVKPGASMKISCKTSGYTFTRVYDNWVNQRPGQGLEWGVSISPRVVDSRKYKTFRGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGSDYSDESAMDVWGQGTSVTVSA。

[0074] CDR region annotation: The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.1: RVYDN; The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.2: SISPRVVDSRKYKTFRG; The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.3: GSDYSDESAMDV; Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.18: GACATCGTGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGAGCACCATCACCTGCGAGGCCGACGAGGAGAGGACCAACCTGAAGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGGGTGATCAACCTGAACAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGAGCAAGGGCAGCGTGGTGAGCACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0075] The amino acid sequence of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.14: DIVLTQSPAIMSASPGEKSTITCEADEERTNLKHWFQQKPGTSPKLWIYRVINLNSGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQSKGSVVSTFGAGTKLEIKRTV。

[0076] CDR region annotation: The amino acid sequence of the complementary determining region CDR-L1 of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.4: EADEERTNLKH; The amino acid sequence of the complementary determining region CDR-L2 of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.5: RVINLNS; The amino acid sequence of the complementary determining region CDR-L3 of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.6: QSKGSVVST.

[0077] So far, the embodiments of the present application have been described in detail. In order to avoid obscuring the concept of the present 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 here based on the above description.

[0078] 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 equivalent substitutions can be made for some technical features without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody combination for detecting feline Cys-C protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 4D5 and monoclonal antibody 3F3; The heavy chain variable region of the monoclonal antibody 4D5 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.1 - SEQ ID NO.3; The light chain variable region of the monoclonal antibody 4D5 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.4 - SEQ ID NO.6; The heavy chain variable region of the monoclonal antibody 3F3 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.7 - SEQ ID NO.9; The light chain variable region of the monoclonal antibody 3F3 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.10 - SEQ ID NO.

12.

2. The monoclonal antibody combination for detecting feline Cys-C protein according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4D5 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 4D5 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting feline Cys-C protein according to claim 2, wherein The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3F3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 3F3 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting feline Cys-C protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4D5 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4D5 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting feline Cys-C protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3F3 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3F3 is shown in SEQ ID NO.

20.

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

7. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting feline Cys-C protein.

8. The application according to claim 7, wherein The tool includes reagents, reagent kits, test strips and antibody chips.

9. The application according to claim 8, wherein The reagent kit includes a double antibody sandwich ELISA kit.

10. The application according to claim 9, wherein The ELISA kit uses monoclonal antibody 4D5 as the coating antibody and monoclonal antibody 3F3 as the labeled antibody.

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