A monoclonal antibody combination for detecting cat Cys-C protein and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody combination for detecting cat Cys-C protein and its application. The monoclonal antibody combination provided by the present invention includes monoclonal antibodies 4D5 and 3F3, and the amino acid sequences of the complementary determining regions (CDRs) of the heavy chain and light chain variable regions are shown in SEQ ID NO.1 to SEQ ID NO.12, respectively. The antibody combination has high specificity and sensitivity, and can effectively identify cat Cys-C recombinant protein and natural cat Cys-C protein. The double-antibody sandwich ELISA detection method constructed based on the antibody combination can still generate a positive signal at a low concentration of 1 ng / mL, and has no obvious cross-reaction, which significantly improves the detection accuracy. The antibody combination can be used to prepare detection kits, test strips, antibody chips and other tools, which are suitable for cat renal function assessment and early auxiliary diagnosis of kidney disease.

Description

Technical Field

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

[0002] In recent years, with the continued increase in the number of dogs and cats kept, the incidence of kidney diseases in pet clinics has also been on the rise, often accompanied by high mortality rates. Studies have shown that the development of chronic renal failure in dogs and cats is closely related to a variety of factors, including age, genetics, underlying diseases, diet, and living environment, and is often the result of the combined effects of multiple pathogenic factors. Many animals with acute nephritis, if not diagnosed and treated promptly and effectively, can easily suffer irreversible kidney damage, which can progress to chronic renal failure and ultimately be life-threatening.

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

[0004] In cats in particular, feline cystatin C (also known as fCys-C or feline Cys-C) has garnered significant attention as a key biomarker of glomerular filtration function due to its excellent stability in plasma. Its concentration is unaffected by factors such as inflammation, infection, tumors, or liver function, enabling it to more accurately and earlier detect abnormal renal function. Consequently, feline Cys-C is widely used to aid in the diagnosis of renal diseases such as acute kidney injury and chronic kidney disease in cats, and can also be used to assess renal function recovery after kidney transplantation, thus possessing significant clinical application value.

[0005] However, the current clinical diagnosis of feline renal failure still relies on a comprehensive evaluation process, including biochemical tests, urinalysis, imaging, and pathological testing. This process is not only complex but also expensive, placing significant financial pressure on pet owners. Furthermore, traditional detection methods lack sensitivity and specificity, making it difficult to meet the needs of early and rapid diagnosis. Therefore, developing a highly sensitive and specific detection method for feline cystatin C, particularly a monoclonal antibody-based immunoassay, would help improve the diagnostic efficiency and accuracy of feline kidney disease and hold significant promise for clinical application. 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:

[0008] 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;

[0009] 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;

[0010] 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;

[0011] 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;

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

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

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

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

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

[0021] In some embodiments, the ELISA kit uses monoclonal antibody 4D5 as a coating antibody and monoclonal antibody 3F3 as a labeling antibody.

[0022] In addition, the double antibody sandwich ELISA detection method is not used for disease diagnosis and treatment.

[0023] Beneficial effects:

[0024] The present invention provides a highly specific and sensitive monoclonal antibody combination targeting feline Cys-C protein, comprising monoclonal antibodies 4D5 and 3F3. Through the design and optimization of the complementarity-determining region (CDR) sequences of their heavy and light chain variable regions, the CDR amino acid sequences of which are shown in SEQ ID NOs. 1 to 12, respectively, this antibody combination is able to efficiently recognize both recombinant and native feline Cys-C proteins, demonstrating excellent binding activity and detection specificity.

[0025] The double-antibody sandwich ELISA assay constructed based on this antibody combination has high sensitivity, producing a positive reaction at a low concentration of 1 ng / mL, and exhibiting no cross-reaction with other unrelated antigens, significantly improving the accuracy of the test results. The present invention also provides the amino acid sequence and encoding nucleotide sequence of the variable regions of the relevant antibodies, laying the foundation for subsequent genetic engineering expression and large-scale production of the antibodies.

[0026] This antibody combination can be used to prepare kits, test strips, antibody chips, and other tools for detecting feline Cys-C protein. It can also be widely used in feline renal function assessment, early diagnosis of acute kidney injury and chronic kidney disease, and has promising clinical application prospects. Compared with traditional detection methods, the immunoassay provided by this invention is simple to operate, cost-effective, and has a rapid response. It overcomes the problems of insufficient sensitivity and severe cross-reactivity in existing technologies, providing a highly efficient diagnostic solution for veterinary clinics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0028] Figure 1 This is the SDS-PAGE identification result of protein purification;

[0029] Figure 2 This is the activity identification diagram of the cat Cys-C recombinant protein;

[0030] Figure 3 This is a test chart of sensitivity and specificity of double antibody sandwich ELISA;

[0031] Figure 4 Figure 2 is a graph showing the binding activity of paired monoclonal antibodies. DETAILED DESCRIPTION

[0032] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0033] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0034] Example 1

[0035] 1. Recombinant protein gene synthesis

[0036] The feline cystatin C protein gene (downloaded from the NCBI database) was synthesized by Qingke Biotechnology after codon optimization and cloned into the pET32a vector. The nucleotide sequence corresponding to the feline cystatin C protein gene is shown in SEQ ID NO. 21:

[0037] ATGTGCGCACTGCCGATCACCTCTTACGGTAACCTGTGCGCTGTTGATCGTGAACACACTATGGCGGGTTCTCTGCGTACTCCATTGTTGCTGCTGGCTGCGGTTGCTCTGACTCTGGCACTGGC TATGAGTCCGGGTACTGGTCGTCGTAACAACAAATCTGCTCTGGTTGGTGCACCGCTGGATGCGGACGTGAACGAAGAAGGTGTACAGCAGGCTCTGAACTTCGCGCTGTCTGAATACAACAAGGC TTCTAACGACGCGTACCACTCTCGTGCGATGCGTGTTGTTCGTGCTCGTAAACAGGTGGTTGCTGGTATGAACTACTTTCTGGACGTGGAAATCGGTCGTACTCGTTGCACCAAATCTCAGCCGA ACCTGGACACCTGTCCGTTCCATGACCAGCCGCACCTGATGCGTAAAACTCTGTGCTCTTTCCAAATCTACACCGTGCCGTGGATGGGTAAGACCTCTCTGGTTAAATCTTCTTGCCAAGATGCA.

[0038] The amino acid sequence corresponding to the cystatin C protein gene is shown in SEQ ID NO.22:

[0039] MCALPITSYGNLCAVDREHTMAGSLRTPLLLLAAVALTLALAMSPGTGRRNNKSALVGAPLDADVNEEGVQALNFALSEYNKASNDAYHSRAMRVVRARKQVVAGMNYFLDVEIGRTRCTKSQPNLDTCPFHDQPHLMRKTLCSFQIYTVPWMGKTSLVKSSCQDA.

[0040] Design seamless cloning primers for the feline Cys-C gene in the pCold-TF vector. Use the synthetic plasmid pET32a-feline Cys-C as a template to amplify the feline Cys-C fragment. Connect the fragment to the vector according to the kit instructions (In-Fusion HD Cloning kits, TaKaRa). Transform BL21 (DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) according to conventional methods. Spread the transformed bacteria on LB agar plates (containing 100 μg / mL ampicillin) and culture at 37°C overnight. Pick a single colony and inoculate it into 5 mL LB medium (containing 100 μg / mL ampicillin) and culture it at 37°C, 220 rpm, and shake overnight. Inoculate 1% of the total volume of the culture medium into LB medium (containing 100 μg / mL ampicillin) and culture it at 37°C, 220 rpm, and shake for about 3 hours until the OD 600 The mixture was cooled to 0.5, and the mixture was cooled at 16°C for 1.5 hours. IPTG was added at a final concentration of 0.1 mM, and the mixture was induced at 16°C and 180 rpm for 16 hours. After that, the bacteria were collected to obtain the feline Cys-C recombinant protein.

[0041] 2. Purification of recombinant protein

[0042] Because the expressed cat Cys-C recombinant proteins all carry histidine tags, GE's AKTA purification instrument and HisTrap TM Purification was performed using an HP affinity chromatography column. 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 column was equilibrated with buffer A. The fermented bacterial broth was then centrifuged at 8,000 rpm for 20 min. The precipitate was resuspended in buffer A and ultrasonically disrupted in ice water for 30 min, followed by 5-second intervals and 5-second intervals. The precipitate was then centrifuged at 12,000 rpm for 30 min. The supernatant was filtered through a 0.22-μm filter and loaded onto the column. The column was washed with buffer A and then gradient eluted with buffer B. Purification was observed by SDS-PAGE protein gel electrophoresis. The target protein peak was selected, dialyzed into buffer A, and concentrated. The protein concentration was determined using a Nanodrop assay. The protein was aliquoted into 1 ml tubes and stored at -20°C.

[0043] 3. Identification of recombinant protein

[0044] The protein purity was determined by SDS-PAGE electrophoresis.

[0045] 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.

[0046] 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%.

[0047] 4. ELISA to identify the activity of recombinant protein

[0048] 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.450 nm value.

[0049] See also Figure 2 , M102202M represents the commercially available feline Cys-C monoclonal antibody M102202M, and Ctrl represents the fNT-proBNP monoclonal antibody M101706M.

[0050] The indirect ELISA method was used to identify the expressed feline Cys-C recombinant protein and its reaction with the commercially available monoclonal antibody M102202M. The feline Cys-C recombinant protein (1ug / ml) was coated. The results showed that the commercially available monoclonal antibody still had a weak positive reaction with the feline Cys-C recombinant protein when diluted to 10ng / ml, indicating that the feline Cys-C recombinant protein had good biological activity and could be used for the next step of the experiment.

[0051] 5. Mouse immunization

[0052] Six-week-old female BALB / c mice were immunized intramuscularly with 20 μg of feline Cys-C recombinant protein mixed with an equal volume of MF59 adjuvant (total volume 200 μl). Immunizations were repeated at two and four weeks using the same protocol. At five weeks, sera were collected from the mice to measure antibody titers using the expressed feline Cys-C recombinant protein. Mice with the highest titers were selected and boosted with 20 μg of feline Cys-C recombinant protein via the tail vein. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0053] 6. Screening of hybridoma cell lines

[0054] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, clones positive for feline Cys-C recombinant protein were screened by indirect ELISA. Positive cells were then cloned to a monoclonal state by limiting dilution, and the cell line was expanded and cryopreserved.

[0055] 7. Screening of positive clones by indirect ELISA

[0056] The microplate was coated with cat Cys-C recombinant protein (expressed in the pCold-TF vector, containing a His tag) (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, and incubated at 4°C overnight. The plate was blocked with 1% gelatin, 150 μL per well, and blocked at 37°C for 2 hours. The plate was washed once with detergent and patted dry. 50 μL of cell culture supernatant was added and reacted at 37°C for 30 minutes. The liquid in the wells was discarded, the plate was washed four times with PBST, patted dry, and 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 minutes, washed four times, patted dry, and 50 μL / well of TMB color development solution was added for color development at room temperature for 10 minutes. Finally, 0.5 M sulfuric acid was added to terminate the reaction, and the OD was measured using a microplate reader. 450 The positive cell lines that reacted only with feline Cys-C recombinant protein but not with other control antigens (fNGAL recombinant protein, synthesized by Qingke Biotechnology) were selected for subsequent experiments.

[0057] The screening process is shown in Table 1.

[0058] Table 1: Indirect ELISA reaction results of hybridoma cell lines to feline Cys-C recombinant protein and control antigen.

[0059]

[0060] Table 1 only shows the results of the screened monoclonal antibodies with better data readings. The remaining data sets are not shown because they are not ideal.

[0061] 8. Preparation of Monoclonal Antibody Ascites

[0062] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0063] 9. Affinity chromatography purification of monoclonal antibodies (Protein G)

[0064] The ascites was centrifuged at 12,000 r / min for 5 minutes, and the supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4). The supernatant was then filtered through a 0.22 μm filter. The filtered sample was pumped at a low speed through a peristaltic pump into a Protein G (Cytiva) purification column equilibrated with binding buffer. The column was connected to an AKTA purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak was flattened. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected and the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, placed in a dialysis bag (MW: 8,000-14,000), and dialyzed against 0.01 M PBS, pH 7.4, at 2-8°C for 14 hours. Transfer the liquid in the dialysis bag to a centrifuge tube and centrifuge at 12,000 r / min for 5 minutes. The supernatant is the purified monoclonal antibody. Measure the concentration using a microspectrophotometer and store in aliquots.

[0065] 10. Double Antibody Sandwich ELISA Pairing

[0066] 10.1. HRP-labeled monoclonal antibodies

[0067] The screened antibodies were labeled with HRP according to the instructions for the G-Biosciences HOOK™ HRP PLUS Labeling Kit (Cat#: 786-313). The labeled antibodies were dialyzed overnight against 0.01M PBS, pH 7.4, and then glycerol was added in a 1:1 ratio. The labeled antibodies were stored in aliquots at -20°C. Specifically, the labeled antibody was diluted with coupling buffer (provided with the kit) to a final concentration of 2 mg / mL. The diluted antibody solution was then added to the HRP tube (provided with the kit) and mixed by pipetting. Incubate at room temperature for 1 hour, mixing regularly during the incubation. The labeling reaction was terminated by adding 50 μL of stop solution and mixing for 15 minutes. The labeled antibodies were dialyzed overnight against PBS, and an equal volume of glycerol was added for cryopreservation.

[0068] 10.2. Establishment of Double Antibody Sandwich Method

[0069] The purified monoclonal antibodies were diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The coating solution was discarded the next day, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 1% BSA at 150 μL / well. The plate was incubated at 37°C for 2 h, the blocking solution was discarded, and the plate was patted dry. The antigen to be tested and the control antigen (fNGAL) were diluted to 50 ng / ml with PBS and added to the enzyme-labeled plate, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times with PBST washing solution, patted dry, and the enzyme-labeled monoclonal antibody diluted to 1000 times with PBS was added, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times, patted dry, and TMB color development solution was added 50 μL / well, and color was developed at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added, 50 μL / well, to terminate the reaction, and the OD was measured with a microplate reader. 450 nm value. The paired monoclonal antibody with the largest P / N value was selected. The results showed that the coating antibody (4D5) and the labeled antibody (3F3) had the largest P / N value, so this paired monoclonal antibody was used for further testing.

[0070] The screening process is shown in Table 2.

[0071] Table 2: Comparison of the reactivity of different monoclonal antibody combinations against fCys-C (feline Cys-C) recombinant protein and control antigen in a double antibody sandwich ELISA.

[0072]

[0073] In Table 2, 1000* indicates 1000-fold dilution, fCys-C indicates feline Cys-C recombinant protein, and fNGAL indicates feline NGAL recombinant protein.

[0074] The antibodies were coated and labeled and paired one by one, and paired antibodies were initially screened out. Monoclonal antibody 4D5 was used as the coating antibody, and monoclonal antibody 3F3 was used as the HRP-labeled antibody.

[0075] 11. Optimization of double antibody sandwich method

[0076] The purified monoclonal antibody 4D5 was added to the microwells at a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL respectively using coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6) 50 μL / well and coated at 4 ° C overnight. The coating solution was discarded the next day and blocked with 1% BSA, 150 μL / well, incubated at 37 ° C for 2 h, and the blocking solution was discarded. The antigen to be tested and the control antigen were diluted with PBS at 50 ng / ml and added to the enzyme-labeled plate, 50 μL / well, incubated at 37 ° C for 35 min, and the plate was washed 4 times with PBST solution. 1000, 2000, 3000 times diluted HRP labeled monoclonal antibody, 50 μL / well, incubate at 37 ° C for 35 minutes, then wash the plate 4 times, pat dry and add TMB color development solution 50 μL / well, develop at room temperature for 10 minutes, finally add 0.5 M sulfuric acid, 50 μL / well, stop the reaction, and measure OD with a microplate reader. 450 nm value. The pairing conditions with the largest P / N value were selected for sensitivity and specificity testing. The screening process is shown in Table 3.

[0077] Table 3: Optimization of the best coating and labeling conditions for double antibody sandwich ELISA.

[0078]

[0079] The optimal reaction conditions are: coating antibody concentration and labeled antibody dilution, based on the pairing conditions with the highest P / N value. The optimal coating antibody concentration is 1ug / ml, and the HRP labeled antibody is diluted 2000 times, which gives the best results for the double antibody sandwich ELISA.

[0080] 12. Double antibody sandwich sensitivity and specificity test

[0081] The optimal reaction conditions were determined as follows: a coating concentration of 1 ug / ml and a 2000-fold dilution of the HRP-labeled monoclonal antibody. Referring to the above detection steps, the feline Cys-C recombinant protein was first serially diluted with PBS buffer solution to concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. At the same time, 50 μL of the fNGAL and fNT-pro BNP recombinant proteins were added to each well at the same concentration for detection to determine the sensitivity and specificity of the detection system for the recombinant proteins.

[0082] according to Figure 3It was found that the double-antibody sandwich ELISA composed of this group of paired antibodies still had a positive reaction when the cat Cys-C recombinant protein was diluted to 1 ng / ml, and did not react with irrelevant antigens. It has good sensitivity and specificity, which is of great significance for the early diagnosis of the renal function status of cats and the auxiliary diagnosis of kidney-related diseases such as acute kidney injury and chronic kidney disease in cats. Figure 3 fNGAL, fCys-C, and fNT-pro BNP represent recombinant feline NGAL, feline Cys-C, and feline NT-pro BNP proteins, respectively. Both recombinant fNGAL and fNT-pro BNP proteins were synthesized and expressed by Qingke Biotechnology.

[0083] Specifically, the feline NT-proBNP 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 NT-proBNP protein gene is shown in SEQ ID NO. 23:

[0084] CATCCGCTGGGTGGTCCAGGTCCAGCGTCTGAGGCTTCTGCGATCCAAGAACTGCTGGACGGTCTGCGTGACACTGTTTCTGAACTGCAAGAAGCGCAGATGGCACTGGGTCCGCTGCAGCAGGGTCACTCTCCGGCTGAAAGCTGGGAAGCTCAGGAAGAACCGCCAGCGCGTGTTCTGGCTCCGCATGACAACGTTCTGCGTGCTCTGCGT.

[0085] The amino acid sequence corresponding to the cat NT-proBNP protein gene is shown in SEQ ID NO. 24:

[0086] HPLGGPGPASEASAIQELLDGLRDTVSELQEAQMALGPLQQGHSPAESWEAQEEPPARVLAPHDNVLRALR.

[0087] The synthetic plasmid pET32a-cat NT-proBNP was transformed into BL21 (DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) according to conventional methods. The transformed bacteria were spread on LB agar plates (containing 100 μg / mL ampicillin) and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C, 220 rpm, and shaken overnight. 1% of the total volume of the culture was inoculated into LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C, 220 rpm, and shaken for about 3 hours until the OD reached 0. 600The mixture was cooled to 0.5, and the temperature was lowered at 16°C for 1.5 hours. IPTG was added at a final concentration of 0.1 mM, and the mixture was induced at 16°C and 180 rpm for 16 hours. After that, the bacteria were collected and the purification test was carried out in the next step.

[0088] 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. 25:

[0089] .

[0090] The amino acid sequence corresponding to the cat NGAL protein gene is shown in SEQ ID NO.26:

[0091] QDSTPNLIPAPPLLLVPVEPDFQNEQFQGKWYFLGLAGNGFNKEKHRRMKMYIANYELNEDNSYNVTSTVAWNQTCHPSTKIFLPNLHLGQFNLGNIERYTGIQNYTSKVVTTDYNQFAILYFKKVHDNQEYIKVILYGRTKEVPSVPKAIFISFIKSLGLTDDHIIFPIPNDECMDK.

[0092] 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 and vector were ligated according to the kit instructions (In-Fusion HD Cloning kits, TaKaRa). BL21(DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) were transformed according to conventional methods. Transformants were plated on LB agar plates (containing 100 μg / mL ampicillin) and cultured overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C with shaking at 220 rpm. The cells were inoculated into LB medium (containing 100 μg / mL ampicillin) at 1% of the total volume of the culture medium, and cultured with shaking at 37°C and 220 rpm for about 3 hours until the OD600 reached 0.5. The cells were then cooled at 16°C for 1.5 hours, and IPTG was added to a final concentration of 0.1 mM. The cells were induced at 16°C and 180 rpm for 16 hours, and the cells were collected.

[0093] 13. Identification of binding activity of paired monoclonal antibodies

[0094] Referring to the aforementioned indirect ELISA method, two monoclonal antibodies, 4D5 and 3F3, were serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml, and the binding activity of feline Cys-C monoclonal antibody was determined using another mouse monoclonal antibody, fNT-proBNP monoclonal antibody M101706M, as a negative control.

[0095] Results see Figure 4 Monoclonal antibody 4D5 and monoclonal antibody 3F3 still had 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 the paired monoclonal antibodies had high binding activity. Figure 4 Here, 3F3 is the monoclonal antibody 3F3, 4D5 is the monoclonal antibody 4D5, and Ctrl is the fNT-proBNP monoclonal antibody M101706M.

[0096] 14. Antibody variable region gene cloning and sequencing

[0097] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0098] mAb variable region sequence

[0099] Monoclonal antibody 3F3

[0100] Heavy chain:

[0101] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.19:

[0102] CAGATGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGACCAGCGGCTACACCTTCACCAACGTGGACCTGAACTGGGTGAACCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCAGCATCAGCCCCGGCGCCGCCAGCAGGAAGTACAA CGAGACCTTCAGGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGAACAGCGCCGTGTACTTCTGCGCCAGGGGCAGCGACTACGGCTACAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0103] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.15:

[0104] QMQLQQSGPELVKPGASMKISCKTSGYTFTNVDLNWVNQRPGQGLEWIGSISPGAASRKYNETFRGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGSDYGYSYAMDYWGQGTSVTVSS.

[0105] CDR region annotation:

[0106] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 3F3 are shown in SEQ ID NO. 7: NVDLN;

[0107] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of the monoclonal antibody 3F3 are shown in SEQ ID NO. 8: SISPGAASRKYNETFRG;

[0108] The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of the monoclonal antibody 3F3 is shown in SEQ ID NO. 9: GSDYGYSYAMDY.

[0109] Light chain:

[0110] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.20:

[0111] GACATCAAGATGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGAGCACCATCACCTGCGAGGCCAGCAGCAGGCTGAACTACAAGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGCACCATCAACCTGGACAG CGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTTCAGCCAGCGTGCCCCACACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0112] The amino acid sequence of the light chain variable region of monoclonal antibody 3F3 is shown in SEQ ID NO.16:

[0113] DIKMTQSPAIMSASPGEKSTITCEASSRLNYKHWFQQKPGTSPKLWIYSTINLDSGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQFSSVPHTFGAGTKLEIKRTV.

[0114] CDR region annotation:

[0115] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of the monoclonal antibody 3F3 are shown in SEQ ID NO. 10: EASSRLNYKH;

[0116] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of the monoclonal antibody 3F3 are shown in SEQ ID NO. 11: STINLDS;

[0117] 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: QQFSSVPHT.

[0118] Monoclonal antibody 4D5:

[0119] Heavy chain:

[0120] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.17:

[0121] GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGACCAGCGGCTACACCTTCACCAGGGTGTACGACAACTGGGTGAACCAGAGGCCCGGCCAGGGCCTGGAGTGGGGCGTGAGCATCAGCCCCAGGGTGGTGGACAGCAGGAAGTA CAAGACCTTCAGGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGAACAGCGCCGTGTACTTCTGCGCCAGGGGCAGCGACTACAGCGACGAGAGCGCCATGGACGTGTGGGGCCAGGGCACCAGCGTGACCGTGAGCGCC.

[0122] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.13:

[0123] EVQLQQSGPELVKPGASMKISCKTSGYTFTRVYDNWVNQRPGQGLEWGVSISPRVVDSRKYKTFRGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGSDYSDESAMDVWGQGTSVTVSA.

[0124] CDR region annotation:

[0125] 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;

[0126] 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;

[0127] 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;

[0128] Light chain:

[0129] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.18:

[0130] GACATCGTGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGAGCACCATCACCTGCGAGGCCGACGAGGAGGACCAACCTGAAGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGGGTGATCAACCTGAAC AGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTGCCAGAGCAAGGGCAGCGTGGTGAGCACCTTCGGCGCCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0131] The amino acid sequence of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO.14:

[0132] DIVLTQSPAIMSASPGEKSTITCEADEERTNLKHWFQQKPGTSPKLWIYRVINLNSGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQSKGSVVSTFGAGTKLEIKRTV.

[0133] CDR region annotation:

[0134] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of the monoclonal antibody 4D5 are shown in SEQ ID NO. 4: EADEERTNLKH;

[0135] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of the monoclonal antibody 4D5 are shown in SEQ ID NO. 5: RVINLNS;

[0136] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of monoclonal antibody 4D5 is shown in SEQ ID NO. 6: QSKGSVVST.

[0137] So far, the various 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. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0138] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents 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 complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of the monoclonal antibody 4D5 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; The heavy chain variable region of the monoclonal antibody 3F3 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively; The light chain variable region of the 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.

2. The monoclonal antibody combination for detecting cat Cys-C protein according to claim 1, characterized in that 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 cat Cys-C protein according to claim 2, characterized in that 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 cat 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 cat 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 cat 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 combination of monoclonal antibodies according to claim 1 in preparing a tool for detecting feline Cys-C protein.

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

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

10. The use according to claim 9, characterized in that The ELISA kit uses the monoclonal antibody 4D5 as the coating antibody and the monoclonal antibody 3F3 as the labeling antibody.

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