Recombinant IFN-gamma monoclonal antibody for minks and application of recombinant IFN-gamma monoclonal antibody

Abstract

The invention belongs to the technical field of monoclonal antibody screening and preparation, and particularly relates to a mink recombinant IFN-gamma monoclonal antibody and application thereof. The amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of light chain and heavy chain variable regions of the mink recombinant IFN-gamma monoclonal antibody are defined, and the mink recombinant IFN-gamma monoclonal antibody can be used as a reagent for detecting the mink IFN-gamma in vitro. Moreover, the mink recombinant IFN-gamma monoclonal antibody provided by the invention can specifically recognize and bind to the mink IFN-gamma, and can effectively bind to an antigen (mink IFN-gamma) at a lower concentration, the effect is better than 1 mu g / mL when the concentration is 0.5 mu g / mL, the content of the mink IFN-gamma can be analyzed through immunological detection, and the sensitivity reaches EC50lt; and 1.131 [mu] g / mL.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monoclonal antibody screening and preparation, and particularly relates to a ferret recombinant IFN-γ monoclonal antibody and application thereof. BACKGROUND

[0002] Ferrets, as an important experimental animal, play an increasingly important role in medical research, virology research and vaccine development. Because of the high similarity of its respiratory system and immune system to humans, ferrets have become an ideal model animal for studying respiratory infectious diseases such as influenza virus, respiratory syncytial virus (RSV) and novel coronavirus (SARS-CoV-2). However, the lack of specific research tools for ferrets, especially the detection means for key cytokines of its immune system (such as Interferon-γ, IFN-γ), seriously restricts the in-depth development of related research.

[0003] IFN-γ is a type II interferon secreted by activated T cells and natural killer (NK) cells, which plays a core role in anti-viral immunity, anti-tumor immunity and immune regulation. It can activate macrophages, enhance antigen presentation, promote Th1-type immune response, and directly inhibit viral replication. In ferret infection models, the dynamic changes of IFN-γ are key indicators for evaluating cellular immune response. For example, after ferrets are infected with canine distemper virus (CDV) or influenza virus, the secretion level of IFN-γ is closely related to the severity of the disease and the protective efficacy of the vaccine. However, at present, the research on ferret IFN-γ mainly faces the following technical bottlenecks: ① Insufficient detection sensitivity: the existing ELISA method has a high lower limit of detection, which is difficult to capture the changes of ferret IFN-γ in the early stage or at a low level. ② Blank of standardized kit: there is no commercialized ferret IFN-γ detection kit, researchers need to establish the method themselves, which leads to non-uniform experimental conditions and poor data comparability.

[0004] Monoclonal antibodies are produced by a single B cell clone and only target a certain specific antigen epitope. This high specificity enables monoclonal antibodies to accurately recognize antigens. In the process of cytokine detection, monoclonal antibodies have the advantages of strong uniformity and high detection sensitivity. Therefore, the development of ferret recombinant IFN-γ monoclonal antibody is of great significance for the detection of ferret IFN-γ. SUMMARY

[0005] The purpose of the present application is to provide a ferret recombinant IFN-γ monoclonal antibody and its application, which specifically detects ferret IFN-γ and has high detection sensitivity.

[0006] The application provides a ferret recombinant IFN-gamma monoclonal antibody, a light chain complementarity determining region CDR1 of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 6, an amino acid sequence of a light chain complementarity determining region CDR2 comprises WAS, and a light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 7. A heavy chain complementarity determining region CDR1 of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 9, a heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 10, and a heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0007] Preferably, a light chain variable region of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 5. A heavy chain variable region of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0008] Preferably, a heavy chain of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 3. A light chain of the ferret recombinant IFN-gamma monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0009] The application also provides a nucleic acid molecule encoding the ferret recombinant IFN-gamma monoclonal antibody.

[0010] Preferably, a sequence encoding the light chain variable region of the ferret recombinant IFN-gamma monoclonal antibody in the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 12, and a sequence encoding the heavy chain variable region of the ferret recombinant IFN-gamma monoclonal antibody in the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0011] The application also provides a biological material for expressing the ferret recombinant IFN-gamma monoclonal antibody, wherein the biological material comprises a recombinant vector or a recombinant cell.

[0012] The application also provides a ferret recombinant IFN-gamma protein for preparing the ferret recombinant IFN-gamma monoclonal antibody, and the ferret recombinant IFN-gamma protein comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0013] The application also provides application of the ferret recombinant IFN-γ monoclonal antibody in one or more of the following aspects: (1) preparing a product for detecting ferret IFN-γ; (2) preparing a product for diagnosing ferret respiratory virus infection; (3) preparing a product for evaluating the efficacy of a vaccine for ferret respiratory virus infection; (4) preparing a product for studying Th1 type immune response of ferret respiratory virus infection.

[0014] The application also provides a kit for detecting ferret IFN-γ, comprising a coating antibody and a detection antibody. The coating antibody is the ferret recombinant IFN-γ monoclonal antibody described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein described in the above technical solution. The detection antibody is a tracer-labeled Fer5-serum-IgG, and the Fer5-serum-IgG is serum obtained after immunizing an animal with the ferret recombinant IFN-γ protein described in the above technical solution.

[0015] The application also provides a method for detecting ferret IFN-γ, comprising the following steps: mixing a test sample, a detection antibody and a coating antibody to form a complex, and then detecting a tracer signal in the complex. The coating antibody is the ferret recombinant IFN-γ monoclonal antibody described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein described in the above technical solution. The detection antibody is a tracer-labeled Fer5-serum-IgG, and the Fer5-serum-IgG is serum obtained after immunizing an animal with the ferret recombinant IFN-γ protein described in the above technical solution.

[0016] Beneficial effects: The application provides a ferret recombinant IFN-gamma monoclonal antibody, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain and the heavy chain variable region of the ferret recombinant IFN-gamma monoclonal antibody are determined, and the ferret recombinant IFN-gamma monoclonal antibody can be used as a reagent for detecting ferret IFN-gamma in vitro. 50 <1.131μg / mL. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0018] Figure 1 The figure is a schematic diagram of a ferret recombinant IFN-gamma expression vector structure. Figure 2 The figure is an SDS-PAGE analysis of ferret recombinant IFN-gamma protein. Figure 3 The figure is a serum titer determination result of ferret recombinant IFN-gamma protein immunized mice; wherein **** represents P <0.0001; Figure 4 The figure is an SDS-PAGE analysis of ferret monoclonal antibody Fer5-IFN-gamma after purification. Figure 5 The figure is a binding activity parameter of ferret monoclonal antibody Fer5-IFN-gamma. Figure 6 The figure is a standard curve and logistic curve fitting of antigen concentration optimization of double antibody sandwich ELISA when 0.5 μg / mL Fer5-serum-IgG-biotin is used as a primary antibody. Figure 7 The figure is a standard curve and logistic curve fitting of antigen concentration optimization of double antibody sandwich ELISA when 1.0 μg / mL Fer5-serum-IgG-biotin is used as a primary antibody. Figure 8 The figure is a result of double antibody sandwich ELISA ferret IFN-gamma serum antibody condition optimization. DETAILED DESCRIPTION

[0019] The application provides a ferret recombinant IFN-γ monoclonal antibody, a light chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 6, an amino acid sequence of a light chain complementarity determining region CDR2 comprises WAS, and a light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 7. A heavy chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 9, a heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 10, and a heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0020] As an embodiment, a light chain variable region of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises an amino acid sequence as shown in SEQ ID NO: 5. As an embodiment, a light chain variable region of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises a nucleotide sequence as shown in SEQ ID NO: 12; and a heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0021] As an embodiment, a light chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises a nucleotide sequence as shown in SEQ ID NO: 13. As an embodiment, a nucleotide sequence of a light chain complementarity determining region CDR2 of the ferret recombinant IFN-γ monoclonal antibody comprises TGGGCATCT. As an embodiment, a light chain complementarity determining region CDR3 of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 14.

[0022] As an embodiment, a heavy chain of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises an amino acid sequence as shown in SEQ ID NO: 3.

[0023] As an embodiment, a heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises an amino acid sequence as shown in SEQ ID NO: 8. As an embodiment, a heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody provided by the application comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0024] As an implementation form, the heavy chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody provided in the present application comprises a nucleotide sequence as shown in SEQ ID NO: 16. As an implementation form, the heavy chain complementarity determining region CDR2 of the ferret recombinant IFN-γ monoclonal antibody provided in the present application comprises a nucleotide sequence as shown in SEQ ID NO: 17. As an implementation form, the heavy chain complementarity determining region CDR3 of the ferret recombinant IFN-γ monoclonal antibody provided in the present application comprises a nucleotide sequence as shown in SEQ ID NO: 18.

[0025] As an implementation form, the light chain of the ferret recombinant IFN-γ monoclonal antibody provided in the present application comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0026] The present application also provides a nucleic acid molecule encoding the ferret recombinant IFN-γ monoclonal antibody provided in the above technical solution.

[0027] As an implementation form, the sequence in the nucleic acid molecule provided in the present application encoding the light chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 12; the sequence in the nucleic acid molecule provided in the present application encoding the heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 15.

[0028] As an implementation form, the sequence in the nucleic acid molecule provided in the present application encoding the light chain of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 19; the sequence in the nucleic acid molecule provided in the present application encoding the heavy chain of the ferret recombinant IFN-γ monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO: 20.

[0029] SEQ ID NO: 19: 5'-ACTCAGAGCCATAAGTTCATGAGCACCAGCCTGGGTGACCGAGTGAGCATCACTTGTAAGGCCAGTCAAGATGTGGGCACTGCTGTGGCCTGGTATCAGCAGAAGCCAGGTCAGAGTCCAAAGCTGCTGATCTATTGGGCATCTGCTCGACATACCGGAGTACCAGATCGGTTCACCGGCAGCGGAAGCGGCACAGATTTCACTCTGACCATCTCTAACGTGCAGAGCGAAGACCTGGCTGACTATTTCTGCCAGCAGTACTCCAGCTTTCCATACACCTTTGGCGGAGGTACTAAGCTGGAAATCAAG-3'; SEQ ID NO: 20: 5'-CAGGTGCAACTGGTGGAGTCTGGACCTGGATTGGTCGCTCCAAGTCAGTCTTTGTCCATCACATGCACCGTGTCTGGCTTCTCTCTGACAGGCTCCGGAGTCAACTGGGTCAGGCAACCACCAGGTAAGGGCTTGGAGTGGCTGGGCATGATCTGGGGAGACGGTACAACAGACTACAACAGCGCTCTGAAGTCCAGGCTGAGCATCAGCAAAGATAACTCCAAGAGCCAGGTCTTCTTGAAGATGAACAGCCTGCAGACAGACGATACCGCTCGATACTACTGTGCACGTGATCTGGTCACCACTGGAATGGACTACTGGGGTCAAGGCACATCTGTCACCGTGTCTAGC-3'; The application further provides a biological material for expressing the ferret recombinant IFN-γ monoclonal antibody according to the above technical solution, wherein the biological material comprises a recombinant vector or a recombinant cell.

[0030] As an embodiment, the recombinant vector according to the application comprises a basic vector and the nucleic acid molecule according to the above technical solution inserted into the basic vector. The application does not have strict requirements on the specific type of the basic vector, which can be used for the expression of the ferret recombinant IFN-γ monoclonal antibody according to the application, for example, can be pcDNA3.1(+). As an embodiment, the recombinant cell according to the application comprises a basic cell and the nucleic acid molecule according to the above technical solution introduced into the basic cell. The application does not have strict requirements on the specific type of the basic cell, which can be used for the expression of the ferret recombinant IFN-γ monoclonal antibody according to the application, for example, can be 293F cells.

[0031] The application further provides a ferret recombinant IFN-γ protein for preparing the ferret recombinant IFN-γ monoclonal antibody according to the above technical solution, wherein the ferret recombinant IFN-γ protein comprises the amino acid sequence as shown in SEQ ID NO: 4.

[0032] As an embodiment, the coding gene of the ferret recombinant IFN-γ protein according to the application comprises the amino acid sequence as shown in SEQ ID NO: 2.

[0033] The application also provides the application of the ferret recombinant IFN-γ monoclonal antibody in one or more of the following: (1) preparing a product for detecting ferret IFN-γ; (2) preparing a product for diagnosing ferret respiratory virus infection; (3) preparing a product for evaluating the efficacy of ferret respiratory virus infection vaccine; (4) preparing a product for studying the Th1 type immune response of ferret respiratory virus infection.

[0034] As an embodiment, the product of the application comprises a kit.

[0035] The application also provides a kit for detecting ferret IFN-γ, comprising a coating antibody and a detection antibody. The coating antibody is the ferret recombinant IFN-γ monoclonal antibody of the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule of the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material of the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein of the above technical solution. The detection antibody is a tracer-labeled Fer5-serum-IgG; the Fer5-serum-IgG is serum obtained after immunizing animals with the ferret recombinant IFN-γ protein of the above technical solution.

[0036] As an embodiment, the tracer of the application comprises one or more of biotin, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold and colored latex; as another embodiment, the tracer of the application is biotin. As an embodiment, the radioisotope of the application comprises one or more of 3H, 14C, 32P, 125I, 131I, 57Co and 51Cr. As an embodiment, the enzyme of the application comprises one or more of β-galactosidase, β-glucosidase, alkaline phosphatase, horseradish peroxidase and malate dehydrogenase. As an embodiment, the fluorescent substance of the application comprises fluorescamine or fluorescein isothiocyanate. As an embodiment, the luminescent substance of the application comprises one or more of luminol, luminol derivatives, luciferin, lucigenin, acridinium ester, peroxyoxalate and dioxetanes. As an embodiment, the colored latex of the application is polyethylene polymerized latex microparticles.

[0037] In one embodiment, the kit of the present invention further includes an IgG secondary antibody. In another embodiment, the IgG secondary antibody of the present invention includes an HRP-streptavidin secondary antibody.

[0038] In one embodiment, the animal described in this invention includes mice. In one embodiment, the immunization is performed three times, with a 14-day interval between adjacent immunizations. In one embodiment, the ferret recombinant IFN-γ protein and aluminum adjuvant described in the above technical solution are mixed at a 1:1 volume ratio before immunization.

[0039] The present invention also provides a method for detecting IFN-γ in ferrets, comprising the following steps: mixing a test sample, a detection antibody and a coating antibody to form a complex, and then detecting the tracer signal in the complex; The coating antibody is the ferret recombinant IFN-γ monoclonal antibody described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material described in the above technical solution, or the ferret recombinant IFN-γ monoclonal antibody prepared using the ferret recombinant IFN-γ protein described in the above technical solution. The detection antibody is tracer-labeled Fer5-serum-IgG; the Fer5-serum-IgG is serum obtained after immunizing animals with the recombinant IFN-γ protein of ferrets using the above-described technical solution.

[0040] As one embodiment, the method of mixing the test sample, detection antibody, and coating antibody to form a complex according to the present invention includes: mixing the test sample and the coating antibody to obtain a coating antibody-IFN-γ complex; mixing the coating antibody-IFN-γ complex and the detection antibody to obtain a detection antibody-coated antibody-IFN-γ complex; and detecting the tracer signal in the detection antibody-coated antibody-IFN-γ complex.

[0041] In one embodiment, the coating concentration of the coated antibody in this invention is 2-5 μg / mL. In another embodiment, the detection concentration of the detection antibody in this invention is 0.5-1 μg / mL. In specific implementation, this invention can detect whether the test sample contains ferret IFN-γ based on the tracer signal, and determine the concentration of ferret IFN-γ in the test sample. The half-maximal effect concentration (EC50) of the ferret recombinant IFN-γ monoclonal antibody provided by this invention... 50 When the concentration of the recombinant IFN-γ monoclonal antibody coating on the ferret is below 1.131 μg / mL, and the concentration is 5 μg / mL, the half-maximal effect concentration (EC50) of the detection antibody described in this invention is... 50 The concentration reached 0.11 μg / mL.

[0042] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0043] In order to further illustrate the present application, a ferret recombinant IFN-γ monoclonal antibody and its application provided by the present application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0044] Example 1 Obtaining of a ferret recombinant IFN-γ protein that can be secreted 1. According to the ferret IFN-γ gene sequence published by NCBI, accession number: NM_001310172.2, the soluble extracellular domain of IFN-γ is fused with the tissue-type plasminogen activator signal peptide gene, and a 6×His tag sequence (catcatcatcatcat) is added at the C terminal to obtain a recombinant IFN-γ protein, the amino acid sequence of which is shown in SEQ ID NO: 1, specifically: MDAMKRGLCCVLLLCGAVFVSPCDLPQDHGLLHWRALMLLQQMRRLSASSCDKYTNDFGFPQEVFDGKQLQKAQALSVIHVTNQKTFHLFCTEASPAPWNTTLLEQLCSGLSEQLGHLAACPLQEAGVGELPLVNGDSILRNYFQRISLYLQEKQYSPCAWEMVRAEIMKPLYASTVLHKRLRSRKAHHHHHHHH*.

[0045] 2. Recombinant protein codon optimization: The codon of the recombinant IFN-γ protein of step 1 is optimized in nucleotide sequence, including: (1) avoiding commonly used enzyme cutting sites; (2) optimizing according to the codon preference of 293F cell transfection expression; (3) in order to improve the transcription efficiency, avoiding too high or too low GC content, keeping at 40%~60%. The optimized coding gene is shown in SEQ ID NO: 2, specifically: 5'-TGCGACTTGCCCCAGGACCACGGGCTGCTGCACTGGAGGGCCCTGATGCTGCTGCAGCAGATGAGGAGGTTGTCAGCCTCTAGCTGTGACAAGTACACCAACGATTTCGGATTCCCACAGGAGGTCTTCGACGGGAAGCAGCTGCAGAAAGCCCAGGCCCTGTCCGTGATCCACGTCACCAACCAGAAGACCTTCCACCTGTTCTGCACCGAGGCCTCCCCCGCTCCCTGGAACACCACCCTCCTCGAACAGTTGTGCTCCGGCCTGTCCGAGCAGCTCGGCCATCTGGCCGCCTGCCCACTCCAGGAGGCCGGAGTTGGCGAGTTGCCACTGGTCAATGGAGACTCTATCCTGAGGAACTACTTCCAGAGGATTTCTTTGTATCTCCAGGAGAAGCAGTACTCCCCCTGTGCCTGGGAAATGGTGAGGGCTGAGATCATGAAGCCCCTGTATGCCAGCACAGTGCTGCACAAGAGGCTGAGGTCCAGGAAG-3'.

[0046] 3. Construction of recombinant expression vector: the optimized coding gene of step 2 is subcloned on pcDNA3.1(+) vector to obtain IFN-γ expression vector, and the structural schematic diagram is shown in Figure 1 .

[0047] 4. Plasmid transfection: after obtaining the IFN-γ expression vector, the IFN-γ expression vector is co-transfected into 100 mL 293F cells to express the recombinant IFN-γ protein, and the cell culture solution is collected after 6 days.

[0048] 5. Culture solution pretreatment: after centrifuging the cell culture solution at 4800 rpm for 10 min in a horizontal centrifuge, the supernatant is filtered by using a 0.45 μm filter to obtain the filtered culture solution.

[0049] 6. Purification: equilibration buffer was made with 20 mM phosphate and 0.5 M NaCl, pH = 7.4; washing buffer was made with 20 mM phosphate, 0.5 M NaCl and 5 mM imidazole, pH = 7.4; elution buffer was made with 20 mM phosphate, 0.5 M NaCl and 250 mM imidazole, pH = 7.4. The purification was performed using AKTA purification chromatography system, first equilibration buffer was used to equilibrate the His affinity column, then the sample was loaded, after washing buffer was used to wash the column until the UV peak was flat, the elution buffer was used to elute the protein, the elution buffer was collected.

[0050] 7. Concentration: after purification, the sample was concentrated using 10 K ultrafiltration column, PBS was used to replace the sample twice, the ultrafiltration sample was collected.

[0051] 8. Results: the ferret recombinant IFN-γ protein was successfully purified, the size of the purified protein was consistent with the expected protein molecular weight, the results were shown in Figure 2 , 196 amino acid residues, the protein size was about 28 kDa, the amino acid sequence was shown in SEQ ID NO: 1.

[0052] Example 2 Mouse immunization and serum titer determination 1. 10 μg of the ferret recombinant IFN-γ protein obtained in Example 1 was diluted with physiological saline, mixed with an equal volume of aluminum adjuvant to prepare a vaccine, a total of 50 μL, 6 six to eight week old Balb / c mice were immunized, once at 0, 14, 28 respectively. 12 days after the third immunization, the mice were bled from the tail vein, the serum was obtained by separation, and the antibody titer was determined.

[0053] 2. Titer detection (indirect ELISA): the ferret recombinant IFN-γ protein of Example 1 was used as the coating antigen, the antigen was diluted with coating buffer and added to the ELISA microreaction plate, 100 ng / well, 100 μL / well, 4°C overnight. The coated ELISA reaction plate was taken out, the liquid was discarded, 1 × PBST was used to wash the plate 5 times, and it was patted dry. 2% bovine serum albumin BSA was added, 200 μL / well, 37°C blocking for 2 h, 1 × PBST was used to wash 5 times and patted dry. Mouse serum (1:100 starting 2-fold gradient dilution) was used as the primary antibody, 1 × PBST was used for 2-fold gradient dilution (1:100 starting), added to the ELISA reaction plate, 100 μL / well. 37°C incubation for 1 h, discard the liquid, wash the plate 5 times with 1 × PBST and pat dry. HRP labeled goat anti-mouse IgG was diluted with 1 × PBST (1:10000) and added to each well, 100 μL / well, 37°C incubation for 1 h, discard the liquid, wash the plate 5 times and pat dry. TMB substrate color developing liquid was added, 50 μL / well, 37°C incubation for 15 min in the dark. Stop solution was added, 50 μL / well, within 5 min, the OD was determined on a microplate reader450 Values minus OD 630 Values, in OD 450 Values minus OD 630 Values minus OD Figure 3 ).

[0054] Example 3 Obtaining of hybridoma cell strain secreting monoclonal antibody against ferret recombinant IFN-γ protein 1. 3 days before cell fusion, the spleen was boosted with 100 μg of protein. The spleen cells of the immunized IFN-γ Balb / c mice were mixed with the logarithmically growing myeloma cells at a ratio of 3:1, and then electrofusion was performed. After fusion, the cells were cultured in HAT semi-solid screening medium for 8-10 days, and then the cells were cloned into Medium E medium screening culture for 2-3 days. The antibody specificity in the cell culture supernatant was detected by indirect ELISA method. The hybridoma cell supernatant was used as the primary antibody, and the HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody. Positive hybridoma cell strains that reacted with the ferret recombinant IFN-γ protein were screened. The positive cells were subcloned in a 96-well plate, and the above steps were repeated twice. Monoclonal cell strains that could stably secrete antibodies were screened, and after expansion culture, they were frozen with 10% DMSO in FBS, and the cell density was 10 6

[0055] 2. The antibody titer in the cell culture supernatant was detected by indirect ELISA method, and the specific steps were as follows: ​Dilute recombinant IFN-γ protein from ferrets to 1 μg / mL, and plate 100 μL / well onto a 96-well ELISA plate. Incubate overnight at 4°C. Wash the plate three times with PBST buffer, then add 100 μL of 5% BSA solution to each well and block at 37°C for 1.5 h. Wash the plate five times with PBST buffer. Prepare a dilution buffer for the culture supernatant in another plate: aspirate 22 μL of culture supernatant from each well, mix with 198 μL of PBS, then transfer 110 μL to the well containing 110 μL of PBS and mix thoroughly. Repeat this dilution process, discarding the last 110 μL of the diluted solution. Add 100 μL of diluted sample to each well of the ELISA plate and block at 37°C for 1 h. Wash the plate 5 times with PBST buffer. Dilute HRP-labeled goat anti-mouse IgG with 1×PBST (1:10000) and add 100 μL / well to each well. Block at 37°C for 1 h. Wash the plate 5 times with PBST buffer. Add 100 μL of TMB chromogenic solution to each well and incubate for 15 min at room temperature in the dark. Then add 50 μL of stop solution to each well to stop the chromogenic reaction. Measure the OD of each well using a microplate reader. 450 Value and OD 630 Value, in OD 450 Value minus OD 630 The values ​​are recorded as measurement results. A curve is plotted using a Logistic four-parameter fit, and the EC50 of the antibody is calculated. 50 Value. As a result, a dominant cell line with high binding activity, Fer5-IFN-γ, was screened out.

[0056] 3. The antibody was purified by affinity chromatography from the culture supernatant of the obtained dominant cell line Fer5-IFN-γ, as follows: Rinse the Protein A affinity chromatography column 3-5 times with 10 column volumes of deionized water at a flow rate of 1 mL / min. Rinse the column 3-5 times with 10 column volumes of equilibration buffer (0.02 M PB + 0.3 M NaCl, pH 7.0) at a flow rate of 1 mL / min until the pH of the eluent remains constant. Dilute the supernatant with 0.02 M PBS, filter through a 0.22 μm filter, and load the sample at a flow rate of 0.6 mL / min. After loading, wash the column with 5-10 column volumes of equilibration buffer and collect the eluent. Elute with 5-10 column volumes of elution buffer (0.1 M glycine, pH 3.0) and collect the eluent. Immediately after elution, add 0.1 column volumes of 1 M Tris-HCl (pH 8.0) to adjust the pH of the elution product to neutral. Wash the column with 5–10 column volumes of pure water, then immediately wash with 5 column volumes of equilibration buffer to neutralize the column. Wash the affinity chromatography column with 5 column volumes of 20% ethanol and store at 4°C. Concentrate the eluted product to approximately 1–5 mL by ultrafiltration using a 10 kDa ultrafiltration tube.

[0057] 4. The subtype of monoclonal antibody Ig was identified using mouse monoclonal antibody subtype identification kit (proteintech), and the subtype of ferret recombinant IFN-γ monoclonal antibody Fer5-IFN-γ was IgG1.

[0058] Example 4 Extraction of monoclonal antibody Fer5-IFN-γ gene and human-mouse chimeric modification 1. Antibody gene extraction: The hybridoma cell strain Fer5-IFN-γ obtained in Example 3 was blown from the cell culture dish, centrifuged at 4800 rpm for 2 min, the supernatant was discarded, 500 μL of PBS solution was added to each tube, the cells were blown off, centrifuged at 4800 rpm for 2 min, and washed once. Total RNA was extracted from the cells using an RNA extraction kit. The total RNA was reverse transcribed into cDNA, and then the antibody V region sequence in the total cDNA was amplified using mouse antibody light and heavy chain specific primers, respectively. After amplification, A ends were added to both ends of the V region fragment according to the kit. Then the kit was used to connect it to the T vector. After transformation into competent cells and heat shock, the cells were cultured in an antibiotic-free medium for 1 h. X-Gal and IPTG were mixed at a ratio of 1:2 and then coated on a solid medium containing ampicillin, 90 μL of bacterial solution was coated on the medium, and cultured for 16 h. After the culture grew, white and large and bright single colonies were selected, cultured in 800 μL of ampicillin medium for 10 h, and then sent for sequencing.

[0059] 2. Amplification of light and heavy chain and human-mouse chimeric monoclonal antibody constant region gene: After sequencing, primers were synthesized at the C and N termini of the light and heavy chain variable regions, and then FLASH PCR enzyme was used to amplify the antibody VH and VL regions.

[0060] 3. Agarose gel electrophoresis: 5.5 μL of 5x Loading Burrfer was added to the PCR reaction product, and electrophoresis was performed using a 1.2% agarose gel at 150 V for 27 min. After electrophoresis, the target band was cut under a 320 nm light source, and the VH, VL, and fragments carrying the heavy and light chain constant regions were recovered according to the DNA gel recovery kit.

[0061] 4. Homologous recombination: Recombination system calculation was performed according to the formula: vector mass (50-100 ng) / vector length (bp) x fragment length (bp) x multiple = x ng (fragment mass). SOSO homologous recombination enzyme was used for recombination at 50°C for 20 min, and then transformed into competent cells. After heat shock, the cells were cultured in an antibiotic-free medium for 1 h, centrifuged at 400 g for 2 min, and then resuspended in 100 μL of antibiotic-free medium. The bacterial solution was spread on solid medium containing ampicillin and cultured for 16 h. On the second day, the bacteria were picked and cultured for 10 h, and then sequenced. The Fer5-G2H expression vector and the Fer5-G2L expression vector were obtained by plasmid extraction from the sequences that were correctly aligned after sequencing. The human-mouse chimeric monoclonal heavy chain complete sequence (SEQ ID NO: 3) and light chain complete sequence (SEQ ID NO: 4) were obtained, and the human-mouse chimeric monoclonal antibody was named Fer5-G2.

[0062] SEQ ID NO: 3: MGWSCIILFLVATATGVHSQVQLVESGPGLVAPSQSLSITCTVSGFSLTGSGVNWVRQPPGKGLEWLGMIWGDGTTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDLVTTGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4: MGWSCIILFLVATATGVHSDIVMTQSHKFMSTSLGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASARHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*.

[0063] Wherein, the specific sequence of variable region and CDR region of monoclonal antibody Fer5-G2 is shown in Table 1.

[0064] Table 1 Sequence of monoclonal antibody Fer5-G2

[0065] Example 5 Transfection and expression purification of monoclonal antibody Fer5-G2 1. Plasmid transfection: after obtaining Fer5-G2H and Fer5-G2L expression vectors in Example 4, Fer5-G2H and Fer5-G2L plasmids were mixed at 1:1 and then transfected into 100 mL 293F cells to express monoclonal antibody Fer5-G2, and the cell culture solution was collected after 6 days.

[0066] 2. Culture solution pretreatment: after centrifuging the cell culture solution at 4800 rpm for 10 min in a horizontal centrifuge, the supernatant was filtered using a 0.45 μm filter to obtain the filtered culture solution.

[0067] 3. Incubation before purification: 10× Buffer A solution was prepared using 0.15 M NaCl and 20 mM Na2HPO4, 10× Buffer was added to the filtered culture solution at a ratio of filtered culture solution: 10× Buffer = 9:1, and 50 μL Protein A filler was added to the filtered culture solution, which was then incubated at 25°C for 2 h.

[0068] 4. Purification: assemble a gravity column, add 200 μL Protein A filler after wetting the filter pad, equilibrate the filler with 10× Buffer to 1×, and then pass 10 mL through the column to equilibrate the filler. After equilibration, pass the incubated sample through the column twice, then wash the column with 10 mL of 1× Buffer, and then elute with 4 mL of 0.1 M glycine (pH = 3.0), and then add 440 μL of Tris-HCl (pH = 8.0) after elution.

[0069] 5. Ultrafiltration: After purification, use 10 KDa ultrafiltration column to concentrate and use PBS buffer to replace twice, collect the ultrafiltrate as the concentrated antibody.

[0070] 6. Results: SDS-PAGE detected the monoclonal antibody Fer5-G2 purified by gravity column affinity chromatography, the purity was more than 99% (Figure 6). Figure 4 ).

[0071] Example 6 Determination of the binding ability of monoclonal antibody Fer5-G2 to IFN-γ antigen The specificity of monoclonal antibody Fer5-IFN-γ was verified by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: The ferret recombinant IFN-γ protein obtained in Example 1 was coated on an enzyme-labeled plate at a concentration of 1 μg / ml, washed and blocked, and then different concentrations of monoclonal antibody Fer5-G2 were added to the enzyme-labeled plate for incubation, washing and adding horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody for incubation, and then washing and adding 3,3',5,5'-TMB substrate solution for color development reaction. Finally, hydrochloric acid solution was added to terminate the reaction, and the OD 450 value and the OD 630 value were determined on an enzyme-labeled instrument, and the OD 450 value minus the OD 630 value was recorded as the measurement result. Logistic four-parameter fitting was used to draw a curve, and the EC 50 value of the antibody was calculated, and the results are shown in Figure 5 .

[0072] According to Figure 5 It can be seen that the monoclonal antibody Fer5-G2 provided by the present application as a detection antibody, the R 2 of the standard curve obtained when the antigen working concentration is 1 μg / ml is 0.99, and the fitting effect is good, indicating that the monoclonal antibody Fer5-G2 can be used as a detection antibody to detect the ferret IFN-γ level, and the detection effect is good. The EC 50 of the monoclonal antibody Fer5-G2 is less than 1.131 μg / mL.

[0073] Example 7 Double antibody sandwich ELISA detection efficiency The double antibody sandwich ELISA detection system was optimized by chessboard titration method, and the specific steps are as follows: 1. Purify the serum by centrifugation to obtain Fer5-serum-IgG, and the steps are as follows: (1) Balance Protein A beads, take 100 μL Protein A beads into 1.5 mL centrifuge tube, add 1 mL PBS, centrifuge and discard the supernatant; (2) Then add 200 μL serum of Example 2 mice after immunization into a new 1.5 mL centrifuge tube, add 1 mL 1xPBS and 100 μL balanced beads, incubate at room temperature for 1 h (shake); (3) Then centrifuge at 2500 g or ≤4000 rpm for 2-5 min, collect the supernatant (equivalent to flow-through); (4) Add 1 mL PBS again, mix well, centrifuge at 2500 g or ≤4000 rpm for 2-5 min, and aspirate the supernatant (collect separately, equivalent to wash waste liquid); (5) Repeat the above steps; (6) Then add 200 μL eluent, mix well by blowing, incubate for 5 min, and centrifuge to collect the supernatant; (7) Repeat step (6) three times, collect the eluent each time, measure the concentration of each collected eluent (use Buffer B as blank), if the concentration is high, continue to repeat step (6); (8) After elution, wash with 1 mL PBS three times, then wash with 1 mL pure water three times, finally resuspend in 20% ethanol, and store at 4°C; (9) Add 5%-10% Tris HCl of elution volume to the eluent for neutralization; (10) Concentrate with ultrafiltration concentration tube, centrifuge 3-5 times, 10 min each time, measure the concentration after concentration; (11) Identify by SDS-PAGE gel electrophoresis.

[0074] 2. Coating antibody concentration gradient: 0.5, 2, 5 μg / mL (Fer5-serum-IgG and Fer5-G2); 100 μL / well, incubate overnight at 4°C; coating buffer is 0.05 mol / L pH 9.6 carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, 950 mL distilled water, adjusted to pH 9.6, and brought to a final volume of 1000 mL); wash the ELISA plate with a BIO-RAD plate washer, adding 300 μL PBST per well, washing 5 times; add blocking buffer (5% skim milk), 200 μL / well, and block at 37°C for 2 h; wash the ELISA plate with a BIO-RAD plate washer, adding 300 μL per well. Wash 5 times with PBST; add IFN-γ antigen (recombinant ferret IFN-γ protein obtained in Example 1) in two or five-fold gradients, starting with an antigen concentration of 1, 10 μg / mL, 50 μL / well, to allow antigen-antibody binding, and incubate at 37°C for 1 h. The negative control is the same volume of PBS. Wash the ELISA plate 5 times with 300 μL PBST per well using a BIO-RAD plate washer; add primary antibody Fer5-serum-IgG-biotin at concentrations of 0.5, 1, 2, 2.5, and 5 μg / mL, 50 μL / well; add HRP-streptavidin secondary antibody, incubate at 37°C for 30 min, 100 μL / well, 37°C for 30 min; wash the ELISA plate 5 times with 300 μL PBST per well using a BIO-RAD plate washer; add 100 μL of substrate TMB per well and incubate in the dark for 15 min; add 50 μL of 2M... The reaction was terminated with H2SO4, and the OD was measured using a microplate reader. 450 Value and OD 630 Value, in OD 450 Value minus OD 630 The value is recorded as the measurement result.

[0075] 3. Using the double-antibody sandwich method described above, the OD of each well was measured. 450 -OD 630 The results of the P / N values ​​showed that the highest P / N values ​​were obtained when Fer5-G2 monoclonal antibody was coated at 5 μg / mL and biotin-labeled serum antibody at a concentration of 0.5–1 μg / mL was used as the detection antibody (Table 2).

[0076] Table 2. Optimization results of the Fer5-G2 monoclonal antibody sandwich ELISA system.

[0077] 4. In order to verify the above results, the primary antibody concentration optimization was carried out using the above double antibody sandwich method, specifically: 100 μL / well, 5 μg / mL Fer5-G2 was used for antibody coating, the IFN-γ antigen concentration was 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 μg / mL, and 50 μL of 1 μg / mL Fer5-serum-IgG-biotin and 0.5 μg / mL Fer5-serum-IgG-biotin were used as primary antibody detection respectively; The standard curve generated with 1 μg / mL Fer5-serum-IgG-biotin as primary antibody was: y=5.0056x+0.0034, R 2 =0.8893, the logistic curve fitting (log2) equation was: Y=0.073+(2.328-0.073) / [1+e^(-2.705*X-6.820)] (R Figure 6 The standard curve generated with 0.5 μg / mL Fer5-serum-IgG-biotin as primary antibody was: y=4.3411x-0.1262, R 2 =0.9671, the logistic curve fitting (log2) equation was: Y=0.036+(2.268-0.036) / [1+e^(-2.702*X-5.571)] (R Figure 7 ).

[0078] According to Figure 6 and Figure 7 , it can be seen that when 0.5 μg / mL Fer5-serum-IgG-biotin is used as primary antibody, the standard curve shows a better linear relationship (R 2 =0.9671), and has a higher goodness of fit than the 1 μg / mL primary antibody concentration (R 2 =0.8893).

[0079] 5. In order to verify the above results, the detection antibody (Fer5-serum-IgG-biotin) optimization was carried out using the above double antibody sandwich method, specifically: 100 μL / well, 5 μg / mL monoclonal antibody Fer5-G2 was used for antibody coating, 1 μg / mL IFN-γ antigen was added, and gradient diluted Fer5-serum-IgG-biotin (2-fold gradient dilution starting from 10 μg / mL) was used as detection antibody. The results are shown in Figure 8 , the EC 50 =0.11 μg / mL.

[0080] According to the above, it can be seen that either the mouse antibody (Fer5-IFN-γ) directly expressed and purified from the supernatant of the hybridoma or the Fer5-G2 after human-mouse chimeric modification can effectively bind to the antigen at a lower concentration, and the effect is better when the concentration of the antibody Fer5-serum-IgG-biotin is 0.5 μg / mL than when the concentration is 1 μg / mL.

[0081] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A recombinant IFN-γ monoclonal antibody of ferrets, characterized in that, The light chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 6, the amino acid sequence of the light chain complementarity determining region CDR2 comprises WAS, and the light chain complementarity determining region CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 7; The heavy chain complementarity determining region CDR1 of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 9, the heavy chain complementarity determining region CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 10, and the heavy chain complementarity determining region CDR3 comprises the amino acid sequence as shown in SEQ ID NO:

11.

2. The recombinant IFN-γ monoclonal antibody of the ferret according to claim 1, characterized by, The light chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 5; The heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO:

8.

3. The recombinant IFN-γ monoclonal antibody of the ferret according to claim 1, characterized by, The heavy chain of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 3; The light chain of the ferret recombinant IFN-γ monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO:

4.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule of claim 4, wherein, The sequence in the nucleic acid molecule encoding the light chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises the nucleotide sequence as shown in SEQ ID NO: 12; and the sequence in the nucleic acid molecule encoding the heavy chain variable region of the ferret recombinant IFN-γ monoclonal antibody comprises the nucleotide sequence as shown in SEQ ID NO:

15.

6. A biological material expressing the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1 to 3, characterized in that, The biological material comprises a recombinant vector or a recombinant cell.

7. A ferret recombinant IFN-γ protein for preparing the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1 to 3, comprising the amino acid sequence as shown in SEQ ID NO:

4.

8. Use of the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1 to 3, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule according to claim 4 or 5, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material according to claim 6, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein according to claim 7, in one or more of the following: (1) preparing a product for detecting ferret IFN-γ; (2) preparing a product for diagnosing ferret respiratory virus infection; (3) preparing a product for evaluating the efficacy of ferret respiratory virus infection vaccine; (4) preparing a product for studying Th1 type immune response of ferret respiratory virus infection.

9. A kit for detecting IFN-γ in ferrets, characterized by, The coated antibody and the detection antibody are included. The coating antibody is the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1-3, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule according to claim 4 or 5, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material according to claim 6, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein according to claim 7. The detection antibody is a tracer-labeled Fer5-serum-IgG; the Fer5-serum-IgG is serum obtained after immunizing an animal with the ferret recombinant IFN-γ protein according to claim 7.

10. A method for detecting IFN-γ in ferrets, comprising contacting a sample with an antibody according to any one of claims 1 to 9. The method comprises the following steps: mixing a test sample, a detection antibody and a coating antibody to form a complex, and then detecting a tracer signal in the complex; The coating antibody is the ferret recombinant IFN-γ monoclonal antibody according to any one of claims 1-3, or the ferret recombinant IFN-γ monoclonal antibody encoded by the nucleic acid molecule according to claim 4 or 5, or the ferret recombinant IFN-γ monoclonal antibody expressed by the biological material according to claim 6, or the ferret recombinant IFN-γ monoclonal antibody prepared by using the ferret recombinant IFN-γ protein according to claim 7. The detection antibody is a tracer-labeled Fer5-serum-IgG; the Fer5-serum-IgG is serum obtained after immunizing an animal with the ferret recombinant IFN-γ protein according to claim 7.

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