Anti-canine IFN-alpha monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an anti-canine IFN-alpha monoclonal antibody and application thereof. The monoclonal antibody comprises a monoclonal antibody 1C1 or an antigen binding part of the monoclonal antibody 1C1 and a monoclonal antibody 2A4 or an antigen binding part of the monoclonal antibody 2A4, and the monoclonal antibody and the antibody combination thereof can be used for detecting the canine interferon after being carefully screened and optimized, and have extremely high specificity and sensitivity. And the ELISA kit developed by the formed antibody combination not only simplifies the detection process, but also improves the detection efficiency. The capture antibody and the detection antibody in the kit can achieve a synergistic effect, high-precision detection of the canine interferon can be achieved, the detection sensitivity is high, the specificity is excellent, and interference of non-specific reaction is effectively avoided. The technology breakthrough shows wide development potential and practical application prospects, and provides a powerful tool for canine health monitoring, disease diagnosis and related research.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a monoclonal antibody against canine IFN-α and application thereof. Background Art

[0002] Interferon is an effective antiviral substance that can inhibit the replication of the genetic material of RNA viruses and DNA viruses, thereby interfering with the proliferation of these viruses in cells. Regarding the antiviral mechanism of interferon, there are clear research results. Under normal physiological conditions, interferon in cells remains silent and is not expressed. However, when the virus invades the body, it triggers the expression mechanism of interferon. Subsequently, interferon will quickly bind to specific receptors on the surface of susceptible cells, and through a series of signal transduction processes, transmit the signal to the cell, thereby activating a series of complex biological reaction systems, showing its powerful antiviral effect. In addition, interferon also plays an active biological role in inhibiting tumor growth and preventing the proliferation of tumor viruses.

[0003] According to the latest international classification standards, interferon is clearly divided into three types: Type I, Type II and Type III. Type I interferon is further subdivided into multiple subtypes such as α, β, τ, ω, etc. based on the differences in its antibody binding antigenicity. As early as 1987, researchers began to explore the canine interferon-α (cIFN-α) gene and successfully achieved the expression of its protein, verifying the significant effectiveness of cIFN-α in the prevention and treatment of pet diseases.

[0004] With the significant improvement in people's quality of life in recent years, the number of pet dogs has increased dramatically, and the related pet medical needs have also increased year by year. In view of the strong antiviral ability of cIFN-α, the applicant unit focuses on the research and development of cIFN-α related preparations for the treatment of canine diseases. At the same time, we used cIFN-α as an antigen to immunize animals and successfully prepared monoclonal antibodies. On this basis, we developed a cIFN-α detection kit suitable for a variety of scenarios, which provides strong support for the convenient and accurate detection of cIFN-α. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a monoclonal antibody against canine IFN-α and its application.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides an anti-canine IFN-α monoclonal antibody 1C1 or an antigen binding site thereof, comprising heavy chain complementary determining regions HC-CDR1, HC-CDR2, HC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 1 to 3 or homologous sequences thereof, and light chain complementary determining regions LC-CDR1, LC-CDR2, LC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 4 to 6 or homologous sequences thereof;

[0008] The homology between the homologous sequence and the original sequence is more than 80%, the same below;

[0009] The heavy chain variable region VH of the monoclonal antibody 1C1 antibody or its antigen-binding site has an amino acid sequence as shown in SEQ ID NO.7 or a homologous sequence thereof, and the light chain variable region VL has an amino acid sequence as shown in SEQ ID NO.8 or a homologous sequence thereof.

[0010] The present invention also provides an anti-canine IFN-α monoclonal antibody 2A4 or an antigen binding site thereof, comprising heavy chain complementary determining regions HC-CDR1, HC-CDR2, HC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 17 to 19 or homologous sequences thereof, and light chain complementary determining regions LC-CDR1, LC-CDR2, LC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 20 to 22 or homologous sequences thereof;

[0011] The heavy chain variable region VH of the monoclonal antibody 2A4 or its antigen-binding site has an amino acid sequence as shown in SEQ ID NO.23 or a homologous sequence thereof, and the light chain variable region VL has an amino acid sequence as shown in SEQ ID NO.24 or a homologous sequence thereof.

[0012] The present invention also provides a use of an anti-canine IFN-α antibody, wherein the antibody is the above-mentioned monoclonal antibody 1C1 or its antigen binding site, and / or the above-mentioned monoclonal antibody 2A4 or its antigen binding site;

[0013] The antibody is applied to at least one biological detection of enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry and immunopharmaceuticals.

[0014] The present invention also provides a nucleotide sequence encoding a monoclonal antibody, which encodes the monoclonal antibody 1C1 or its antigen binding site as described above, or the monoclonal antibody 2A4 or its antigen binding site as described above;

[0015] The nucleotide sequence includes heavy chain HC-CDR1, HC-CDR2, HC-CDR3 encoding monoclonal antibody 1C1 or its antigen binding site, and the nucleotide sequence thereof is shown in SEQ ID NO.9-11 or its homologous sequence; and light chain LC-CDR1, LC-CDR2, LC-CDR3 encoding monoclonal antibody 1C1 or its antigen binding site, and the nucleotide sequence thereof is shown in SEQ ID NO.12-14 or its homologous sequence;

[0016] The nucleotide sequence includes a heavy chain variable region encoding monoclonal antibody 1C1 or its antigen binding site, the nucleotide sequence of which is shown in SEQ ID NO.15 or its homologous sequence; and a light chain variable region encoding monoclonal antibody 1C1 or its antigen binding site, the nucleotide sequence of which is shown in SEQ ID NO.16 or its homologous sequence;

[0017] The nucleotide sequence includes heavy chain HC-CDR1, HC-CDR2, HC-CDR3 encoding monoclonal antibody 2A4 or its antigen binding site, and the nucleotide sequence thereof is shown in SEQ ID NO.25-27 or its homologous sequence; and light chain LC-CDR1, LC-CDR2, LC-CDR3 encoding antibody 2A4, and the nucleotide sequence thereof is shown in SEQ ID NO.28-30 or its homologous sequence;

[0018] The nucleotide sequence includes a heavy chain variable region encoding monoclonal antibody 2A4 or its antigen binding site, the nucleotide sequence of which is shown in SEQ ID NO.31 or its homologous sequence; and a light chain variable region encoding antibody 2A4, the nucleotide sequence of which is shown in SEQ ID NO.32 or its homologous sequence.

[0019] The nucleotide sequence is used for constructing expression vectors, preparing recombinant antibodies, and preparing gene therapy products and vaccines.

[0020] The present invention also provides a vector, which comprises the above-mentioned nucleotide sequence encoding the monoclonal antibody 1C1 or its antigen binding site, or the monoclonal antibody 2A4 or its antigen binding site.

[0021] The present invention also provides a hybridoma cell, which is obtained by fusing cells capable of expressing canine IFN-α with spleen cells, and the hybridoma cell secretes monoclonal antibody 1C1 or monoclonal antibody 2A4.

[0022] The present invention also provides a host cell, which has been transfected or transformed to contain the above expression vector and express the above monoclonal antibody 1C1 or its antigen binding site, or monoclonal antibody 2A4 or its antigen binding site.

[0023] The present invention also provides a product for detecting canine IFN-α, the composition of the product includes a capture antibody and a detection antibody;

[0024] The capture antibody is the above-mentioned monoclonal antibody 1C1 or its antigen binding site, and the detection antibody is the above-mentioned monoclonal antibody 2A4 or its antigen binding site;

[0025] The capture antibody is fixed to a solid phase carrier, and the capture antibody binds to the antigen;

[0026] The products for detecting canine IFN-α include Elisa kits, test strips or biochips;

[0027] The method for detecting canine IFN-α comprises a double antibody sandwich detection.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention provides monoclonal antibodies against canine IFN-α, including monoclonal antibody 1C1 or its antigen binding site and monoclonal antibody 2A4 or its antigen binding site. The monoclonal antibodies and their antibody combinations are carefully screened and optimized, and can be used for the detection of canine interferon, with extremely high specificity and sensitivity. The highly specific antibody combination formed by the above two monoclonal antibodies can accurately identify and bind to the target interferon, effectively avoid the interference of nonspecific reactions, and improve the accuracy of detection. And the ELISA kit developed by the formed antibody combination not only simplifies the detection process, but also improves the detection efficiency. The capture antibody and the detection antibody in the kit can work synergistically, and can achieve high-precision detection of canine interferon, which not only has high detection sensitivity, but also has excellent specificity, and effectively avoids the interference of nonspecific reactions. This technological breakthrough shows broad development potential and practical application prospects, and provides a powerful tool for canine health monitoring, disease diagnosis and related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a WB detection image of the recombinant protein in Example 1;

[0031] Figure 2 This is the sensitivity analysis diagram of double antibody sandwich Elisa. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0033] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0034] Example 1

[0035] Preparation of canine interferon antigen

[0036] The amino acid sequence of canine interferon-α (cIFN-α) (GenBank sequence number: AEI30863.1) was retrieved and obtained through the NCBI database, and then reverse translation was performed to synthesize the corresponding gene sequence. In order to achieve the expression of cIFN-α, the yeast expression system pPICZαA vector was used for construction, and Pichia pastoris X33 was used as the host cell to successfully express the target protein IFN-α. According to the prediction, the molecular weight of the protein is about 21.35kDa. However, in the actual recombinant protein expression process, due to the influence of glycosylation modification, it was observed that the main protein bands were 2, such as Figure 1 As shown, the recombinant protein was used in subsequent mouse immunization experiments.

[0037] Example 2

[0038] Mouse immunization

[0039] The experiment used Balb / c mice (purchased from the Experimental Animal Management Center of Southern Medical University) as the immunization subjects, all of which were healthy mice of 6 weeks old. The experiment selected cIFN-α prepared in Example 1 as the immunogen, and a total of 20 mice were immunized. The entire immunization process was divided into three times:

[0040] First immunization: Inject the immunogen cIFN-α into mice at a dose of 50 μg / mouse;

[0041] Second immunization: 3 weeks after the first immunization, the immunogen dose was halved to 25 μg / mouse;

[0042] Third immunization: 2 weeks after the second immunization, mice were immunized with 25 μg of the immunogen per mouse.

[0043] One week after the third immunization, blood samples were collected from the mice by eyelid bleeding. Subsequently, the antibody titer of the mice after immunization was detected by indirect ELISA method to evaluate the strength of their immune response.

[0044] According to the test results shown in Table 1, mice with higher antibody titers (high serum dilution multiples and large OD values) were selected as experimental subjects for subsequent spleen cell fusion experiments.

[0045] Table 1 Chessboard method detection results

[0046]

[0047] Example 3

[0048] Screening of monoclonal antibody cell lines

[0049] 1. Mouse spleen cell fusion

[0050] 1.1 Spleen cell collection: Remove the spleen under sterile conditions, peel off the membranous tissue, etc., place it in an incomplete culture medium, use a syringe to extract the incomplete culture medium and inject it into the spleen to release spleen cells, grind the remaining spleen tissue with a cell sieve, collect cells, and count them;

[0051] 1.2SP2 / 0 collection: SP2 / 0 cells are round and translucent. Collect the cells in a 50 mL centrifuge tube and count them.

[0052] 1.3 Adjust the ratio of SP2 / 0 and spleen cells to 1:10-1:3;

[0053] 1.4 Take out the PEG1450 preheated at 37℃, rotate the centrifuge tube, add 1mL of PEG solution within 60s, and let it stand for 1min; slowly add 3mL of DMEM medium within 3min, then gradually increase the dropping speed, and add DMEM medium to a total volume of 30mL within 5min; put it in a 37℃ water bath and let it stand for 5min.

[0054] 1.5 Centrifuge at 1000 rpm for 5 min, remove the supernatant, add appropriate amount of HAT medium to resuspend the cells, plate 200 μL per well in a 96-well cell culture plate (the 96-well plate had been pre-added with peritoneal macrophages the day before, 10,000 cells / well), and culture in a 37°C 5% carbon dioxide incubator.

[0055] 2. Indirect ELISA screening positive wells

[0056] 2.1 Coating: Dilute cIFN-α protein with 1× coating buffer to a 2 μg / mL coating solution, add 50 μL of coating solution to each well of the test plate, and incubate at 2-8°C for at least 12 hours;

[0057] 2.2 Blocking: Wash the coated plate 4 times with TBST, remove the remaining solution, add blocking solution, 100 μL per well, seal, and place at 37°C for 2 hours; wash the plate 4 times with TBST and set aside;

[0058] 2.3 Sample incubation: Take the supernatant of the fused cell culture plate for detection, use the immunized mouse serum as the positive control, the cell culture medium as the negative control, and PBS as the blank control. Load 100 μL into each well and incubate at 37°C for 1 hour.

[0059] 2.4 Secondary antibody incubation: Wash the test plate 4 times with TBST, dilute the goat anti-mouse secondary antibody (Celvi, HRP*Goat Anti-Mouse IgG (H+L)) with blocking solution at 1:10000, load 100 μL per well, incubate at 37°C for 1 hour;

[0060] 2.5 Color development and termination: Wash the test plate 4 times with TBST, remove the remaining liquid, add color development solution, 100 μL per well, and leave at room temperature for 10 minutes; add 50 μL of stop solution to all wells of the ELISA reaction plate;

[0061] 2.6 Reading: The microplate reader reads the absorbance at 450nm;

[0062] 2.7 Select the wells with higher test values ​​as suspected positive wells. After repeated testing the next day, the wells with higher positive values ​​are regarded as positive wells.

[0063] After the above detection, 9 positive wells including 1A1, 1C1, 5E10, 4F10, 3H11, 2A4, 8A9, 12B3 and 10C12 were screened.

[0064] 3. Monoclonalization

[0065] 3.1 Round 1 monoclonal cloning: Collect cells from the above positive wells, dilute the cells to single cell / 200 μL using monoclonal cell culture medium, add the cell suspension to a 96-well cell culture plate, and after 8 days of culture, detect using the above indirect ELISA method;

[0066] 3.2 Second round of monocloning: Analyze the results of the first round of subcloning ELISA test. If the positive well rate is 100%, it can be determined that the cell purity is sufficient. Otherwise, select the positive wells for this round of monocloning. The second round of monocloning operation is the same as the first round of monocloning.

[0067] 3.3 After multiple rounds of monocloning of the above positive well cells, if the purity of the hybridoma cells reaches 100%, the monocloning can be terminated.

[0068] The cell lines obtained by the above screening were named 1A1, 1C1, 5E10, 4F10, 3H11, 2A4, 8A9, 12B3 and 10C12 cell lines according to the plate number and well number, and the corresponding antibodies were named 1A1, 1C1, 5E10, 4F10, 3H11, 2A4, 8A9, 12B3 and 10C12 antibodies.

[0069] Example 4

[0070] Screening of paired antibodies

[0071] The cell lines were injected into the peritoneal cavity of mice, with 5 mice injected for each cell line, and the ascites was collected and the antibodies were purified using a protein A affinity chromatography column. The purified antibodies were labeled with biotin and used as detection antibodies, and the purified unlabeled antibodies were used as coating antibodies. The sandwich antibody pairing combination was screened according to the following sandwich Elisa experimental process and with reference to the specific operation details such as the indirect Elisa plate washing in Example 3.

[0072] 4.1 The capture antibody was coated at 100 ng / well and incubated at 2-8°C for at least 12 hours;

[0073] 4.2 Canine interferon 5 μg / mL × 50 μL, incubate at 37℃ for 1 hour;

[0074] 4.3 Load 100 μL of detection antibody into each well (the titer of the detection antibody needs to be adjusted to be consistent before loading) and incubate for 1 hour;

[0075] 4.4HRP-labeled streptavidin (CellVision, C050109-HRP-SA) was diluted 1:4000, 100 μL was loaded into each well, and incubated at 37°C for 1 h;

[0076] 4.5 Color development and termination;

[0077] 4.6 Reading: The absorbance value was read at 450 nm using a microplate reader.

[0078] Table 2 Chessboard method detection results

[0079]

[0080]

[0081] As can be seen from Table 2, 1C1 as the capture antibody and 2A4 as the detection antibody have the best detection effect. 1C1 and 2A4 antibodies can be used as an antibody combination for the detection of cIFN-α.

[0082] The selected antibody combination was tested as follows:

[0083] 1. Specificity detection

[0084] In order to test the specificity of the combination of 1C1 and 2A4 antibodies, the present invention selected 2 healthy dog ​​sera, 2 canine parvovirus-infected dog sera, 2 feline calicivirus-infected cat sera, and 2 swine fever-infected domestic pig sera as test samples, set PBS as a negative control, and 1 μg / mL cIFN-α solution as a positive control. According to the sandwich Elisa experimental process in Example 4, the sample detection was performed, and the detection results are as shown in the following table:

[0085] Table 3 Specificity detection OD values

[0086]

[0087]

[0088] According to the standard of S / N ≥ 2.1 (S is the OD value of the test sample, N is the OD value of the negative sample) as a positive well, otherwise as a negative well, it can be seen from Table 3 that the S / N of healthy dog ​​serum and canine parvovirus-infected dog serum is > 2.1, while the S / N of feline calicivirus-infected cat serum and swine fever-infected domestic pig serum is < 2, indicating that the combination of 1C1 and 2A4 antibodies is specific for cIFN-α detection.

[0089] Based on the above pairing experiment results and specific detection results, the antibody combination of 1C1 and 2A4 can be developed as a detection reagent for cIFN-α. To this end, antibody sequencing and optimization of the detection conditions of the antibody combination were subsequently carried out.

[0090] 2. Antibody Sequence Determination

[0091] The purified 1C1 and 2A4 hybridoma cell lines were amplified and collected to obtain more than 1×10 6 After cell lysis, mRNA was extracted and reverse transcribed into cDNA. After antibody gene amplification, it was cloned into a vector. After Sanger sequencing, the nucleotide sequence of the antibody variable region was obtained. After bioinformatics analysis, the capture antibody related sequence was obtained as shown in Table 4, and the detection antibody related sequence was obtained as shown in Table 5:

[0092] Table 4 Capture antibody information

[0093]

[0094]

[0095] Table 5 Information on detection antibodies

[0096]

[0097]

[0098] Note: The naming of the relevant amino acids and nucleic acids in the capture antibody and detection antibody in Table 4 and Table 5 are the same, but the sequences are different;

[0099] The amino acid sequence of the above SEQ ID NO.5 is: KVS;

[0100] The nucleotide sequence of SEQ ID NO.13 is: AAAGTTTCC;

[0101] The amino acid sequence of SEQ ID NO.21 is: WAS;

[0102] The nucleotide sequence of the above SEQ ID NO.29 is: TGGGCATCC.

[0103] The capture antibody and the detection antibody also include sequences with more than 80% homology to the original sequence.

[0104] 3. Optimization of detection conditions

[0105] 3.1 Determination of the optimal working concentration of antibodies

[0106] 1) Dilute the purified 1C1 monoclonal antibody to different concentrations (1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL);

[0107] The conjugated 2A4 monoclonal antibody was diluted to 4 dilutions (1:1600, 1:3200, 1:6400, and 1:12800) starting from 1:1600;

[0108] 2) 1C1 monoclonal antibody was coated at different concentrations, 100 μL / well, at 4°C overnight.

[0109] 3) Wash the plate with buffer 3 times and pat dry;

[0110] 4) Antigen incubation: 0.01 μg / mL cIFN-α, 100 μL / well, incubate at 37°C for 1 h;

[0111] 5) Wash the plate as in step 3);

[0112] 6) Add 4 dilutions of labeled monoclonal antibody, 100 μL / well, and incubate at 37°C for 1 h;

[0113] 7) Wash the plate, same as step 3);

[0114] 8) HRP-labeled streptavidin (CellVision, C050109-HRP-SA) diluted 1:4000, 100 μL / well, incubated at 37°C for 1 h;

[0115] 9) Wash the plate, same as step 3);

[0116] 10) Color display and reading.

[0117] According to the above steps, the results are shown in Table 6. It was finally concluded that 4 μg / mL of 1C1 monoclonal antibody was the optimal coating concentration, and 1:6400 was the optimal detection concentration of 2A4 monoclonal antibody. Under this condition, the antigen detection value was the highest.

[0118] Table 6 Determination of optimal antibody concentration by chessboard titration method

[0119]

[0120]

[0121] 3.2 Determination of antibody incubation time

[0122] According to the above optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, different antigen incubation times (15min, 30min, 45min, 1h, 1.5h) were set, and other conditions were fixed. 0.01μg / mL cIFN-α was tested. The results are shown in the following table:

[0123] Table 7 Determination of optimal antigen incubation time

[0124] Incubation time 15min 30min 45min 1h 1.5h S / N 7.65±3.38 17.36±1.36 20.11±0.96 23.33±1.08 20.13±1.23

[0125] As can be seen from Table 7, the optimal antigen incubation time determined by the S / N value is 1 h.

[0126] According to the above-mentioned optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, as well as the optimal antigen incubation time, different detection antibody incubation times (15min, 30min, 45min, 1h, 1.5h) were set to detect 0.01μg / mL cIFN-α. The results are shown in the following table:

[0127] Table 8 Determination of optimal detection antibody incubation time

[0128] Incubation time 15min 30min 45min 1h 1.5h S / N 9.97±2.29 19.53±1.27 24.61±1.35 21.06±2.01 19.95±2.31

[0129] As can be seen from Table 8, the optimal detection antibody incubation time determined by the S / N value is 45 min.

[0130] According to the above-mentioned optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, as well as the optimal antigen and detection antibody incubation time, different HRP-labeled streptavidin incubation times were set to detect 0.01 μg / mL cIFN-α. The results are shown in the following table:

[0131] Table 9 Determination of optimal streptavidin incubation time

[0132] Incubation time 15min 30min 45min 1h 1.5h S / N 16.31±4.53 25.06±2.03 24.69±1.84 21.27±0.95 20.57±1.34

[0133] As shown in Table 9, the optimal HRP-labeled streptavidin incubation time was determined to be 30 min and 45 min according to the S / N value.

[0134] 4. Sensitivity test

[0135] The above paired antibodies and optimized conditions were used, i.e., 4 μg / mL of 1C1 monoclonal antibody and 1:6400 dilution of 2A4 monoclonal antibody were used to detect cIFN-α. cIFN-α was prepared into different concentrations for sensitivity testing. The test results are shown in the following table:

[0136] Table 10 Sensitivity test results

[0137]

[0138] As shown in Table 10, when the concentration of cIFN-α is as low as 0.078 ng / mL, S / N>2.1, it can be seen that the above paired antibodies can detect 78 pg / mL of cIFN-α, and the sensitivity is relatively high. As shown in Table 10, there is a good linear relationship in the range of antigen concentration of 0.078-2.5 ng / mL, and the equation y=0.771x+0.1559, R2=0.9933 can be generated within this range. Figure 2 As shown, the horizontal axis represents the antigen concentration value, and the vertical axis represents the absorbance value.

[0139] 5. Detection of drug residue after in vivo injection of recombinant cIFN-α preparation

[0140] In order to detect the detection effect of the double antibody combination after the above-mentioned optimized experimental conditions, further tests were conducted. The highly active interferon α recombinant protein in the applicant's previous patent CN112079912B (corresponding to SEQID NO.3 in the CN112079912B text) was injected into dogs 1 and 2 weighing about 10 kg at a dose of 0.1 mg / dog. After injection, venous blood samples were collected at 1h, 2h, 4h, 8h, 16h, 24h and 48h respectively, and the supernatant was centrifuged for detection, and PBS was used as a negative control.

[0141] The antibody combination of 1C1 monoclonal antibody at 4 μg / mL and 2A4 monoclonal antibody at a dilution factor of 1:6400 was used to detect the residual amount of recombinant cIFN-α preparation after in vivo injection. The test results are as follows:

[0142] Table 11 Results of drug residue detection after in vivo injection of recombinant cIFN-α preparation

[0143]

[0144] After the recombinant protein was injected into the dog's body, as time went on, degradation increased and the residual amount decreased. After a certain period of time, the level of interferon-α in the dog's serum was close to the basal expression level. As can be seen from Table 11, as time went on, the OD value gradually decreased. The test results were consistent with expectations, indicating that the dual antibody combination (1C1 monoclonal antibody + 2A4 monoclonal antibody) of the present invention can be used for the detection of residual drug dosage after in vivo injection of cIFN-α preparations, and has good practical value.

Claims

1. An anti-canine IFN-α monoclonal antibody 1C1 or an antigen binding site thereof, characterized in that: The monoclonal antibody 1C1 antibody or its antigen binding site includes heavy chain complementary determining regions HC-CDR1, HC-CDR2, HC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 1 to 3 or their homologous sequences, and light chain complementary determining regions LC-CDR1, LC-CDR2, LC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 4 to 6 or their homologous sequences.

2. The anti-canine IFN-α monoclonal antibody 1C1 or its antigen binding site according to claim 1, characterized in that: The heavy chain variable region VH of the monoclonal antibody 1C1 antibody or its antigen-binding site has an amino acid sequence as shown in SEQ ID NO.7 or a homologous sequence thereof, and the light chain variable region VL has an amino acid sequence as shown in SEQ ID NO.8 or a homologous sequence thereof.

3. An anti-canine IFN-α monoclonal antibody 2A4 or an antigen binding site thereof, characterized in that: The monoclonal antibody 2A4 or its antigen binding site includes heavy chain complementary determining regions HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 17 to 19 or their homologous sequences, and light chain complementary determining regions LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in SEQ ID NOs. 20 to 22 or their homologous sequences.

4. The anti-canine IFN-α monoclonal antibody 2A4 or its antigen binding site according to claim 3, characterized in that: The heavy chain variable region VH of the monoclonal antibody 2A4 or its antigen-binding site has an amino acid sequence as shown in SEQ ID NO.23 or a homologous sequence thereof, and the light chain variable region VL has an amino acid sequence as shown in SEQ ID NO.24 or a homologous sequence thereof.

5. An application of an antibody against canine IFN-α, characterized in that: The antibody comprises the monoclonal antibody 1C1 or the antigen binding site thereof according to claim 1 or 2, and / or the monoclonal antibody 2A4 or the antigen binding site thereof according to claim 3 or 4; The anti-canine IFN-α antibody is applied to at least one biological detection of enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry and immune medicine.

6. A nucleotide sequence encoding a monoclonal antibody, characterized in that: It encodes the monoclonal antibody 1C1 or its antigen binding site according to claim 1 or 2, or the monoclonal antibody 2A4 or its antigen binding site according to claim 3 or 4; The nucleotide sequence is used for constructing expression vectors, preparing recombinant antibodies, and preparing gene therapy products and vaccines.

7. A carrier, characterized in that The vector comprises the nucleotide sequence encoding the monoclonal antibody according to claim 6.

8. A hybridoma cell, characterized in that The hybridoma cell is obtained by fusing cells capable of expressing canine IFN-α with spleen cells, and the hybridoma cell secretes the monoclonal antibody 1C1 according to claim 1 or 2, or the monoclonal antibody 2A4 according to claim 3 or 4.

9. A host cell, characterized in that It has been transfected or transformed to contain the vector of claim 7 and expresses the monoclonal antibody 1C1 or its antigen binding site of claim 1 or 2, or the monoclonal antibody 2A4 or its antigen binding site of claim 3 or 4.

10. A product for detecting canine IFN-α, characterized in that: The composition of the product includes capture antibody and detection antibody; The capture antibody is the monoclonal antibody 1C1 described in claim 1 or 2, and the detection antibody is the monoclonal antibody 2A4 described in claim 3 or 4; The capture antibody is fixed to a solid phase carrier, and the capture antibody binds to the antigen; The products for detecting canine IFN-α include Elisa kits, test strips or biochips; The method for detecting canine IFN-α comprises a double antibody sandwich detection.

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