Monoclonal antibody against canine ifn-alpha and use thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody against canine IFN-alpha and application thereof. The monoclonal antibody comprises a monoclonal antibody 1C1 or an antigen binding site thereof and a monoclonal antibody 2A4 or an antigen binding site thereof, and the monoclonal antibody and the antibody combination thereof are carefully screened and optimized, can be used for detection of canine interferon, and have extremely high specificity and sensitivity. An ELISA kit developed by the antibody combination not only simplifies a detection process, but also improves detection efficiency. The capture antibody and the detection antibody in the kit can synergistically act, can realize high-precision detection of the canine interferon, are not only high in detection sensitivity, but also excellent in specificity, effectively avoid interference of non-specific reactions. The technical breakthrough exhibits broad development potential and practical application prospect, and provides a powerful tool for canine health monitoring, disease diagnosis and related research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody against canine IFN-alpha and application thereof. BACKGROUND

[0002] Interferon is an effective antiviral substance, which can inhibit the replication process of the genetic material of RNA viruses and DNA viruses, and further interfere with the proliferation of these viruses in cells. At present, there are clear research results on the antiviral mechanism of interferon. Under normal physiological conditions, the interferon in the cell remains silent and does not express. However, when viruses invade the body, the expression mechanism of interferon will be triggered. Subsequently, interferon will rapidly combine with specific receptors on the surface of susceptible cells, transmit signals to the cells through a series of signal transduction processes, and then activate a series of complex biological reaction systems, thereby showing its strong antiviral efficacy. In addition, interferon also plays a positive biological role in inhibiting tumor growth and preventing tumor virus proliferation.

[0003] According to the latest international classification standard, interferon is clearly divided into Type I, Type II and Type III, and Type I interferon is further divided into alpha, beta, tau and omega subtypes according to the differences in antibody binding antigenicity. As early as 1987, researchers began to explore the canine interferon-alpha (cIFN-alpha) gene, and successfully expressed the protein, verifying the significant effect of cIFN-alpha in the prevention and treatment of pet diseases.

[0004] With the significant improvement of people's quality of life in recent years, the number of pet dogs has increased dramatically, and the demand for pet medical treatment has also increased year by year. In view of the strong antiviral ability of cIFN-alpha, the applicant unit focuses on the research and development of cIFN-alpha related preparations for the treatment of canine diseases. At the same time, we use cIFN-alpha as an antigen to immunize animals, successfully prepare monoclonal antibodies, and develop cIFN-alpha detection kits suitable for various scenes on this basis, providing strong support for the convenient and accurate detection of cIFN-alpha. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide a monoclonal antibody against canine IFN-alpha and application thereof.

[0006] The technical content of the present application is as follows:

[0007] The application provides a monoclonal antibody 1C1 or an antigen binding site thereof against canine IFN-alpha, which comprises heavy chain complementary determining regions HC-CDR1, HC-CDR2 and HC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 1-3 or homologous sequences thereof, and light chain complementary determining regions LC-CDR1, LC-CDR2 and LC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 4-6 or homologous sequences thereof.

[0008] The homologous sequences have a homology of more than 80% with the original sequences, and the same applies below.

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

[0010] The application also provides a monoclonal antibody 2A4 or an antigen binding site thereof against canine IFN-alpha, which comprises heavy chain complementary determining regions HC-CDR1, HC-CDR2 and HC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 17-19 or homologous sequences thereof, and light chain complementary determining regions LC-CDR1, LC-CDR2 and LC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 20-22 or homologous sequences thereof.

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

[0012] The application also provides an antibody against canine IFN-alpha, which is the monoclonal antibody 1C1 or the antigen binding site thereof, and / or the monoclonal antibody 2A4 or the antigen binding site thereof.

[0013] The antibody is applied to at least one biological detection in enzyme-linked immunoassay, immunofluorescence, flow cytometry, immunohistochemistry and immunodrug.

[0014] The application also provides a nucleotide sequence encoding a monoclonal antibody, which encodes the monoclonal antibody 1C1 or the antigen binding site thereof, or the monoclonal antibody 2A4 or the antigen binding site thereof.

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

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

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

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

[0019] The nucleotide sequence is used in the construction of an expression vector, the preparation of a recombinant antibody, the preparation of a gene therapy product, and the preparation of a vaccine.

[0020] The present application also provides a vector comprising the above-mentioned nucleotide sequence encoding monoclonal antibody 1C1 or an antigen binding site thereof, or monoclonal antibody 2A4 or an antigen binding site thereof.

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

[0022] The present application also provides a host cell that has been transfected or transformed to contain the above-mentioned expression vector and express the above-mentioned monoclonal antibody 1C1 or an antigen binding site thereof, or monoclonal antibody 2A4 or an antigen binding site thereof.

[0023] The application also provides a product for detecting canine IFN-α, and the composition of the product comprises a capture antibody and a detection antibody.

[0024] The capture antibody is the monoclonal antibody 1C1 or an antigen binding site thereof, and the detection antibody is the monoclonal antibody 2A4 or an antigen binding site thereof.

[0025] The capture antibody is immobilized on a solid carrier, and the capture antibody is combined with the antigen.

[0026] The product for detecting canine IFN-α comprises an Elisa kit, a detection test strip or a biochip.

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

[0028] The application has the following beneficial effects:

[0029] The application provides monoclonal antibodies against canine IFN-α, including the monoclonal antibody 1C1 or an antigen binding site thereof and the monoclonal antibody 2A4 or an antigen binding site thereof, and the combination of the monoclonal antibodies and the antibodies thereof is carefully screened and optimized, and can be used for detecting canine interferon and has extremely high specificity and sensitivity. The high-specificity antibody combination formed by the two monoclonal antibodies can accurately recognize and bind the target interferon, effectively avoids the interference of non-specific reactions, and improves the accuracy of detection. The ELISA kit developed by the 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 synergistically act to realize high-precision detection of canine interferon, have high detection sensitivity and excellent specificity, and effectively avoid the interference of non-specific reactions. This technical breakthrough has broad development potential and practical application prospect, and provides a powerful tool for canine health monitoring, disease diagnosis and related research. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a WB detection graph of the recombinant protein in Example 1.

[0031] Figure 2 It is a double antibody sandwich Elisa sensitivity analysis graph. DETAILED DESCRIPTION

[0032] The application will be further described in detail through specific implementation examples and the description of the drawings, and it should be understood that the examples are only used to illustrate the application and are not used to limit the protection scope of the application, and various equivalent modifications of the application made by those skilled in the art after reading the application all fall within the scope defined by the claims of the application.

[0033] All raw materials and reagents of the present application are conventional market raw materials and reagents unless otherwise specified.

[0034] Example 1

[0035] Preparation of canine interferon antigen

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

[0037] Example 2

[0038] Mouse immunization

[0039] Balb / c strain mice (purchased from the experimental animal management center of Southern Medical University) were used as immunization objects in the experiment, and all were 6-week-old healthy mice. The cIFN-a prepared in Example 1 was selected as the immunogen for immunization of a batch of 20 mice, and the whole immunization process was divided into three times:

[0040] First immunization: according to the dose of 50 μg per mouse, the immunogen cIFN-a was injected into the mice;

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

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

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

[0044] According to the detection results shown in Table 1, the mice with higher antibody titer (high serum dilution multiple and large OD value) were selected as the experimental objects for the subsequent spleen cell fusion experiment.

[0045] Table 1 Checkerboard detection results

[0046]

[0047] Example 3

[0048] Screening of monoclonal antibody cell strains

[0049] 1. Mouse spleen cell fusion

[0050] 1.1 Spleen cell sampling: Under sterile conditions, the spleen was removed, and the membranous tissue was peeled off and placed in incomplete medium. The spleen cells were separated by injecting incomplete medium into the spleen with a syringe. The remaining spleen tissue was ground, and the cells were collected and counted.

[0051] 1.2 SP2 / 0 collection: The SP2 / 0 was round and transparent, and the cells were collected in a 50 mL centrifuge tube and counted.

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

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

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

[0055] 2. Indirect ELisa screening of positive wells

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

[0057] 2.2 Blocking: Wash the coated plate with TBST 4 times, remove the remaining liquid, add blocking solution to each well, 100 μL, add seal, and incubate at 37°C for 2 hours; wash the plate with TBST 4 times and reserve;

[0058] 2.3 Sample incubation: Take the supernatant of the cell culture plate after fusion for detection, use the serum of the immunized mouse as a positive control, use cell culture medium as a negative control, and use PBS as a blank control, add 100 μL per well, incubate at 37°C for 1 h;

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

[0060] 2.5 Color development and termination: Wash the plate 4 times with TBST, after removing the remaining liquid, add color developing solution, 100 μL per well, and place at room temperature for 10 min; add 50 μL of termination solution to all wells of the ELISA reaction plate;

[0061] 2.6 Reading: Read the absorbance value at 450 nm using a microplate reader;

[0062] 2.7 Select the wells with high detection values as suspected positive wells, and repeat the detection the next day. Wells with high positive values are considered as positive wells.

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

[0064] 3. Monoclonalization

[0065] 3.1 First round of monoclonalization: The cells in the above positive wells were collected and diluted to a single cell per 200 μL using a monoclonal cell culture solution. The cell suspension was added to a 96-well cell culture plate, and after 8 days of culture, the above indirect ELisa method was used for detection;

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

[0067] 3.3 After the above positive well cells are subjected to multiple rounds of monoclonalization, the hybridoma cell purity reaches 100%, and the monoclonalization is terminated.

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

[0069] Example 4

[0070] Screening of paired antibodies

[0071] The cell strains are injected into the abdominal cavity of mice, 5 mice for each cell strain, and the ascites are collected and purified by protein A affinity chromatography column. The purified antibodies are labeled with biotin and used as detection antibodies, and the purified antibodies without labeling are used as coating antibodies. According to the sandwich Elisa experimental procedure and referring to the specific operation details of the indirect Elisa plate washing in Example 3, the screening of the sandwich antibody pairing combination is carried out.

[0072] 4.1 The capture antibody is coated at 100 ng / well, and incubated at 2-8℃ for at least 12 hours;

[0073] 4.2 Dog interferon 5 μg / mL x 50 μL, 37℃ incubation for 1 h;

[0074] 4.3 The detection antibody is loaded at 100 μL per well (the titer needs to be adjusted before loading the detection antibody), and incubated for 1 h;

[0075] 4.4 HRP-labeled streptavidin (Selleck, C050109-HRP-SA) is diluted at 1:4000, and loaded at 100 μL per well, and incubated at 37℃ for 1 h;

[0076] 4.5 Color development and termination;

[0077] 4.6 Reading: the absorbance value is read at 450 nm by the enzyme label instrument.

[0078] Table 2 Checkerboard detection results

[0079]

[0080] As shown in Table 2, 1C1 as the capture antibody and 2A4 as the detection antibody have the optimal detection effect, and the 1C1 and 2A4 antibodies can be used as an antibody combination for the detection of cIFN-α.

[0081] The selected antibody combination is detected as follows:

[0082] 1. Specific detection

[0083] In order to test the specificity of the 1C1 and 2A4 antibody combination, the present application selects 2 healthy dog serum, 2 canine parvovirus infected dog serum, 2 feline calicivirus infected cat serum, and 2 swine fever infected pig serum as detection samples, sets PBS as negative control, and 1 μg / mL cIFN-α solution as positive control. According to the sandwich Elisa experimental procedure in Example 4, the sample detection is carried out, and the detection results are as follows:

[0084] Table 3 Specificity detection OD value

[0085]

[0086] According to the standard of S / N≥2.1 (S is the OD value of the sample, and N is the OD value of the negative sample), the S / N of the serum of healthy dogs and the serum of dogs infected with canine parvovirus is greater than 2.1, and the S / N of the serum of cats infected with feline calicivirus and the serum of pigs infected with swine fever is less than 2, indicating that the antibody combination of 1C1 and 2A4 has specificity for cIFN-α detection.

[0087] Based on the above pairing experiment results and specificity detection results, the antibody combination of 1C1 and 2A4 can be developed as a detection reagent for cIFN-α. Therefore, subsequent antibody sequencing and detection condition optimization of the antibody combination are carried out.

[0088] 2. Antibody sequence determination

[0089] The purified 1C1 and 2A4 hybridoma cell strains are expanded, and more than 1×10 6 After cell lysis, mRNA is extracted and reverse transcribed into cDNA. After amplification of the antibody gene, it is cloned into a vector. After Sanger sequencing, the variable region nucleotide sequence of the antibody is obtained. Through bioinformatics analysis, the capture antibody related sequence is obtained as shown in Table 4, and the detection antibody related sequence is obtained as shown in Table 5:

[0090] Table 4 Information of capture antibody

[0091]

[0092] Table 5 Information of detection antibody

[0093]

[0094] Note: The naming method of the related amino acids and nucleic acids in the capture antibody and the detection antibody in Table 4 and Table 5 is the same, and the sequences are different;

[0095] The amino acid sequence of SEQ ID NO. 5 is KVS;

[0096] The nucleotide sequence of SEQ ID NO. 13 is AAAGTTTCC;

[0097] The amino acid sequence of SEQ ID NO. 21 is WAS;

[0098] The nucleotide sequence of SEQ ID NO. 29 is TGGGCATCC.

[0099] The capture antibody and the detection antibody also include sequences with more than 80% homology to the original sequence.

[0100] 3. Detection condition optimization

[0101] 3.1 Determination of optimal working concentration of antibody

[0102] 1) Dilute the purified 1C1 monoclonal antibody into different concentrations (1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL);

[0103] Dilute the conjugated 2A4 monoclonal antibody by 4 dilution ratios (1:1600, 1:3200, 1:6400 and 1:12800) starting from 1:1600;

[0104] 2) Coat the 1C1 monoclonal antibody at different concentrations respectively, 100 μL / well, 4°C overnight.

[0105] 3) Wash the plate, wash the plate with buffer for 3 times, pat dry;

[0106] 4) Antigen incubation: 0.01 μg / mL cIFN-α, 100 μL / well, 37°C incubation for 1 h;

[0107] 5) Wash the plate according to step 3);

[0108] 6) Add 4 dilution ratios of labeled monoclonal antibody, 100 μL / well, 37°C incubation for 1 h;

[0109] 7) Wash the plate according to step 3);

[0110] 8) HRP labeled streptavidin (Sino Biological, C050109-HRP-SA) 1:4000 dilution, 100 μL / well, 37°C incubation for 1 h;

[0111] 9) Wash the plate according to step 3);

[0112] 10) Color development, read the results.

[0113] According to the above steps, the results are shown in Table 6, and finally it is concluded that the optimal coating concentration of 1C1 monoclonal antibody is 4 μg / mL, and the optimal detection concentration of 2A4 monoclonal antibody is 1:6400 dilution ratio, and under this condition, the antigen detection value is the highest.

[0114] Table 6 Determination of optimal working concentration of antibody by checkerboard titration method

[0115]

[0116] 3.2 Determination of antibody incubation time

[0117] According to the above optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, different antigen incubation times (15 min, 30 min, 45 min, 1 h, 1.5 h) were set, other conditions were fixed, 0.01 μg / mL cIFN-α was detected, and the results are shown in the following table:

[0118] Table 7 Determination of optimal antigen incubation time

[0119]

[0120] As can be seen from Table 7, according to the S / N value, the optimal antigen incubation time is 1 h.

[0121] According to the above optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, and the optimal antigen incubation time, different detection antibody incubation times (15 min, 30 min, 45 min, 1 h, 1.5 h) were set, and 0.01 μg / mL cIFN-α was detected, and the results are shown in the following table:

[0122] Table 8 Determination of optimal detection antibody incubation time

[0123]

[0124] As can be seen from Table 8, according to the S / N value, the optimal detection antibody incubation time is 45 min.

[0125] According to the above optimal 1C1 monoclonal antibody coating concentration and 2A4 monoclonal antibody detection concentration, and the optimal antigen and detection antibody incubation time, different HRP-labeled streptavidin incubation times were set, and 0.01 μg / mL cIFN-α was detected, and the results are shown in the following table:

[0126] Table 9 Determination of optimal streptavidin incubation time

[0127]

[0128] As can be seen from Table 9, according to the S / N value, the optimal HRP-labeled streptavidin incubation time is 30 min and 45 min.

[0129] 4. Sensitivity test

[0130] The above-mentioned paired antibodies and optimized conditions were used, i.e. 4 μg / mL of 1C1 monoclonal antibody, 1:6400 dilution of 2A4 monoclonal antibody were used for detecting cIFN-α, cIFN-α was prepared into different concentrations, and a sensitivity test was performed, and the test results are shown in the following table:

[0131] Table 10 Sensitivity test results

[0132]

[0133] As can be seen from the results 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-mentioned paired antibodies can detect 78 pg / mL of cIFN-α, and the detection sensitivity is high. As shown in Table 10, there is a good linear relationship in the range of 0.078-2.5 ng / mL of the antigen concentration, and the equation y = 0.771x + 0.1559, R2= 0.9933 can be generated in this range, as shown in Figure 2 the horizontal coordinate represents the antigen concentration value, and the vertical coordinate represents the absorbance value.

[0134] 5. Detection experiment of drug residue after injection of recombinant cIFN-α preparation in vivo

[0135] In order to detect the detection effect of the double antibody combination after the above-mentioned optimization experiment, further testing was carried out. The high-activity α-interferon recombinant protein (corresponding to SEQ ID NO. 3 in CN112079912B) in the previous patent CN112079912B of the applicant was injected into dogs 1 and 2 weighing about 10 kg, and the injection was carried out at a dose of 0.1 mg per dog. Blood samples were taken at 1 h, 2 h, 4 h, 8 h, 16 h, 24 h and 48 h after injection, and the supernatant was obtained by centrifugation. PBS was used as a negative control.

[0136] The antibody combination of 4 μg / mL of 1C1 monoclonal antibody and 1:6400 dilution of 2A4 monoclonal antibody was used to detect the drug residue after injection of recombinant cIFN-α preparation in vivo, and the detection results are as follows:

[0137] Table 11 Detection results of drug residue after injection of recombinant cIFN-α preparation in vivo

[0138]

[0139] After the recombinant protein is injected into the dog, the degradation increases and the residual amount decreases with the extension of time. After a certain time, the α-interferon level in the serum of the dog approaches the basic expression amount, and as can be seen from Table 11, the OD value gradually decreases with the extension of time, and the detection results are consistent with the expectation, indicating that the double antibody combination (1C1 monoclonal antibody + 2A4 monoclonal antibody) of the application can be used for the detection of drug residue after injection of cIFN-α preparation in vivo, and has good practical value.

Claims

1. A monoclonal antibody 1C1 against canine IFN-α or an antigen-binding site thereof, characterized in that, The monoclonal antibody 1C1 or its antigen-binding site comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, HC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 1-3, and light chain complementarity determining regions LC-CDR1, LC-CDR2, LC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 4-6.

2. The monoclonal antibody 1C1 or an antigen-binding portion thereof against canine IFN-α according to claim 1, characterized by, The monoclonal antibody 1C1 or its antigen-binding site comprises heavy chain variable region VH, the amino acid sequence of which is shown in SEQ ID NO. 7, and light chain variable region VL, the amino acid sequence of which is shown in SEQ ID NO.

8.

3. A monoclonal antibody 2A4 or an antigen-binding portion thereof against canine IFN-α, characterized in that, The monoclonal antibody 2A4 or its antigen-binding site comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, HC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 17-19, and light chain complementarity determining regions LC-CDR1, LC-CDR2, LC-CDR3, the amino acid sequences of which are shown in SEQ ID NO. 20-22.

4. The monoclonal antibody 2A4 or an antigen-binding portion thereof against canine IFN-α according to claim 3, characterized by The monoclonal antibody 2A4 or its antigen-binding site comprises heavy chain variable region VH, the amino acid sequence of which is shown in SEQ ID NO. 23, and light chain variable region VL, the amino acid sequence of which is shown in SEQ ID NO.

24.

5. Use of an antibody against canine IFN-α, characterized in that, The antibody comprises the monoclonal antibody 1C1 or its antigen-binding site of claim 1 or 2, and / or the monoclonal antibody 2A4 or its antigen-binding site of claim 3 or 4. The antibody against canine IFN-α is used for preparing an enzyme-linked immunoassay, immunofluorescence, flow cytometry, immunohistochemical detection reagent.

6. A nucleotide sequence encoding a monoclonal antibody, characterized in that, The nucleotide sequence encodes 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. The nucleotide sequence is used for constructing an expression vector and preparing a recombinant antibody.

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

8. A host cell, characterized in that, The cell has been transfected or transformed to comprise 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.

9. A product for detecting canine IFN-α, characterized by, The composition of the product comprises a capture antibody and a detection antibody. The capture antibody is the monoclonal antibody 1C1 of claim 1 or 2, and the detection antibody is the monoclonal antibody 2A4 of claim 3 or 4. The capture antibody is immobilized on a solid carrier, and the capture antibody binds to the antigen. The product for detecting canine IFN-α comprises an Elisa kit, a test strip, or a biochip. The detection method for detecting canine IFN-α comprises a double antibody sandwich assay.

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