An antibody composition for detecting interleukin-29 and its application

Abstract

The present invention discloses an antibody composition for detecting interleukin-29 and its application. The antibody composition comprises a capture antibody A1001 and a detection antibody A1004. The antibody composition of the present invention is used to detect IL-29 or a fusion protein containing IL-29, has high specificity and sensitivity, reduces detection costs, improves detection efficiency, and realizes rapid and efficient detection of IL-29 or a fusion protein containing IL-29.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an antibody composition for detecting interleukin 29 and an application thereof. Background Art

[0002] Interleukin 29 (IL-29) is a cytokine with antiviral activity that was first reported in 2003. It is highly similar to the IL-10 cytokine family in terms of amino acid sequence. Because it has interferon-like antiviral activity, it also belongs to the type III interferon or IFN-λ family, also known as IFN-λ1.

[0003] IL-29, similar to IFN-α / β (type I interferon), activates the JAK-STAT signaling pathway by binding to the corresponding receptors, inducing the transcription of downstream interferon-stimulated genes (ISGs), thereby stimulating the expression of proteins related to antiviral response, including antiviral proteins such as MxA, OASL and PKR. In vitro studies have shown that human IL-29 can reduce the replication and induced cytopathic effects of viruses, including cytomegalovirus (CMV), hepatitis B virus (HBV), encephalomyocarditis virus (EMCV), and vesicular stomatitis virus (VSV).

[0004] Although IL-29 has similar antiviral activity to IFN-α / β, since IFN-α / β receptors are widely distributed on various types of cells, IFN-α / β binding to receptors has not only antiviral effects, but also immunomodulatory, immunosuppressive and anti-cell proliferation effects. Therefore, clinical use has very obvious side effects, including flu-like syndrome, erythropenia and leukopenia, bone marrow cell reduction, and mental disorders (depression, delusion, anxiety, etc.). One of the receptors of IL-29 (IL-28R1) is specifically expressed only in specific tissues, such as keratinocytes, bronchial epithelial cells and hepatocytes. The specificity of the receptor determines the specificity of cell binding to IL-29, so the clinical side effects of IL-29 are significantly lower than those of IFN-α / β.

[0005] In modern large-scale and intensive pig farms, outbreaks of viral infectious diseases, such as blue ear disease, porcine epidemic diarrhea, and African swine fever, have caused significant economic losses to pig farms. Porcine IL-29 can effectively inhibit the replication of viruses such as porcine reproductive and respiratory syndrome virus (PRRSV), vesicular stomatitis virus (VSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), and porcine epidemic diarrhea virus (PEDV). In ileal organoids, IL-29 is more effective than IFN-α in inducing ISGs expression and inhibiting PEDV infection. In addition, porcine IL-29 can be used to treat porcine viral infectious diseases without affecting pigs' diet, energy conversion, etc., and its side effects are significantly lower than those of porcine IFN-α.

[0006] The accuracy and linearity of the existing ELISA kits for detecting porcine IL-29 cannot meet the requirements for detecting porcine albumin / porcine IL-29 fusion protein, and there is still a lack of accurate and stable detection antibodies and detection methods. Summary of the invention

[0007] In order to solve the above technical problems and to accurately and effectively detect IL-29 protein or its fusion protein, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides an antibody composition or antigen-binding fragment composition for detecting interleukin-29, wherein the antibody composition comprises a capture antibody A1001 and a detection antibody A1004; the antigen-binding fragment composition comprises an antigen-binding fragment of antibody A1001 and an antigen-binding fragment of antibody A1004.

[0009] Preferably, the heavy chain variable region (VH) complementarity determining regions 1-3 (CDR1-3) of the capture antibody A1001 or its antigen-binding fragment have more than 85% homology with the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively.

[0010] Preferably, the light chain variable region (VL) complementarity determining regions 1-3 (CDR1-3) of the capture antibody A1001 or its antigen-binding fragment have more than 85% homology with the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively.

[0011] Preferably, the heavy chain variable region (VH) complementarity determining regions 1-3 (CDR1-3) of the detection antibody A1004 or its antigen-binding fragment have more than 85% homology with the amino acid sequences shown in SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, respectively.

[0012] Preferably, the light chain variable region (VL) complementarity determining regions 1-3 (CDR1-3) of the detection antibody A1004 or its antigen-binding fragment have more than 85% homology with the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47, respectively.

[0013] Furthermore, the above 85% homology is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0014] More preferably, the amino acid sequences of the complementarity determining regions 1-3 (CDR1-3) of the heavy chain variable region (VH) of the capture antibody A1001 or its antigen-binding fragment are as shown in SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively.

[0015] More preferably, the amino acid sequences of the complementary determining regions 1-3 (CDR1-3) of the light chain variable region (VL) of the capture antibody A1001 or its antigen-binding fragment are as shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively.

[0016] More preferably, the amino acid sequences of the heavy chain variable region (VH) complementarity determining regions 1-3 (CDR1-3) of the detection antibody A1004 or its antigen-binding fragment are as shown in SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, respectively.

[0017] More preferably, the amino acid sequences of the complementary determining regions 1-3 (CDR1-3) of the light chain variable region (VL) of the detection antibody A1004 or its antigen-binding fragment are as shown in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47, respectively.

[0018] Preferably, the antibody or antigen-binding fragment thereof of the present invention is a whole antibody, a bi- or multi-specific antibody, a bi- or multi-specific single-chain antibody, a single-chain antibody (scFv), Fab, Fab', F(ab')2, a linear antibody or an Fv antibody, and any polypeptide comprising an antibody binding domain or a homologous antibody binding domain. Among them, the antibody binding domain may include a complete heavy chain and / or light chain CDR, a complete antibody heavy chain and / or light chain variable region, a complete full-length heavy chain and / or light chain, or a single, two, three, four, five or six CDRs from the antibody. A single-chain antibody comprises a heavy chain variable region and a light chain variable region.

[0019] Preferably, the amino acid sequences of FR1-4 of the heavy chain variable region of A1001 are shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively.

[0020] Preferably, the amino acid sequences of FR1-4 of the light chain variable region of A1001 are shown as SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively.

[0021] Preferably, the amino acid sequences of FR-4 of the heavy chain variable region of A1004 are shown as SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40, respectively.

[0022] Preferably, the amino acid sequences of FR1-4 of the light chain variable region of A1004 are shown as SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48, respectively.

[0023] Preferably, the antibody composition further comprises an A1001 heavy chain variable region signal peptide, an A1001 light chain region signal peptide, an A1004 heavy chain variable region signal peptide and an A1004 light chain region signal peptide.

[0024] Furthermore, the amino acid sequence of the signal peptide of the A1001 heavy chain variable region is shown as SEQ ID NO:1, and the amino acid sequence of the signal peptide of the A1001 light chain variable region is shown as SEQ ID NO:9; the amino acid sequence of the signal peptide of the A1004 heavy chain variable region is shown as SEQ ID NO:33, and the amino acid sequence of the signal peptide of the A1004 light chain variable region is shown as SEQ ID NO:41.

[0025] Preferably, capture antibody A1001 and detection antibody A1004 further include a light chain constant region and a heavy chain constant region. Optionally, the types of capture antibody A1001 and detection antibody A1004 are independently selected from IgM, IgE, IgA, IgD or IgG. It is understood that the type of antibody can be converted by the constant region sequence.

[0026] Preferably, the antibody composition or antigen-binding fragment composition specifically binds to IL-29.

[0027] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody composition or antigen-binding fragment composition described in aspect 1. It is understood that due to the degeneracy of codons, nucleic acid sequences capable of expressing the same protein have multiple forms.

[0028] Preferably, CDR1-3 of the heavy chain variable region of A1001 have more than 80% homology with the DNA sequences shown in SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, respectively.

[0029] Preferably, CDR1-3 of the light chain variable region of A1001 has more than 80% homology with the DNA sequences shown in SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, respectively.

[0030] Preferably, CDR1-3 of the heavy chain variable region of A1004 have more than 80% homology with the DNA sequences shown in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 55, respectively.

[0031] Preferably, CDR1-3 of the light chain variable region of A1004 has more than 80% homology with the DNA sequences shown in SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, respectively.

[0032] More preferably, the DNA sequences of CDR1-3 of the heavy chain variable region of A1001 are shown as SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, respectively.

[0033] More preferably, the DNA sequences of CDR1-3 of the light chain variable region of A1001 are shown as SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, respectively.

[0034] More preferably, the DNA sequences of CDR1-3 of the heavy chain variable region of A1004 are shown as SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 55, respectively.

[0035] More preferably, the DNA sequences of CDR1-3 of the light chain variable region of A1004 are shown as SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, respectively.

[0036] Preferably, the DNA sequences of FR1-4 of the heavy chain variable region of A1001 are shown as SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively.

[0037] Preferably, the DNA sequences of FR1-4 of the light chain variable region of A1001 are shown as SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32, respectively.

[0038] Preferably, the DNA sequences of FR1-4 of the heavy chain variable region of A1004 are shown as SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:56, respectively.

[0039] Preferably, the DNA sequences of FR1-4 of the light chain variable region of A1004 are shown as SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64, respectively.

[0040] Preferably, the signal peptide DNA sequence of the A1001 heavy chain variable region is as shown in SEQ ID NO: 17 or a DNA sequence having more than 80% homology thereto.

[0041] Preferably, the DNA sequence of the signal peptide of the A1001 light chain variable region is as shown in SEQ ID NO: 25 or a DNA sequence having more than 80% homology thereto.

[0042] Preferably, the DNA sequence of the signal peptide of the A1004 heavy chain variable region is as shown in SEQ ID NO: 49 or a DNA sequence having more than 80% homology thereto.

[0043] Preferably, the DNA sequence of the signal peptide of the A1004 light chain variable region is as shown in SEQ ID NO: 57 or a DNA sequence having more than 80% homology thereto.

[0044] Furthermore, the above-mentioned 80% or more homology is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0045] More preferably, the signal peptide DNA sequence of the A1001 heavy chain variable region is as shown in SEQ ID NO:17.

[0046] More preferably, the DNA sequence of the signal peptide of the A1001 light chain variable region is as shown in SEQ ID NO:25.

[0047] More preferably, the DNA sequence of the signal peptide of the A1004 heavy chain variable region is as shown in SEQ ID NO:49.

[0048] More preferably, the DNA sequence of the signal peptide of the A1004 light chain variable region is as shown in SEQ ID NO:57.

[0049] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule described in the second aspect.

[0050] In a fourth aspect, the present invention provides a hybridoma cell, wherein the hybridoma cell is capable of producing the antibody composition or antigen-binding fragment composition described in the first aspect.

[0051] In a fifth aspect, the present invention provides a method for preparing the hybridoma cells described in the fourth aspect, the method comprising immunizing a non-human animal with porcine IL-29, collecting spleen cells of the non-human animal after immunization with IL-29, and fusing the collected spleen cells with myeloma cells to obtain hybridoma cells.

[0052] Preferably, the non-human animal is a non-human mammal, and more preferably a rodent.

[0053] In a specific embodiment of the present invention, the non-human animal is a rat or a mouse.

[0054] In a sixth aspect, the present invention provides a method for preparing the antibody composition or antigen-binding fragment composition described in the first aspect, after culturing the hybridoma cells obtained in the fourth aspect, screening antibodies with high affinity for IL-29, and then obtaining the antibody composition or antigen-binding fragment composition through pairing screening.

[0055] In a seventh aspect, the present invention provides a pharmaceutical composition comprising at least one of the antibody composition or antigen-binding fragment composition described in the first aspect, the nucleic acid molecule described in the second aspect, or the hybridoma cell described in the fourth aspect.

[0056] In an eighth aspect, the present invention provides a kit for detecting interleukin-29, the kit comprising the antibody composition or antigen-binding fragment composition described in the first aspect.

[0057] Preferably, the kit further comprises a reagent for antibody detection. More preferably, the reagent is an ELISA reagent, such as: IL-29 standard, immunosorbent, horseradish peroxidase, diluent, color developing solution, etc.

[0058] In a ninth aspect, the present invention provides use of the antibody composition or antigen-binding fragment composition described in the first aspect, the nucleic acid molecule described in the second aspect, the hybridoma cell described in the fourth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of a product for detecting interleukin-29.

[0059] Preferably, the product includes livestock and poultry meat, animal blood, animal tissue fluid, etc. using interleukin 29. By detecting the level of interleukin 29 in the product, the health status of the animal and the effect of using interleukin 29 can be evaluated.

[0060] In the tenth aspect, the present invention provides the use of the antibody composition or antigen-binding fragment composition described in the first aspect, the nucleic acid molecule described in the second aspect, the hybridoma cell described in the fourth aspect, or the pharmaceutical composition described in the seventh aspect in detecting interleukin-29 or a fusion protein containing interleukin-29.

[0061] Preferably, the application includes monitoring and evaluating the therapeutic effect of IL-29 or a fusion protein containing IL-29 on animal viral diseases, and evaluating the severity of related diseases.

[0062] Furthermore, the animal diseases include porcine reproductive and respiratory syndrome, vesicular stomatitis, pseudorabies, classical swine fever and porcine epidemic diarrhea.

[0063] Beneficial effects of the present invention:

[0064] 1. Provide a specific antibody combination targeting IL-29, which can specifically and efficiently capture and detect IL-29 under the conditions of 2.5 μg / mL of capture antibody A1101 and 0.5 μg / mL of detection antibody A1004.

[0065] 2. The detection sensitivity of the present invention for IL-29 is as low as 10 ng / mL, which reduces the detection cost and improves the detection efficiency.

[0066] 3. The present invention can efficiently monitor and evaluate the therapeutic effect of IL-29 or a fusion protein containing IL-29 on animal viral diseases and the health level of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Shown is the standard curve in Example 7. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

[0069] Example 1 Antibody Preparation and Screening

[0070] (1) Recombinantly expressed antigen (porcine IL-29), molecular weight 20 kDa, purity 84%.

[0071] (2) Five Balb / c mice (M1101#-M1105#) were immunized three times. Serum was collected before and after the three immunizations to detect antibody titers. The samples were coated with 1 μg / mL porcine IL-29 (A) or His-labeled irrelevant protein (B), and serum at different dilution ratios was added. Finally, HRP-labeled goat anti-mouse IgG antibody was used for detection. The results are shown in Table 1. The antibody titer in the serum of M1103# mice was the highest, and the signal-to-noise ratio (signal value at dilution multiple / signal value of blank control) was above 2.1 when diluted 512,000 times. At the same time, the antibodies in this serum had the weakest binding to irrelevant albumin.

[0072] Table 1 Mouse serum antibody detection results

[0073]

[0074] Note: Titer is the dilution multiple of serum when the signal-to-noise ratio is greater than 2.1; 3rd is the serum after the third immunization, and NC is the serum before immunization.

[0075] (3) M1103# mouse spleen cells were fused with myeloma cells to prepare 10 positive mother clones, and a round of subcloning was performed. The supernatants of these 10 positive clones were subjected to binding experiments using the ELISA method. 1 μg / mL of porcine IL-29 (A) or His-labeled irrelevant protein (B) was used for coating, and cell supernatants of different dilution ratios were added. Finally, HRP-labeled goat anti-mouse IgG antibody was used for detection. The results are shown in Table 2. The antibody titers in the supernatants of the 10 cells were all high, and the signal-to-noise ratios were all above 2.1 when diluted 2430 times. In addition, the antibodies in the supernatants of the 10 cells all bound weakly to irrelevant albumin.

[0076] Table 2 Antibody detection signal values ​​in positive clone cells

[0077]

[0078] Note: The titer is the dilution multiple of the cell supernatant when the signal-to-noise ratio is greater than 2.1.

[0079] Example 2 Detection of antibody affinity of positive cloned cell lines

[0080] The affinity of the supernatants of the 10 cell strains obtained in Example 1 was ranked using the ELISA method. The specific process is as follows:

[0081] After coating with porcine IL-29 and adding supernatant of different dilution multiples, detection was performed with HRP-labeled goat anti-mouse IgG antibody. EC was calculated by fitting based on the detected signal value and response curve using the four-parameter method. 50Signal value and calculated EC 50 As shown in Table 3, the highest affinity (EC 50 The supernatants of cells C1001, C1003, and C1004 were obtained from the supernatants of cells C1001, C1003, and C1004, respectively.

[0082] Table 3 Antibody affinity test results of positive clone cell lines

[0083]

[0084] Example 3 Antibody Binding Epitope Analysis of Positive Clone Cell Lines

[0085] The supernatants of the above 10 positive clones were subjected to epitope classification using the ELISA method. The specific process is as follows:

[0086] The binding epitopes of the supernatants of the 10 cell strains were divided into the following 4 categories (see Table 4). Combined with the affinity data in Example 2, two supernatants with high affinity and different epitopes were selected for pairing. At least one antibody with the highest affinity was selected in each epitope classification, and then paired in pairs, namely C1001, C1003, C1005, C1006 and C1010.

[0087] Table 4 Antibody binding epitopes of positive clone cell lines

[0088]

[0089] Example 4 Antibody pairing experiment

[0090] (1) The five cell supernatants with the highest affinity in Example 3 were purified to obtain five antibodies with a purity of >98% (named A1001, A1003, A1005, A1006 and A1010, respectively), and labeled with HRP (named A1001-HRP, A1003-HRP, A1005-HRP, A1006-HRP and A1010-HRP, respectively). The five unlabeled antibodies were coated (2.5 μg / mL) as capture antibodies, and the HRP-labeled antibodies (1 μg / mL) and a polyclonal antibody prepared by Biosun (porcine albumin / porcine IL-29 fusion protein-HRP polyclonal antibody) were used as detection antibodies to detect 0-100 ng / mL of porcine IL-29. As shown in Table 5, when A1001 and A1003 were used as capture antibodies and A1004-HRP and A1006-HRP were used as detection antibodies, the signal-to-noise ratio was high and the maximum absolute value that the signal value could reach was high.

[0091] Table 5 Antibody detection results of capture antibody and detection antibody pairs

[0092]

[0093] (2) The sensitivity of the above two pairs of antibodies was tested. 2.5 μg / mL of antibody was used as the capture antibody, and different concentrations of HRP-labeled antibody were used as the detection antibody to detect 0-2000 pg / mL of porcine IL-29. Among the multiple antibody pairs tested, the signal-to-noise ratio of the combination of A1001 and A1004-HRP or A1003 and A1004-HRP was the best, and the optimal concentration of the detection antibody was 0.5 μg / mL.

[0094] Table 6 Antibody pair screening results

[0095]

[0096] Example 5 Screening for the best antibody pair for detecting porcine albumin / porcine IL-29 fusion protein

[0097] A1001 was diluted to 2.5 μg / mL and added to the ELISA plate, and incubated overnight at 4°C. The next day, it was washed and 5% BSA was added, and it was blocked at 37°C for 2 hours. Then the porcine albumin / porcine IL-29 fusion protein standard was added to the ELISA plate and incubated for 1 hour. Then 0.5 μg / mL of A1004-HRP or A1006-HRP was added, incubated at 37°C for 1 hour, and washed several times to remove unbound substances. Subsequently, TMB colorimetric solution was added and incubated in the dark for 20 minutes, and finally the stop solution was added and the OD value at 450 nm was read. The results are shown in Table 7. Among the two groups of antibody pairs tested, the signal-to-noise ratio of the combination of A1001 and A1004-HRP was better than that of the combination of A1001 and A1006-HRP, and it was selected as the final antibody pair for detection.

[0098] Table 7 Screening results of porcine albumin / porcine IL-29 fusion protein detection antibody pairs

[0099]

[0100] Example 6 Preparation of a detection kit using A1001 and A1004-HRP

[0101] A1001 and A1004-HRP were used as detection antibody pairs to prepare an enzyme-linked immunosorbent assay kit for detecting porcine albumin / porcine IL-29 fusion protein or porcine IL-29 protein levels. The specific steps are as follows:

[0102] 1) Dilute the capture antibody to 2 μg / mL with coating solution, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight.

[0103] 2) Spin dry the liquid in the wells, pat dry the residual liquid, add 300 μL / well of washing solution, discard the washing solution immediately after 1 min, spin dry, and repeat this operation four times.

[0104] 3) Add blocking solution at 300 μL / well and block at 37℃ for 2 hours. Spin the solution in the wells to dry, pat dry the residual liquid, add washing solution at 300 μL / well, discard the washing solution immediately after 1 min, spin dry, and repeat this operation four times.

[0105] 4) Dilute the standard with PBS or serum to the working concentration and add to the ELISA plate, 100 μL per well, and incubate at 37°C for 1 h.

[0106] 5) Spin dry the liquid in the wells, pat dry the remaining liquid on absorbent paper, add washing solution at 300 μL / well, discard the washing solution immediately after 1 min, spin dry, and repeat this operation four times.

[0107] 6) Dilute the detection antibody to 0.1 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 37°C for 1 h.

[0108] 7) Spin dry the liquid in the wells, pat dry the residual liquid, add washing solution at 300 μL / well, discard the washing solution immediately after 1 min, spin dry, and repeat this operation four times.

[0109] 8) Add 100 μL / well of TMB colorimetric solution to the ELISA plate and incubate at 37°C in the dark for 20 min.

[0110] 9) Add 50 μL / well of the stop solution into the ELISA plate and read the OD value at 450 nm.

[0111] Example 7 Using a kit to detect the concentration of porcine albumin / porcine IL-29 fusion protein in serum after administration

[0112] The concentration of porcine albumin / porcine IL-29 fusion protein in the blood of animals after administration was detected using the kit prepared in Example 6 and the detection method established using A1001 and A1004-HRP in Example 5. The specific process is as follows:

[0113] Six 10 kg Sanyuan pigs were intramuscularly injected with porcine albumin / porcine IL-29 fusion protein or saline. Blood was collected after administration, and serum was separated and tested according to the law. A1001 was diluted to 2 μg / mL and added to the ELISA plate and incubated overnight at 4°C. The next day, it was washed and 5% BSA was added, and blocked at 37°C for 2 hours. Then the standard and 10-200 times diluted serum (to reduce the matrix effect of serum) were added to the ELISA plate and incubated for 1 hour. Then 0.1 μg / mL of A1004-HRP was added and incubated at 37°C for 1 hour, and washed several times to remove unbound substances. Subsequently, TMB colorimetric solution was added and incubated in the dark for 20 minutes. Finally, the stop solution was added and the OD value at 450nm was read. The standard curve data is shown in Table 8.

[0114] Table 8 Standard curve data

[0115]

[0116] like Figure 1 As shown, the detection range is 10~320 ng / mL.

[0117] The results of the blood drug concentrations of various animals are shown in Table 9, indicating that the combination of A1001 and A1004-HRP can effectively detect the concentration of porcine albumin / porcine IL-29 fusion protein.

[0118] Table 9 Concentration of porcine albumin / porcine IL-29 fusion protein in animal blood medicine

[0119]

[0120] Example 8 Sequence Determination

[0121] The variable region amino acids and corresponding coding DNAs of A1001 and A1004 were sequenced, and the results are shown in Tables 10 to 13.

[0122] Table 10A1001 variable region amino acid sequence

[0123]

[0124] Table 11 DNA sequence of A1001

[0125]

[0126] Table 12 Variable region amino acid sequence of A1004

[0127]

[0128] Table 13 Variable region DNA sequences of A1004

[0129]

[0130] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. An antibody composition or antigen-binding fragment composition for detecting interleukin-29, characterized in that: The antibody composition includes a capture antibody A1001 and a detection antibody A1004; the antigen-binding fragment composition includes an antigen-binding fragment of antibody A1001 and an antigen-binding fragment of antibody A1004; The capture antibody A1001 or its antigen-binding fragment comprises heavy chain variable region (VH) complementary determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively, and light chain variable region (VL) complementary determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively; The detection antibody A1004 or its antigen-binding fragment comprises heavy chain variable region (VH) complementary determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, respectively, and light chain variable region (VL) complementary determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47, respectively.

2. The antibody composition or antigen-binding fragment composition according to claim 1, characterized in that: The amino acid sequences of FR1-4 of the heavy chain variable region of A1001 are shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively; The amino acid sequences of FR1-4 of the light chain variable region of A1001 are shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively; The amino acid sequences of FR1-4 of the heavy chain variable region of A1004 are shown in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40, respectively; The amino acid sequences of FR1-4 of the light chain variable region of A1004 are shown in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48, respectively.

3. The antibody composition or antigen-binding fragment composition according to claim 1, characterized in that: The antibody pair further comprises an A1001 heavy chain variable region signal peptide, an A1001 light chain region signal peptide, an A1004 heavy chain variable region signal peptide, and an A1004 light chain region signal peptide; The signal peptide amino acid sequence of the A1001 heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of the signal peptide of the A1001 light chain variable region is shown in SEQ ID NO: 9; The amino acid sequence of the signal peptide of the A1004 heavy chain variable region is shown in SEQ ID NO: 33; The amino acid sequence of the signal peptide of the A1004 light chain variable region is shown in SEQ ID NO:

41.

4. The antibody composition or antigen-binding fragment composition according to any one of claims 1 to 3, characterized in that: The antibody composition or antigen-binding fragment composition specifically binds to IL-29.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody composition or antigen-binding fragment composition according to any one of claims 1 to 4.

6. A carrier, characterized in that: The vector comprises the nucleic acid molecule of claim 5.

7. A hybridoma cell, characterized in that: The hybridoma cell can produce the antibody composition or antigen-binding fragment composition according to any one of claims 1 to 4.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises at least one of the antibody composition or antigen-binding fragment composition according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, or the hybridoma cell according to claim 7.

9. A kit for detecting interleukin-29, characterized in that: The kit comprises the antibody composition or antigen-binding fragment composition according to any one of claims 1 to 4.

10. Use of the antibody composition or antigen-binding fragment composition according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the hybridoma cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a product for detecting interleukin-29.

Images