A porcine IL-15 monoclonal antibody, its preparation method and application

Abstract

The present invention provides a porcine IL-15 monoclonal antibody, a preparation method thereof and an application, which relates to the technical field of immunological detection. The present invention prepares a porcine IL-15 monoclonal antibody by using a hybridoma cell line with the preservation number of CCTCC NO: C2024127, and establishes a double antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in biological samples by means of this antibody. This method has high sensitivity, specificity and accuracy, provides an important tool for porcine IL-15 related research, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly relates to a porcine IL-15 monoclonal antibody, a preparation method thereof, and an application thereof. Background Art

[0002] Interleukin-15 (IL-15) is a pleiotropic cytokine discovered by Grabstein in 1994 when detecting the culture supernatant of the simian kidney epithelial cell line CV-1 / EBNA, and is produced by various cells such as stromal cells, monocytes, macrophages, dendritic cells, epithelial cells, etc. The mRNA of IL-15 exists in various immune or non-immune cells and tissues, but its protein is mainly limited to monocytes, macrophages, and dendritic cells. IL-15 belongs to a 14- to 15-ku member of the 4α-helical bundle cytokine family and signals by binding to the IL-15Rα / IL-2Rβ / γc heterotrimeric receptor, where the IL-15 receptor α (IL-15Rα, CD215) is the specific receptor for IL-15, IL-2Rβ (CD122, also known as IL-15Rβ) is shared with IL-2, and γc (CD132) is shared with IL-4, IL-7, IL-9, and IL-21.

[0003] IL-15 is a pleiotropic cytokine that can promote the proliferation of natural killer cells (NK cells), NK-T, γδT, and memory CD8 T cells, and plays a key role especially in maintaining the survival, homeostatic proliferation, and renewal of CD8 T cells. In addition, IL-15 is a hematopoietic factor for generating NK cells, can replace the bone marrow environment to promote the differentiation and maturation of NK cells, and regulate NK cell activation and maintain NK cell homeostasis. IL-15 is an important immune molecule for studying the interaction between pathogenic microorganisms and hosts and evaluating the cellular immune status.

[0004] However, there is a lack of detection methods related to porcine IL-15 in the prior art. Summary of the Invention

[0005] In view of this, the present invention provides a porcine IL-15 monoclonal antibody, a preparation method thereof, and an application thereof. The present invention uses the hybridoma cell line with the preservation number of CCTCC NO: C2024127 to prepare a porcine IL-15 monoclonal antibody, and establishes a double-antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in biological samples through this antibody. This method has high sensitivity, specificity, and accuracy, provides an important tool for porcine IL-15-related research, and has good application prospects.

[0006] The specific monoclonal antibody of porcine IL-15 of the present invention is prepared using the hybridoma cell line with the preservation number of CCTCC NO: C2024127.

[0007] The preparation method of the monoclonal antibody of porcine IL-15 of the present invention includes the following steps:

[0008] S21. Extract the nucleic acid of porcine inguinal lymph node tissue, reverse transcribe it into cDNA and then perform PCR amplification. The primers for porcine IL-15 are:

[0009] e porcine IL-15U: 5′-CTAGAATTCGCCACCATGAGAATTTTAAAAC-3′

[0010] e porcine IL-15L: 5′-GACCTCGAGTCAAGAAGGGTTGATG-3′;

[0011] Digest the PCR product, pET28α(+), and pcDNA3.1(+) with restriction enzymes, ligate the digested PCR product with the plasmid to construct the recombinant plasmids pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15. Transform the pET-28α-porcine IL-15 recombinant plasmid into Escherichia coli Rosetta(DE3) competent cells, and transfect the pcDNA3.1-porcine IL-15 recombinant plasmid into PK-15 cells.

[0012] S22. Recombinant porcine IL-15 protein. After immunizing mice with the purified porcine IL-15 protein and then boosting the immunity, prepare a spleen cell suspension and fuse the spleen cells with SP2 / 0 myeloma cells. Screen the hybridoma cell line that stably secretes the specific monoclonal antibody of porcine IL-15 (preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: C2024127, and the classification name is hybridoma cell line Pocine-IL-15-2C10), prepare ascites, and purify the antibody using the octanoic acid-ammonium sulfate method to complete the preparation of the monoclonal antibody of porcine IL-15. Preferably, the restriction enzymes are EcoR I and Xho I.

[0013] Preferably, after the construction of the recombinant plasmid, perform enzyme digestion and sequencing on the recombinant plasmid.

[0014] Preferably, transfect the pcDNA3.1-porcine IL-15 recombinant plasmid using the liposome transfection method.

[0015] Preferably, use SDS-PAGE and Western Blot analysis to prove the expression of porcine IL-15 protein in Escherichia coli.

[0016] Preferably, after screening out the hybridoma cell line stably secreting porcine IL-15 specific monoclonal antibody, at least one of subcloning, antibody titer determination, monoclonal antibody class / subclass identification, monoclonal antibody identification, and specificity detection is performed. More preferably, Western Blot is used to confirm the expression of recombinant porcine IL-15 protein and the specificity of the monoclonal antibody, and the intracellular fluorescence signal is observed through a fluorescence microscope to verify the expression of porcine IL-15 in PK-15 cells and the binding ability of the monoclonal antibody; the specificity of the monoclonal antibody to porcine IL-15 is ensured through cross-reactivity detection, and non-specific binding to other cytokines is avoided.

[0017] The present invention also provides a double antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in a biological sample, and this method is performed using the porcine IL-15 monoclonal antibody described above.

[0018] Preferably, the biological sample is at least one of serum, tissue fluid, and cell culture.

[0019] The method for establishing the porcine IL-15 double antibody sandwich ELISA detection of the present invention includes the following steps:

[0020] Preferably, the double antibody includes a capture antibody and an enzyme-labeled antibody that can specifically bind to porcine IL-15.

[0021] Preferably, the capture antibody is monoclonal antibody 2D10, and the enzyme-labeled antibody is monoclonal antibody 2C10.

[0022] The method for establishing and evaluating the porcine IL-15 double antibody sandwich ELISA detection of the present invention includes the following steps: S11, determination of the affinity of the monoclonal antibody;

[0023] S12, performing a pairing test of the monoclonal antibody;

[0024] S13, determination of the antibody coating concentration;

[0025] S14, determination of the blocking solution;

[0026] S15, determination of the dilution factor of the enzyme-labeled monoclonal antibody;

[0027] S16, determination of the lowest detectable concentration of porcine IL-15;

[0028] S17, evaluation of the specificity of the double antibody sandwich ELISA method;

[0029] S18, evaluation of the repeatability of the double antibody sandwich ELISA method.

[0030] Preferably, the pairing test steps of the monoclonal antibody are as follows: Label the purified monoclonal antibody, select the unlabeled antibody as the capture antibody, coat an ELISA plate, incubate, wash the ELISA plate, add a blocking solution for blocking treatment, add recombinant porcine IL-15 protein, set up a negative control, incubate, wash the plate, add HRP-labeled antibodies in different combinations, incubate, add a chromogenic solution for color development, add a stop solution to terminate the reaction, and select the optimal antibody combination according to the ratio of the D 450nm value of the positive and negative controls (P / N value).

[0031] Preferably, the antibody coating concentration is the concentration at which the P / N value is the highest. In some specific embodiments of the present invention, the antibody coating concentration is 5 μg / mL.

[0032] Preferably, the determination method of the blocking solution is as follows: At a specific antibody coating concentration, after blocking treatment with different blocking solutions, perform ELISA and calculate the P / N value. The one with the largest P / N value is the optimal blocking solution. In some specific embodiments of the present invention, the blocking solution is 2% BSA-PBST.

[0033] Preferably, the determination method of the dilution factor of the enzyme-labeled monoclonal antibody: Perform sandwich ELISA on enzyme-labeled antibodies with different dilution factors, calculate the P / N value, and the one with the largest P / N value is the optimal dilution factor. In some specific embodiments of the present invention, the dilution factor of the enzyme-labeled antibody is 1:2000.

[0034] In addition, in addition to the above-mentioned porcine IL-15 double antibody sandwich ELISA detection method, the porcine IL-15 monoclonal antibody of the present invention can also be used in methods such as Western blot, immunofluorescence, immunohistochemistry, flow cytometry, etc.

[0035] Compared with the prior art, the beneficial technical effects of the present invention:

[0036] The porcine IL-15 double antibody sandwich ELISA detection method established by the present invention can detect porcine IL-15 protein as low as 31.25 ng / mL, and has no cross-reaction with other cytokines or proteins (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, TNF-α). The average coefficients of variation of the within-batch and between-batch repeatability experiments of the method are 2.56% and 3.39% respectively.

[0037] The porcine IL-15 double antibody sandwich ELISA detection method of the present invention can quantitatively detect porcine IL-15 in samples such as serum, tissue fluid, and cell culture, has high sensitivity, specificity, and accuracy, provides an important tool for porcine IL-15-related research, and has good application prospects. Description of the Drawings

[0038] The present invention will be further described below in conjunction with the accompanying drawings.

[0039] Figure 1 For the identification of recombinant plasmid pET-28α-porcine IL-15 and the identification of porcine IL-15 protein expression. Among them, A is the amplification of porcine IL-15 gene, PCR and enzyme digestion identification of recombinant plasmid. M is 5000bp DNA ladder. 1, 2 and 5 are PCR products of empty plasmid pET28α(+), recombinant plasmid pET28α-porcine IL-15 and porcine lymphoid tissue respectively. 3 and 4 are double enzyme digestion products of pET28α-porcine IL-15 and pET28α(+) by EcoR I-Xho I; B is SDS-PAGE analysis of recombinant protein induced expression. M is 10-250ku protein molecular weight standard. 1 and 2 are porcine IL-15 protein expression bacteria before and after induction respectively; C is Western blot identification of porcine IL-15 recombinant protein. M is 8-180ku protein molecular weight standard. 1 is the reaction result of the induced product of empty plasmid pET-28α(+) transformed into E.coli and His-tag antibody. 2 is the reaction result of the expressed porcine IL-15 protein and His-tag antibody;

[0040] Figure 2 For the identification of recombinant plasmid pcDNA3.1-porcine IL-15 and the identification of porcine IL-15 expression in PK-15. Among them, A is the identification of pcDNA3.1-porcine IL-15. M is 5000bp DNA ladder. 1 and 2 are PCR products of empty plasmid pcDNA3.1(+) and recombinant plasmid pcDNA3.1-porcine IL-15 respectively. 3 and 4 are double enzyme digestion products of pcDNA3.1-porcine IL-15 and pcDNA3.1(+) by EcoRI-Xho I; B is Western blotting identification. M is 8-180ku protein molecular weight standard. 1 and 2 are the reaction results of cells transfected with pcDNA3.1-porcine IL-15 and porcine IL-15 antiserum and porcine IL-15 antibody negative serum respectively. 3 is the reaction result of cells transfected with empty plasmid pcDNA3.1(+) and porcine IL-15 antiserum. C is IFA identification (100×);

[0041] Figure 3 For the titer of monoclonal antibody. Among them, A is cell culture supernatant. B is ascites.

[0042] Figure 4The Western Blotting identification results of monoclonal antibodies, where M is the 10-250 ku protein molecular weight standard; 1-9 are monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4, and D5 respectively, 10 is the positive mouse serum, and 11 and 12 are the negative mouse serum and SP2 / 0 cell supernatant respectively;

[0043] Figure 5 The indirect immunofluorescence identification (100×) results of monoclonal antibodies, where A-G are monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3D5 respectively, H is the positive mouse serum of porcine IL-15 antibody, and I is the negative mouse serum;

[0044] Figure 6 The specific detection results of monoclonal antibodies;

[0045] Figure 7 The affinity curve of monoclonal antibodies;

[0046] Figure 8 The P / N values of different antibody working concentrations and blocking solutions; where A, B, and C are the coating concentration of the capture antibody 2D10, the blocking solution, and the dilution of the enzyme-labeled antibody 2C10 respectively;

[0047] Figure 9 The linear range of the standard curve and the sensitivity analysis results of sandwich ELISA; where A is the linear range of the standard curve and B is the sensitivity analysis result;

[0048] Figure 10 The specific detection results of sandwich ELISA;

[0049] Figure 11 The quantitative detection results of IL-15, where A is Western blotting; B is the gray value analysis of the reaction bands, and 1-16 are DCs isolated from different porcine lymph node samples. Detailed implementation manners

[0050] The present invention provides a method for establishing a porcine IL-15 double-antibody sandwich ELISA detection. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the protection scope of the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0051] In the present invention, the term "and / or" describes the relationship between associated objects and indicates that there can be three relationships. For example, A and / or B can represent the following situations: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural.

[0052] In the present invention, "at least one kind" means one kind or more than one kind, and "a plurality of kinds" means two or more than two kinds.

[0053] In the present invention, many terms are used. Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0054] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0055] In the embodiments of the present invention, SP2 / 0 myeloma cells, Escherichia coli (E. coli) Rosetta (DE3) competent cells and PK-15 cells are involved. These Escherichia coli are commercially available products, so no additional preservation evidence and genetic resource registration materials are required, and plasmids pET28α(+) and pcDNA3.1(+).

[0056] Restriction endonucleases (EcoR I and Xho I) were purchased from Takara Bio (Dalian) Co., Ltd.; Gel Extraction Kit and Plasmid Extraction Kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; T4 DNA Ligase was from Thermo Scientific Co., Ltd.; Freund's complete adjuvant and Freund's incomplete adjuvant were products of Sigma Company; Dulbecco’s Modified Eagle Medium was from GIBCO Company; Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG and His-tag Protein Purification Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Solarbio); Mouse Monoclonal Antibody Ig Class / Subclass / Identification ELISA Kit and Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG were purchased from Suzhou BioLong Science & Technology Co., Ltd.; Multicolor Prestained Protein Marker and ECL Chemiluminescence Detection Kit were purchased from Shanghai Yeasen Biotech Co., Ltd.

[0057] SPF-grade BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0058] Example 1 Preparation, Identification and Specificity Detection of Porcine IL-15 Monoclonal Antibody

[0059] 1.1 Gene cloning, protein expression and identification of porcine IL-15

[0060] Extract nucleic acid from porcine inguinal lymph node tissue, reverse transcribe cDNA and then perform PCR (annealing temperature is 55 °C, amplified fragment size is 498 bp).

[0061] The primers for porcine IL-15 are as follows:

[0062] e porcine IL-15U: 5′-CTAGAATTCGCCACCATGAGAATTTTAAAAC-3′

[0063] e porcine IL-15L: 5′-GACCTCGAGTCAAGAAGGGTTGATG-3′.

[0064] Digest the purified PCR products, plasmid pET28α(+) and pcDNA3.1(+) with EcoR I and Xho I restriction endonucleases respectively; ligate the digested target gene with different vectors at 16 °C for 16 h using T4 DNA ligase to construct pET-28α-porcine IL-15 recombinant plasmid and pcDNA3.1-porcine IL-15 recombinant plasmid, and identify the recombinant plasmids by restriction enzyme digestion and sequencing (completed by Sangon Biotech Co., Ltd.).

[0065] Transform the pET-28α-porcine IL-15 recombinant plasmid into Escherichia coli Rosetta(DE3) competent cells, induce activation, spread the bacterial solution on kanamycin-resistant LB (Kana+ / LB) plates; pick positive single colonies and inoculate them into Kana+ / LB liquid medium, shake culture at 37 °C until the logarithmic growth phase, induce culture with 0.25 mmol / L IPTG at 32 °C for 6 h, collect the bacteria and lyse them by ultrasound, analyze the protein expression by SDS-PAGE, and identify the expressed protein by Western blot.

[0066] The Western blot procedure: Transfer the proteins in the gel to a PVDF membrane; block the membrane overnight at 4 °C; after washing the membrane, add a mouse monoclonal antibody against His tag diluted 1:5000 and incubate at room temperature for 2 h; wash the membrane, add goat anti-mouse IgG-HRP diluted 1:5000 and incubate at room temperature for 1.5 h; wash the membrane, add CEL substrate chromogenic solution and observe the reaction of the antibody with the expressed protein.

[0067] Using the liposome transfection method, the pcDNA3.1-porcine IL-15 recombinant plasmid was transfected into PK-15 cells. 48 h after transfection, the expression of porcine IL-15 was detected by indirect immunofluorescence assay (IFA). The specific method was as follows: PK-15 cells were fixed with pre-cooled absolute methanol, and then reacted successively with porcine IL-15 mouse polyclonal antibody (1:200) and FITC-labeled goat anti-mouse IgG (diluted 1:300 times). The green fluorescence in the cells was observed under a fluorescence microscope to identify the expression of porcine IL-15.

[0068] The results showed that the porcine IL-15 gene ( Figure 1 A) consistent with the expected size (498 bp) was amplified from piglet lymph nodes by RT-PCR in the present invention. The constructed porcine IL-15 recombinant expression plasmids pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15 were identified by PCR, and target fragments consistent with the expected size could be amplified. After double digestion with the restriction enzymes EcoR I-Xho I, DNA fragments of the same size as the target gene could be released, while the empty plasmid control plasmids pET28α(+) and pcDNA3.1(+) did not release the target bands ( Figure 1 A and Figure 2 A).

[0069] The sequencing results showed that the cloned target gene sequence was consistent with the reference sequence. It indicated that the pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15 recombinant plasmids were successfully constructed. The Escherichia coli Rosetta(DE3) transformed with the recombinant plasmid pET-28α-porcine IL-15 was induced with IPTG at 32 °C for 6 h, and SDS-PAGE showed the target recombinant protein of the expected size (about 21 ku) ( Figure 1 B).

[0070] Western blot identification showed that after the expressed protein reacted with the anti-His tag antibody, reaction bands consistent with the expected size appeared in the corresponding lanes ( Figure 1 C). It indicated that the porcine IL-15 protein was expressed.

[0071] PK-15 cells transfected with the recombinant plasmid pcDNA3.1-porcine IL-15 were collected and identified by Western blot. After reaction with porcine IL-15 antiserum, reaction bands of the expected size appeared in the corresponding lanes, while no reaction bands appeared in the corresponding lanes of the negative control ( Figure 2 B).

[0072] IFA identification showed that specific green fluorescence reacting with porcine IL-15 antiserum appeared in PK-15 cells transfected with pcDNA3.1-porcine IL-15, while no green fluorescence appeared after reaction with porcine IL-15 antibody negative serum( Figure 2 C). It indicated that porcine IL-15 was expressed in PK-15 cells.

[0073] 1.2 Screening of hybridoma cell lines stably secreting porcine IL-15 specific monoclonal antibodies and identification of monoclonal antibody classes / subclasses

[0074] The expressed recombinant porcine IL-15 protein was purified using a His-tag protein purification kit. SPF mice aged 6 - 8 weeks were subcutaneously inoculated with the protein (50 μg / mouse) through the back, and inoculation was carried out continuously 3 times at an interval of 14 days. The mouse with the highest antibody level was selected and boosted immunized by intraperitoneal injection of 80 μg porcine IL-15 protein 3 days before cell fusion. The spleen of the boosted immunized mouse was aseptically removed to prepare a spleen cell suspension; under the action of PEG4000, the spleen cells were fused with SP2 / 0 myeloma cells. On the 9th day after fusion, the culture supernatant of the cell clone wells was collected, and ELISA was used to detect porcine IL-15 specific antibodies to screen hybridoma cell lines stably secreting porcine IL-15 specific monoclonal antibodies.

[0075] During ELISA detection, the enzyme-linked immunosorbent assay plate was coated with 1 μg / mL of recombinant porcine IL-15 protein, and at the same time, an antigen control coated with the induced expression product of Escherichia coli transformed with empty plasmid was set up to exclude non-specific reactions. The dilution factor of goat anti-mouse IgG-HRP was 1:10000.

[0076] The selected positive hybridoma cells were subcloned continuously 3 - 5 times to obtain hybridoma cell lines stably secreting porcine IL-15 specific monoclonal antibodies (deposit number: CCTCC NO: C2024127). The culture supernatant of the positive hybridoma cells was serially diluted 10 2 -10 4 times, and ELISA was used to detect the titer of the monoclonal antibodies secreted by the hybridoma cells.

[0077] The antibody type secreted by the hybridoma cell line was detected using a mouse monoclonal antibody Ig class / subclass / identification ELISA kit. At the same time, negative and positive controls were set up. After color development, the D 450nm value was measured. When the D 450nm value of the test sample < 0.15, it was determined to be negative; when the D 450nm of the test sample > negative control D 450nm + 0.15, it was determined to be positive. Subsequently, ascites was prepared by the in vivo induction method, the antibody was purified by the octanoic acid ammonium sulfate method, and the titer of the ascites monoclonal antibody was determined by ELISA.

[0078] The results showed that the fusion rate of the spleens of immunized mice and SP2 / 0 myeloma cells was 33%, and the antibody positive rate was 99%. After 3 to 5 subclonings, a total of 9 hybridoma cell lines (1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4, and 3D5) that stably secreted porcine IL-15-specific monoclonal antibodies were obtained. Identified by a mouse monoclonal antibody Ig class / subclass / identification ELISA kit, all 9 monoclonal antibodies reacted with anti-IgG2b antibody and anti-κ light chain antibody, belonging to the IgG2b class κ chain, that is, IgG2bκ. The antibody titers in the culture supernatants of the 9 hybridoma cell lines were between 1:3200 and 1:12800( Figure 3 A). Ascites were prepared from the hybridoma cell lines 2C10, 2D10, 2F3, and 3D5, and the antibody titers in the ascites were between 400000 and 3200000( Figure 3 B).

[0079] 1.3 Identification of monoclonal antibodies

[0080] After SDS-PAGE electrophoresis of the recombinant porcine IL-15 protein, Western blot was used to identify the monoclonal antibodies, and the induced product of Escherichia coli transformed with the empty plasmid pET28α(+) was used as a negative serum control. When performing Western blot identification, the monoclonal antibody to be tested was diluted 1:500 times, and the goat anti-mouse IgG-HRP was diluted 1:5000 times.

[0081] Using the liposome transfection method, the eukaryotic expression plasmid pCDNA3.1-porcine IL-15 was transfected into PK-15 cells. 48 h after transfection, the monoclonal antibodies were identified by IFA. When identifying, the dilution factor of the monoclonal antibody to be tested was 1:200, and the dilution factor of the FITC-labeled goat anti-mouse IgG was 1:300. After the reaction, the results were observed under a fluorescence microscope.

[0082] 1.4 Specificity detection of monoclonal antibodies

[0083] The enzyme-linked immunosorbent assay (ELISA) plates were coated with 9 different proteins, namely IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, and TNF-α, respectively. The cross-reactivity of the prepared porcine IL-15-specific monoclonal antibodies with the above protein antigens was detected by indirect ELISA to verify the specificity of the monoclonal antibodies.

[0084] Western blot identification showed that when monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4, and 3D5 reacted with porcine IL-15 protein respectively, a clear specific reaction band appeared in the corresponding lane, while no reaction band appeared in the corresponding lane after reacting with the induced product of the empty plasmid-transformed bacteria ( Figure 4 ). Further IFA results showed that obvious green fluorescence appeared in the cells after PK-15 cells transfected with pcDNA3.1-porcine IL-15 recombinant plasmid reacted with each strain of monoclonal antibody ( Figure 5 ).

[0085] ELISA results showed that the obtained monoclonal antibodies specifically bound only to porcine IL-15 protein and had no cross-reaction with protein antigens ( Figure 6 ), indicating that the monoclonal antibodies obtained in the present invention had good specificity.

[0086] Example 2 Establishment and evaluation of porcine IL-15 double antibody sandwich ELISA detection method

[0087] 2.1 Determination of the affinity of monoclonal antibodies

[0088] The affinity of monoclonal antibodies was identified by the indirect ELISA method. The specific method was as follows: The enzyme-linked immunosorbent assay (ELISA) plates were coated with porcine IL-15 protein at 0.5 and 0.25 μg / mL respectively. The selected monoclonal antibodies 2C10, 2D10, 2F3, and 3D5 were diluted to 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 μg / mL respectively. After color development, the D 450nm value was measured, and the antibody-antigen binding reaction curve was plotted. By the graphical method, the antibody concentration (Ab) corresponding to half of the maximum D 450nm value (i.e., 50% D 450nm ) at each antigen concentration (Ag) was taken, and the affinity constant (Kaff) was calculated.

[0089] Kaff = (n - 1) / 2(nAb′ - Ab)

[0090] In the formula: Ab and Ab′ represent the antibody (Ab) concentrations (mol / L) that produce half of the absorbance value when the antigen (Ag) concentration is 0.5 μg / mL and 0.25 μg / mL respectively, and n = Ag / Ag′;

[0091] The average value of the Kaff values was taken as the affinity constant Kaff of the monoclonal antibody, and the dissociation constant Kd of the monoclonal antibody was the reciprocal of the affinity constant Kaff.

[0092] According to the D values of each strain of monoclonal antibody at different dilution multiples measured by ELISA after reacting with porcine IL-15 protein450nm Based on the values and the Kaff formula, it was found that the dissociation constants of the monoclonal antibodies 2C10, 2D10, 2F3, and 3D5 were 8.32, 12.4, 8.33, and 8.32 nmol / L respectively, indicating that the four monoclonal antibodies had a high affinity for porcine IL-15( Figure 7 ).

[0093] 2.2 Pairing test of monoclonal antibodies

[0094] Referring to the instructions of the LinKine TM horseradish peroxidase-conjugated kit, four purified monoclonal antibodies (2C10, 2D10, 2F3, 3D5) were labeled. Then, the four antibodies were paired pairwise using sandwich ELISA. The specific method was as follows: Unlabeled 2C10, 2D10, 2F3, or 3D5 was used as the capture antibody to coat the enzyme-linked immunosorbent assay (ELISA) plate (0.5 μg / well), and the plate was incubated at 37 °C for 1 h and then transferred to 4 °C overnight; the plate was washed 3 times, 5 min each time; blocking solution was added and the plate was blocked at 37 °C for 1 h; 1 μg / mL of recombinant porcine IL-15 protein was added and the reaction was carried out at 37 °C for 1 h, and other proteins were set as negative controls; after washing the plate, different HRP-labeled antibodies were added and incubated at 37 °C for 1 h (combination pairing); TMB substrate chromogenic solution was added and the color was developed in the dark at room temperature for 15 min, 2 mol / L of concentrated sulfuric acid was added to terminate the reaction, and the D 450nm value was measured. The detection results are shown in Table 1.

[0095] Table 1 Combination pairing and detection results of monoclonal antibodies

[0096]

[0097]

[0098] As can be seen from Table 1, after the combination of 2D10 and HRP-2C10, the ratio of the positive to negative control D 450nm value (P / N value) was the highest (12.61). Therefore, 2D10 was selected as the capture antibody and HRP-2C10 as the enzyme-labeled antibody.

[0099] 2.3 Determination of antibody coating concentration

[0100] The ELISA plate was coated with monoclonal antibody 2D10 at 0.1, 0.5, and 1 μg / mL respectively. Using 1 μg / mL of recombinant porcine IL-15 protein as the positive sample, after reacting with HRP-2C10 (diluted 1:4000 times), TMB was added and the color was developed in the dark at room temperature for 15 min, and the D 450nm value was measured and the P / N value was calculated.

[0101] The results showed that the P / N value was the largest when 2D10 was coated at 5 μg / mLFigure 8 A), so the antibody coating concentration was 5 μg / mL.

[0102] 2.4 Determination of blocking solution

[0103] Using the determined antibody coating concentration, after blocking with PBST (0.01 mol / L PBS containing 0.05% Tween-20, pH 7.4) containing 5% skim milk powder, 5% BSA, 2% BSA, 5% calf serum, and 1% gelatin respectively, ELISA was performed and the P / N value was calculated.

[0104] The results showed that the P / N was the highest when the blocking solution was 2% BSA-PBST, so 2% BSA-PBST was selected as the blocking solution ( Figure 8 B).

[0105] 2.5 Determination of the dilution factor of enzyme-labeled monoclonal antibody

[0106] The enzyme-labeled antibody was diluted 1:1000, 1:2000, 1:4000, and 1:8000 times, and sandwich ELISA was performed to calculate the P / N value.

[0107] The results showed that the P / N value was higher when HRP-2C10 was diluted 1:2000 than that of other dilution degrees ( Figure 8 C), so the enzyme-labeled antibody was diluted 1:2000.

[0108] 2.6 Establishment of standard curve and sensitivity analysis of sandwich ELISA

[0109] Using the absolute quantification method, porcine IL-15 standards at different concentrations (1000, 500, 250, 125, 62.5, 31.2, 15.6, 0 ng / mL) were detected by sandwich ELISA. With the standard concentration as the abscissa and the D 450nm value as the ordinate, a standard curve was plotted, the regression equation was obtained and the correlation coefficient (R 2 ) was calculated to determine the linear range of the standard curve.

[0110] The standard curve plotted according to the results showed that when the porcine IL-15 protein concentration was between 15 and 1000 ng / mL, the D 450nm value had a good linear relationship with the sample concentration, and R 2 was 0.99 ( Figure 9 A).

[0111] Porcine IL-15 (2 μg / mL) was serially diluted two-fold (a total of 8 dilution levels) for sandwich ELISA, and other proteins were used as negative controls. The detection limit of the established double-antibody sandwich ELISA method was determined as the maximum dilution level with a P / N value > 2.1, and the lowest detectable concentration of porcine IL-15 was calculated based on the standard curve.

[0112] The results showed that when the concentration of porcine IL-15 was 31.25 ng / mL, the P / N value = 6.05 > 2.1 ( Figure 9 B), indicating that the sensitivity of the established sandwich ELISA was 31.25 ng / mL.

[0113] 2.7 Specificity test

[0114] According to the established ELISA procedure, the established double-antibody sandwich ELISA method was used to simultaneously detect porcine IL-15 and other cytokines or proteins (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, TNF-α).

[0115] The results showed that except for the porcine IL-15 samples, the detection results of the other proteins were all negative ( Figure 10 ), indicating that this method did not cross-react with other cytokines or proteins.

[0116] 2.8 Repeatability test

[0117] ELISA was performed using the 2D10 capture antibody from the same batch (intra-group repeat) and 3 batches (inter-group repeat) to detect 6 lymph node tissue fluids and 2 negative protein samples, and the coefficient of variation (CV) of the detection results within and between groups was calculated to evaluate the repeatability of the method. The results are shown in Tables 2 and 3.

[0118] Table 2 Intra-batch repeatability experiment

[0119]

[0120] Table 3 Inter-batch repeatability experiment

[0121]

[0122]

[0123] The average CV of the intra-batch repeatability test was 2.51%. The average CV of the inter-batch repeatability test was 3.15%, indicating that the sandwich ELISA established in the present invention had good repeatability.

[0124] The IL-15 gene was amplified from porcine lymph nodes in the present invention. Its coding region is 498 bp in total, and the identity with the reference sequence (GenBank ID: EU234503) is 96%, indicating that the IL-15 gene was successfully obtained in the present invention.

[0125] The fusion rate of mouse spleen cells and SP / 20 cells is directly related to the screening of hybridoma cells. The cell fusion rate in the present invention is 33%, and the antibody positive rate is 99%, which is higher than the fusion rate (24.91%) of mouse anti-human IL-15 specific monoclonal antibodies in the prior art, and the antibody positive rate is 1.0%.

[0126] Such a high antibody positive rate may affect the specificity of the detection results. Therefore, in the present invention, the induced product of Escherichia coli transformed with an empty plasmid was used as the coating antigen to re-screen the positive hybridoma cells screened for the IL-15 protein antigen. After 3 to 5 rounds of cloning and screening, 9 hybridoma cell lines were obtained, and the monoclonal antibodies secreted by them were all of the IgG2bκ type. This antibody type is different from the subclass (IgM) of mouse anti-human IL-15 specific monoclonal antibodies in the prior art, and the subclasses (IgG1 and IgG2a) of 4 mouse anti-human IL-15 specific monoclonal antibodies.

[0127] In order to identify and prove the specificity of the monoclonal antibody obtained in the present invention in binding to the IL-15 protein, the present invention also constructed an IL-15 eukaryotic expression plasmid, and further identified the obtained monoclonal antibody using PK-15 cells transfected with the IL-15 expression plasmid. Further, through cross-reactions with other cytokines or proteins, it was proved that the obtained monoclonal antibody specifically binds to IL-15.

[0128] ELISA is one of the most commonly used methods for detecting antigens and antibodies. In order to provide a commercial detection tool for porcine IL-15, in the present invention, after mutual pairing tests among monoclonal antibodies, 2D10 was finally selected as the capture antibody and 2C10 as the enzyme-labeled antibody to establish a porcine IL-15 protein quantitative sandwich ELISA method. The drawing of the standard curve and its linear range are related to the results of quantitative detection, indicating that the standard curve established with the recombinant porcine IL-15 protein standard prepared in the present invention has a good linear relationship with the OD value of ELISA (R 2 = 0.99), and has a similar linear relationship with the standard curves of commercial cytokine kits such as IL-4, IL-10, IFN-γ, and TNF-α.

[0129] In summary, the IL-15 sandwich ELISA established in the present invention can detect porcine IL-1 at a minimum concentration of 31.25 ng / mL, has good specificity and sensitivity, and can be applied to the quantitative detection of porcine IL-15 in biological samples. The preparation of the porcine IL-15-specific monoclonal antibody, the establishment and evaluation of the sandwich ELISA method in the present invention can provide important material support for the research on the infection immunity related to porcine IL-15.

[0130] Example 3 Application of the Porcine IL-15 Double Antibody Sandwich ELISA Detection Method

[0131] Inguinal lymph nodes of 16 piglets were collected, minced, and 500 μg / mL of type IV collagenase and 200 μg / mL of DNase I were added, followed by digestion for 15 min; the digested cell suspension was filtered through a 70 nm filter and centrifuged at 1000 r / min for 10 min; the cell pellet was collected and resuspended in 10% FBS 1×RPMI-1640 nutrient solution, and dendritic cells (DCs) were isolated by the adherent method. Then, the DC cells were stimulated with 10 μg / mL of LPS for 18 h, and the cell supernatant and cells were collected respectively. The concentration of IL-15 in the collected supernatant was detected by the porcine IL-15 double antibody sandwich ELISA detection method established in the present invention, and the content of IL-15 in DCs was detected by Western blot method.

[0132] Main steps of the Western blot assay: After SDS-PAGE of the inguinal lymph node tissue, the protein was transferred to a PVDF membrane; the membrane was blocked overnight at 4°C; after washing the membrane, 2D10 monoclonal antibody diluted 1:50000-fold was added and incubated at room temperature for 2 h; after washing the membrane, goat anti-mouse IgG-HRP diluted 1:5000-fold was added and incubated at room temperature for 1.5 h; after washing the membrane, CEL substrate chromogenic solution was added to observe the reaction of the antibody with the expressed protein. The results are shown in Table 4 and Figure 11 as follows.

[0133] Table 4 Detection Results of IL-15 Content

[0134]

[0135] As can be seen from Table 4 and Figure 11 it can be known that the content of IL-15 expressed by porcine DCs detected by the porcine IL-15 double antibody sandwich ELISA detection method established in the present invention is between 1.4 and 56 ng / mL, which is consistent with the Western blot detection results. This result indicates that the porcine IL-15 double antibody sandwich ELISA detection method can be used for the quantitative detection of IL-15.

[0136] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A porcine IL-15 monoclonal antibody, characterized in that: Prepared using the hybridoma cell line with the preservation number of CCTCC NO: C2024127.

2. A double-antibody sandwich ELISA detection method for non-diagnostic quantitative detection of porcine IL-15 content in biological samples, characterized in that, Performed using the porcine IL-15 monoclonal antibody described in claim 1.

3. A method for establishing and evaluating the detection method according to claim 2, characterized in that, Including the following steps: S11. Determination of the affinity of the monoclonal antibody; S12. Conducting the pairing test of the monoclonal antibody; S13. Determination of the antibody coating concentration; S14. Determination of the blocking solution; S15. Determination of the dilution factor of the enzyme-labeled monoclonal antibody; S16. Determination of the lowest detectable concentration of porcine IL-15; S17. Evaluating the specificity of the double antibody sandwich ELISA method; S18. Evaluating the repeatability of the double antibody sandwich ELISA method.

4. The method according to claim 3, characterized in that The double antibody includes a capture antibody and an enzyme-labeled antibody that specifically binds to porcine IL-15.

5. The method according to claim 3, wherein The antibody coating concentration is the concentration at which the P / N value is the highest.

6. The method according to claim 3, wherein The method for determining the blocking solution is as follows: At a specific antibody coating concentration, after blocking treatment with different blocking solutions, ELISA is performed and the P / N value is calculated. The blocking solution with the largest P / N value is the optimal blocking solution.

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