A monoclonal antibody combination for detecting human il-10 protein and application thereof

Abstract

This invention relates to the field of biological detection, and more particularly to a monoclonal antibody combination for detecting human IL-10 protein and its application. The antibody combination includes 8E10 and 10D12. The amino acid sequences of the complementarity-determining region (CDR) of the heavy chain variable region of monoclonal antibody 8E10 correspond to SEQ ID NO.1-SEQ ID NO.3, and the amino acid sequences of the CDR of the light chain variable region correspond to SEQ ID NO.4-SEQ ID NO.6. The amino acid sequences of the CDR of the heavy chain variable region of monoclonal antibody 10D12 correspond to SEQ ID NO.7-SEQ ID NO.9, and the amino acid sequences of the CDR of the light chain variable region correspond to SEQ ID NO.10-SEQ ID NO.12. This antibody combination ensures the reproducibility of raw material production, providing a reliable foundation for the preparation of highly sensitive and stable ELISA kits and other in vitro diagnostic tools, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a combination of monoclonal antibodies for detecting human IL-10 protein and its application. Background Technology

[0002] Interleukin-10 (IL-10) is an important pleiotropic immunomodulatory cytokine that plays a central role in regulating inflammation and maintaining immune homeostasis. Human IL-10 is encoded by the IL-10 gene located on the long arm of chromosome 1. Its natural biologically active form is a soluble homodimer of approximately 36 kDa, composed of two monomers. Each monomer has a typical six-α-helix structure and maintains conformational stability through intrachain disulfide bonds. Classified according to the structural characteristics of cytokine receptors, IL-10 belongs to the class II cytokine family and mediates signal transduction through its specific receptor IL-10R (also a class II cytokine receptor, CRF2).

[0003] The biological effects of IL-10 begin with the binding of the IL-10 homodimer to the heterotetramer IL-10 receptor complex (IL-10R) on the cell surface, thereby activating the JAK1 / TYK2–STAT3 signaling pathway and regulating the expression of various immune-related genes. IL-10's role in the immune system is a double-edged sword: on the one hand, it is a potent anti-inflammatory and immunosuppressive cytokine; on the other hand, it also possesses immunostimulatory properties. The different sources of IL-10, the target cell types, and the site and timing of secretion are key factors in activating multiple signal transduction pathways, triggering either inhibitory or activating signaling pathways in different microenvironments.

[0004] During acute infection, IL-10 helps limit the intensity of the inflammatory response, prevents immune-mediated tissue damage, and promotes inflammation resolution and tissue repair after pathogen clearance. However, when IL-10 expression or signaling is persistently enhanced, it may suppress effective anti-infective immune responses, induce immune tolerance and immune escape, and promote long-term pathogen survival, thereby driving the formation of chronic or latent infections. For example, IL-10 can suppress pathogenic inflammatory responses in acute Toxoplasma gondii infection, while in chronic infections such as Toxoplasma gondii, Leishmaniasis, EBV, HIV, and hepatitis B virus, its high expression is closely related to suppressed T cell function and enhanced pathogen replication.

[0005] At the cellular level, IL-10 has a significant inhibitory effect on myeloid cells such as monocytes, macrophages, and dendritic cells, which typically highly express IL-10R. IL-10 suppresses immune function by blocking the synthesis of pro-inflammatory cytokines (such as IL-1, IL-6, IFN-γ, and TNF-α) in T cells, monocytes, and macrophages, and by inhibiting the expression of cell surface molecules involved in antigen presentation and co-stimulation. Simultaneously, under specific immune conditions, IL-10 can also produce immune-enhancing effects on lymphocyte populations, such as promoting B cell survival and antibody production, or improving CD8 expression in certain chronic infections and tumor environments. + T cell function reflects its role in immune stimulation.

[0006] Given its crucial role in inflammation regulation and immune homeostasis, IL-10 has become an important biomarker for assessing the progression of infectious diseases, monitoring the immune status of sepsis, determining the activity of autoimmune diseases, and studying the tumor immune microenvironment. The types of samples for IL-10 testing are quite broad. Clinically, this mainly includes serum, plasma, and whole blood. In research, it can also be used to detect IL-10 levels in peripheral blood mononuclear cell culture supernatant, cell culture supernatant, tissue homogenate, and inflammation-related fluids (such as cerebrospinal fluid, pleural effusion, and ascites).

[0007] Currently, quantitative detection of IL-10 primarily employs enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and multifactorial detection platforms. Among these, double-antibody sandwich ELISA remains one of the most widely used techniques for cytokine detection due to its advantages such as mature methodology, ease of operation, relatively low cost, high throughput, stable results, and ease of standardization. This method relies on two high-quality monoclonal antibodies that recognize different antigenic epitopes, serving as the capture antibody and the detection antibody, respectively. Its detection performance (such as sensitivity and specificity) largely depends on the affinity, epitope, and stability of the antibodies themselves. Therefore, developing monoclonal antibodies with high affinity, strong specificity, and well-defined sequences targeting different epitopes of human IL-10 is of great significance for establishing a stable, sensitive, and reproducible double-antibody sandwich ELISA detection system. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a monoclonal antibody combination for detecting human IL-10 protein and its application, which solves the technical problems of unclear antibody epitope information and difficulty in constructing a stable detection system in the prior art.

[0010] (II) Technical Solution

[0011] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0012] In a first aspect, this application provides a monoclonal antibody combination for detecting human IL-10 protein, the monoclonal antibody combination comprising monoclonal antibody 8E10 and monoclonal antibody 10D12;

[0013] The heavy chain variable region of the monoclonal antibody 8E10 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3.

[0014] The light chain variable region of the monoclonal antibody 8E10 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6.

[0015] The heavy chain variable region of the monoclonal antibody 10D12 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.9.

[0016] The light chain variable region of the monoclonal antibody 10D12 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.12.

[0017] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO. 14.

[0018] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO. 16.

[0019] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO. 18.

[0020] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.20.

[0021] In some embodiments, the monoclonal antibody combination specifically recognizes recombinant human IL-10 protein and natural human IL-10 protein.

[0022] Secondly, the application of the combination of monoclonal antibodies provided in this application in the preparation of tools for detecting human IL-10 protein.

[0023] In some embodiments, the tool includes reagents, kits, test strips, and antibody chips.

[0024] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0025] In some embodiments, the ELISA kit uses monoclonal antibody 8E10 as the coating antibody and monoclonal antibody 10D12 as the labeling antibody.

[0026] (III) Beneficial Effects

[0027] This invention utilizes hybridoma technology to screen monoclonal antibodies 8E10 and 10D12, which have clearly defined and non-interfering binding epitopes, enabling them to form a highly efficient double-antibody sandwich pair with high specificity and affinity. Experimental data show that this combination can specifically recognize recombinant and native human IL-10 protein and exhibits no cross-reactivity with various irrelevant cytokines, effectively avoiding false-positive interference in clinical testing. The detection system has high sensitivity and a low detection limit for recombinant human IL-10 protein. This invention provides the amino acid sequence information of the antibodies, wherein the amino acid sequences of the complementarity-determining region (CDR) of the heavy chain variable region of monoclonal antibody 8E10 correspond to SEQ ID NO.1-SEQ ID NO.3, and the amino acid sequences of the CDR of the light chain variable region correspond to SEQ ID NO.4-SEQ ID NO.6; the amino acid sequences of the CDR of the heavy chain variable region of monoclonal antibody 10D12 correspond to SEQ ID NO.7-SEQ ID NO.9, and the amino acid sequences of the CDR of the light chain variable region correspond to SEQ ID NO.10-SEQ ID NO.12. This well-defined antibody combination not only ensures the reproducibility of raw material production, but also provides a reliable material basis for the preparation of in vitro diagnostic tools such as highly sensitive and stable ELISA kits, and has broad application prospects. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Image showing the results of SDS-PAGE protein identification;

[0030] Figure 2 The image shows the identification results of the purified monoclonal antibody.

[0031] Figure 3 The results of the specificity and sensitivity of the double-antibody sandwich ELISA for recombinant IL-10 protein are shown in the figure.

[0032] Figure 4 This is a diagram illustrating the activity of paired antibodies. Detailed Implementation

[0033] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0034] This invention utilizes hybridoma technology to successfully prepare monoclonal antibodies capable of specifically recognizing human IL-10 protein. Based on this, antibody pairings with complementary properties and different binding sites were screened and obtained. These monoclonal antibodies can be used to construct ELISA detection systems suitable for the quantitative detection of IL-10 protein in samples such as serum, plasma, and cell culture supernatants. Validation has shown that this system exhibits high sensitivity, strong specificity, low background, and good reproducibility, meeting the accuracy and consistency requirements of both research and clinical testing. It provides reliable antibody raw materials for the development of IL-10-related immunodiagnostic reagents.

[0035] The double-antibody sandwich ELISA detection method described in this application is not intended for disease diagnosis and treatment.

[0036] 1. Expression of recombinant IL-10 protein

[0037] Referring to the IL-10 gene sequence, the gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is shown in SEQ ID NO.21:

[0038] ATGAGTCCGGGTCAGGGCACCCAGAGTGAAAATAGTTGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGATGCATTTTCACGTGATGCATTTTCACGTGTGAAAACCTTTTTCCAGATGAAAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGTCTGCTGGAAGATTTTAAAGGCTATCTGGGTTGTCAGGCCCTGAGCGAAATGATTCAGTTTTATCTGGAAGAAGTGATGCCGCAGGC AGAAAATCAGGACCCTGATATTAAGGCACATGTTAATAGCCTGGGTGAAAATCTGAAAACCCTGCGCCTGCGTCTGCGTCGCTGTCATCGTTTTCTGCCGTGCGAAAATAAGAGTAAAGCCGTTGAACAGGTTAAAAATGCCTTTAATAAGCTGCAGGAAAAAGGTATCTATAAAGCAATGAGCGAATTTGATATCTTCATTAATTACATCGAGGCCTATATGACCATGAAAATTCGTAAT.

[0039] The amino acid sequence is shown in SEQ ID NO.22:

[0040] MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0041] The recombinant plasmid pET32a-IL-10 was transformed into BL21(DE3) competent cells and induced to express the gene using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600The concentration of the solution is 0.6-0.9, preferably 0.7. IPTG is added to a final concentration of 0.1 mM, and the cells are collected after induction at 16°C and 180 rpm for 16 hours.

[0042] 2. Purification of recombinant proteins

[0043] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TM Purification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second sonication intervals. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 M protein marker, 1 is the sample flow-through, 2 is the IL-10 recombinant protein purified by elution with 100mM imidazole, 3 is the IL-10 recombinant protein purified by elution with 500mM imidazole. A clear main band is visible between 33-43kDa, consistent with the expected size (the estimated molecular weight of recombinant IL-10 is 37 kDa). The purified IL-10 can be used for subsequent mouse immunization and monoclonal antibody screening.

[0044] 3. Screening of IL-10 monoclonal antibodies - mouse immunization

[0045] Mice were immunized with high-purity human IL-10 recombinant protein, and monoclonal antibody screening was performed using IL-6 recombinant protein expressed by the pET32a vector (expressed by the applicant using E. coli) as a reverse screening antigen. Specifically, purified IL-10 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 20 μg / mouse. At weeks 4, 8, and 12, booster immunizations were performed subcutaneously at multiple sites with an equal volume of Freund's incomplete adjuvant at the same dose. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for intraperitoneal booster immunization with 20 μg of IL-10 recombinant protein, and the spleens of these mice were harvested three days later for hybridoma cell preparation.

[0046] Referring to the human IL-6 amino acid sequence (NP_000591.1), Val30-Met212 was selected for recombinant expression, with Met added at the N-terminus. The gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is SEQ ID NO. 23:

[0047] .

[0048] The amino acid sequence is SEQ ID NO.24:

[0049] MVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM.

[0050] The recombinant plasmid pET32a-IL-6 was transformed into BL21(DE3) competent cells and induced to express the plasmid using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600 The concentration was 0.6-0.9, preferably 0.7. After adding IPTG to a final concentration of 0.1 mM, the cells were collected at 37°C and 200 rpm for 4 hours to obtain recombinant IL-6 protein.

[0051] 4. Screening of hybridoma cells

[0052] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones that reacted positively with recombinant IL-10 protein were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET32a vector containing a His tag, background components needed to be screened to select specific cell lines targeting IL-10 protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0053] 5. Screening positive clones using indirect ELISA method

[0054] To efficiently screen for IL-10-specific monoclonal antibodies, human IL-10 recombinant protein expressed in E. coli (IL-10(E.Coli)) and human IL-10 recombinant protein expressed in 293 cells (nearshore, CX04, IL-10(293)) were used for detection to obtain monoclonal antibodies that could react with antigens expressed in both different systems. At the same time, IL-6 recombinant protein was used for reverse screening to remove immune background. Specifically, the two recombinant IL-10 proteins and other recombinant proteins of the pET32a vector (pET32a-IL-6, His tag) were coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, diluted to 1L of pure water), with a coating concentration of 1μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 2% sucrose + 3% BSA, 150μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. 50μL of cell culture supernatant was added, and a negative control was prepared by diluting another mouse monoclonal antibody (Sino-Pharmaceutical IL-17A monoclonal antibody: 10247-M237) to 1μg / mL, and reacted at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewand, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with the IL-10 recombinant protein but not with the control recombinant protein were selected for subsequent experiments. The screening results are shown in Table 1.

[0055] Table 1: Screening results of monoclonal antibodies

[0056]

[0057] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0058] 6. Purification and Identification of Monoclonal Antibodies

[0059] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, preferably 4°C, and collect the supernatant. Filter the supernatant through defatted cotton, add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, incubate the precipitate overnight at 2–8°C, and centrifuge at 12000 rpm for 30 minutes at 2–8°C to collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl (pH 9) and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS (pH 7.4) for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0060] The purified monoclonal antibody was diluted to 1 μg / ml, and another mouse monoclonal antibody (Sino-Pharmaceutical IL-17A monoclonal antibody: 10247-M237) was diluted to the same concentration as a negative control. The binding activity of the antibody to recombinant human IL-10 protein (expressed in E. coli and 293 cells) and recombinant control antigen IL-6 protein was detected using an indirect ELISA method. Specific results are shown below. Figure 2 The results showed that the selected monoclonal antibody specifically binds to human IL-10 recombinant protein and does not react with the control antigen IL-6 recombinant protein, and can be used for subsequent testing.

[0061] 7. Establishment of double-antibody sandwich ELISA

[0062] 7.1 HRP-labeled monoclonal antibodies

[0063] The selected antibody was diluted to a final concentration of 2 mg / mL using carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). 2 mg of HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 1 mg of the diluted antibody solution was added to the matching tube containing HRP and mixed by pipetting. The tube was incubated at room temperature for 1 hour, with regular mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0064] 7.2 Establishment of the double-antibody sandwich method

[0065] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was patted dry and blocked with 3% sucrose and 2% BSA at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. The test antigen IL-10 recombinant protein (E. coli) and the control antigen protein IL-6 were diluted to 10 ng / ml with PBS and added to the microplate at 50 μL / well. The plate was incubated at 37°C for 35 min. The plate was washed four times with PBST wash buffer, patted dry, and then 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS was added. The plate was incubated at 37°C for 35 min, washed four more times, and then 50 μL / well of TMB chromogenic buffer was added. The plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD was measured using a microplate reader. 450 nm value.

[0066] Using IL-10 as the positive antigen (i.e., recombinant IL-10 protein) and IL-6 as the negative antigen, a sandwich assay was performed. The P / N ratio was calculated, and the monoclonal antibody combination with the highest P / N ratio (highest positive detection value and low negative detection value) was selected as the optimal pair. The results are shown in Table 2. 8E10 as the coating antibody and 10D12 as the labeling antibody resulted in the highest P / N ratio when detecting recombinant proteins.

[0067] Table 2: OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value analysis

[0068]

[0069] “ " " indicates the dilution factor."

[0070] 8. Optimization of the double-antibody sandwich ELISA method

[0071] The purified monoclonal antibody 8E10 was diluted with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, and then diluted to 50 μL / well. Coating was performed overnight at 4°C. The coating buffer was discarded the next day, and the wells were blocked with 3% sucrose and 2% BSA at 150 μL / well. The wells were incubated at 37°C for 2 hours, and the blocking buffer was discarded. The test antigen (IL-10 recombinant protein) and control antigen (I...) were then added... L-6 recombinant protein was diluted 10 ng / ml with PBS and added to an ELISA plate at 50 μL / well. The plate was incubated at 37°C for 35 min. The plate was washed four times with PBST wash buffer. HRP-labeled monoclonal antibody 10D12, diluted 1000, 2000, 3000, and 4000 times with PBS, was added at 50 μL / well. The plate was incubated at 37°C for 35 min. The plate was washed four more times. After drying, 50 μL of TMB chromogenic buffer was added to each well, and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD was measured using an ELISA reader. 450 nm value. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The screening results are shown in Table 3.

[0072] Table 3: Results of Optimization of Dual Antibody ELISA Conditions

[0073]

[0074] Table 3 shows that the P / N ratio was highest when the HRP-labeled antibody was coated at 1 μg / ml and diluted 2000 times, making it the optimal combination.

[0075] 9. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting recombinant IL-10 protein

[0076] After determining the optimal reaction conditions, including a coating monoclonal antibody concentration of 1 μg / mL and a 2000-fold dilution of the HRP-labeled monoclonal antibody, and following the above detection steps, recombinant human IL-10 protein was first serially diluted with PBS buffer to obtain concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL. Recombinant human IL-1β protein (Chongqing Tansheng, FAP-BC006, expressed in 293 cells), recombinant human IL-2 protein (Nearshore, GMP-CD66, expressed in 293 cells), and recombinant human IL-4 protein (Nearshore, CX03) were also analyzed. The following recombinant proteins were expressed in 293 cells: human IL-6 (expressed by the applicant using *E. coli*), human IL-7 (expressed by Kaika Biotechnology, IL7-HE001, *E. coli*), human IL-8 (expressed by the applicant using *E. coli*), human IL-11 (expressed by *Pichia pastoris*, C006), human IL-17A (expressed by *Pichia pastoris*, C774, 293 cells), and human IL-33 (expressed by Yiqiao Shenzhou, 10368-HNAE, *E. coli*). 50 μL of each protein was added to each well for detection at the same concentration to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 3 It can be seen that the double-antibody sandwich ELISA test composed of this group of paired antibodies still showed a weak positive reaction when the recombinant human IL-10 protein was diluted to 10 pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0077] Referring to the IL-8 gene sequence, the gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is SEQ ID NO.25:

[0078] ATGGAAGGCGCCGTGCTGCCGCGCAGCGCTAAAGAACTGCGCTGCCAGTGCATTAAGACCTATAGCAAACCGTTTCATCCGAAATTCATTAAGGAACTGCGTGTGATTGAAAGTGGCCCGCATTGTGCAAATACCGAAATTATTGTGAAACTGAGCGATGGTCGTGAACTGTGTCTGGACCCTAAAGAAAATTGGGTTCAGCGCGTGGTGGAAAAATTTCTGAAACGCGCAGAAAATAGC.

[0079] The amino acid sequence is SEQ ID NO.26:

[0080] MEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0081] The recombinant plasmid pET32a-IL-8 was transformed into BL21(DE3) competent cells and induced to express the gene using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600 The concentration of the sample was 0.6-0.9, preferably 0.7. IPTG was added to a final concentration of 0.1 mM, and the cells were collected after induction at 30°C and 200 rpm for 4 hours to obtain the recombinant IL-8 protein.

[0082] 10. Identification of binding activity of paired monoclonal antibodies

[0083] Following the aforementioned indirect ELISA method, paired monoclonal antibodies were serially diluted to 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Another murine-derived unrelated monoclonal antibody, Yiqiao Shenzhou IL-17A monoclonal antibody (10247-M237), was used as a negative control. The binding activity of the antibodies to recombinant human IL-10 protein was measured. Results are as follows... Figure 4 The results showed that it still reacted positively with IL-10 at a dilution of 1 ng / ml, indicating high antibody activity.

[0084] 11. Light and heavy chain variable region sequences of paired monoclonal antibodies

[0085] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by PCR in three rounds. The PCR products were purified by gel excision and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequence of the variable region of the light and heavy chains of the monoclonal antibody.

[0086] Encapsulated monoclonal antibody 8E10:

[0087] Light chain variable region nucleotide sequence:

[0088] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.18:

[0089] GACATCCAGATGATTCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTGTGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACAAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATC CACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGCAAGCAATCTTATGATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0090] Light chain variable region amino acid sequence:

[0091] The amino acid sequence of the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.14:

[0092] DIQMIQSPSSLAVSAGEKVTVSCKSSQSLLNSRTRKNYLAWYQQKQGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDLYTFGGGTKLEIKRTV.

[0093] CDR area annotation:

[0094] The amino acid sequence CDR-L1 of the complementarity-determining region of the light chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 4:

[0095] CDR-L1: KSSQSLLNSRTRKNYLA;

[0096] The amino acid sequence CDR-L2 of the complementarity-determining region of the light chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 5:

[0097] CDR-L2: Wastres;

[0098] The amino acid sequence CDR-L3 of the complementarity-determining region of the light chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 6:

[0099] CDR-L3: KQSYDLYT.

[0100] Heavy chain variable region nucleotide sequence:

[0101] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.17:

[0102] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTAACTATTTCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTTGGTCGCAGTCATTAATAGTAATGGTGGTTTCACCTACTATCCA GACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCTTGTATTACTGTGCAAGACATGAGGTCGGTGATTACGACCTCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACAGTCTCCTCA.

[0103] Heavy chain variable region amino acid sequence:

[0104] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.13;

[0105] EVKLVESGGGLVKLGGSLKLSCAASGFAFSNYFMSWVRQTPEKRLELVAVINSNGGFTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALYYCARHEVGDYDLWYFDVWGAGTTVTVSS.

[0106] CDR area annotation:

[0107] The amino acid sequence CDR-H1 of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 1:

[0108] CDR-H1: NYFMS;

[0109] The amino acid sequence CDR-H2 of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 2:

[0110] CDR-H2: VINSNGGFTYYPDTVKG;

[0111] The amino acid sequence CDR-H3 of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 8E10 is shown in SEQ ID NO. 3:

[0112] CDR-H3: HEVGDYDLWYFDV.

[0113] Labeled monoclonal antibody 10D12:

[0114] Light chain variable region nucleotide sequence:

[0115] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.20:

[0116] GAAATTGTGCTCACTCAGTCTCCATCCTCCCTGACTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCCAGTCCAGTCAGAGCCTTTTAAATAATAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATCATAGCTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATCAAACGTACGGTG.

[0117] Light chain variable region amino acid sequence:

[0118] The amino acid sequence of the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.16:

[0119] EIVLTQSPSSLTMSVGQKVTMSCQSSQSLLNNSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHHSFPLTFGAGTKLEIKRTV.

[0120] CDR area annotation:

[0121] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 10;

[0122] CDR-L1: QSSQSLLNNSNQKNYLA;

[0123] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 11;

[0124] CDR-L2: FASTRES;

[0125] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 12;

[0126] CDR-L3: QQHHSFPLT.

[0127] Heavy chain variable region nucleotide sequence:

[0128] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.19:

[0129] GAGGTGCAGCTGCAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCTTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATAAACTGGGTAAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAGCCCCTACACTGGAAAGCCAACA TATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAGATTATTACGGAAGTAGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0130] Heavy chain variable region amino acid sequence:

[0131] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.15:

[0132] EVQLQESGPELKKPGETVKISCKASGYTFTNYGINWVKQAPGKGLKWMGWISPYTGKPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCADYYGSSWFAYWGQGTLVTVSA.

[0133] CDR area annotation:

[0134] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 7;

[0135] CDR-H1: NYGIN;

[0136] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 8;

[0137] CDR-H2: WISPYTGKPTYADDFKG;

[0138] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 10D12 is shown in SEQ ID NO. 9;

[0139] CDR-H3: YYGSSWFAY.

[0140] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any manner.

Claims

1. A combination of monoclonal antibodies for detecting human IL-10 protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 8E10 and monoclonal antibody 10D12; The heavy chain variable region of the monoclonal antibody 8E10 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 8E10 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 10D12 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 10D12 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination for detecting human IL-10 protein according to claim 1, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting human IL-10 protein according to claim 2, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting human IL-10 protein according to claim 3, characterized by, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8E10 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting human IL-10 protein according to claim 4, characterized by, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 10D12 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting human IL-10 protein according to claim 5, characterized by, The monoclonal antibody combination specifically recognizes recombinant human IL-10 protein and native human IL-10 protein.

7. The use of a combination of monoclonal antibodies based on claim 1 in the preparation of a tool for detecting human IL-10 protein.

8. Use according to claim 7, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

9. The application according to claim 8, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The ELISA kit uses monoclonal antibody 8E10 as the coating antibody and monoclonal antibody 10D12 as the labeling antibody.

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