A monoclonal antibody combination for detecting human il-33 protein and application thereof

Abstract

The application relates to the field of biological detection, in particular to a monoclonal antibody combination for detecting human IL-33 protein and application. The antibody combination comprises 1B3 and 4A10, the heavy chain variable region complementarity determining region amino acid sequence of the monoclonal antibody 1B3 corresponds to SEQ ID NO. 1-SEQ ID NO. 3, the light chain variable region CDR amino acid sequence corresponds to SEQ ID NO. 4-SEQ ID NO. 6; the heavy chain variable region CDR amino acid sequence of the monoclonal antibody 4A10 corresponds to SEQ ID NO. 7-SEQ ID NO. 9, and the light chain variable region CDR amino acid sequence corresponds to SEQ ID NO. 10-SEQ ID NO. 12. The antibody combination guarantees raw material production repeatability, provides a reliable basis for preparing in-vitro diagnostic tools such as ELISA kits with high sensitivity and high stability, and has a wide application prospect.

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-33 protein and its application. Background Technology

[0002] Interleukin-33 (IL-33) belongs to the interleukin-1 (IL-1) family and is a pro-inflammatory protein. Its structure is highly similar to IL-1β, a subfamily of human IL-1. IL-33 protein possesses transcriptional regulatory properties. Related studies have shown that IL-33 is a protein with dual functions, acting as both a cytokine and an intracellular nuclear factor. It is primarily highly expressed in endothelial cells, epithelial cells, and fibroblast-like cells. As an "alarm hormone," IL-33 is released when cells or tissues are damaged, indicating the occurrence of inflammation or injury, and activating immune cells expressing the ST2 receptor (IL-1RL1), thereby rapidly initiating an immune response.

[0003] IL-33 signaling relies on its complex formation with ST2 and its co-receptor IL-1RAcP to activate the NF-κB and MAPK signaling pathways, thereby inducing downstream immune responses. IL-33 can activate type 2 innate lymphoid cells (ILC2), Th2 helper T cells, mast cells, and eosinophils, promoting the secretion of Th2 cytokines such as IL-4, IL-5, and IL-13, playing important roles in Th2 immune responses, inflammation regulation, tissue repair, and fibrosis. Furthermore, IL-33 participates in innate immune regulation and is closely related to various diseases, including asthma, allergic diseases, inflammatory bowel disease, autoimmune diseases, and cardiovascular diseases, making it an important immunomodulatory factor and a potential disease biomarker and intervention target. Therefore, accurate and sensitive detection of IL-33 levels in serum, plasma, tissue homogenates, or cell culture supernatants is of great significance for studying disease mechanisms, assessing inflammatory states, and monitoring treatment efficacy.

[0004] Currently, methods for detecting IL-33 mainly include immunological methods (such as ELISA, Western blotting, and flow cytometry), molecular biological methods (such as qRT-PCR), and biochips. While each method has its advantages, they also have limitations in practical applications: qRT-PCR can only reflect mRNA levels and cannot directly reflect protein quantity or secretory activity; Western blotting and flow cytometry are complex to operate, difficult to standardize, and low-abundance samples are easily affected by background noise; biochips, although enabling high-throughput detection, have limited sensitivity and quantitative accuracy, are highly dependent on antibodies or probes, and batch-to-batch variations may affect result consistency. In contrast, enzyme-linked immunosorbent assay (ELISA) remains the primary method for detecting IL-33 due to its ability to directly quantify proteins, ease of operation, and standardization.

[0005] However, some existing IL-33 detection products still have shortcomings in terms of antibody raw materials. Some reagents use polyclonal antibodies or monoclonal antibodies with unclear epitope information, which are prone to non-specific binding or background interference. At the same time, the pairing effect between different antibodies is not ideal, limiting the detection sensitivity, especially in the detection of low abundance samples. In addition, unclear antibody sequences or inconsistent sources can also cause performance fluctuations between different batches of kits, seriously affecting the reliability of the detection results and making it difficult to meet the needs of high-standard scientific research and clinical testing. Therefore, obtaining human IL-33 monoclonal antibodies with high affinity, high specificity and clear sequences, and screening antibody pairing combinations suitable for sandwich ELISA are key to building a stable, reliable and high-performance IL-33 immunoassay system. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] 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-33 protein and its application, which solves the technical problems of non-specific binding and limited sensitivity of antibody raw materials in the prior art.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, this application provides a monoclonal antibody combination for detecting human IL-33 protein, the monoclonal antibody combination comprising monoclonal antibody 1B3 and monoclonal antibody 4A10;

[0011] The heavy chain variable region of the monoclonal antibody 1B3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0012] The light chain variable region of the monoclonal antibody 1B3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0013] The heavy chain variable region of the monoclonal antibody 4A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0014] The light chain variable region of the monoclonal antibody 4A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0015] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO. 14.

[0016] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO. 16.

[0017] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.18.

[0018] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.20.

[0019] In a further implementation, the monoclonal antibody combination specifically recognizes both recombinant human IL-33 protein and native human IL-33 protein.

[0020] Secondly, this application provides the use of the combination of the above-mentioned monoclonal antibodies in the preparation of a tool for detecting human IL-33 protein.

[0021] In further implementations, the tools include reagents, kits, test strips, and antibody chips.

[0022] In a further implementation, the kit includes a double-antibody sandwich ELISA kit.

[0023] In a further implementation, the ELISA kit uses monoclonal antibody 1B3 as the coating antibody and monoclonal antibody 4A10 as the labeling antibody.

[0024] (III) Beneficial Effects

[0025] The monoclonal antibody assemblies for detecting human IL-33 protein provided by this invention include monoclonal antibodies 1B3 and 4A10. The heavy and light chain variable region sequences of monoclonal antibody 1B3 are shown in SEQ ID NO. 1-6, and the heavy and light chain variable region sequences of monoclonal antibody 4A10 are shown in SEQ ID NO. 7-12. Monoclonal antibodies 1B3 and 4A10 in this antibody assemblies can specifically recognize recombinant and native IL-33 proteins, possessing clear epitope information and ensuring the reproducibility of raw material production. When applied to the preparation of in vitro diagnostic tools such as double-antibody sandwich ELISA kits, this assemblies exhibit high affinity and high specificity, low background signal, and can achieve high sensitivity and high stability detection of human IL-33 protein, providing a reliable raw material basis for the development of related immunodiagnostic reagents and showing broad application prospects. Attached Figure Description

[0026] 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.

[0027] Figure 1 The image shows the identification results of the purified monoclonal antibody;

[0028] Figure 2 The results of the specificity and sensitivity of the double-antibody sandwich ELISA for recombinant IL-33 protein are shown in the figure.

[0029] Figure 3 This is a diagram illustrating the activity of paired antibodies. Detailed Implementation

[0030] 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.

[0031] This invention utilizes hybridoma technology to successfully prepare monoclonal antibodies capable of specifically recognizing human IL-33 protein, and obtains antibody pairings with different binding sites and complementary properties. These monoclonal antibodies can be used to construct enzyme-linked immunosorbent assay (ELISA) detection systems, suitable for the quantitative detection of IL-33 in samples such as serum, plasma, and cell culture supernatants. Verification has shown that this antibody pairing system exhibits good detection sensitivity and specificity, with low background signal, meeting the accuracy and consistency requirements of scientific research and clinical testing, and providing a reliable raw material basis for the development of human IL-33-related immunodiagnostic reagents.

[0032] The double-antibody sandwich ELISA detection method described in this application is not intended for disease diagnosis and treatment.

[0033] 1. IL-33 recombinant protein

[0034] The recombinant IL-33 protein is a product of Sinocare, catalog number 10368-HNAE. Its amino acid sequence is derived from NP_254274.1 (Ser 112-Thr 270), expressed by Escherichia coli, and has been identified as having high purity and biological activity.

[0035] 2. Screening of IL-33 monoclonal antibodies - mouse immunization.

[0036] Mice were immunized with recombinant human IL-33 protein, and other IL proteins such as IL-1β and IL-10 were used as screening antigens for monoclonal antibody selection. Specifically, recombinant human IL-33 protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and administered subcutaneously at multiple sites to immunize 6-week-old female BALB / c mice at a dose of 20 μg / mouse. At weeks 4 and 8, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. At week 12, a dose of 15 μg / mouse was administered intramuscularly with a water-soluble adjuvant. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 15 μg of recombinant IL-33 protein via intraperitoneal pulse, and the spleens of these mice were harvested three days later for hybridoma cell preparation.

[0037] 3. Screening of hybridoma cells.

[0038] 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-33 protein but not with IL-1β or IL-10 were selected by indirect ELISA. The positive cells were cloned into monoclonal states by limiting dilution, and then the cell lines were expanded and cryopreserved.

[0039] 4. Screening for positive clones using indirect ELISA:

[0040] IL-33 and IL-1β belong to the IL-1 family. To improve the screening efficiency of IL-33-specific monoclonal antibodies, IL-1β and other interleukins such as IL-10 are used for screening. Specifically, IL-33 recombinant protein, IL-1β, and IL-10 are coated in microplates (coating buffer is 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 solution is discarded, and the plates are blocked with 3% sucrose + 2% BSA, 150μL per well, and incubated at 37°C for 2 hours. Then, the plates are washed once with PBST washing buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. Add 50 μL of cell culture supernatant. Simultaneously, use another unrelated mouse monoclonal antibody (Sino-US TREM-2 monoclonal antibody: 11084-MM08) as a negative control. Dilute the unrelated antibody 1 μg / ml and add it to the detection plate. Incubate at 37℃ for 30 min. Discard the liquid from the wells, wash the plate 4 times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37℃ for 30 min, wash 4 more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for color development at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to stop the reaction. Measure OD using a microplate reader. 450 nm value. Positive cell lines that reacted with the IL-33 recombinant protein but not with the control recombinant protein were selected for subsequent experiments. The screening results are shown in Table 1.

[0041] Table 1: Screening Results of Monoclonal Antibodies

[0042]

[0043] 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.

[0044] 5. Purification and Identification of Monoclonal Antibodies

[0045] 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 through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. 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.

[0046] The purified monoclonal antibody was diluted to 1 μg / ml, and another mouse irrelevant monoclonal antibody (Sino-US TREM-2 monoclonal antibody: 11084-MM08) was used as a negative control at the same concentration. The binding activity of the antibody to recombinant human IL-33 protein and irrelevant antigen was detected by indirect ELISA. See below for specific results. Figure 1 The results showed that the selected monoclonal antibody specifically binds to recombinant human IL-33 protein and does not react with control antigens IL-1β and IL-10 recombinant protein, and can be used for subsequent testing.

[0047] 6. Establishment of the double-antibody sandwich ELISA

[0048] 6.1 HRP-labeled monoclonal antibodies

[0049] 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.

[0050] 6.2 Establishment of the double-antibody sandwich method

[0051] 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 2% sucrose + 3% 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. Dilute the test antigen IL-33 protein and the control antigen IL-1β protein to 100 ng / ml with PBS and add 50 μL / well to an ELISA plate. Incubate at 37°C for 35 min. Wash the plate four times with PBST wash buffer, blot dry, add 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS, incubate at 37°C for 35 min, wash four more times, blot dry, add 50 μL / well of TMB chromogenic buffer, and develop at room temperature for 10 min. Finally, add 50 μL of TMB stop solution to stop the reaction. Measure the OD using an ELISA reader. 450 nm value.

[0052] Using IL-33 as the positive antigen (i.e., recombinant IL-33 protein) and IL-1β protein as the negative antigen, a sandwich sieve method was employed for detection. 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 pairing. The results are shown in Table 2. 1B3 as the coating antibody and 4A10 as the labeling antibody resulted in the highest P / N ratio when detecting the recombinant protein.

[0053] Table 2: OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value analysis

[0054]

[0055] “ " " indicates the dilution factor."

[0056] 7. Optimization of the double-antibody sandwich ELISA method

[0057] The purified monoclonal antibody 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 50 μL / well was incubated 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-33 protein) and control antigen (IL-1β) were then coated with PBS according to the specified concentrations. Add 100 ng / ml diluted HRP-labeled monoclonal antibody to each well (50 μL / well), incubate at 37°C for 35 min, wash four times with PBST buffer, add 50 μL / well of HRP-labeled monoclonal antibody diluted 1000, 2000, 3000, or 4000 times with PBS, incubate at 37°C for 35 min, wash four more times, blot dry, add 50 μL / well of TMB chromogenic buffer, incubate at room temperature for 10 min, and finally add 50 μL of TMB stop solution to terminate the reaction. Measure OD using a microplate 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.

[0058] Table 3: Results of Optimization of Dual Antibody ELISA Conditions

[0059]

[0060] Table 3 shows that the P / N ratio was highest when the HRP-labeled antibody was coated at 2 μg / ml and diluted 2000 times, making it the optimal combination.

[0061] 8. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting recombinant IL-33 protein

[0062] After determining the optimal reaction conditions, including a coating monoclonal antibody concentration of 2 μg / mL and a 2000-fold dilution of the HRP-labeled monoclonal antibody, and following the above detection steps, recombinant human IL-33 protein was first serially diluted with PBS buffer to concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL. Simultaneously, 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) were also tested. The following recombinant proteins were expressed in CX03 and 293 cells: human IL-6 recombinant protein (expressed by the applicant using E. coli), human IL-7 recombinant protein (Kaikai Biotechnology, IL7-HE001, expressed in E. coli), human IL-8 recombinant protein (expressed by the applicant using E. coli), human IL-10 recombinant protein (expressed by the applicant using E. coli), human IL-11 recombinant protein (nearshore, C006, expressed in Pichia pastoris), and human IL-17A recombinant protein (nearshore, C774, 293 cells). 50 μL of each protein was added to each well for detection to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 2 It can be seen that the double-antibody sandwich ELISA composed of this group of paired antibodies still showed a weak positive reaction when the recombinant human IL-33 protein was diluted to 100 pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0063] IL-6 recombinant protein expression

[0064] 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. 21:

[0065] .

[0066] The amino acid sequence is SEQ ID NO.22:

[0067] MVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM.

[0068] IL-8 recombinant protein expression

[0069] 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.23:

[0070] ATGGAAGGCGCCGTGCTGCCGCGCAGCGCTAAAGAACTGCGCTGCCAGTGCATTAAGACCTATAGCAAACCGTTTCATCCGAAATTCATTAAGGAACTGCGTGTGATTGAAAGTGGCCCGCATTGTGCAAATACCGAAATTATTGTGAAACTGAGCGATGGTCGTGAACTGTGTCTGGACCCTAAAGAAAATTGGGTTCAGCGCGTGGTGGAAAAATTTCTGAAACGCGCAGAAAATAGC.

[0071] The amino acid sequence is SEQ ID NO.24:

[0072] MEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0073] 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.

[0074] IL-10 recombinant protein expression

[0075] 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.25:

[0076] ATGAGTCCGGGTCAGGGCACCCAGAGTGAAAATAGTTGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGATGCATTTTCACGTGATGCATTTTCACGTGTGAAAACCTTTTTCCAGATGAAAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGTCTGCTGGAAGATTTTAAAGGCTATCTGGGTTGTCAGGCCCTGAGCGAAATGATTCAGTTTTATCTGGAAGAAGTGATGCCGCAGGC AGAAAATCAGGACCCTGATATTAAGGCACATGTTAATAGCCTGGGTGAAAATCTGAAAACCCTGCGCCTGCGTCTGCGTCGCTGTCATCGTTTTCTGCCGTGCGAAAATAAGAGTAAAGCCGTTGAACAGGTTAAAAATGCCTTTAATAAGCTGCAGGAAAAAGGTATCTATAAAGCAATGAGCGAATTTGATATCTTCATTAATTACATCGAGGCCTATATGACCATGAAAATTCGTAAT.

[0077] The amino acid sequence is shown in SEQ ID NO.26:

[0078] MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0079] 9. Identification of binding activity of paired monoclonal antibodies

[0080] 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. An unrelated murine monoclonal antibody, Yiqiao Shenzhou TREM-2 murine monoclonal antibody (11084-MM08), was used as a negative control. The binding activity of the antibodies to recombinant human IL-33 protein was measured. Results are as follows... Figure 3 The results showed that it still reacted positively with IL-33 at a dilution of 1 ng / ml, indicating high antibody activity.

[0081] 10. Light and heavy chain variable region sequences of paired monoclonal antibodies

[0082] 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 two rounds of PCR. AgeⅠ / BsiWI and AgeⅠ / SalⅠ restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37℃ for 14 h to obtain the gene sequence of the variable region of the light and heavy chains of the monoclonal antibody.

[0083] Encapsulated monoclonal antibody 1B3:

[0084] Light chain variable region nucleotide sequence:

[0085] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.18:

[0086] GACATCCTGATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACATAATTATGGAAACACCTATTTAGAATGGTACCTGCAGAGACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAA CCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTATTGCTTTCAGGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAACGTACGGTG.

[0087] Light chain variable region amino acid sequence:

[0088] The amino acid sequence of the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.14:

[0089] DILMTQSPLSLPVSLGDQASISCRSSQSLVHNYGNTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRTV.

[0090] CDR area annotation:

[0091] The amino acid sequences of the CDR-L1 complementarity-determining region of the light chain variable region of the monoclonal antibody 1B3 are shown in SEQ ID NO.4:

[0092] CDR-L1: RSSQSLVHNYGNTYLE;

[0093] The amino acid sequences of the CDR-L2 complementation-determining region of the light chain variable region of the monoclonal antibody 1B3 are shown in SEQ ID NO. 5:

[0094] CDR-L2: KVSNRFS;

[0095] The amino acid sequences of the CDR-L3 complementarity-determining region of the light chain variable region of the monoclonal antibody 1B3 are shown in SEQ ID NO. 6:

[0096] CDR-L3: FQGSHVPWT.

[0097] Heavy chain variable region nucleotide sequence:

[0098] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.17:

[0099] GAAGTGCAGCTGTTGGAGACTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAATTTGGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGACTGGAGTGGGTCGCATACATTAGTGGTGGCAGTACTACCATCTACTATGCA GACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAATCCCTGGACACACCCAGTATTACTATGCTATGGCCTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0100] Heavy chain variable region amino acid sequence:

[0101] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.13:

[0102] EVQLLETGGGLVQPGGSRKLSCAASGFTFSNLGMHWVRQAPEKGLEWVAYISGGSTTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARIPGHTQYYYAMAYWGQGTSVTVSS.

[0103] CDR area annotation:

[0104] The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 1B3 is shown in SEQ ID NO.1:

[0105] CDR-H1: NLGMH;

[0106] The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 1B3 is shown in SEQ ID NO.2:

[0107] CDR-H2: YISGGSTTIYYADTVKG;

[0108] The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 1B3 is shown in SEQ ID NO.3:

[0109] CDR-H3: IPGHTQYYYAMAY.

[0110] Labeled monoclonal antibody 4A10:

[0111] Light chain variable region nucleotide sequence:

[0112] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.20:

[0113] GAAATTGTGCTCACTCAGTCTCCACTCACTTTGTCGGTTACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAGACTGATGGAGAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAA ACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGGATTTATTATTGCTGGCAAGGTACACATTCTCCGTTAACGTTCGGTGCTGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0114] Light chain variable region amino acid sequence:

[0115] The amino acid sequence of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.16:

[0116] EIVLTQSPLTLSVTIGQPASISCKSSQSLLETDGETYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHSPLTFGAGTKLEIKRTV.

[0117] CDR area annotation:

[0118] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO. 10:

[0119] CDR-L1: KSSQSLLETDGETYLN;

[0120] The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO. 11:

[0121] CDR-L2: LVSKLDS;

[0122] The amino acid sequence of the complementarity-determining region (CDR-L3) of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO. 12:

[0123] CDR-L3: WQGTHSPLT.

[0124] Heavy chain variable region nucleotide sequence:

[0125] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.19:

[0126] CAGCGTGAGCTGCAGGAGTCTGGACCTGAGCTGGTGACACCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCCTCCGGAAACATTTTCACTGACTACATCATTCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTCTCCTCACAATGGGGGTAC TGGCTATAATTGGAAGTTCAAGAACAAGGCCACATTGACTGTAGACACTTCCTCCACCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCTGTCTATTACTGTGCAAGATTGGGGCCTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0127] Heavy chain variable region amino acid sequence:

[0128] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.15:

[0129] QRELQESGPELVTPGASVKISCKASGNIFTDYIIHWVKQSHGKSLEWIGYISPHNGGTGYNWKFKNKATLTVDTSSTTAYMELRSLTSEDSAVYYCARLGPLDYWGQGTTLTVSS.

[0130] CDR area annotation:

[0131] The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 4A10 is shown in SEQ ID NO.7:

[0132] CDR-H1: DYIIH;

[0133] The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 4A10 is shown in SEQ ID NO. 8:

[0134] CDR-H2: YISPHNGGTGYNWKFKN;

[0135] The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 4A10 is shown in SEQ ID NO. 9:

[0136] CDR-H3: LGPLDY.

[0137] 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 monoclonal antibody combination for detecting human IL-33 protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 1B3 and monoclonal antibody 4A10; The heavy chain variable region of the monoclonal antibody 1B3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 1B3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 4A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 4A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting human IL-33 protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting human IL-33 protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting human IL-33 protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1B3 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting human IL-33 protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4A10 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting human IL-33 protein according to claim 5, characterized in that, The monoclonal antibody combination specifically recognizes both recombinant human IL-33 protein and native human IL-33 protein.

7. The use of a combination of monoclonal antibodies based on claim 1 in the preparation of a tool for detecting human IL-33 protein.

8. The application 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 1B3 as the coating antibody and monoclonal antibody 4A10 as the labeling antibody.

Images