A monoclonal antibody combination for detecting human il-6 protein and use thereof

Abstract

The present application relates to the field of biological detection, and particularly relates to a monoclonal antibody combination for detecting human IL-6 protein and application. The combination comprises monoclonal antibodies 3A10 and 2G1, and the light and heavy chain variable region complementarity determining region amino acid sequences are shown in SEQ ID NO. 1-12 respectively. The present application defines the complete variable region sequence and coding nucleotide sequence of the antibody. The combination specifically recognizes human IL-6 recombinant and natural proteins, and has no cross reaction with IL-11. A diabody sandwich ELISA kit based on the combination, with 3A10 as a coating antibody and 2G1 as a labeled antibody, has high sensitivity and high specificity. The present application solves the problems of insufficient specificity and low sensitivity of existing reagents, and provides reliable core raw materials for IL-6 standardized detection, and is suitable for reagent kit, test strip and antibody chip preparation.

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-6 protein and its application. Background Technology

[0002] Human interleukin-6 (IL-6) is a multifunctional cytokine belonging to the IL-6 cytokine family, which also includes other cytokines such as interleukin-11 (IL-11). They share gp130 as a signal transduction pathway. The IL-6 protein exhibits a typical four-alpha helix bundle conformation and can regulate cell growth and differentiation in various tissues. Various cell types, including monocytes / macrophages, T lymphocytes, B lymphocytes, fibroblasts, and endothelial cells, can secrete IL-6 upon stimulation. When the body is in a state of infection, tissue damage, or inflammation, the expression level of IL-6 is significantly increased under the induction of Toll-like receptor signaling and inflammatory factors such as IL-1 and TNF-α; while in healthy individuals, its serum level is usually maintained at a low level. Furthermore, IL-6 is involved in hematopoiesis, bone metabolism, and cancer progression, and has been identified as playing a crucial role in guiding the transition from innate immunity to adaptive immunity.

[0003] Abnormal changes in IL-6 levels are closely related to the occurrence and development of various diseases. For example, in chronic inflammatory diseases such as rheumatoid arthritis, persistently elevated IL-6 can amplify inflammatory responses and cause tissue damage. In patients with severe infections or systemic inflammatory response syndromes, IL-6 often serves as an indicator of inflammation intensity. Furthermore, in certain tumor microenvironments, IL-6 is also associated with tumor cell survival and altered immune regulation. Therefore, reliable detection of IL-6 is not only valuable for disease monitoring but also provides important evidence for research into related mechanisms.

[0004] Currently, IL-6 protein levels are primarily detected using immunological methods, with common sample types including serum, plasma, cell culture supernatant, and tissue homogenate. Chemiluminescent immunoassay and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques, with double-antibody sandwich ELISA being widely used in cytokine detection due to its relatively mature operation and ease of quantitative analysis. However, in practical applications, existing IL-6 detection reagents still suffer from insufficient antibody specificity, susceptibility to cross-reactivity, and low sensitivity, making it difficult to meet the needs of standardized reagent development. Therefore, obtaining human IL-6 monoclonal antibodies with high affinity, high specificity, and well-defined sequences, and screening for the optimal paired antibody combinations, is crucial for developing high-performance, standardized ELISA reagents. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] 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-6 protein and its application, which solves the technical problems of insufficient antibody specificity, easy cross-reactivity and low sensitivity in existing IL-6 detection reagents, making it difficult to meet the requirements of standardized reagent development.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a monoclonal antibody combination for detecting human IL-6 protein, the monoclonal antibody combination comprising monoclonal antibody 3A10 and monoclonal antibody 2G1;

[0010] The heavy chain variable region of the monoclonal antibody 3A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0011] The light chain variable region of the monoclonal antibody 3A10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0012] The heavy chain variable region of the monoclonal antibody 2G1 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0013] The light chain variable region of the monoclonal antibody 2G1 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO. 14.

[0015] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO. 16.

[0016] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO. 18.

[0017] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO. 20.

[0018] In some embodiments, the monoclonal antibody combination specifically recognizes recombinant human IL-6 protein and native human IL-6 protein.

[0019] Secondly, this application provides the use of the combination of the monoclonal antibodies in the preparation of a tool for detecting human IL-6 protein; the tool is used to detect human IL-6 protein in a sample selected from any one of serum, plasma, cell culture supernatant and tissue homogenate, and the detection is not used for the diagnosis of disease.

[0020] In some embodiments, the tool includes reagents, kits, test strips, and antibody chips.

[0021] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0022] In some embodiments, the ELISA kit uses monoclonal antibody 3A10 as the coating antibody and monoclonal antibody 2G1 as the labeling antibody.

[0023] (III) Beneficial Effects

[0024] This invention provides a monoclonal antibody combination for detecting human IL-6 protein, the core of which lies in the pairing of monoclonal antibodies 3A10 and 2G1, which specifically recognize different epitopes. The heavy and light chain variable regions of monoclonal antibody 3A10 respectively contain complementarity-determining regions (CDRs) defined by SEQ ID NO. 1-3 and SEQ ID NO. 4-6, while the heavy and light chain variable regions of monoclonal antibody 2G1 respectively contain complementarity-determining regions defined by SEQ ID NO. 7-9 and SEQ ID NO. 10-12.

[0025] The combination exhibits high specificity. Through screening, antibodies 3A10 and 2G1 can specifically recognize recombinant and native human IL-6 proteins, while showing no cross-reactivity with IL-11 and other common cytokines such as IL10, effectively solving the technical challenges of non-specific binding and cross-interference in existing detection reagents.

[0026] The combination exhibited good sensitivity. When a double-antibody sandwich ELISA system was constructed using monoclonal antibody 3A10 as the coating antibody and monoclonal antibody 2G1 as the labeling antibody, the detection P / N ratio was improved, enabling accurate capture of trace amounts of IL-6 in the sample, thus meeting the needs of early inflammation monitoring and detection of low-abundance samples.

[0027] This invention specifies the exact amino acid and nucleotide sequences of the antibodies (SEQ ID NO. 1-20), resulting in antibody combinations with well-defined sequences and uniform structures. This provides reliable core raw materials for the preparation of high-performance, standardized IL-6 detection kits, test strips, or antibody chips, and lays the foundation for large-scale stable production, demonstrating promising prospects for clinical applications and scientific research development. 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 recombinant IL-6 protein;

[0031] Figure 3 The image shows the identification results of the purified monoclonal antibody;

[0032] Figure 4 The results of specificity and sensitivity of double-antibody sandwich ELISA for recombinant IL-6 protein are shown in the figure.

[0033] Figure 5 This is a diagram illustrating the activity of paired antibodies. Detailed Implementation

[0034] 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.

[0035] This invention successfully prepared a monoclonal antibody capable of specifically recognizing human interleukin-6 (IL-6) protein using hybridoma technology, and further screened for antibody pairs with complementary properties and different binding sites. The ELISA detection method established based on this method has been verified to have the advantages of high sensitivity and no cross-reactivity with related cytokines. This invention provides reliable core raw materials and solutions for the accurate detection of IL-6, and has broad application prospects in clinical auxiliary detection, mechanism research, and drug development.

[0036] The double-antibody sandwich ELISA detection method described in this application is not intended for disease diagnosis and treatment.

[0037] Example 1

[0038] 1. Expression of recombinant IL-6 protein

[0039] 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:

[0040] ATGGTACCCCCAGGAGAAGATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAACAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAACAAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAACCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAAATG。

[0041] The amino acid sequence is SEQ ID NO.22:

[0042] MVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM。

[0043] 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.

[0044] 2. Purification of recombinant IL-6 protein

[0045] 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 , Figure 1 In the diagram, M represents the protein marker, number 1 indicates sample flow-through, number 2 indicates recombinant IL-6 protein eluted with 100 mM imidazole, and number 3 indicates recombinant IL-6 protein eluted with 500 mM imidazole. Electrophoresis of the 500 mM imidazole elution peak showed a clear main band between 33-43 kDa, while the estimated molecular weight of the pET32a-IL-6 recombinant protein was approximately 38 kDa, consistent with the expected size, indicating successful expression and effective purification of the target protein.

[0046] 3. Identification of recombinant IL-6 protein

[0047] The purified recombinant IL-6 protein was coated onto an ELISA plate, and its reaction with the IL-6 positive antibody was identified by indirect ELISA. The IL-6 antibody was a commercially available mouse monoclonal antibody (Beyotime, AD1307). The recombinant protein was first coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. IL-6 positive clonal antibody was diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of the antigen-coated microplate. A negative control was prepared by diluting another unrelated mouse monoclonal antibody (Sino-Pharmaceutical TREM-2 monoclonal antibody: 11084-MM08) at the same concentration. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-mouse IgG secondary antibody (Solepro, diluted 5000 times with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 . Figure 2 The purified recombinant IL-6 protein showed a significant positive reaction with commercial IL-6 monoclonal antibody. This recombinant protein (antigen) can be used for subsequent mouse immunization and monoclonal antibody screening.

[0048] 4. Screening of IL-6 monoclonal antibodies - mouse immunization

[0049] Mice were immunized with high-purity recombinant human IL-6 protein. Human IL-10 protein expressed via pET32a vector and the IL-6 family cytokine human IL-11 were used as screening antigens for monoclonal antibody selection. Specifically, purified recombinant IL-6 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 15 μg / mouse. At weeks 4, 6, and 10, booster immunizations were administered 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 15 μg of recombinant IL-6 protein. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0050] 5. Screening of hybridoma cells

[0051] 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 human IL-6 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 human IL-6 protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0052] 6. Screening positive clones using indirect ELISA method

[0053] Since the recombinant IL-6 protein used for immunization is expressed via the pET32a vector and contains a His tag, a reverse screening process using the recombinant IL-10 protein, also expressed via the pET32a vector, was performed to remove vector background. Furthermore, other cytokines in the IL-6 cytokine family, such as IL-11, may cause cross-interference during actual detection. To improve screening efficiency and obtain a specific monoclonal antibody against IL-6, both IL-10 and IL-11 proteins were used for reverse screening. Specifically, the recombinant IL-6 protein and IL-10 (pET32a-IL-10, His tag, expressed in E. coli) and IL-11 (nearshore, C006, expressed in Pichia pastoris) of the pET32a vector were coated in microplates (coating buffer was carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water), with a coating concentration of 1 μg / mL, and incubated overnight at 4°C; the coating buffer was discarded the next day, and the plates were blocked with 2% sucrose + 3% BSA, 150 μL per well, and incubated at 37°C for 2 hours, followed by washing once with PBST washing buffer (PBS containing 0.05% Tween-20, pH 7.4), and then 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 recombinant IL-6 protein but not with control recombinant proteins IL-10 and IL-11 were selected for subsequent experiments.

[0054] 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] 7. 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, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PBS, 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 unrelated mouse monoclonal antibody (Sino-Clinical TREM-2 monoclonal antibody: 11084-MM08) was used as a negative control, diluted to 1 μg / ml. The binding activity of the antibody to recombinant human IL-6 protein and control antigens IL-10 and IL-11 recombinant proteins was detected using the aforementioned indirect ELISA method. Specific results are shown below. Figure 3The results showed that the selected monoclonal antibody specifically binds to recombinant human IL-6 protein and does not react with recombinant IL-10 and IL-11 proteins, and can be used for subsequent testing.

[0061] 8. Establishment of double-antibody sandwich ELISA

[0062] 8.1 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] 8.2 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 recombinant IL-6 antigen and the recombinant IL-11 control antigen 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. 450nm value. Using IL-6 as the positive antigen (i.e., recombinant IL-6 protein) and IL-11 as the negative antigen, a sandwich sieve was used 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. Monoclonal antibody 3A10 as the coating antibody and monoclonal antibody 2G1 as the labeling antibody showed the highest P / N ratio when detecting recombinant protein samples.

[0066] Table 2. OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value

[0067]

[0068] “ " " indicates the dilution factor."

[0069] 9. Optimization of the double-antibody sandwich ELISA method

[0070] The purified monoclonal antibody 3A10 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 incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose and 2% BSA at 150 μL / well. The plates were incubated at 37°C for 2 h, and the blocking buffer was discarded. The test antigen (IL-6 recombinant protein) and control antigen (IL-11 recombinant protein) were diluted with PBS at 10 ng / mL and added to the microplate at 50 μL / well. The plates were incubated at 37°C for 35 min, washed four times with PBST wash buffer, and then coated with PBS. HRP-labeled monoclonal antibody 2G1 diluted 1000, 2000, 3000, and 4000 times, 50 μL / well, was incubated at 37°C for 35 min. After washing the plate four times and patting it dry, 50 μL / well of TMB chromogenic buffer was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction, and the OD was measured 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.

[0071] Table 3. Results of Optimization of Double Antibody ELISA Conditions

[0072]

[0073] Table 3 shows that the P / N ratio was highest when the HRP-labeled antibody was coated at 0.5 μg / ml and diluted 2000 times, making it the optimal combination.

[0074] 10. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting recombinant IL-6 protein.

[0075] After determining the optimal reaction conditions, including a coating monoclonal antibody concentration of 0.5 μg / ml and a 2000-fold dilution of the HRP-labeled monoclonal antibody, and following the above detection steps, the recombinant human IL-6 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. Simultaneously, recombinant human IL-1β protein (Chongqing Tansheng, FAP-BC006, 293 cell expression), recombinant human IL-2 protein (Jinyan, GMP-CD66, 293 cell expression), recombinant human IL-4 protein (Jinyan, CX03, 293 cell expression), recombinant human IL-7 protein (Kaikai Biotechnology, IL7-HE001, E. coli expression), recombinant human IL-8 protein (expressed by the applicant using E. coli), recombinant human IL-10 protein (expressed by the applicant using E. coli), recombinant human IL-11 protein (Jinyan, C006, Pichia pastoris expression), recombinant human IL-17A protein (Jinyan, C774, 293 cell expression), and recombinant human IL-33 protein (Yiqiao Shenzhou, 10368-HNAE, E. coli expression) were added to each well at the same concentration (50 μL) for detection to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 4 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-6 protein was diluted to 10 pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0076] The amino acid sequence of recombinant human IL-8 protein is SEQ ID NO.23:

[0077] MEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0078] The nucleotide sequence of the recombinant human IL-8 protein is SEQ ID NO.24:

[0079] ATGGAAGGCGCCGTGCTGCCGCGCAGCGCTAAAGAACTGCGCTGCCAGTGCATTAAGACCTATAGCAAACCGTTTCATCCGAAATTCATTAAGGAACTGCGTGTGATTGAAAGTGGCCCGCATTGTGCAAATACCGAAATTATTGTGAAACTGAGCGATGGTCGTGAACTGTGTCTGGACCCTAAAGAAAATTGGGTTCAGCGCGTGGTGGAAAAATTTCTGAAACGCGCAGAAAATAGC.

[0080] The amino acid sequence of recombinant human IL-10 protein is SEQ ID NO.25:

[0081] MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0082] The nucleotide sequence of recombinant human IL-10 protein is SEQ ID NO.26:

[0083] ATGAGTCCGGGTCAGGGCACCCAGAGTGAAAATAGTTGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGATGCATTTTCACGTGATGCATTTTCACGTGTGAAAACCTTTTTCCAGATGAAAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGTCTGCTGGAAGATTTTAAAGGCTATCTGGGTTGTCAGGCCCTGAGCGAAATGATTCAGTTTTATCTGGAAGAAGTGATGCCGCAGGC AGAAAATCAGGACCCTGATATTAAGGCACATGTTAATAGCCTGGGTGAAAATCTGAAAACCCTGCGCCTGCGTCTGCGTCGCTGTCATCGTTTTCTGCCGTGCGAAAATAAGAGTAAAGCCGTTGAACAGGTTAAAAATGCCTTTAATAAGCTGCAGGAAAAAGGTATCTATAAAGCAATGAGCGAATTTGATATCTTCATTAATTACATCGAGGCCTATATGACCATGAAAATTCGTAAT.

[0084] 11. Identification of binding activity of paired monoclonal antibodies

[0085] 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 TREM-2 monoclonal antibody: 11084-MM08, was used as a negative control. The binding activity of the antibodies to recombinant human IL-6 protein was measured. Results are as follows: Figure 5 The results showed that it still reacted positively with IL-6 at a dilution of 1 ng / ml, indicating high antibody activity.

[0086] 12. Light and heavy chain variable region sequences of paired monoclonal antibodies

[0087] 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 3 rounds of PCR. 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 variable region gene sequences of the light and heavy chains of the monoclonal antibody.

[0088] This application presents a monoclonal antibody combination comprising monoclonal antibodies 3A10 and 2G1, the amino acid sequences of their light and heavy chain variable regions complementarity-determining regions (CDMRs) as shown in SEQ ID NO. 1-12, respectively. This invention defines the complete variable region sequence and encoding nucleotide sequence of the antibodies. The combination specifically recognizes recombinant and native human IL-6 proteins and exhibits no cross-reactivity with IL-11, etc. A double-antibody sandwich ELISA kit constructed based on this combination, using 3A10 as the coating antibody and 2G1 as the labeling antibody, demonstrates high sensitivity and high specificity. This invention solves the problems of insufficient specificity and low sensitivity in existing reagents, providing a reliable core material for the standardized detection of IL-6, and is suitable for the preparation of kits, test strips, and antibody chips.

[0089] Encapsulated monoclonal antibody 3A10:

[0090] Light chain variable region nucleotide sequence:

[0091] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.18:

[0092] GACATTTGTGATGTCACAGTCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGTATTGGTTATTACAGAGGCCAGGCCAGTTCCCAAAGCGCCTAATCTATCTGGTGTCTAA ACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGATTATTACTGCGTGCAAGGTACACATTTTCCTTTCACGTTCGGCTCGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0093] Light chain variable region amino acid sequence:

[0094] The amino acid sequence of the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.14:

[0095] DIVMSQSPLTLSVTIGQPASISCKSSQSLLYSNGKTYLYWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGDYYCVQGTHFPFTFGSGTKLEIKRTV.

[0096] CDR area annotation:

[0097] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 4:

[0098] CDR-L1: KSSQSLLYSNGKTYLY;

[0099] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 5:

[0100] CDR-L2: LVSKLDS;

[0101] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 6:

[0102] CDR-L3: VQGTHFPFT.

[0103] Heavy chain variable region nucleotide sequence:

[0104] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.17:

[0105] GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGACTTCTGGCTACACCTTCACAGGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATTTTAATATTAAGTA CAATGAGAAGTTCCAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAACCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGGGGGGGTTACTACGGCTACGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0106] Heavy chain variable region amino acid sequence:

[0107] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.13:

[0108] EFQLQQSGPELVKPGASVRISCKTSGYTFTGYYIHWVKQRPGQGLEWIGWIYPGNFNIKYNEKFQGKATLTADKSSSTTYMQLSSLTSEDSAVYFCARGGVTTATFPYWGQGTLVTVSS.

[0109] CDR area annotation:

[0110] The amino acid sequence of CDR-H1 in the complementarity-determining region of the heavy chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 1:

[0111] CDR-H1: GYYIH;

[0112] The amino acid sequence of the CDR-H2 complementarity-determining region of the heavy chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 2:

[0113] CDR-H2: WIYPGNFNIKYNEKFQG;

[0114] The amino acid sequence of CDR-H3 in the complementarity-determining region of the heavy chain variable region of monoclonal antibody 3A10 is shown in SEQ ID NO. 3:

[0115] CDR-H3: GGVTTATFPY.

[0116] Labeled monoclonal antibody 2G1:

[0117] Light chain variable region nucleotide sequence:

[0118] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.20:

[0119] GATGTCCAGATGATTCAGTCTCCAGCTTCACTGTCTGCATCTGTGGGAGAAACTGTCACCATCACATGTGGAGCAAATGAGAATCTTTACGGTGCTTTAAATTGGTATCAGCGGAAGCAGGGAAAATCTCCTCAGCTCCTGATCTATGGTGCAACCAACTTGGCA GATGGCATGTCATCGAGGTTCAGTGGCAGTGGATCTGGTAGACAGTATTCTCTCAAGATCAGTAGCCTGCATCCTGACGATGTTGCAACGTATTACTGTCAAAATATATTCAGTAGACCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0120] Light chain variable region amino acid sequence:

[0121] The amino acid sequence of the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.16:

[0122] DVQMIQSPASSLSASVGETVTITCGANENLYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQYSLKISSLHPDDVATYYCQNIFSRPYTFGGGTKLELKRTV.

[0123] CDR area annotation:

[0124] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 10:

[0125] CDR-L1: GANENLYGALN;

[0126] The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 11:

[0127] CDR-L2: GATNLAD;

[0128] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 12:

[0129] CDR-L3: QNIFSRPYT.

[0130] Heavy chain variable region nucleotide sequence:

[0131] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.19:

[0132] CAGGTTCAGCTGCAACAGTCTGGGGCTGAACTGGTGAAGCCTGGGACTTCAGTGAAGCTGTCCTGCAAGGCTTCCGGCTACACCTTCACCAACAACTGGTTGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATCAATCCTAGCGACGGTCGTATTTA CTACAATGAGAAGTTCAAGACCAGGGCCACACTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGCCACATAACTGGGACGGGGAAGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0133] Heavy chain variable region amino acid sequence:

[0134] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO. 15:

[0135] QVQLQQSGAELVKPGTSVKLSCKASGYTFTNNWLHWVKQRPGQGLEWIGEINPSDGRIYYNEKFKTRATLTVDKSSSTAYMQLSSSLTSEDSAVYYCHITGTGKFAYWGQGTLVTVSS.

[0136] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 7:

[0137] CDR area annotation:

[0138] CDR-H1: NNWLH;

[0139] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 8:

[0140] CDR-H2: EINPSDGRIYYNEKFKT;

[0141] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 2G1 is shown in SEQ ID NO. 9:

[0142] CDR-H3: TGTGKFAY.

[0143] 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-6 protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 3A10 and monoclonal antibody 2G1; The heavy chain variable region of the monoclonal antibody 3A10 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 3A10 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 2G1 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 2G1 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-6 protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting human IL-6 protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting human IL-6 protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3A10 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting human IL-6 protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G1 is shown in SEQ ID NO.

20.

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

7. The use of the combination of monoclonal antibodies according to claim 1 in the preparation of a tool for detecting human IL-6 protein; the tool is used to detect human IL-6 protein in a sample selected from any one of serum, plasma, cell culture supernatant and tissue homogenate, and the detection is not used for the diagnosis of disease.

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 3A10 as the coating antibody and monoclonal antibody 2G1 as the labeling antibody.

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