A monoclonal antibody against human interleukin-6 and a preparation method and use thereof

Abstract

The application discloses a kind of anti-human interleukin-6 monoclonal antibody and its preparation method and purposes, it is related to human interleukin-6 detection field.Specifically, the monoclonal antibody provided by the application can specifically recognize human interleukin-6, and the positive coincidence rate and negative coincidence rate and total coincidence rate of the detection reagent constructed using the antibody and Roche control reagent are all 100%, and the stability is significantly better than existing reference kit.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to monoclonal antibodies against interleukin-6, their preparation methods, and uses. Background Technology

[0002] Interleukin-6 (IL-6) is a secreted protein encoded by a single gene. Its precursor contains a 212-amino acid sequence. During post-translational modification, the N-terminal signal peptide is cleaved to form a mature polypeptide composed of 184 amino acid residues, with a molecular weight of approximately 22-27 kDa. A 1989 study reported the presence of immune response complexes with a molecular weight of approximately 60-70 kDa in the body fluids of patients with acute bacterial infections, suggesting that IL-6 may form complexes with binding proteins during infection to participate in the immune response.

[0003] Under physiological and pathological conditions, IL-6 expression is strictly regulated. When the body experiences internal or external injury, surgery, stress response, infection, brain death, tumor development, or other acute inflammatory responses, IL-6 is rapidly synthesized and released. Its serum / plasma concentration can significantly increase in the early stages of inflammation, and the magnitude of the increase is positively correlated with the severity of inflammation. Clinical studies have shown that preoperative and postoperative serum IL-6 concentrations in surgical patients can effectively predict the risk of postoperative complications. In intensive care unit (ICU) patients, continuous monitoring of serum or plasma IL-6 levels can effectively assess the severity of systemic inflammatory response syndrome (SIRS) and provide important reference for the prognosis of sepsis and septic shock. Furthermore, IL-6 has been proven to serve as an early warning indicator of sepsis, with its elevation occurring earlier than C-reactive protein (CRP) and procalcitonin (PCT), exhibiting higher timeliness. In chronic inflammatory diseases, IL-6 levels are also closely related to disease activity; for example, in patients with rheumatoid arthritis, IL-6 concentration can reflect the severity of joint inflammation.

[0004] Currently, clinical detection methods for IL-6 mainly include immunological detection technologies such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and electrochemiluminescent immunoassay (ECLIA). However, existing anti-IL-6 monoclonal antibodies for detection still have the following shortcomings in practical applications: First, the detection sensitivity and linear range of some antibodies are limited, making it difficult to meet the requirements for simultaneous and accurate quantification of low concentrations of IL-6 (such as normal physiological levels) and high concentrations of IL-6 (such as in patients with severe infections); Second, some antibodies have low recognition efficiency for complexes formed by IL-6 and soluble IL-6 receptors (sIL-6R) or IL-6 binding proteins, which may lead to deviations between the detection results and the actual active IL-6 level; Third, the antibodies have insufficient anti-interference ability in complex clinical samples (such as hemolyzed and lipemic samples), affecting the robustness of the detection results; Fourth, the batch-to-batch consistency of some commercially available antibodies is poor, which is not conducive to establishing a standardized detection system.

[0005] Therefore, developing a novel anti-IL-6 monoclonal antibody with high affinity, high specificity, wide detection range, good anti-interference ability and excellent batch stability is of great clinical value and has broad application prospects for improving the sensitivity and accuracy of IL-6 clinical diagnosis, realizing early warning and dynamic monitoring of inflammatory diseases (especially sepsis), and guiding clinical treatment decisions and prognostic assessment. Summary of the Invention

[0006] To improve upon this invention, a monoclonal antibody against interleukin-6 was designed and screened. The antibody was then validated against existing positive control kits and other highly efficient detection antibodies, fully demonstrating its detection advantages. Specifically, the technical solutions are as follows.

[0007] On the one hand, this application provides a binding molecule that resists interleukin-6.

[0008] In some embodiments, the binding molecule is an antibody against interleukin-6 or its antigen-binding fragment.

[0009] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region.

[0010] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13. In some specific embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13.

[0011] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:20; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:19; and in some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:20; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:19. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown in any of the following groups: (1-1) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:13. (1-2) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:20, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:19.

[0012] In some specific implementations, the CDR shown is defined according to the numbering system of Kabat, IMGT, Chothia, AbM, or Contact.

[0013] In some specific implementations, the CDR shown is defined according to Kabat's numbering system.

[0014] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:1, 2, and 3, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively.

[0015] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

[0016] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:7, 8, and 9, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:7, 8, and 9, respectively.

[0017] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively.

[0018] In some specific embodiments, the antibody or its antigen-binding fragment includes any one of the following groups: (2-1) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, 2, and 3, respectively; (2-2) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, 8, and 9, respectively.

[0019] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:13; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13; in some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13.

[0020] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:20, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:19. In some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19. In some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:20, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:19.

[0021] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown in any of the following groups: (3-1) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:13; (3-2) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:19.

[0022] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a recombinant antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0023] In some embodiments, the antibody or its antigen-binding fragment further includes an immunoglobulin Fc region. In some specific embodiments, the Fc is selected from IgG1, IgG2, IgG3, or IgG4.

[0024] In some embodiments, the light chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:17. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:17. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:17.

[0025] In some embodiments, the light chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:24, and the heavy chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:23. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:24, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:23. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:24, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:23.

[0026] The present invention also provides a biomaterial.

[0027] In some embodiments, the biomaterial includes any one of the following: (4-1) A nucleic acid molecule, said nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any of the places above; (4-2) Expression vectors / vector sets containing any of the nucleic acid molecules described above; (4-3) Host cells containing any of the nucleic acid molecules or expression vectors / vector groups described above.

[0028] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in any of the above claims.

[0029] In some embodiments, the method includes culturing host cells as described in any of the preceding embodiments. In some specific embodiments, the cultured host cells express an antibody or an antigen-binding fragment thereof. In some specific embodiments, the method further includes isolating the antibody or the antigen-binding fragment thereof. In some specific embodiments, the method further includes purifying the antibody or the antigen-binding fragment thereof.

[0030] The present invention also provides an anti-interleukin-6 composition.

[0031] In some embodiments, the composition comprises the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.

[0032] The present invention also provides the use of an antibody or antigen-binding fragment thereof as described in any of the preceding claims, or a composition as described in any of the preceding claims.

[0033] In some implementations, the application includes: for preparing products for anti-interleukin-6, or for detecting interleukin-6 not for the purpose of disease diagnosis and treatment; said products are detection kits, test strips, or detection chips.

[0034] In some specific implementations, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip.

[0035] In some specific embodiments, the product includes the antibody as a labeling antibody as described in group (1-1) and the antibody as a coating antibody as described in group (1-2); in some specific embodiments, the product includes the antibody as a labeling antibody as described in group (2-1) and the antibody as a coating antibody as described in group (2-2); in some specific embodiments, the product includes the antibody as a labeling antibody as described in group (3-1) and the antibody as a coating antibody as described in group (3-2).

[0036] In some specific embodiments, the product includes the antibody as described in group (1-1) as a coating antibody and the antibody as described in group (1-2) as a labeled antibody; in some specific embodiments, the product includes the antibody as described in group (2-1) as a coating antibody and the antibody as described in group (2-2) as a labeled antibody; in some specific embodiments, the product includes the antibody as described in group (3-1) as a coating antibody and the antibody as described in group (3-2) as a labeled antibody.

[0037] In some implementations, the interleukin-6 described in any of the above embodiments is human interleukin-6.

[0038] In some implementations, any of the antibodies described above are monoclonal antibodies. Attached Figure Description

[0039] Figure 1 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the IL-6 antigen.

[0040] Figure 2 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the 7G1 antibody.

[0041] Figure 3 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the 8E6 antibody.

[0042] Figure 4 This indicates the results of the correlation analysis. Among them, Figure 4 A represents the Roche-assigned clinical sample test T / C ratio data. Figure 4 B is the linear fitting curve. Detailed Implementation

[0043] The following description of specific embodiments further illustrates this application, but this is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products; the methods described, unless otherwise specified, are all conventional techniques in the art.

[0044] Example 1: Expression of recombinant IL-6 protein Recombinant IL-6 protein was prepared using genetic engineering methods, including the following steps: (1) Obtaining the target gene: Total RNA was extracted from human peripheral blood mononuclear cells induced by the stimulant. Using the total RNA as a template, cDNA fragment encoding the mature peptide of human IL-6 was amplified by RT-PCR. (2) Construction of recombinant expression vector: The cDNA fragment obtained in step (1) is cloned into the expression vector (pET series vector), so that the cDNA fragment is under the control of the promoter (T7 promoter) to construct the recombinant expression vector; (3) Obtaining engineered bacteria: The recombinant expression vector obtained in step (2) is transformed into the host bacteria, positive transformants are screened, and recombinant engineered bacteria are obtained; (4) Induction of expression: The engineered bacteria (Escherichia coli BL21(DE3)) obtained in step (3) were cultured and an inducer (isopropyl thio-β-D-galactoside) was added under suitable conditions to induce the expression of recombinant IL-6 protein; (5) Purification: Collect the bacterial cells after induction and expression, lyse the bacterial cells, separate the fraction containing IL-6 recombinant protein, purify by nickel column affinity chromatography, collect the eluent, transfer the eluent containing the target protein into a dialysis bag, and dialyze overnight in PBS buffer (pH 7.4) at 4°C, changing the dialysis buffer 3 times during the process; after dialysis, the sample is concentrated by centrifugation at 4°C and 4,000×g in an ultrafiltration centrifuge tube, aliquoted and stored at -20°C.

[0045] (6) Identification of the purified product: The purified protein was analyzed by SDS-PAGE electrophoresis to identify its molecular weight and purity. The results showed that the target protein appeared as a single protein band at approximately 23 kDa (see [link to study]). Figure 1 According to gel scanning analysis, its purity is over 90%, which meets the requirements of subsequent experiments.

[0046] Example 2: Preparation of anti-human IL-6 mouse monoclonal antibody 1. Animal immunization Five female Balb / c mice aged 6-8 weeks were selected and immunized with the recombinant human IL-6 protein expressed above as an immunogen. For the first immunization, 50 μg of the recombinant human IL-6 protein was mixed with Freund's complete adjuvant per mouse, emulsified to a water-in-oil state using a double-syringe method, and then injected subcutaneously at multiple sites (200 μL / mouse) along both sides of the spine, bilateral groins, and bilateral axillae. A second immunization was administered 14 days after the first, with the same immunogen dose but with Freund's incomplete adjuvant. The injection method and dose were the same as the first immunization. A third immunization was administered 15 days after the second, with the immunogen and Freund's incomplete adjuvant emulsified and injected intraperitoneally at the same dose. Seven days after the third immunization, blood was collected from the tail and serum was separated. Antibody titers were detected using ELISA (Table 1), with a titer of 1:100,000 or higher being the inclusion criterion. Three days before cell fusion, mice with qualified titers were selected and 100 μg / mouse of recombinant human IL-6 protein was dissolved in sterile phosphate buffer (without adjuvant) and injected intraperitoneally to boost immunization in preparation for cell fusion.

[0047] Table 1. Antibody titer test results

[0048] 2. Construction of B cells (1) Preparation of myeloma cells This embodiment uses the SP2 / 0 myeloma cell line, which was adaptively passaged to the logarithmic growth phase before fusion. On the day of fusion, cells were gently blown off the flask wall with a bent dropper and collected in a 50 mL centrifuge tube. The tube was centrifuged at 1000 r / min for 5-10 min. After discarding the supernatant, the pellet was resuspended in 30 mL of culture medium and washed once by centrifugation using the same method. Finally, the supernatant was discarded, and the pellet was resuspended in 20 mL of culture medium for later use.

[0049] (2) Preparation of spleen cells Immunized BALB / c mice were used. Blood was collected by enucleation and serum was separated as a positive control. Mice were euthanized by neck dislocation after blood collection and immersed in 75% alcohol for 5 min. The spleen was aseptically removed in a laminar flow hood and washed in a culture dish containing 10 mL of culture medium, removing surrounding connective tissue. The spleen was punctured with a sterile syringe needle and repeatedly perfused until the spleen tissue turned white. The spleen cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, and the precipitate was resuspended in 10 mL of culture medium. A small amount was diluted and used for cell counting.

[0050] (3) Cell fusion and cloning The spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 10:1 and fused using polyethylene glycol (PEG) as a fusion agent. After fusion, the cells were cultured in HAT selective medium. After 10 days of culture, the hybridoma cell culture supernatant was screened using ELISA. Positive clones were cloned using the limiting dilution method. After three rounds of screening, 15 positive hybridoma cell lines that stably secreted specific antibodies were finally obtained.

[0051] (4) Screening of hybridoma cells (ELISA method) IL-6 antigen was coated into 96-well ELISA plates at a concentration of 5 μg / mL and a volume of 50 μL / well, and incubated overnight at 4°C. The next day, the liquid in the wells was discarded, the plates were washed three times and blotted dry. Then, 100 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 2 h, followed by washing twice and blotting dry. Culture supernatant from 15 hybridoma cell lines was added to each well at 50 μL, with positive, negative, and blank controls included. The plates were incubated at 37°C for 30 min, washed three times, and blotted dry. Next, 50 μL of HRP-labeled goat anti-mouse IgG (diluted 1:20000) was added to each well, and the plates were incubated at 37°C for 30 min, washed three times again, and blotted dry. For color development, 100 μL of TMB substrate solution was added to each well, and the plates were incubated at room temperature for 15 min, then stopped with 50 μL / well of 2 mol / L dilute hydrochloric acid. The OD values ​​of each well were read at dual wavelengths of 450 nm and 630 nm. The experimental results showed that the OD values ​​of the blank wells were below 0.02, and the OD values ​​of the negative wells were below 0.1. Furthermore, the positive and negative controls showed good differentiation, confirming that the obtained hybridoma cells could secrete antibodies that specifically recognize the IL-6 antigen (Table 2). In addition, the titers of mouse ocular serum, detected using the same method, were all above 1:100,000, meeting the standards for fusion experiments.

[0052] Table 2. OD value measurement results

[0053] 3. Production and purification of monoclonal antibodies a) Induction and collection of ascites From 15 established positive hybridoma cell lines, two clones with the strongest antibody secretion capacity were selected for mouse ascites preparation. Five Balb / c mice weighing approximately 25 g were pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin; two weeks later, the ascites was further treated with 1×10⁻⁶... 6 Hybridoma cells were injected into the peritoneal cavity of mice at a dose of 1 cell per mouse. When the mice's abdomens became noticeably distended and signs of ascites appeared, the ascites fluid was collected, centrifuged at 10,000 rpm for 10 min, cell debris and precipitate were discarded, and the supernatant was collected and stored at -20℃ for later use.

[0054] b) Purification of antibodies in ascites fluid After thawing the frozen ascites fluid, centrifuge and filter the supernatant through a 0.22 μm filter membrane. Purify the antibody using a Protein A affinity chromatography column, following the product instructions. Collect the eluted target antibody fraction and dialyze it in 20 mM PBS (pH 7.4) for 72 h. After dialysis, sterilely filter through a 0.22 μm filter membrane, aliquot, and store at -20°C for subsequent identification experiments.

[0055] 4. Characterization of purified monoclonal antibodies According to conventional sequencing, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 7G1 antibody are shown in SEQ ID NO:1-3, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:4-6, as detailed in Table 3 below.

[0056] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 8E6 antibody are shown in SEQ ID NO:7-9, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:10-12. See Table 3 below for details.

[0057] Table 3. Distribution of antibody CDR sequences (Kabat numbers) ; Quantification of antibody protein concentration: The absorbance of the purified antibody at wavelengths of 280 nm and 260 nm was measured using ultraviolet spectrophotometry. 280 A 260 According to the formula, protein content (mg / mL) = (A 280 The antibody concentration in the sample is calculated by multiplying the dilution factor by 1.35. The antibody concentration of 7G1 is 5 mg / ml, and the antibody concentration of 8E6 is 4.8 mg / ml.

[0058] Antibody molecular weight determination: The purified antibody was analyzed by SDS-PAGE electrophoresis to determine its molecular weight. The results showed that the 7G1 antibody heavy chain was approximately 55 kDa, and the light chain was approximately 25 kDa (see...). Figure 2 The 8E6 antibody heavy chain is approximately 55 kDa, and the light chain is approximately 25 kDa (see...). Figure 3 ).

[0059] Antibody titer assessment: The titer of the purified ascites monoclonal antibody was determined by indirect ELISA. The results (see Table 4) showed that the titer of the purified antibody was higher than 1:100000.

[0060] Table 4. Results of Indirect ELISA assay

[0061] Example 3: Application of 7G1 and 8E6 antibodies in interleukin-6 chemiluminescent reagent 1. Preparation of monoclonal antibodies coated with magnetic microparticles: Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 37℃, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 7G1 monoclonal antibody at a ratio of 10 μg antibody to 1 mg of magnetic microparticles. The conjugation process was carried out at 25℃ for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25℃ for 2 h and then stored at 2-8℃ for later use.

[0062] 2. Preparation of alkaline phosphatase-labeled monoclonal antibodies (1) Take 1.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing 0.15 M sodium chloride, and add 0.04 mg TR to it. React at 37 °C for 1 h.

[0063] (2) Take 1 mg of 8E6 antibody, adjust the concentration to 1 mg / mL, add 0.034 mg SMCC, and react at 37℃ for 1 h.

[0064] (3) Add the alkaline phosphatase that has completed the reaction to the 2 antibody after SMCC treatment, mix them evenly at a mass ratio of 1:1, and react at 25℃ for 1h. After the reaction is completed, add 0.1mL of phosphate buffer containing 0.15M sodium chloride, 2% cysteine, and pH=7.4, let stand for 1h, add 1mL of glycerol, and store at -20℃ for later use.

[0065] 3. Reagent performance testing Magnetic microparticles coated with 7G1 antibody were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride, and alkaline phosphatase-labeled 8E6 antibody was diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride before testing. The test samples were Roche Diagnostics' thyroglobulin assay kit (electrochemiluminescence immunoassay; catalog number: 09015612190, batch number: 89377901). The results of the 79 clinical samples tested are shown below.

[0066] Test samples: A total of 79 clinical human serum samples were collected, including 25 low-value serum samples, 30 medium-value serum samples, and 24 high-value serum samples. The concentrations covered the measurement range of the kit. All samples showed no hemolysis, lipemia, or jaundice.

[0067] 4. Experimental Results Using Roche's reagent kit interpretation criteria as a reference, with a reference range of <7 pg / ml, the interpretation results of this reagent kit are shown in Table 5. The positive concordance rate, negative concordance rate, and total concordance rate of the reagent described in this invention and Roche's reagent are all 100%. All indicators show that the results of "test reagent" and "Roche reagent" are completely consistent with each other, with no difference. Kappa=1, indicating perfect consistency.

[0068] Table 5. Chemiluminescent microparticle immunoassay results of test reagents and Roche reagents ; Clinical serum samples at low, medium, and high concentration levels were selected, and each sample was measured 10 times using both kits. The coefficient of variation (CV) was calculated. As shown in Table 6, the CV values ​​of the kit of this invention at each concentration level were comparable to those of the Roche kit.

[0069] Table 6. Precision Analysis Results ; All 79 clinical samples were tested in parallel using the luminescent antibody pairing system of this invention and the Roche reference kit, respectively, following standard operating procedures. For the Roche kit, the testing procedure was as follows: 30 μL of sample was added to a reaction vessel with 50 μL of magnetic microparticle-coated 7G1 antibody working solution, mixed, and incubated at 37°C for 5 min. Then, alkaline phosphatase-labeled 8E6 antibody working solution was added, mixed, and incubated at 37°C for 5 min. Magnetic separation and washing were then performed, followed by the addition of 200 μL of substrate solution. The luminescence value in the reaction vessel was measured using an instrument, and the IL-6 concentration of each sample was recorded. Linear regression analysis was performed with the Roche kit result as the x-axis and the system result as the y-axis. The results are shown in Table 7. The linear correlation coefficient was 0.990, indicating consistent detection ranges. The clinical detection results of the two systems were highly consistent and met the requirements for clinical IL-6 detection.

[0070] Table 7. Results of Correlation and Detection Range Analysis ; Accelerated stability test: The self-developed reagent of this invention and the Roche reagent were divided into two portions. One portion was placed at 4°C and the other portion was placed in an oven at 37°C for a 7-day accelerated thermal stability test. After 7 days of acceleration, the reagents were taken out and tested according to their respective standard operating procedures (correlation test). High, medium and low concentration samples were tested separately, and each concentration was tested twice in parallel. The luminescence value (RLU) was recorded and the average value was calculated. The luminescence value retention rate at 37°C and 4°C was compared. The results are shown in Table 8 below. The stability of the reagent of this invention is better than that of the positive control under both low temperature refrigeration and high temperature accelerated conditions.

[0071] Table 8. Results of Luminescence Value Analysis ; In summary, the chemiluminescence detection kit constructed from the IL-6 antibody pair provided by this invention exhibits highly consistent results with the currently mainstream Roche imported kits used in clinical practice. Furthermore, this invention's kit demonstrates superior precision in high-value samples. Additionally, the self-developed reagent exhibits higher luminescence value retention under both refrigerated and accelerated storage conditions, demonstrating better storage stability than Roche reagents. The antibody pair described demonstrates excellent clinical application performance in the chemiluminescence platform, fully meeting the clinical needs for interleukin-6 quantitative detection.

[0072] Example 4: Application of 7G1 and 8E6 antibodies in IL-6 immunofluorescence assay strips 1. Preparation of coating membrane The IL6 7G1 antibody and goat anti-mouse IgG were diluted to 1 mg / mL with 10 mM phosphate buffer at pH 7.4, and then streaked onto the T and C lines of the nitrocellulose membrane at a volume of 1 μL / cm. The membranes were then dried in an oven at 37°C for 20 hours before use.

[0073] 2. Preparation of fluorescent binding pads 100 μL of 200 nm carboxyl fluorescent microspheres (1%) were added to 2 mL of MES buffer, followed by 100 μL of 20 mg / mL EDC solution and 300 μL of 20 mg / mL NHS solution. The mixture was stirred at room temperature for 1 h to activate the microspheres. After centrifugation at 10,000 rpm for 10 min to remove the supernatant, 2 mL of MES buffer was added. The mixture was then sonicated in a water bath for 10-20 s. 400 μg of IL68E6 antibody was added to the solution in one go. The mixture was stirred at room temperature for 2 h to react. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 6 min to remove the supernatant. 1 mL of microsphere storage solution was added. The mixture was sonicated on ice for 7 min and stirred at room temperature for 2 h. The mixture was then incubated at 37 °C overnight. The storage concentration of the microspheres was 1 mg / mL (0.1%).

[0074] The IL6 8E6 fluorescent microspheres, with a storage concentration of 1 mg / mL, were diluted to 0.5 mg / mL with microsphere preservation solution. The microspheres were then sprayed onto the treated conjugate pads using an XYZ three-dimensional gold-spraying spectrometer at a spray rate of 2 μL / cm, and dried in a 37°C oven for 20 hours. The dried conjugate pads were then cut to 300 mm × 10 mm dimensions and labeled as IL6 8E6 fluorescent conjugate pads for later use.

[0075] 3. Reagent performance testing The prepared IL6 7G1 antibody-coated membrane was combined with the IL6 8E6 antibody fluorescent binding pad, and after attaching the sample pad, it was cut into 3mm test strips and placed in a cartridge. The Roche-assigned IL-6 clinical serum samples were then tested using an immunofluorescence quantitative PCR instrument. The test results are as follows: Correlation analysis steps: ① Collect clinical serum samples and perform measurements using Roche's "Elecsys IL-6" electrochemiluminescence assay kit in conjunction with Roche's "cobas e801" instrument; the measured values ​​should include high, medium and low values.

[0076] ② Using the clinical serum samples with the above-assigned high, medium, and low values, and with the immunofluorescence quantitative detection instrument, the self-made fluorescent test strips were used to determine the T / C ratio of the samples.

[0077] ③ Using an Excel spreadsheet, with T / C as the ordinate and Roche-assigned concentration as the abscissa, fit a linear equation and calculate the linear equation and correlation coefficient R. 2 .

[0078] 4. Experimental Results The results are as follows Figure 4 As shown, the linear correlation coefficient R 2 The result was 0.9913. The test results of the self-made IL-6 immunofluorescence test strip were highly consistent with those of the Roche IL-6 electrochemiluminescence kit, which can meet the clinical testing requirements.

[0079] The relevant sequences of this invention are as follows: > 7G1 antibody heavy chain variable region EVQLQQSGPELVKPGASVKMSCKASGYTFTAYVIHWVKQKPGQGLEWIGYINPYNDGTKYNENFKGKATLTSDKSSNTAYMDLTSLTSEDSAVYYCARRPFFGNHAMDYWGQGTAVTVSS SEQ ID NO:13 > 7G1 antibody light chain variable region DIVLTQSPAIMSASPGEKVTMTCRASSSVTSSYLHWYQQKSGDSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELK SEQ ID NO:14 > 7G1 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:15 > 7G1 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:16 > 7G1 antibody heavy chain EVQLQQSGPELVKPGASVKMSCKASGYTFTAYVIHWVKQKPGQGLEWIGYINPYNDGTKYNENFKGKATLTSDKSSNTAYMDLTSLTSEDSAVYYCARRPFFGNHAMDYWGQGTAVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:17 > 7G1 antibody light chain DIVLTQSPAIMSASPGEKVTMTCRASSSVTSSYLHWYQQKSGDSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:18 > 8E6 antibody heavy chain variable region QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKFYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRADYGTSYDYALDYWGPGTSVTVSS SEQ ID NO:19 > 8E6 antibody light chain variable region DIVMTQTPKFLLVSAGDRVTITCKASQSVSDDVAWYQQKPGQSPKVLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQGYRSPWTFGGGTKLEIK SEQ ID NO:20 > 8E6 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:21 > 8E6 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:22 > 8E6 antibody heavy chain QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKFYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRADYGTSYDYALD YWGPGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:23 > 8E6 Antibody Light Chain DIVMTQTPKFLLVSAGDRVTITCKASQSVSDDVAWYQQKPGQSPKVLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQGYRSPWTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:24 The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An antibody against human interleukin-6 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment includes any one of the following groups of light chain variable regions and heavy chain variable regions: (1-1) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:

13. (1-2) The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:20, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:

19. The CDR shown is defined according to the Kabat numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: (2-1) The amino acid sequences are as shown in SEQ ID NO:4, 5, and 6, respectively, for LCDR1, LCDR2, and LCDR3, and the amino acid sequences are as shown in SEQ ID NO:1, 2, and 3, respectively, for HCDR1, HCDR2, and HCDR3; or (2-2) The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, 8, and 9, respectively.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment includes any one of the following groups of light chain variable regions and heavy chain variable regions: (3-1) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:13; (3-2) The light chain variable region with an amino acid sequence as shown in SEQ ID NO:20, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:

19.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv, or dsFv.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, It also includes the immunoglobulin Fc region, wherein the Fc is selected from IgG1, IgG2, IgG3 or IgG4.

6. A biomaterial, characterized in that, The biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5; (2) An expression vector / vector group containing the nucleic acid molecules described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the expression vector / vector group described in (2).

7. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1-5, characterized in that, The method includes culturing the host cells as described in claim 6 to express an antibody or an antigen-binding fragment thereof; and optionally includes isolating or purifying the antibody or the antigen-binding fragment thereof.

8. A composition for resisting human interleukin-6, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

9. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-6, or the composition as described in claim 8, characterized in that, Products used to prepare anti-interleukin-6, wherein the products are detection kits, test strips or detection chips.

10. The application according to claim 9, characterized in that, The detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit. The test strip is a colloidal gold test strip or an ELISA test strip.

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