Interleukin-6 antibodies and uses thereof

Abstract

The application discloses an antibody of interleukin-6 and application thereof, and provides the antibody with high binding activity and affinity to interleukin-6, and can realize specific, sensitive, reliable and accurate detection of trace interleukin-6 in a sample through a double-antibody sandwich method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular, to an antibody of interleukin-6 and application thereof. BACKGROUND

[0002] Interleukin-6, abbreviated as IL-6, is a kind of interleukin, a kind of multifunctional cytokine, which was first discovered by Japanese scholar Kishimoto Chuzo in 1973 and named IL-6 in 1988. IL-6 is a glycoprotein in chemical nature, composed of a single-chain polypeptide, about 5 kb long, a single copy gene with 5 exons and 4 introns.

[0003] IL-6 has a wide range of biological effects. It can promote T cell development, stimulate cytotoxic T cell response, induce B cell proliferation and differentiation and produce antibodies; in addition, IL-6 is also involved in hematopoietic regulation, inflammatory response, and plays a complex bidirectional regulation role in tumor occurrence and development (with anti-cancer and pro-cancer potential). Mechanistically, IL-6 needs to bind to IL-6 receptor (IL-6R) to exert its biological function. IL-6R is composed of two transmembrane glycoprotein subunits: one is the ligand binding subunit (gp80), which can bind IL-6 with low affinity; the other is the signal transduction subunit (gp130), which cannot directly bind IL-6, but can bind IL-6 / IL-6R complex with high affinity, mediating signal transmission. In addition, there is a soluble IL-6 receptor (sIL-6R) in the human body, which is a fragment produced by proteolysis of membrane-bound IL-6R.

[0004] The clinical significance of interleukin-6 is as follows: (1) Early inflammatory marker: IL-6 rapidly rises within 1-2 hours after infection or tissue damage, earlier than CRP and PCT, and is one of the most sensitive indicators of early inflammation, which can be used to identify infection and assess inflammation activity; (2) Sepsis and severe warning: IL-6 level is positively correlated with the severity of infection, >500 pg / mL often indicates septic shock or poor prognosis; in COVID-19 severe patients, IL-6 significantly increases, becoming one of the key indicators of cytokine storm (CRS), guiding whether to start immunosuppressive treatment (such as tocilizumab); (3) Virus and bacteria infection differentiation: IL-6 can be elevated in both viral and bacterial infections, combined with PCT detection can improve the accuracy of differential diagnosis, making up for the insensitivity of PCT to viral infection; (4) Tumor related: IL-6 is elevated in various malignant tumors (such as gastric cancer, colorectal cancer, lung cancer), which may be related to tumor-related inflammation, immune escape and other mechanisms, and has certain auxiliary diagnosis and prognosis evaluation value. SUMMARY

[0005] In view of the problems of the prior art, the present application provides a monoclonal antibody for interleukin-6 detection, a kit and application thereof, the antibody of the present application has high binding activity and affinity to interleukin-6, and can realize specific, sensitive, reliable and accurate detection of trace interleukin-6 in a sample through a double-antibody sandwich method. The present application is realized through the following technical solutions:

[0006] The present application provides a monoclonal antibody or antigen-binding fragment thereof for interleukin-6, which comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises:

[0007] HCDR1 with the amino acid sequence shown in SEQ ID No: 1;

[0008] HCDR2 with the amino acid sequence shown in SEQ ID No: 2;

[0009] HCDR3 with the amino acid sequence shown in SEQ ID No: 3;

[0010] The light chain variable region VL comprises:

[0011] LCDR1 with the amino acid sequence shown in SEQ ID No: 4;

[0012] LCDR2 with the amino acid sequence shown in SEQ ID No: 5;

[0013] LCDR3 with the amino acid sequence shown in SEQ ID No: 6.

[0014] In the specific embodiment of the present application, the heavy chain variable region VH of the antibody or antigen-binding fragment thereof is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO. 7.

[0015] In the specific embodiment of the present application, the light chain variable region VL of the antibody or antigen-binding fragment thereof is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO. 8.

[0016] In the specific embodiment of the present application, the heavy chain variable region VH of the antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region VL of the antibody has the amino acid sequence shown in SEQ ID NO. 8.

[0017] Further, the antigen binding fragment is selected from at least one of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dsFv fragment, a scFv fragment, a sc(Fv)2 fragment and a Diabody.

[0018] Further, the antibody is a full-length antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO. 9, and the light chain amino acid sequence of which is shown in SEQ ID NO. 10.

[0019] The second aspect of the present application provides an antibody conjugate, which is coupled by the antibody and a label or a solid phase carrier.

[0020] Further, the label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a nanoparticle label and a radioactive label; and the solid phase carrier is selected from one or more of a microsphere, a plate and a membrane.

[0021] The third aspect of the present application provides an interleukin-6 detection reagent or kit, which comprises the above-mentioned antibody or conjugate thereof.

[0022] The fourth aspect of the present application provides an interleukin-6 detection reagent or kit, which comprises a capture reagent and a detection reagent, each of which comprises an antibody or conjugate thereof of interleukin-6 with a different sequence, and the antibody or conjugate thereof of interleukin-6 is selected from the above-mentioned antibody or conjugate thereof.

[0023] The fifth aspect of the present application provides a use of the above-mentioned antibody or conjugate thereof in the preparation of an interleukin-6 detection product.

[0024] The sixth aspect of the present application provides a method for detecting interleukin-6 for non-diagnostic purposes, which comprises the following steps: using the above-mentioned antibody or conjugate thereof to form an immune complex with an antigen in a sample.

[0025] The seventh aspect of the present application provides a nucleic acid molecule, which encodes the above-mentioned antibody or antigen binding fragment thereof of interleukin-6.

[0026] The eighth aspect of the present application provides a vector or host cell, which comprises the above-mentioned nucleic acid molecule.

[0027] The present application has the beneficial effect that the antibody provided by the present application has high binding activity and affinity to interleukin-6, and can realize specific, sensitive, reliable and accurate detection of trace interleukin-6 in a sample through a double antibody sandwich method, which has good clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Interleukin-6 reference material relatedness test results. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Unless specific conditions are specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. Unless the manufacturers of the reagents or instruments are specified, the conventional products that can be obtained by market purchase are adopted.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. Materials, methods, and examples are illustrative only and not limiting. Unless specific conditions are specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. Unless the manufacturers of the reagents or instruments are specified, the conventional products that can be obtained by market purchase are adopted.

[0031] A first object of the present application is to provide an antibody or conjugate thereof of interleukin-6 of high quality, said antibody or conjugate thereof of interleukin-6:

[0032] The first antibody comprises: HCDR1, HCDR2 and HCDR3 having an amino acid sequence as shown in SEQ ID No: 1-3; LCDR1, LCDR2 and LCDR3 having an amino acid sequence as shown in SEQ ID No: 4-6;

[0033] In the present application, "CDR", "CDRs" or "complementarity determining region" refers to the highly variable regions of the heavy and light chains of immunoglobulins, referring to the region comprising one or more or even all of the major amino acid residues that play a role in the binding of the antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the detailed description of the present application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0034] In the present application, the heavy chain complementarity determining region is denoted as HCDR, which comprises HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted as LCDR, which comprises LCDR1, LCDR2 and LCDR3.

[0035] Methods for defining CDRs are well known in the art and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system set forth in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). See also Chothia et al., J Mol Biol 196:901-917 (1987) for the "Chothia definition". Other CDR definition methods can not strictly follow one of the above schemes but will still overlap at least a portion of the CDR regions defined by the Kabat definition, although they can be shortened or lengthened according to prediction or experimental results for particular residues or groups of residues.

[0036] In the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 are defined by the Kabat system.

[0037] In the present application, the antibody conjugate is conjugated by an antibody with a label or a solid phase carrier. The label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a nanoparticle label, a radioactive label; the solid phase carrier is selected from one or more of a microsphere, a plate or a membrane.

[0038] In an alternative embodiment, the above-mentioned label refers to a kind of substance with a property that can be directly observed by the naked eye or detected or probed by an instrument, such as luminescence, color development, radioactivity, etc., through which the qualitative or quantitative detection of the corresponding target can be achieved. It includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0039] In actual use, the skilled person in the art can select a suitable label according to the detection conditions or actual needs, and no matter what kind of label is used, it all falls within the protection scope of the present application.

[0040] In optional embodiments, the fluorescent dye includes, but is not limited to, fluorescein-based dyes and their derivatives (e.g., including, but not limited to, fluorescein isothiocyanate (FITC), hydroxycoumarin (FAM), tetra-chloro-fluorescein (TET), and the like or analogs thereof), rhodamine-based dyes and their derivatives (e.g., including, but not limited to, rhodamine red (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof), Cy series dyes and their derivatives (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or analogs thereof), Alexa series dyes and their derivatives (e.g., including, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or analogs thereof), and protein-based dyes and their derivatives (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0041] In optional embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0042] In optional embodiments, the radioisotope includes, but is not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0043] In optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacyanin, ruthenium bis-2-pyridyl, acridinium ester and its derivatives, dioxetane and its derivatives, luminol and its derivatives, and peroxyoxalate and its derivatives.

[0044] In optional embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0045] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0046] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver and colloidal selenium.

[0047] In optional embodiments, the colloidal metal is colloidal gold.

[0048] In optional embodiments, the antibody conjugate further comprises a solid support coupled to the antibody or antigen-binding fragment thereof.

[0049] In optional embodiments, the solid support is selected from the group consisting of microspheres, plates and membranes.

[0050] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0051] In the present application, the antibody is a full-length antibody or an antigen-binding fragment thereof; the antigen-binding fragment is selected from at least one of the group consisting of Fab fragment, F(ab')2 fragment, Fv fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment and Diabody.

[0052] The amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

[0053] In the present application, the "framework region" or "FR region" includes heavy chain framework region and light chain framework region, and refers to the region of the antibody heavy chain variable region and light chain variable region except CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0054] In the present application, the heavy chain variable region is obtained by connecting the CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; and the light chain variable region is obtained by connecting the CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In optional embodiments, the antibody further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0056] It is noted that in other embodiments, the framework region amino acid sequences of the antibodies of the present application can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region.

[0057] In optional embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0058] In optional embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0059] In optional embodiments, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD, or a combination of segments of multiple constant regions.

[0060] In optional embodiments, the heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0061] In optional embodiments, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0062] In optional embodiments, the light chain constant region is selected from a kappa type or a lambda type light chain constant region.

[0063] In optional embodiments, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock, or human.

[0064] In optional embodiments, the species origin of the constant region is mouse.

[0065] It is noted that in other embodiments, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the above constant region.

[0066] The heavy chain amino acid sequence of the first antibody in the embodiments of the present application is shown in SEQ ID NO. 9; and the light chain amino acid sequence is shown in SEQ ID NO. 10.

[0067] A second object of the present application is to provide an interleukin-6 detection reagent or kit, which comprises the above first antibody.

[0068] In some kits of the detection methodology, a capture reagent and a detection reagent are included, the capture reagent and the detection reagent including the first antibody or its conjugate.

[0069] For example, the above-mentioned kit can be used in the detection involving the specific binding of interleukin-6 and its antibody, such as immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence method, latex immunoturbidimetry, etc. For example, in the fluorescent immunochromatography, the antigen in the sample is combined with the fluorescently labeled first antibody on the binding pad, and the solution is subjected to chromatography by the siphon effect of the water absorption pad and moves towards the water absorption pad. When the complex moves to the detection line, it is combined with the coated antibody on the T line to form a "sandwich" type complex and is enriched on the T line. In the chemiluminescence method, the antigen in the sample is combined with the first antibody coated on the magnetic beads, and after washing, the second antibody (usually horseradish peroxidase HRP or alkaline phosphatase AP) labeled with enzyme is combined to form a "solid phase antibody-antigen-enzyme-labeled antibody" sandwich complex. It can be understood that the first and second antibodies here are not specific to a certain sequence of the present application, and in theory, as long as the two antibodies forming the "double-antibody sandwich" immunocomplex bind to different epitopes of the antigen, therefore, the two antibodies in the capture reagent and the detection reagent are not specific and can be interchanged in theory.

[0070] A third object of the present application is to provide the use of the first antibody or its conjugate in the preparation of a product for detecting interleukin-6.

[0071] The technical solutions of the present application are further described below through some specific examples.

[0072] Example 1 Preparation of interleukin-6 monoclonal antibody

[0073] 1. Mouse immunization and antibody detection

[0074] Twenty to twenty-five 6-8 week old SPF female BALB / c mice (Hunan Slaik Jingda Experimental Animal Co., Ltd.) were selected, and Freund's complete adjuvant was mixed with interleukin-6 (Chongqing Aisheng Biological Engineering Co., Ltd.) at a concentration of 1 mg / ml in equal volumes and emulsified. The emulsified antigen was used to immunize 6-8 week old SPF healthy BALB / c mice, and each mouse was injected with 500 μg of antigen protein by subcutaneous injection at multiple points on the back. Two weeks after the initial immunization, the antigen protein was mixed with incomplete Freund's adjuvant and emulsified, and each mouse was injected with 200 μg of antigen protein by subcutaneous injection on the back. Three weeks later, blood was collected from the tail vein, centrifuged to collect the supernatant, and the serum titer was detected by ELISA. The serum titer was detected every two weeks. After three immunizations, the serum titer was detected one week after the last immunization, and the serum titer after dilution by 1 million times was as high as 1.5 or more. The serum titer was screened to be 10 6The spleen cells of the above mice were taken for cell fusion.

[0075] 2. Screening of hybridoma antibodies

[0076] In the antibody screening stage, an ELISA plate was first coated with interleukin-6, and after potential target antibodies bound to the interleukin-6, a goat anti-mouse-HRP was used to develop the ELISA plate with an ELISA developing solution, and hybridoma clones with high reactivity were screened. Five strains of antibodies with high reactivity and specificity were screened, numbered 1-5#.

[0077] 3. Production and purification of monoclonal antibodies

[0078] Fifteen 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response of the mice. One week after injection, 0.5 ml of interleukin-6 monoclonal antibody 1-5# hybridoma cells (1×10 6 The number of cells was about 1×10

[0079] Example 2 Pairing evaluation of antibodies

[0080] The purified 1-5# antibodies were used as coating antibodies (naked antibodies) and HRP-labeled antibodies (Chongqing Aisheng Biological Engineering Co., Ltd., item number DAA070) to recognize interleukin-6 protein in a double-antibody sandwich manner, and antibodies that could be used for pairing were screened, and the evaluation data are as follows.

[0081] Table 1 Antibody pairing data

[0082]

[0083] From the above data, it can be seen that the 1# and 3# antibodies with higher reactivity with OD values above 1.1 were selected for further testing.

[0084] Example 3 Cloning of antibody gene sequences and performance testing

[0085] 1. Subtype identification and gene sequence cloning of monoclonal antibodies

[0086] The SBA Clonotyping System-HRP kit of Southern Biothech Company was used to identify the subtypes of the heavy and light chains of the 1# and 3# monoclonal antibodies of interleukin-6 according to the instructions. The specific operation was as follows:

[0087] a. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M carbonate and bicarbonate buffer, pH 9.5), add 100 μL / well to the enzyme-labeled plate, and coat overnight at 4°C. Wash the plate 3 times with PBS buffer containing 0.05% Tween-20 (washing buffer);

[0088] b. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at 1:1, add 100 μL / well to the enzyme-labeled plate, and incubate at 37°C for 30 min. Dilute the corresponding enzyme-labeled antibody (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at 1:3000 with diluent;

[0089] c. After washing the plate 3 times with washing buffer, add 100 μL of diluted enzyme-labeled antibody per well, and incubate at 37°C for 30 min. After washing the plate again 3 times, add color developing solution, and after about 5 min (depending on the reaction strength), add 2 M sulfuric acid to stop the reaction, and read the OD450 absorbance value. According to the antibody subtype results, use the method based on the RACE technology route to clone the antibody gene sequence. Collect the hybridoma cells in good growth condition, use a total RNA extraction kit to obtain the total RNA of the hybridoma cells, and according to the operation method of the SMARTer RACE instruction manual of Takara Company, reverse transcribe the mRNA into cDNA, and amplify the full-length sequence of the target antibody.

[0090] Table 2 Subtype of monoclonal antibody

[0091]

[0092] Stability determination

[0093] The above antibody was subjected to 37°C heat acceleration for 14 days in a predetermined buffer (PBS, 0.05% ProClin™300), the accelerated antibody was evaluated by ELISA indirect method, the test results were shown as the relative deviation of the 4°C storage detection value, and the heat acceleration stability of the antibody was identified; in addition, the antibody was repeatedly frozen and thawed 5 times at -20°C, the test results were shown as the relative deviation of the 0d detection value of 4°C storage, and the freeze-thaw stability of the antibody was tested, and the test results were as follows (the detection method of this experiment was consistent with that of "activity identification").

[0094] Table 3 Stability study

[0095]

[0096] It can be known from the test result that the antibody 1# and 3# provided by the application have good thermal acceleration stability and freeze-thaw stability, and the stability of the 1# is better, and the 1# can be further researched.

[0097] 3. Functional detection

[0098] A. Biotin-labeled antibody

[0099] Biotin (NHS-LC-LC-Biotin from Thermo Scientific, 21343) is weighed, dissolved into 5.677 mg / mL by using DMSO, 0.3 mg of the antibody is taken, the volume of the biotin solution required for each 1 mg of the antibody is 3.3 μL, the 1# antibody is added into the biotin and mixed, the antibody is connected with the biotin, and reaction is carried out at 25 ℃ for 4 h in the dark.

[0100] B. Alkaline phosphatase-labeled antibody

[0101] Substitute 0.3 mg of labeled antibody (Chongqing Aisens Biological Engineering Co., Ltd., Item No. DAA070) into TSE (pH 8.5) buffer solution, and substitute 0.66 mg of alkaline phosphatase (ALP) (from sigma, ALPI12G) into ALP dialysis buffer (pH 7.6) solution, mix well to determine the concentration. Dissolve Traut's Reagent 2-Iminothiolane·HCl (abbreviated as 2-IT, from Thermo Scientific, 26101) weighed into TSE (pH 8.5) solution to 13.76 mg / mL, and dissolve Sulfo-SMCC (from Thermo Scientific, PG82085) weighed into purified water to 3.7 mg / mL, both of which are prepared and used within 10 minutes after preparation. Calculate the volume of 2-IT solution required according to the volume of 2-IT solution required for each 1 mg of antibody. Add 2-IT solution to the antibody, mix well, and place in a (25±2) °C environment for static reaction for 20 min, which is the antibody activation. After activation, substitute the activated antibody into TSE (pH 7.3) solution to determine the antibody concentration. At the same time, calculate the volume of Sulfo-SMCC solution required according to the volume of SMCC solution required for each 1 mg of ALP. Add Sulfo-SMCC solution to the desalted ALP, mix well, and place in a (25±2) °C environment for static reaction for 20 min. After activation, substitute the activated ALP into TSMZ (pH 7.3) solution to determine the ALP concentration. Dilute the desalted antibody with TSE (pH 7.3) solution to 0.3 mg / mL, and dilute the desalted ALP with TSMZ (pH 7.3) solution to 0.4 mg / mL. Calculate the volume of antibody and ALP used for connection according to the mass ratio of antibody to ALP of 1:0.91. Mix the antibody and ALP according to the calculated volume, and place in a 2~8 °C environment for static reaction for 12-20 h. Dissolve maleimide in DMSO to 9.7 mg / mL, and dilute 10 times with TSMZ (pH 7.3) to obtain the termination solution. Add the termination solution required for 1 mL of antibody-ALP conjugate to terminate the reaction.

[0102] Performance evaluation

[0103] (1) Linearity experiment

[0104] Determination reagents, including R1, R2 and streptavidin magnetic bead solution:

[0105] The R1 includes: biotin-labeled antibody, using concentration: 1 μg / mL;

[0106] The R2 includes: alkaline phosphatase labeled antibody, using a concentration of 1 μg / mL.

[0107] The specific experimental operation steps are as follows:

[0108] a. Experimental preparation: R1, R2 working solution each 5 mL, magnetic bead solution 4 mL, calibration WRS A~WRS H.

[0109] b. Put each component into the kit, then put the kit into the full-automatic chemiluminescence immunoassay analyzer (EXI1800, Zhongyuan Huiji Biotechnology Co., Ltd.), select IL-6 project (sample 20 μL, R1 50 μL, R2 50 μL, magnetic bead solution 30 μL) to perform the experiment.

[0110] Table 4: Results of calibration experiment

[0111]

[0112] From the data in Table 4 above, it can be seen that when the 1# antibody is used as a coating antibody in the interleukin-6 detection reagent, it has a good detection linear range (see Figure 1 ).

[0113] From the above data, it can be seen that when the 1# antibody is paired with the commercial labeled antibody and applied in the IL-6 chemiluminescence immunoassay reagent, it has good detection precision and good clinical detection value.

[0114] The 1# antibody of the present application relates to the antibody part of interleukin-6 amino acid sequence as shown in Table 5 below (defined by Kabat system).

[0115] Table 5: Amino acid sequence table

[0116]

[0117] All data, reagents and steps herein should be understood as illustrative rather than limiting, although the present application has been described in connection with the above specific embodiments, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope and protection of the present application.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof to interleukin-6, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL, said heavy chain variable region VH comprising: a HCDR1 of an amino acid sequence as set forth in SEQ ID No: 1; a HCDR2 of an amino acid sequence as set forth in SEQ ID No: 2; a HCDR3 of an amino acid sequence as set forth in SEQ ID No: 3; and said light chain variable region VL comprising: a LCDR1 of an amino acid sequence as set forth in SEQ ID No: 4; a LCDR2 of an amino acid sequence as set forth in SEQ ID No: 5; a LCDR3 of an amino acid sequence as set forth in SEQ ID No:

6.

2. The antibody or antigen-binding fragment thereof of claim 1, said antibody or antigen-binding fragment thereof having a heavy chain variable region VH of an amino acid sequence as set forth in SEQ ID NO. 7; and a light chain variable region VL of an amino acid sequence as set forth in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof of claim 1, said antigen-binding fragment being selected from at least one of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dsFv fragment, a scFv fragment, a sc(Fv)2 fragment, and a diabody, or said antibody being a full-length antibody having a heavy chain amino acid sequence as set forth in SEQ ID NO. 9; and a light chain amino acid sequence as set forth in SEQ ID NO.

10.

4. An antibody conjugate coupled from the antibody or antigen-binding fragment thereof of any one of claims 1-3 to a label or a solid support.

5. The antibody conjugate of claim 4, said label being selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a nanoparticle label, a radioactive label; and said solid support being selected from one or more of a microsphere, a plate, or a membrane.

6. An assay reagent or kit for interleukin-6, said reagent or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody conjugate of claim 4 or 5.

7. An assay reagent or kit for interleukin-6, said reagent or kit comprising a capture reagent and a detection reagent, each comprising an antibody or conjugate thereof to interleukin-6 of different sequence, and wherein at least one reagent comprises the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody conjugate of claim 4 or 5.

8. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody conjugate of claim 4 or 5, in the manufacture of a product for detecting interleukin-6.

9. A method for detecting interleukin-6 for non-diagnostic purposes, comprising the steps of forming an immunocomplex between the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody conjugate of claim 4 or 5, and an antigen in a sample, and detecting the presence or amount of said immunocomplex. ​ ​ ​ ​ ​ ​ ​ 10. A nucleic acid molecule expressing the antibody or antigen-binding fragment thereof of any one of claims 1-3, or a vector or host cell comprising the nucleic acid molecule.

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