CN118515763B - Antibody of IL-6, detection kit and application thereof - Google Patents
Antibody of IL-6, detection kit and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/248—IL-6
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
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- Chemical & Material Sciences (AREA)
- Immunology (AREA)
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Abstract
The invention relates to the technical field of antibodies, in particular to an antibody of IL-6, a detection kit and application thereof. The antibody provided by the invention has high specificity to IL-6, has no cross reaction with other cytokines, has high affinity to IL-6, can realize high specificity, high sensitivity, high accuracy, high precision and high stability detection of IL-6, and is excellent in quantitative detection of IL-6. The IL-6 detection kit provided by the invention can realize quantitative detection of IL-6 in samples with different matrixes, is not interfered by the matrixes, has higher sensitivity, specificity, accuracy and precision, is simple and quick to operate, has low cost, and has better application value in detection practice of IL-6.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody of IL-6, a detection kit and application thereof.
Background
Inflammation is part of the immune response and plays an important role in the body's protection against pathogens such as viruses, bacteria, fungi and other parasites. However, inappropriate activation of inflammatory processes is the root cause of many common pathological conditions; for example, autoimmune diseases can result when the immune system misrecognizes and attacks autologous cells or tissues as pathogens. Furthermore, studies have shown that when the microenvironment of pro-inflammatory cytokine production favors cancer progression, tumor proliferation and metastasis may result.
Cytokine storm or Cytokine Release Syndrome (CRS) is characterized by an aggressive and inadequate anti-inflammatory response leading to an imbalance in immune response homeostasis. Key indicators identified in the study of cytokine storm pathology include TNF- α, interferon, IL-1- β, MCP-1 (CCL 2), most importantly interleukin-6 (IL-6). T-cell-based activation or lysis of immune cells induces release of IFN-gamma or TNF-alpha, resulting in activation of macrophages, dendritic cells, other immune cells and endothelial cells. After activation of these cells, the pro-inflammatory cytokines are further released. Macrophages and endothelial cells produce large amounts of IL-6, activate T cells and other immune cells and form a positive feedback loop leading to a storm of cytokines, thereby inducing the release of more cytokines and chemokines, while at the same time up-regulating acute phase proteins. Therefore, it is important to detect the IL-6 content efficiently and accurately. However, the existing IL-6 quantitative detection reagent generally has the problems of insufficient stability, large inter-plate variation coefficient and interference among different matrixes.
Disclosure of Invention
The invention provides an antibody of IL-6, a detection kit and application thereof.
Specifically, the invention provides the following technical scheme.
In a first aspect, the invention provides an antibody to IL-6, or an antigen-binding fragment thereof, which is any one of the following (1) - (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1.2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is DVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of the CDR2 is LGS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 10.
The IL-6 antibody or antigen binding fragment thereof with the heavy chain variable region CDR and the light chain variable region CDR can specifically bind to IL-6, has no cross reaction with other cytokines, has higher affinity to IL-6, is not interfered by a matrix when being used for quantitative detection of IL-6, and can realize higher sensitivity, specificity, accuracy and precision.
Preferably, the antibody or antigen binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12;
(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
Preferably, the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2, fv, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.
In some embodiments of the invention, an antibody having clone number 5D9 is provided, the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of which are set forth in SEQ ID NO: 1. 2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is DVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 5. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12.
In some embodiments of the invention, an antibody having clone number 5D5H10 is provided, the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of which are set forth in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of the CDR2 is LGS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 10. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
The antibody or antigen binding fragment thereof of the IL-6 provided by the invention can specifically bind the IL-6, has no cross reaction with other cytokines such as interleukin, has higher affinity to the IL-6, and can realize IL-6 detection with higher sensitivity, specificity, accuracy and precision.
In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof to IL-6 as described above.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the production of the antibodies or antigen binding fragments thereof are within the scope of the invention.
In a third aspect, the present invention provides a biological material comprising a nucleic acid molecule as described above; the biological material is an expression cassette, a vector or a host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling an antibody or antigen binding fragment thereof to a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radiolabel.
In a fifth aspect, the invention provides an antibody composition for IL-6 comprising the antibodies in (1) and (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1.2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is DVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of the CDR2 is LGS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 10.
The antibodies in (1) and (2) above bind to specific IL-6 epitope, respectively.
The antibody composition can be used as a pairing antibody for detecting IL-6 by a double-antibody sandwich ELISA (enzyme-linked immunosorbent assay), and two antibodies in the antibody composition are respectively used as a capture antibody and a detection antibody in the double-antibody sandwich ELISA. Wherein the detection antibody may also carry a detectable label.
In a sixth aspect, the invention provides a detection kit comprising an antibody or antigen-binding fragment thereof to IL-6 as described above, or comprising said antibody conjugate, or an antibody composition comprising said IL-6.
The kit is a detection kit of IL-6. In some embodiments of the invention, the detection kit is an ELISA detection kit, preferably a double antibody sandwich ELISA detection kit. The kit has the advantages of simple operation, low cost, high sensitivity, high specificity, high accuracy, high precision and the like, and can be used for accurately and rapidly detecting the content of IL-6.
Based on the antibody or antigen binding fragment thereof or antibody conjugate or antibody composition, the content of IL-6 in body fluid such as serum and plasma, cell culture fluid, cell products and other samples can be specifically detected by a double-antibody sandwich ELISA method, and the antibody has higher sensitivity, specificity, accuracy and stability.
In the above kit, the antibody or antigen-binding fragment thereof may further comprise a detectable label; the kit may further comprise a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof.
For ease of detection, the kit may also contain other reagents for ELISA detection, including but not limited to IL-6 standards, PBST washes, blocking solutions, chromogenic solutions, stop solutions, and the like.
In a seventh aspect, the invention provides the use of an antibody or antigen binding fragment thereof to said IL-6 or said nucleic acid molecule or said biological material or said antibody conjugate or said antibody composition or said detection kit for any of the following:
(1) Use of IL-6 for detecting the presence or level of IL-6 in a sample for non-disease diagnostic purposes;
(2) Use in the preparation of a product for detecting the presence or level of IL-6 in a sample.
In the above (1), the use comprises detecting the presence or level of IL-6 in a sample using the antibody or antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit. In the detection for the purpose of non-disease diagnosis, the sample may be a cell culture solution obtained by in vitro culture, a cell preparation, or the like, which is not derived from a human or animal.
The method for detecting IL-6 using the antibody or antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit may use detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc.
In some embodiments of the invention, the antibody or antigen binding fragment thereof, antibody conjugate, antibody composition or detection kit is used to detect IL-6 in a double antibody sandwich ELISA method.
In the above (2), the product may be a detection reagent or kit for non-diagnostic purposes or a detection reagent or kit for diagnostic purposes.
In an eighth aspect, the invention provides a method for detecting IL-6 for non-disease diagnostic purposes, the method comprising: detecting IL-6 in a sample to be detected using the antibody or antigen-binding fragment thereof to IL-6 or the antibody conjugate or the antibody composition to IL-6 or the detection kit.
The detection can be performed by enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and other detection methods.
In the present invention, the IL-6 is human IL-6.
The beneficial effects of the invention at least comprise: the antibody provided by the invention can be specifically combined with IL-6, has no cross reaction with other cytokines such as interleukin, has high affinity to IL-6, can realize high-specificity, high-sensitivity, high-accuracy, high-precision and high-stability detection of IL-6, has excellent performance in quantitative detection of IL-6, is an ideal detection antibody for the content of IL-6, and can be used for clinical detection of IL-6.
Based on the antibody, the invention develops a detection kit of IL-6, and the detection kit can realize quantitative detection of IL-6 in samples with different matrixes, is not interfered by the matrixes, has higher sensitivity, specificity, accuracy and precision, is simple and quick to operate, has low cost, and has better application value in detection practice of IL-6.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard curve of the detection of IL-6 content by the double antibody sandwich ELISA method in example 4 of the present invention.
Detailed Description
In the specific embodiment of the invention, the hybridoma cell strain expressing the antibody is obtained by performing mouse immunization by taking the in vitro constructed human IL-6 protein as an immunogen, and performing cell fusion and screening and subcloning on hybridoma cells. Experiments prove that the antibody can specifically recognize human IL-6 protein and does not generate cross reaction with other cytokines such as IL-2, IL-7, IL-15 and the like. The amino acid sequence of the antibody and the nucleotide sequence of the encoding gene are further obtained through hybridoma sequencing.
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of antibodies that specifically bind human IL-6
1. Immunization of mice: mice were immunized with human IL-6 protein (purchased from Acrobiosystems) as an immunogen. After the immunization, the immune response level was determined by detecting serum of the immunized animal by ELISA. After conventional immunization is completed, if the immunized animal is able to reach the level of immune response against the immunogen, cell fusion may be performed.
2. Screening: the supernatant of the fused cells was screened by ELISA to select cells positive for the specific binding of human IL-6 protein and non-binding to proteins such as IL-2, IL-7, IL-15, IL-10, IL-21, GM-CSF and TNF-alpha.
3. Cloning and expanding culture: positive master clone cells were transferred to 24 well plates for expansion culture. Supernatants were collected from each of the expanded clones and tested by ELISA.
4. Subcloning: subcloning positive parent clone by limiting dilution method and subcloning screening by indirect ELISA method. The supernatant of subcloned cells was screened using the BLI method, and cells with high affinity and specific binding to human IL-6 protein were selected.
5. Hybridoma cell antibody gene sequencing: total RNA of the hybridoma cells is extracted, and the RNA is reversely transcribed into cDNA through an RT-PCR reaction. Cloning antibody light chain and heavy chain sequences, constructing the antibody light chain and heavy chain sequences on a T vector, and then carrying out DNA sequencing analysis to obtain antibody gene sequences.
6. Antibody production and purification: and (3) transfecting the antibody gene sequence obtained in the step (5) into HEK293 cells, performing amplification culture, purifying the antibody by adopting a protein A/G affinity chromatography method, and storing the purified antibody in Phosphate Buffer (PBS) by adopting a dialysis method.
EXAMPLE 2 specificity analysis of human IL-6 antibodies
Cell culture supernatants of 10 test-positive hybridoma cells were obtained by cell fusion and screening according to the method of example 1, and the 10 human IL-6 antibodies were specifically analyzed by ELISA, as described below.
1. IL-2, IL-7, IL-15, IL-10, IL-21, GM-CSF and TNF-alpha proteins were diluted to 2. Mu.g/mL with PBS and added to wells of an microplate of 100. Mu.L per well. The plates were sealed with a plate and left to stand overnight at 4 ℃.
2. The wells were discarded, the ELISA plates were dried by pipetting, washed with PBST wash solution for 300. Mu.L/Kong Jinpao s, the ELISA plates were dried by pipetting, and the plates were washed 3 times.
3. Mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. Cell supernatants from the different clones were added separately to the ELISA plates at 100. Mu.L per well. The plates were sealed, incubated at 37℃and then washed.
5. HRP-Anti-Mouse IgG was diluted to 0.05. Mu.g/mL with sample dilution, 100. Mu.L was added to each well, membrane sealed with sealing plate, incubated at 37℃and then washed.
6. 100 Mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
7. 50 Mu L of stop solution is added into each hole, and the ELISA plate is gently shaken until color development is uniform.
8. Absorbance values at 450nm and 630nm were read with a microplate reader, and the absorbance value (OD value) was subtracted from the OD 630 value with OD 450, and the detection results are shown in table 1.
9. Clones that bound strongly to human IL-6 protein, and did not bind to IL-2, IL-7, IL-15, IL-10, IL-21, GM-CSF and TNF-alpha proteins were selected for subcloning. Selected clones include 5D9, 6D4D5, 8E11C3, 13F5C12, 9D8D1, 13F5D11, 8E11F12, 9D8H9, 8E11G4 and 5D5H10, wherein antibodies of the above clones, including antibodies with clone numbers 5D9 and 5D5H10, all exhibited high specificity, bound strongly to human IL-6 protein, and did not cross-react with other cytokines.
TABLE 1 ELISA detection of the supernatants of different clone cells for OD values
Example 3 affinity analysis of human IL-6 antibodies
After cell subcloning was performed on 10 clones selected in example 2, 10 cell supernatants were obtained, and monoclonal antibodies generated by the 10 clones were subjected to affinity analysis using the BLI technique, and the analysis method and results were as follows.
1. The AMC biosensor in the first column containing 0.02% Tween-20 and 0.1% BSA 1X PBS solution (pH 7.4) running for 60s, this step is the baseline step.
2. The sensors were run in cell supernatants of different clones, respectively, for a period of time until 200s were solidified on the sensors.
3. The biosensor was run in 1 XPBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 60s, this step being the baseline step.
4. IL-6 protein was diluted to 200nM with 1 XPBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA, and the sensor was run for 60s during which time antibodies from different clones on the sensor would bind to IL-6 protein.
5. The sensor was run in 1 XPBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 90s, during which the IL-6 protein was dissociated from the sensor.
6. The sensor was regenerated in 10mM Glycine-HCl (pH 1.5) solution for 5s, then neutralized in 1 XPBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 5s, and regenerated three times in succession.
The results of the detection are shown in Table 2, in which antibodies of 10 clones including the antibodies of clone numbers 5D5D9 and 5D5H10 all exhibited high affinity.
TABLE 2 affinity values for BLI detection of different antibodies
EXAMPLE 4 identification and functional analysis of human IL-6 specific antibodies
The sensitivity analysis was performed on 10 human IL-6 antibodies in examples 2 and 3 by using a double antibody sandwich ELISA method and a checkerboard method, and finally two antibodies (clone No. 5D5D9,5D5H10) with higher detection sensitivity were screened out, wherein the sensitivity of the antibodies with clone numbers of 5D5D9 and 5D5H10 to IL-6 detection when paired for detection by using the double antibody sandwich ELISA method was 2.344pg/mL, which is significantly higher than that of the other antibodies paired with clone numbers.
Further, a quantitative detection experiment of human IL-6 was performed by using antibodies of clone numbers 5D5D9 and 5D5H10, wherein 5D5H10 was a coated antibody and 5D5D9 was a detection antibody, as described below.
1. The quantitative detection experimental result (figure 1) of the human IL-6 shows that the antibody of clone numbers 5D5D9 and 5D5H10 can be used for quantitatively detecting the human IL-6 by adopting a double-antibody sandwich ELISA method, so that the content of the IL-6 protein in the matrix is obtained, and the linear correlation is good.
2. The quantitative detection experimental results (Table 3) of human IL-6 show that the antibodies of clone numbers 5D5D9 and 5D5H10 are used for quantitative detection of human IL-6 by a double antibody sandwich ELISA method, and the quantitative detection is not interfered by a matrix.
TABLE 3 double antibody sandwich ELISA method for detecting recovery of IL-6 in different matrices
3. The quantitative detection method of the human IL-6 by using the antibodies of clone numbers 5D5D9 and 5D5H10 and adopting a double-antibody sandwich ELISA method (Table 4) shows that the quantitative detection method has high accuracy, high precision and good stability, and can accurately and rapidly quantify the IL-6 protein in the matrix.
TABLE 4 quantitative human IL-6 detection methodological validation results
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
- An antibody or antigen-binding fragment thereof to il-6, wherein the antibody or antigen-binding fragment thereof is any one of the following (1) - (2):(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1.2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is DVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of the CDR2 is LGS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 10.
- 2. The antibody or antigen-binding fragment thereof to IL-6 according to claim 1, wherein the antibody or antigen-binding fragment thereof is any one of the following (1) - (2):(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12;(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
- 3. The antibody or antigen-binding fragment thereof of IL-6 of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2, fv, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.
- 4. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof to IL-6 according to any one of claims 1 to 3.
- 5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is an expression cassette, a vector or a host cell.
- 6. An antibody conjugate, which is characterized in that the antibody or antigen binding fragment thereof of the IL-6 as defined in any one of claims 1 to 3 is conjugated with a label, wherein the label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
- An antibody composition for il-6, characterized in that the antibody composition comprises the antibodies in (1) and (2) below:(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1.2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is DVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of the CDR2 is LGS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: shown at 10.
- 8. A test kit comprising the antibody or antigen-binding fragment thereof to IL-6 of any one of claims 1-3, or comprising the antibody conjugate of claim 6, or comprising the antibody composition to IL-6 of claim 7.
- 9. Use of an antibody or antigen binding fragment thereof to IL-6 according to any one of claims 1 to 3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or an antibody conjugate according to claim 6 or an antibody composition to IL-6 according to claim 7 or a detection kit according to claim 8, as follows:(1) Use of IL-6 for detecting the presence or level of IL-6 in a sample for non-disease diagnostic purposes;(2) Use in the preparation of a product for detecting the presence or level of IL-6 in a sample.
- 10. A method for detecting IL-6 for non-disease diagnostic purposes, comprising: detecting IL-6 in a sample to be detected using the antibody or antigen-binding fragment thereof of IL-6 according to any one of claims 1 to 3 or the antibody conjugate of claim 6 or the antibody composition of IL-6 according to claim 7 or the detection kit of claim 8.
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