Anti-human inflammatory factor interleukin 6 monoclonal antibody and application thereof

Abstract

The invention discloses an anti-human inflammatory factor interleukin 6 monoclonal antibody and an application thereof. When the monoclonal antibody is prepared, an adopted immunogen is a human inflammatory factor interleukin 6 antigen, and an amino acid sequence of the human inflammatory factor interleukin 6 antigen comprises a sequence as shown in SEQ ID NO: 1. A group of monoclonal antibodies specifically combined with antigens is constructed based on the IL-6 antigens. The IL-6 monoclonal antibody has strong binding capacity with an IL-6 antigen in a clinical sample, has no cross reaction with other proteins, has good specificity and affinity compared with a commercial antigen, can be effectively applied to ELISA and a dry-type immunofluorescence method, has good detection linearity, precision, stability and accuracy, and can be widely applied to the field of immunoassay. The antibody can be combined with the antigen to serve as a detection reagent raw material combination, and has clinical detection advantages.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a monoclonal antibody against human inflammatory factor interleukin 6 and application thereof. BACKGROUND

[0002] Interleukin-6 (IL-6) as a pleiotropic cytokine plays a central role in immune response, inflammatory response and host defense. The dynamic changes of its serum / plasma level are closely related to the occurrence and development of various diseases. In recent years, the value of IL-6 detection in clinical diagnosis, prognosis evaluation and treatment monitoring has become increasingly prominent, and it has become an important biomarker for the management of inflammatory diseases.

[0003] IL-6 as a key mediator of early inflammatory response is rapidly released when pathogens such as bacteria and viruses invade, and its detection can achieve early identification and typing of infectious diseases. Studies have shown that the serum IL-6 level of patients with bacterial infection (such as sepsis, pneumonia) often increases exponentially, and the peak value can reach more than 1000 pg / mL, while most viral infections (such as common cold) only show mild increase or no significant change. This difference provides an important basis for clinical differentiation of infection types. In addition, dynamic monitoring of IL-6 can help evaluate the severity of infection: if the IL-6 level of patients with severe infection is continuously higher than 500 pg / mL, it indicates that the risk of poor prognosis significantly increases.

[0004] Compared with C-reactive protein (CRP) or procalcitonin (PCT), the biological characteristics of IL-6 make it have unique advantages in early diagnosis of infection. IL-6 gene is rapidly activated after stimulation by pathogens (the transcription peak appears within 1 hour), and the serum concentration can be detected to increase significantly 2-4 hours after the onset of the disease, while CRP usually shows significant changes only after 6-12 hours. This time difference makes IL-6 a "quick alarm" for emergency infection screening, especially suitable for diseases such as neonatal sepsis and severe pneumonia that require urgent intervention.

[0005] Although there are many IL-6 detection products on the market now, the existing detection products have limited sensitivity and insufficient detection ability for low concentration IL-6, which cannot meet the capture of early trace inflammatory signals. And the quality of different manufacturers is uneven, and the specificity is limited, which may have cross-reactions with IL-6 family cytokines (such as IL-11, IL-27), resulting in false positive results. In order to solve the problem of uneven quality of existing IL-6 detection reagents, it is still the research focus in the field to develop IL-6 protein detection reagents and detection systems with good specificity, good repeatability, high titer, and good correlation with clinical detection results. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the present application provides an anti-human inflammatory factor interleukin 6 monoclonal antibody and an application thereof. The present application performs sequence analysis and combined screening on an IL-6 related antigen epitope to obtain a recombinantly expressed IL-6 antigen, and ensures the stability and specificity of the recombinant expression. A group of detection antibodies for the antigen are prepared based on the above high-sensitivity IL-6 antigen, and the detection antibodies have good consistency compared with a commercially available kit, and can be used as a new raw material for in vitro diagnostic reagents such as colloidal gold, immunochromatography, chemiluminescence, and the like.

[0007] To achieve the object of the present application, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a human inflammatory factor interleukin 6 antigen, and an amino acid sequence of the human inflammatory factor interleukin 6 antigen comprises a sequence as shown in SEQ ID NO: 1.

[0009] The present application performs sequence analysis and combined screening on an IL-6 related antigen epitope to obtain an IL-6 antigen with a specific sequence, explores antigen binding site information, and performs prokaryotic expression system sequence optimization on the obtained antigen, so that the obtained antigen is similar to a commercial antigen and the specificity is ensured.

[0010] In a second aspect, the present application provides a nucleic acid molecule, and the nucleic acid molecule encodes the human inflammatory factor interleukin 6 antigen of the first aspect.

[0011] Preferably, a nucleotide sequence of the nucleic acid molecule comprises a sequence as shown in SEQ ID NO: 2.

[0012] In a third aspect, the present application provides a recombinant expression vector, and the recombinant expression vector contains the nucleic acid molecule of the second aspect.

[0013] Preferably, after the recombinant expression vector is transfected, transduced or transformed into a host cell, the host cell expresses the human inflammatory factor interleukin 6 antigen of the first aspect.

[0014] In a fourth aspect, the present application provides a recombinant expression strain, and the recombinant expression strain contains at least one copy of the recombinant expression vector of the third aspect, or at least one copy of the nucleic acid molecule of the second aspect.

[0015] In a fifth aspect, the present application provides an anti-human inflammatory factor interleukin 6 monoclonal antibody, and the monoclonal antibody comprises a heavy chain variable region and a light chain variable region.

[0016] The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 5;

[0017] The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 5;

[0018] The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 5;

[0019] The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 5.

[0020] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the anti-human inflammatory factor interleukin 6 monoclonal antibody is shown in SEQ ID NO: 10;

[0021] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the anti-human inflammatory factor interleukin 6 monoclonal antibody is shown in SEQ ID NO: 10;

[0022] In the present application, the immunogen used in the preparation of the anti-human inflammatory factor interleukin 6 monoclonal antibody is the human inflammatory factor interleukin 6 antigen of the first aspect.

[0023] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the anti-human inflammatory factor interleukin 6 monoclonal antibody is shown in SEQ ID NO: 10;

[0024] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the anti-human inflammatory factor interleukin 6 monoclonal antibody is shown in SEQ ID NO: 10;

[0025] The preparation process of the human inflammatory factor interleukin 6 antigen and the related monoclonal antibody in the present application is shown in the schematic diagramFigure 1

[0026] In a sixth aspect, the present application provides a nucleic acid molecule encoding the anti-human inflammatory factor interleukin 6 monoclonal antibody of the fifth aspect.

[0027] In a seventh aspect, the present application provides an expression vector containing the nucleic acid molecule of the sixth aspect.

[0028] In an eighth aspect, the present application provides a reference product for detecting human inflammatory factor interleukin 6, the reference product containing the human inflammatory factor interleukin 6 antigen of the first aspect.

[0029] In a ninth aspect, the present application provides the use of any one or a combination of at least two of the human inflammatory factor interleukin 6 antigen of the first aspect, the anti-human inflammatory factor interleukin 6 antigen monoclonal antibody of the fifth aspect, the nucleic acid molecule of the sixth aspect, the expression vector of the seventh aspect, or the reference product for detecting human inflammatory factor interleukin 6 of the eighth aspect in the preparation of a product for detecting human inflammatory factor interleukin 6.

[0030] In the present application, the above-mentioned monoclonal antibody can be used as a detection or capture antibody alone, or can be used in the form of an antibody pair in combination, and can be used as a raw material for preparing a human IL-6 ELISA detection kit or a dry immunofluorescence kit.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The present application performs sequence analysis and combination screening on IL-6 related antigen epitopes to obtain a recombinant IL-6 antigen with a specific sequence, thereby ensuring its specificity; the present application also constructs a group of monoclonal antibodies that specifically bind to the antigen based on the above-mentioned high-sensitivity IL-6 antigen; the IL-6 monoclonal antibody pair has strong binding ability and strong affinity to the IL-6 antigen in a clinical sample, has high detection sensitivity, and can be combined as a detection reagent raw material after binding to the antigen, thereby having clinical detection advantages. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a schematic diagram of the preparation process of the IL-6 recombinant protein antigen and related monoclonal antibody of the present application.

[0034] Figure 2 Figure 2 is an electrophoretogram of the target IL-6 protein nucleic acid (about 636 bp) and plasmid nucleic acid (4368 bp) after double enzyme digestion.

[0035] Figure 3 Figure 3 is an SDS-PAGE diagram of the IL-6 protein.

[0036] ​Figure 4 Figure for Western blot identification of IL-6 protein.

[0037] Figure 5 Figure for SDS-PAGE electrophoresis of IL-6 monoclonal antibody.

[0038] Figure 6 Figure for affinity detection of IL-6 monoclonal antibody.

[0039] Figure 7 Figure for linearity of IL-6 monoclonal antibody for clinical detection. DETAILED DESCRIPTION

[0040] To further illustrate the technical means adopted by the present application and its effects, the present application is further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0041] If a specific technique or condition is not specified in the embodiments, the technique or condition described in the literature in the art, or according to the product manual is used. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0042] Example 1

[0043] This embodiment provides a human inflammatory factor interleukin 6 antigen.

[0044] 1. PCR amplification and sequencing of IL-6 gene

[0045] Objective: To amplify the target sequence and complete the sequencing comparison to prove the consistency of the actual sequence with the design.

[0046] The IL-6 nucleic acid expression sequence (SEQ ID NO: 2) screened and optimized was used as a template for PCR amplification. The PCR was performed in a 50 μL reaction system, and the reaction system was as follows:

[0047] dd H2O 39.1 μL, primer (10 μmol / L) 2 μL, expression sequence DNA 1 μL, Platinum PFX DNA Polymerase 0.4 μL, 10×PFX Amplification Buffer 5 μL, dNTP Mix (10 mM) 1.5 μL, MgSO4 (50 mM) 1.0 μL were added.

[0048] The PCR reaction conditions were as follows: 94℃ for 5 min; 94℃ for 15 s, 55℃ for 30 s, 68℃ for 1 min, 35 cycles; 72℃ for 5 min; 4℃ storage.

[0049] 1% agarose gel electrophoresis was used to detect the PCR results, and the gel was weighed and recovered according to the instructions of the DNA gel recovery kit. 5-10 μL of the gel recovery product was detected by agarose gel electrophoresis to determine the recovery efficiency. 2 μL was used to determine the gene concentration, and the rest was stored at -20°C for subsequent plasmid construction.

[0050] 2. Construction and sequencing of recombinant plasmid

[0051] Objective: To prepare a recombinant plasmid that can express recombinant protein, which can be used for subsequent expression of recombinant protein in host cells.

[0052] The PCR-amplified IL-6 gene fragment SEQ ID NO: 2 was ligated to the linear pTrcHis2C vector digested with BamHI and EcoRI to construct a recombinant plasmid. The recombinant plasmid was double-digested with BamHI and EcoRI, and the digestion products were identified by 1% agarose gel electrophoresis. Sequencing primers were designed according to the length of the gene, and sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3500XL gene analyzer. The sequencing results were spliced and compared.

[0053] The electrophoresis results are shown in Figure 2 , Figure 2 The target IL-6 Da protein nucleic acid (about 636 bp) and plasmid nucleic acid (4368 bp) were electrophoresed after double digestion.

[0054] 3. Expression and purification of IL-6 protein

[0055] Objective: To prepare the target recombinant protein and obtain high-purity protein through purification.

[0056] The constructed IL-6-pTrcHis2C recombinant plasmid was transformed into competent cells Rosetta (DE3), and positive strains were selected using Amp resistance. The colonies containing the recombinant expression plasmid were cultured in SOB medium (containing Amp 50 μg / mL, MgCl210 mmol / L) at 37°C, 220 rpm. When the OD 600 was 0.4-0.8, IPTG was added to a final concentration of 1 mmol / L. After induction at 37°C for 12 h, the bacteria were centrifuged, broken, and the supernatant was analyzed by SDS-PAGE.

[0057] After induction of IL-6 protein, most of the protein was expressed in soluble form, which was analyzed by SDS-PAGE. The soluble expressed protein was purified by protein purification liquid chromatograph. The IL-6 expressing bacteria were collected, resuspended with 1x Ni column buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and centrifuged after being broken and lysed. The supernatant was used for subsequent purification using a Ni-separation column.

[0058] The results of SDS-PAGE analysis are shown in Figure 3 , Figure 3 The SDS-PAGE of IL-6 protein is shown in Figure 2, lane 2 is the target protein band, and lane 3 is the IL-6 protein standard. The target band is about 30 kDa, and the purity is 98%. The amino acid sequence of the IL-6 protein is shown in SEQ ID NO: 1.

[0059] 4. Identification of IL-6 protein

[0060] Objective: To determine the consistency of the prepared recombinant protein with the target designed protein.

[0061] The purified IL-6 protein was transferred to a PVDF membrane activated in methanol after SDS-PAGE gel electrophoresis, blocked with PBS containing 5% skim milk at room temperature for 2 h, and then incubated with a commercial IL-6 monoclonal antibody (IL-6 antibody 2706SPTN-5, M Company, U.S.A.) at room temperature for 1 h. After washing with TBS-T (20 mM Tris buffer saline containing 0.1% Tween 20), 10 mL of goat anti-mouse IgG-HRP conjugate secondary antibody was added and incubated at room temperature for 1 h. After washing, HRP substrate was added, and the protein band was detected by gel imaging.

[0062] The Western blot identification of IL-6 protein is shown in Figure 4 , Figure 4 The Western blot identification of IL-6 protein is shown in Figure 2, lane 2 is the IL-6 protein standard result (WHO standard), and lane 3 is the target IL-6 recombinant protein result. The Western blot result shows that the IL-6 protein described in the present application can specifically bind to the commercial monoclonal antibody, and an obvious band can be seen at 30 kDa, which is similar in size to the protein standard. The recombinant IL-6 protein has good antigenicity.

[0063] Example 2

[0064] This example provides a monoclonal antibody of human inflammatory factor interleukin 6.

[0065] Objective: To prepare a detection monoclonal antibody using the prepared recombinant antigen, obtain mouse ascites containing the antibody using the hybridoma antibody preparation method, and purify and identify the antibody.

[0066] After emulsifying IL-6 antigen with Freund's complete adjuvant, five 6-week-old female BALB / c mice were subcutaneously injected with 30 μg each. On days 15 and 30, the mice were injected with a second and third dose of emulsified antigen containing Freund's incomplete adjuvant and IL-6, respectively. On day 45, a shock immunization was performed using the purified antigen. Antibody production in the mice was detected using ELISA. Once the antibody titer reached the required level, B cells were harvested from the mouse spleen and fused with SP20 cells to prepare hybridoma cells using the PEG fusion method. Hybridoma cells were screened using HAT medium, cloned, and then screened using ELISA for hybridoma cells that could produce anti-IL-6 specific antibodies. Positive hybridoma cells were injected into the peritoneal cavity of mice to obtain ascites, which was then purified using a Protein-A affinity chromatography kit. These monoclonal antibodies can be detected using ELISA and Western blot with the recombinant IL-6 protein mentioned in this invention.

[0067] IL-6 monoclonal antibody SDS-PAGE electrophoresis image as shown below Figure 5 As shown, lane 1 is the protein molecular weight marker, lane 2 is monoclonal antibody 1, lane 3 is monoclonal antibody 2, and lanes 4 and 5 are commercial monoclonal antibody pairs (IL-6 antibody 2706SPTN-5, M Company USA and IL-6 antibody 2707SPTN-5, M Company USA).

[0068] 2. Sequencing results of monoclonal antibodies

[0069] Monoclonal antibody 1:

[0070] Heavy chain CDR1: SEQ ID NO:3: GYTFTDYYMK.

[0071] Heavy chain CDR2: SEQ ID NO:4: YIRNKANGYTPTYNHKFKG.

[0072] Heavy chain CDR3: SEQ ID NO:5: YNYAMDY.

[0073] Light chain CDR1: SEQ ID NO:6: KSSQSLLYSDGKTYLN.

[0074] Light chain CDR2: SEQ ID NO:7: LVSKLDS.

[0075] Light chain CDR3: SEQ ID NO:8: WQGTHFPLT.

[0076] The heavy chain variable region of monoclonal antibody 1 is set forth in SEQ ID NO: 9.

[0077] The light chain variable region of monoclonal antibody 1 is set forth in SEQ ID NO: 10.

[0078] The heavy chain of monoclonal antibody 1 is set forth in SEQ ID NO: 11.

[0079] The light chain of monoclonal antibody 1 is set forth in SEQ ID NO: 12.

[0080] Monoclonal antibody 2:

[0081] Heavy chain CDR1 : SEQ ID NO: 13: GFTFSDYY.

[0082] Heavy chain CDR2: SEQ ID NO: 14: IRNNVYNYAT.

[0083] Heavy chain CDR3: SEQ ID NO: 15: TVNLQLNLVVVPAPMRSFYTMNQ.

[0084] Light chain CDR1 : SEQ ID NO: 16: QDINRY.

[0085] Light chain CDR2: RAN.

[0086] Light chain CDR3: SEQ ID NO: 17: LQYDDFPYT.

[0087] The heavy chain variable region of monoclonal antibody 2 is set forth in SEQ ID NO: 18.

[0088] The light chain variable region of monoclonal antibody 2 is set forth in SEQ ID NO: 19.

[0089] The heavy chain of monoclonal antibody 2 is set forth in SEQ ID NO: 20.

[0090] The light chain of monoclonal antibody 2 is set forth in SEQ ID NO: 21.

[0091] 3. Potency verification of monoclonal antibodies

[0092] Dilute 10x ELISA coating buffer to 1x, dilute the IL-6 recombinant protein antigen according to the application to 0.5 μg / mL with 1x ELISA coating buffer, add the diluted sample coating solution to the enzyme-labeled plate, 100 μL per well; seal the enzyme-labeled plate with a sealing film, and coat overnight at 4°C; after washing the plate, add ELISA blocking solution to the enzyme-labeled plate, 200 μL per well, and block at 37°C for 1 h. Dilute the commercial monoclonal antibodies 1 and 2 (2706M Company, 2707M Company) and the monoclonal antibodies 1 and 2 according to the application by a ratio of 1:2000 to 1:256000 by doubling, a total of 8 concentrations; dilute the negative control sample (PBS) by 64000 times with 1x ELISA enzyme-labeled diluent; 100 μL per well, 37°C, and incubate for 1 h; dilute the HRP-labeled donkey anti-mouse secondary antibody by a ratio of 1:10000 with 1x ELISA washing solution, add to the enzyme-labeled plate, 100 μL per well, and incubate at 37°C for 30 min; repeat the plate washing step. After washing the plate, add TMB substrate to the enzyme-labeled plate, 100 μL per well, and incubate at room temperature in the dark for 15 min; and add ELISA termination solution to the enzyme-labeled plate, 100 μL per well, to terminate the reaction. Detect the absorbance value at 450 nm single wavelength; and analyze the absorbance value data to obtain the antibody titer.

[0093] The ELISA results show that the two monoclonal antibodies provided by the application have the following antigen binding titers: the ELISA titer of monoclonal antibody 1 is 1:2560000, and the ELISA titer of monoclonal antibody 2 is 1:2560000; both are better than the ELISA titers of the two commercial antibodies, 1:1280000, and the titer of monoclonal antibody 1 according to the application is numerically better than that of monoclonal antibody 2.

[0094] As can be seen from the results, the two monoclonal antibodies mentioned in the application have better antigen binding titers than the commercial antibodies.

[0095] Table 1 is a comparison of the ELISA titers of IL-6 monoclonal antibodies 1 and 2 and two commercial monoclonal antibodies for detecting the IL-6 recombinant protein antigen.

[0096] Table 1

[0097]

[0098]

[0099] 4. Specificity of monoclonal antibodies

[0100] The specificity of the monoclonal antibodies is detected by cross-reaction, and the steps are as follows:

[0101] (1) Dilute 10× ELISA coating buffer to 1×, and use 1× ELISA coating buffer to dilute the IL-6 recombinant protein antigen, human IL-6 international standard (WHO21 / 308, NIBSC), human IL-11 international standard (WHO92 / 788, NIBSC), human IL-27 recombinant protein (90235ES10, Y Company), and mouse IL-6 international standard (WHO93 / 730, NIBSC) described in this invention to 0.5 μg / mL. Add the diluted sample coating solution to the microplate, 100 μL per well; seal the microplate with sealing film and coat overnight at 4℃; after washing the plate, add ELISA blocking solution to the microplate, 200 μL per well, and block at 37℃ for 1 h.

[0102] (2) Add 1 μg / mL of commercial IL-6 monoclonal antibody, monoclonal antibody 1 and monoclonal antibody 2 described in this invention to the blocked detection wells using 1×ELISA enzyme dilution buffer, 100 μL per well, and incubate at 37°C for 1 h. Dilute HRP-labeled donkey anti-mouse secondary antibody with 1×ELISA washing buffer at a ratio of 1:10000, add 100 μL to the enzyme-labeled plate, and incubate at 37°C for 30 min. Repeat the washing steps. After washing, add 100 μL of TMB substrate to the enzyme-labeled plate, incubate at room temperature in the dark for 15 min, and add 100 μL of ELISA stop solution to the enzyme-labeled plate to stop the reaction. Detect the absorbance at a single wavelength of 450 nm; and analyze the absorbance data to determine the antibody specificity.

[0103] ELISA results showed that the two monoclonal antibodies provided by this invention and the two commercial monoclonal antibodies bound to the recombinant protein and human IL-6 international standard described in this invention, but did not bind to human IL-11 international standard, human IL-27 recombinant protein and mouse IL-6 international standard.

[0104] The results show that the two monoclonal antibodies mentioned in this invention bind to the human IL-6 protein and the IL-6 international standard described in this invention, and do not cross-react with other IL-6 similar proteins, indicating good specificity.

[0105] Table 2 shows the ELISA results of IL-6 monoclonal antibodies 1 and 2 and two commercial monoclonal antibodies in detecting IL-6 recombinant protein antigen, international standard and other similar antigens.

[0106] Table 2

[0107]

[0108]

[0109] 5. Affinity detection of monoclonal antibodies

[0110] Affinity detection was performed using Bio-Layer Interferometry (BLI). The Octet Molecular Interaction System was turned on and allowed to warm for 40 minutes. AMC2 sensors were pre-wet for 10 minutes in 1x PBS buffer to activate the surface. Two monoclonal antibodies provided by the present application and commercial monoclonal antibody 2706 (M company) were diluted to 5 μg / mL with 1x PBS buffer (containing 0.02% Tween + 1 mg / mL BSA). IL-6 international standard (WHO 21 / 308, NIBSC) was diluted to 5 μg / mL with the same buffer, and then 3-fold gradient dilution was performed (a total of 7 concentration gradients). The diluted antibodies were added to the sample wells (250 μL per well) of a black 96-well plate. The pre-wetted sensors were immersed in the antibody solution for the loading step, so that the antibodies were immobilized on the sensor surface (target immobilization height: 1-2 nm). The sensors were first immersed in the buffer to balance and establish the baseline. The sensors were then transferred to the antigen gradient wells, and the antigen binding signal was monitored in real time (target signal value: 0.3-0.6 nm). Finally, the sensors were moved back to the buffer to monitor the antigen dissociation process. The binding / dissociation curves were recorded and curves with signal values <0.01 nm were removed. Ensure that the curve distribution is uniform and concentration-dependent between gradients. The highest concentration of antigen should show a saturation trend (curve bending). The instrument software was used to fit the binding kinetics data (1:1 binding model). Keep ≥4 valid concentration points, and the fitting degree R 2 >0.95. Calculate the affinity constant KD (equilibrium dissociation constant), and the smaller the KD, the higher the affinity (excellent antibody binding is usually in the picomolar KD≈10 -10 M).

[0111] The BLI results are shown in Figure 6 (including three upper, middle and lower graphs), Figure 6 for IL-6 monoclonal antibody affinity detection. Figure 6 The upper graph in FIG. 1 is the affinity result of monoclonal antibody 1 described in the present application, the middle graph is the affinity result of monoclonal antibody 2 described in the present application, and the lower graph is the result of commercial monoclonal antibody 2706. The BLI results show that the affinity KD values of the two monoclonal antibodies described in the present application are 1.487E-10 and 1.472E-10, and the affinity of the commercial monoclonal antibody 2706 is 1.654E-10, all of which are in the picomolar level, and the affinity of the monoclonal antibodies described in the present application is lower than the KD value of the commercial monoclonal antibody.

[0112] As can be seen from the results, the binding titers of the two monoclonal antibodies mentioned in the present application and the commercial antibody to IL-6 standard are similar, both in the picomolar level, and the monoclonal antibodies 1 and 2 described in the present application have stronger affinity than the commercial monoclonal antibody.

[0113] Example 3

[0114] The present embodiment carries out the function verification of the monoclonal antibody. The commercial human IL-6 dry fluorescence immunoassay kit (X company) is used as a control to detect the function of the monoclonal antibody prepared by the present application.

[0115] 1. Antibody clinical test results

[0116] The performance of the monoclonal antibody is evaluated by using a dry fluorescence immunoassay analyzer. The clinical serum samples and enterprise reference materials are all Roche assigned values, and the comparison results come from the commercial human IL-6 dry fluorescence immunoassay kit (X company).

[0117] 100 μL of serum sample with known concentration (Roche value) is vertically dropped into the sample well of the detection card, and the timing is started. The detection is carried out by using the matched instrument at 15 minutes.

[0118] The dry fluorescence immunoassay results show that the monoclonal antibody provided by the present application has the following results in combination with the clinical serum sample of the commercial human IL-6 dry fluorescence immunoassay kit: in the detection process of the seven serum standard samples, the signal value of the monoclonal antibody provided by the present application is in the range of 0.176-7.675; the signal value of the commercial human IL-6 dry fluorescence immunoassay kit is in the range of 0.107-4.12, the signal value has correlation with the serum concentration, and the numerical value of the monoclonal antibody provided by the present application is better than that of the commercial human IL-6 dry fluorescence immunoassay kit.

[0119] It can be known from the results that the monoclonal antibody provided by the present application has good combination with the clinical serum, and can be used for detecting the clinical sample.

[0120] Table 3 is the comparison results of the dry fluorescence signal values of the IL-6 monoclonal antibody pair and the commercial human IL-6 dry fluorescence immunoassay kit after detecting IL-6 (which has been assigned by Roche detection) in the clinical serum.

[0121] Table 3

[0122]

[0123] 2. Antibody pair accuracy test results

[0124] The performance of the monoclonal antibody is evaluated by using a dry fluorescence immunoassay analyzer. The clinical serum samples and enterprise reference materials are all Roche assigned values.

[0125] 9 portions of the monoclonal antibody pair (monoclonal antibody 1 and monoclonal antibody 2) of the present application and the commercial dry fluorescence immunoassay kit are used to measure the reference materials with IL-6 concentrations of 1675.0 pg / mL, 198.5 pg / mL and 57.07 pg / mL, each concentration is measured for 3 times, and the accuracy is calculated. The calculation formula is as follows:

[0126]

[0127] B i Accuracy; X i Test results; T - Reference product labeled value.

[0128] The accuracy determination results show that the monoclonal antibody provided by the application has the following results in combination with the commercial human IL-6 dry fluorescence immunoassay kit for clinical serum samples: during the detection of three concentration reference products, the relative error of the monoclonal antibody provided by the application is in the range of -2.89% to 3.37%, and the average error of the three concentrations is 0.56%, 2.39% and -2.10% respectively; the relative error of the commercial human IL-6 dry fluorescence immunoassay kit is in the range of 3.63 to 8.77%, and the average error of the three concentrations is 6.24%, 7.28% and 5.81% respectively. The monoclonal antibody described in the application is superior to the commercial human IL-6 dry fluorescence immunoassay kit in accuracy.

[0129] Table 4 is the accuracy comparison results of the IL-6 monoclonal antibody pair and the commercial human IL-6 dry fluorescence immunoassay kit for detecting three concentrations of reference products.

[0130] Table 4

[0131]

[0132] 3. Antibody pair sensitivity test results

[0133] The performance of the monoclonal antibody was evaluated by a dry fluorescence immunoassay analyzer. The clinical serum samples and enterprise reference products were all Roche assigned values.

[0134] Take the serum of healthy people in the clinic as a standby sample, and mix and inactivate it. Centrifuge at 15000 r / min for 30 min at 4℃, take the supernatant, repeat centrifugation once, take the supernatant, and add a preservative, which is a blank sample.

[0135] The blank sample was repeatedly determined 10 times by the interleukin 6 (IL-6) kit in one run. The determination results were recorded in the table. The mean value of the 10 results and the standard deviation SD. The blank mean value plus twice the standard deviation Report the detection limit of the method.

[0136] The sensitivity determination results show that the monoclonal antibody provided by the application has the following repeated determination results in combination with the commercial human IL-6 dry-type fluorescence immunoassay kit for a blank sample: in the process of ten repeated detections, the detection value of the monoclonal antibody provided by the application is in the range of 0.21-2.66, the average detection value is 1.55, and the detection limit is 3.11; the detection value of the commercial human IL-6 dry-type fluorescence immunoassay kit is in the range of 1.25-5.47, the average detection value is 2.53, and the detection limit is 5.48. The monoclonal antibody provided by the application has a lower detection limit than the commercial human IL-6 dry-type fluorescence immunoassay kit, and is superior to the commercial monoclonal antibody in sensitivity.

[0137] Table 5 is the sensitivity comparison results of the IL-6 monoclonal antibody pair in combination with the commercial human IL-6 dry-type fluorescence immunoassay kit for ten blank samples.

[0138] Table 5

[0139]

[0140] 4. Antibody pair precision test

[0141] The performance of the monoclonal antibody was evaluated by a dry-type fluorescence immunoassay analyzer. The clinical serum sample and the enterprise reference were both assigned by Roche.

[0142] Using 30 portions of the monoclonal antibody pair of the application and the commercial human IL-6 dry-type fluorescence immunoassay kit, IL-6 reference with a concentration of 1675.0 pg / mL, 198.5 pg / mL and 57.07 pg / mL was respectively detected, each concentration was repeated for 10 times, and the average value of 10 test results of each concentration was calculated and the standard deviation SD according to formula (2), (3) and (4).

[0143]

[0144] In the formula: The average value of the test results; SD is the standard deviation of the measurement results; CV is the coefficient of variation; x i is a single measurement value; i is 1, 2, 3, …, n.

[0145] The precision determination results show that the monoclonal antibody pair provided by the application has the following repeated determination results with the commercial monoclonal antibody pair and the reference product: when the reference product with a detection concentration of 1675 pg / mL is detected for ten times, the average detection value of the monoclonal antibody provided by the application is 1745.69 pg / mL, and the CV value is 2.34 %; the average detection value of the commercial monoclonal antibody pair is 1492.60 pg / mL, and the CV value is 8.09 %. When the reference product with a detection concentration of 198.5 pg / mL is detected for ten times, the average detection value of the monoclonal antibody provided by the application is 203.07 pg / mL, and the CV value is 4.61 %; the detection value of the commercial human IL-6 dry fluorescence immunoassay kit is 201.02 pg / mL, and the CV value is 7.19 %. The average detection value is 2.53, and the detection limit is 5.48. When the reference product with a detection concentration of 57.07 pg / mL is detected for ten times, the average detection value of the monoclonal antibody provided by the application is 58.64 pg / mL, and the CV value is 4.72 %; the detection value of the commercial human IL-6 dry fluorescence immunoassay kit is 50.60 pg / mL, and the CV value is 9.64 %. The CV value of the monoclonal antibody pair provided by the application is lower than that of the commercial human IL-6 dry fluorescence immunoassay kit, and the precision is better than that of the commercial monoclonal antibody pair.

[0146] Table 6 is the precision comparison results of the IL-6 monoclonal antibody pair and the commercial human IL-6 dry fluorescence immunoassay kit combined with three concentration reference products.

[0147] Table 6

[0148]

[0149] 5. Antibody pair linear range evaluation

[0150] The monoclonal antibody performance is evaluated by a dry fluorescence immunoassay analyzer. The clinical serum samples and enterprise reference products are all Roche assigned values.

[0151] Using 30 parts of the monoclonal antibody pair and the commercial antibody pair provided by the application, the reference products with IL-6 concentrations of 7.79 pg / mL, 25.92 g / mL, 57.07 pg / mL, 116.70 pg / mL, 198.50 pg / mL, 394.80 pg / mL, 816.00 pg / mL, 1675.00 pg / mL, 2556.00 pg / mL and 3441.00 pg / mL are determined, and the correlation coefficient r is calculated by continuously repeating the test for 3 times.

[0152] Linear range determination results Figure 7)showed that the monoclonal antibody pair provided by the present application had the following repeated determination results with the reference product of the commercial monoclonal antibody pair: the r value of the monoclonal antibody pair provided by the present application was 0.9994; and the r value of the commercial monoclonal antibody pair was 0.993. The monoclonal antibody pair provided by the present application was more accurate than the commercial monoclonal antibody pair in the linear range.

[0153] Tables 7 and 8 are respectively the linear range detection results of the IL-6 monoclonal antibody pair in combination with ten concentration reference products of the commercial human IL-6 dry fluorescence immunoassay kit.

[0154] Table 7

[0155]

[0156] Table 8

[0157]

[0158] In summary, the IL-6 monoclonal antibody pair screened by the present application is superior to the commercial human IL-6 dry fluorescence immunoassay kit in potency verification, clinical testing, accuracy testing, sensitivity testing, precision testing and linear range. The IL-6 monoclonal antibody pair has strong binding ability with IL-6 antigen in a clinical sample, has high detection sensitivity, and can be combined with the antigen as a detection reagent raw material, and has clinical detection advantages.

[0159] The applicant declares that the detailed method of the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, i.e. it does not mean that the present application must rely on the above-mentioned detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A human inflammatory cytokine interleukin 6 antigen, characterized in that, The amino acid sequence of the human inflammatory factor interleukin 6 antigen comprises the sequence as shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the human inflammatory factor interleukin 6 antigen according to claim 1. Preferably, the nucleotide sequence of the nucleic acid molecule comprises the sequence as shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule according to claim 2. Preferably, the recombinant expression vector, after transfecting, transducing or transforming a host cell, enables the host cell to express the human inflammatory factor interleukin 6 antigen according to claim 1.

4. A recombinant expression strain, characterized in that, The recombinant expression strain contains at least one copy of the recombinant expression vector according to claim 3, or at least one copy of the nucleic acid molecule according to claim 2.

5. An anti-human interleukin 6 monoclonal antibody, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 3, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 5; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 6, the amino acid sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO: 7, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 8; Or, the amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 13, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 15; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 16, the amino acid sequence of CDR2 of the light chain variable region is RAN, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO:

17.

6. The anti-human interleukin 6 monoclonal antibody according to claim 5, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 10; Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 19; Preferably, the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain of the anti-human inflammatory factor interleukin 6 monoclonal antibody is as shown in SEQ ID NO: 12; Or, the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO:

21.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human inflammatory factor interleukin 6 monoclonal antibody according to claim 5 or 6.

8. An expression vector, characterized by, The expression vector contains the nucleic acid molecule according to claim 7.

9. A reference for detecting human inflammatory cytokine interleukin 6, characterized in that, The reference contains the human inflammatory factor interleukin 6 antigen according to claim 1.

10. Use of any one or a combination of at least two of the human inflammatory factor interleukin 6 antigen according to claim 1, the anti-human inflammatory factor interleukin 6 antigen monoclonal antibody according to claim 5 or 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8 or the reference for the detection of human inflammatory factor interleukin 6 according to claim 9 for the manufacture of a product for the detection of human inflammatory factor interleukin 6.

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