Enzyme-linked immunosorbent assay kit for soluble IL-6 protein content detection

Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting the content of soluble IL-6 (interleukin-6) protein. The enzyme-linked immunosorbent assay kit comprises two anti-human IL-6 monoclonal antibodies, namely, IL-6-1C7 and IL-6-2E11. The amino acid sequence of a heavy chain variable region of the IL-6-1C7 monoclonal antibody is SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the IL-6-1C7 monoclonal antibody is SEQ ID NO.2; the amino acid sequence of a heavy chain variable region of the IL-6-2E11 is SEQ ID NO.3, and the amino acid sequence of a light chain variable region of the IL-6-2E11 is SEQ ID NO.4. The enzyme linked immunosorbent assay kit provided by the invention can effectively detect the content of soluble IL-6, and detection by using the kit finds that serum of healthy people contains soluble IL-6 protein with a certain basic level, the content of soluble IL-6 in serum of patients with inflammatory and autoimmune diseases and the like is obviously increased and is related to clinical staging and pathology of the patients, so that the content of soluble IL-6 in serum of patients with inflammatory and autoimmune diseases is obviously reduced. The detection of the content of the soluble IL-6 in the body fluid of the inflammatory patient has clinical value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical products, and in particular relates to an enzyme-linked immunosorbent assay kit for detecting the content of soluble IL-6 protein. Background Art

[0002] Interleukin 6 (IL-6) is a cytokine that has both pro-inflammatory and anti-inflammatory properties. In humans, it is encoded by the IL-6 gene. During infection and after trauma, especially in patients with inflammation caused by burns or other tissue damage, IL-6 is secreted by T cells and macrophages to promote immune response. Studies have shown that IL-6 is required for mice to resist infection with Streptococcus pneumoniae, indicating that IL-6 plays a role in resisting infection.

[0003] IL-6 can promote the progression of inflammatory and autoimmune diseases in many diseases such as diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, Behcet's disease and rheumatoid arthritis. Patients with advanced / metastatic cancer have higher levels of IL-6 in their blood. Therefore, the content of IL-6 in the test also plays an important clinical role in the diagnosis and treatment of specific diseases. Summary of the invention

[0004] The purpose of the present invention is to provide an enzyme-linked immunosorbent assay kit for detecting the content of soluble IL-6 protein, thereby making up for the deficiencies of the prior art.

[0005] The present invention first provides a monoclonal antibody against human IL-6 protein, wherein the first anti-human IL-6 monoclonal antibody is an IL-6-1C7 antibody, comprising a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of the IL-6-1C7 is as follows:

[0006] EVQLQQSGAELAKPGASVKMSCKASGYAFSNYWMHWVKQRPGQGLEWIG YIIPTTGYADYNQKFKDKATLTADKSSSTAYMQLTSLTSEDSAVYFCAKLLYGTYDF WGQGTTLTVSS(SEQ ID NO:1);

[0007] The amino acid sequence of the IL-6-1C7 light chain variable region (mVL) is as follows:

[0008] DIVLTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCAQSTHVPPLTFGA GTKLELK(SEQ ID NO:2);

[0009] The second anti-human IL-6 monoclonal antibody is an IL-6-2E11 antibody, comprising a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region (mVH) of the IL-6-2E11 is as follows:

[0010] QVKLQESDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYIS PGNGDTMYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKIIYYGNFGDHF DYWGQGTTLTVSS(SEQ ID NO:3);

[0011] The amino acid sequence of the light chain variable region (mVL) of IL-6-2E11 is the same as SEQ ID NO.4, as follows:

[0012] DIVITQSPLSLPVSLGDQASISCRSGQSLVHSNGNTYLHWHLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK (SEQ ID NO: 4).

[0013] The monoclonal antibody provided by the present invention is used to prepare a kit for detecting human IL-6 protein;

[0014] In another aspect, the present invention also provides a kit for detecting human IL-6 protein, which contains the monoclonal antibody.

[0015] The kit is used for detecting soluble IL-6 protein in body fluids.

[0016] The monoclonal antibodies screened by the present invention and the prepared enzyme-linked immunosorbent assay kit can effectively detect the soluble IL-6 content. The kit of the present invention was used to detect that a certain basic level of soluble IL-6 protein (~5pg / ml) was contained in the serum of healthy people, while the soluble IL-6 content in the serum of patients with inflammatory diseases, autoimmune diseases, etc. was significantly increased, indicating that it is correlated with the clinical stage and pathology of IL-6 patients. The detection of soluble IL-6 content in the body fluids of inflammatory patients has clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the ELISA kit of the present invention for detecting the soluble IL-6 protein content in a sample. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the embodiments.

[0019] Example 1: Preparation of anti-human IL-6 monoclonal antibody hybridoma cells

[0020] 1.1 Immunization of mice

[0021] BALB / c mice were immunized 4 times with IL-6 fusion recombinant protein, with the first three immunizations separated by 14 days. The antibody titer in the blood of mice was measured 5-7 days after the third immunization. If the titer was good, a fourth booster immunization was performed.

[0022] 1.2 Culture of trophoblast cells

[0023] 1.2.1 Remove the spleen of 7-8 week old BALB / c mice in a sterile clean bench and grind it in a 200 mesh sterile sieve to obtain a single cell suspension.

[0024] 1.2.2 The single cell suspension was washed twice with RPMI-1640 medium, and the cells were resuspended with an appropriate amount of RPMI-1640 medium containing 15% FBS. The cell solution was evenly dropped into a 96-well culture plate using a sterile Pasteur glass pipette. The volume of each well was about 80ul, and the cells were cultured in a cell culture incubator overnight.

[0025] 1.3 Cell fusion and screening

[0026] 1.3.1 Take out the spleen of 7-8 week old BALB / c mice in a sterile clean bench. Grind in a 200 mesh sterile sieve to obtain a single cell suspension. Resuspend and wash twice in 1640 basal medium preheated at 37°C at 1400 rpm / 5 min.

[0027] 1.3.2 Mix the collected spleen cells and SP2 / 0 cells in the logarithmic growth phase in a centrifuge tube at a ratio of approximately 5:1, and add preheated RPMI-1640 basal medium to mix thoroughly. Then centrifuge once at 1400rpm / 5min, discard the supernatant and flick the bottom of the tube with your fingers to mix the two cells.

[0028] 1.3.3 Add 1 ml of preheated PEG solution to the two mixed cells, and gently shake the centrifuge tube to accelerate the fusion of the two cells.

[0029] 1.3.4 After fusion is complete, add 14 ml of RPMI-1640 basal medium to terminate the fusion process. Centrifuge and remove the supernatant.

[0030] 1.3.5 Add RPMI-1640 complete medium to resuspend the fused cells, then use Pasteur glass to place the 96-well culture plate containing the trophoblast cells, approximately 80 μl per well, and culture in an incubator. Replace the medium with HT medium every 3-4 days, and then take a small amount of supernatant from each well for ELISA detection.

[0031] 1.3.6 Select the wells with higher antibody detection values ​​for subcloning. Perform ELISA test after each subcloning. Usually, it takes 3-4 subclones to obtain cells with stable antibody secretion phenotype. The cells after the last subclone are frozen.

[0032] Example 2: Determination of the light and heavy chain variable region sequences of anti-human IL-6 monoclonal antibodies

[0033] The method for determining the heavy chain and light chain variable regions of anti-human IL-6 monoclonal antibodies comprises the following steps:

[0034] 2.1 Acquisition of hybridoma cell cDNA

[0035] RNA is obtained from the target hybridoma cell, and the obtained RNA is reverse transcribed into cDNA by reverse transcription technology. The heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cell are cloned by PCR using specifically designed upstream and downstream primers.

[0036] 2.2 The heavy chain variable region (mVH) and light chain variable region (mVL) were connected to the pJET cloning vector respectively. Subsequently, the connected products were transformed into DH5a competent bacteria, and the transformed bacterial solution was evenly spread on LB solid culture medium.

[0037] 2.3 Select colonies with clear edges and good growth on LB solid culture medium for sequencing identification.

[0038] 2.4 According to the sequencing results, the candidate light and heavy chain variable region sequences were retained, and the variable region sequences of the light and heavy chains that could be connected to the expression vector were cloned again by PCR, and the variable region sequences were connected to the expression vector, and then the connection products were transformed into DH5a. The transformed bacterial solution was evenly coated on LB solid medium and cultured overnight.

[0039] 2.5 Select bacteria with good growth for sequencing, and compare the two sequencing results to obtain transformed bacteria with the correct sequence, and extract the plasmid after expanding the culture.

[0040] 2.6 The expression vectors containing the heavy chain and light chain variable region genes of the cloned antibody were co-transfected into the eukaryotic expression cells HEK293.

[0041] 2.7 HEK293 cells were cultured in suspension using serum-free medium, and the medium was replaced with serum-free medium during transfection.

[0042] 2.7 The supernatant containing the target antibody was detected using an ELISA kit, and the results were good.

[0043] The heavy chain variable region of anti-human IL-6-1C7 monoclonal antibody was obtained by the above method:

[0044] EVQLQQSGAELAKPGASVKMSCKASGYAFSNYWMHWVKQRPGQGLEWIGY IIPTTGYADYNQKFKDKATLTADKSSSTAYMQLTSLTSEDSAVYFCAKLLYGTYDFW GQGTTLTVS;

[0045] IL-6-1C7 monoclonal antibody light chain variable region:

[0046] DIVLTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCAQSTHVPPLTFGAGTKLE LK;

[0047] IL-6-2E11 monoclonal antibody heavy chain variable region

[0048] QVKLQESDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISP GNGDTMYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKIIYYGNFGDHFD YWGQGTTLTVSS;

[0049] IL-6-2E11 monoclonal antibody light chain variable region

[0050] DIVITQSPLSLPVSLGDQASISCRSGQSLVHSNGNTYLHWHLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEI K.

[0051] The invention extracts the variable regions of the heavy and light chains of the antibody from hybridoma cells expressing anti-human IL-6 monoclonal antibodies, retains candidate light and heavy chain variable region sequences according to sequencing results, amplifies the light and heavy chain variable region sequences matching the expression vector by PCR, connects the PCR product with the expression vector pretreated with double enzyme digestion, co-transfects the eukaryotic expression cell line HEK293 with the expression vectors connected with the light and heavy chain variable regions of the target monoclonal antibody, collects the supernatant after culture and contains the target antibody, indicating that the obtained heavy and light chain variable region sequences are correct.

[0052] Example 3: ELISA kit for detecting soluble IL-6 protein content:

[0053] 3.1 Composition of the kit

[0054] The ELISA kit of the present invention comprises the IL-6 coated antibody (IL-6-1C7) prepared in Example 1 coated on an ELISA plate, a biotin-labeled IL-6 detection antibody (IL-6-2E11), a soluble IL-6 protein standard (ThermoFisher), horseradish peroxidase (HRP), a sample diluent, a washing solution (PBST), a color developing solution (TMB) and a stop solution.

[0055] 3.2 Sample collection and processing

[0056] 3.2.1 Collect a batch of serum from inflammatory patients in the hospital, store it in a -80℃ refrigerator after packaging, and avoid repeated freezing and thawing.

[0057] 3.3.2 Before testing serum samples, equilibrate the samples at room temperature for half an hour and vortex to mix them. Take an appropriate amount of patient serum and dilute it 2-fold with the sample diluent.

[0058] 3.3 Determination of soluble IL-6 protein content

[0059] 3.3.1 Use coating solution (1×PBS) to dilute the coating antibody IL-6-1C7 (1 μg / ml), then add the coating antibody to a 96-well ELISA plate (50 μl / well) and incubate at 4°C overnight.

[0060] 3.3.2 On the next day, wash the plate once with washing solution (PBST), then add 300 μl of 3% bovine serum albumin blocking solution to each well and block at 37°C for 1 h.

[0061] 3.3.3 Remove the blocking solution, use a pipette to add 100 μl of the diluted serum sample to be tested and the gradient diluted soluble IL-6 protein standard (starting from 100 pg / ml, with a total of 7 concentrations, and the last empty sample dilution solution is used as a blank control, and each gradient is repeated 3 times), and incubate at room temperature with shaking at 500 rpm for 1 hour.

[0062] 3.3.4 Wash the plate 6 times with PBST buffer, add biotin-labeled detection antibody IL-6-2E11 (100 μl / well), and incubate at room temperature with shaking at 500 rpm for 0.5 h.

[0063] 3.3. Wash the plate 6 times with PBST solution, add horseradish peroxidase (HRP, 100 μl / well), and incubate at room temperature with shaking at 500 rpm for 0.5 h.

[0064] 3.3.5 Wash the plate 6 times with PBST solution, add color developing solution TMB (100 μl / well), incubate at room temperature with shaking for 10 min, then add stop solution (100 μl / well) to terminate the color reaction, and measure the OD450 and OD630 values ​​of each well using an enzyme reader.

[0065] 3.3.6 The standard curve is prepared by multiplying the concentration of IL-6 standard by the dilution factor of the sample (×2) as the horizontal axis and subtracting the corresponding measured OD450 from OD630 as the vertical axis (e.g. Figure 1 ), and obtain the calculation formula, and calculate the content of soluble IL-6 in the sample according to the OD difference of the sample to be tested.

[0066] The kit of the present invention can achieve the following indicators according to the methodological identification:

[0067] Standard curve linearity: R 2 ≥0.99; minimum detection limit ≤2.0pg / ml.

[0068] The enzyme-linked immunosorbent assay kit of the present invention can effectively detect the soluble IL-6 content. The kit was used to detect and find that a certain basic level of soluble IL-6 protein (~5pg / ml) is contained in the serum of healthy people, and the soluble IL-6 content in the serum of patients with inflammatory diseases, autoimmune diseases, etc. is significantly increased, which is correlated with the clinical stage and pathology of the patients. The detection of the soluble IL-6 content in the body fluids of inflammatory patients has clinical value.

Claims

1. A monoclonal antibody against human IL-6 protein, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO:

2.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is SEQ ID NO:

4.

3. Use of the monoclonal antibody according to claim 1 or claim 2 in preparing a kit for detecting human IL-6 protein.

4. A kit for detecting human IL-6 protein, characterized in that: The kit comprises the monoclonal antibody according to claim 1 and / or claim 2.

5. The kit according to claim 4, characterized in that The kit is used for detecting soluble IL-6 protein in body fluids.

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