An enzyme-linked immunosorbent assay kit for detecting soluble IL-6 protein content

Abstract

The application discloses an enzyme-linked immunoassay kit for soluble IL-6 protein content detection, which comprises two anti-human IL-6 monoclonal antibodies, IL-6-1C7 and IL-6-2E11. The amino acid sequence of the heavy chain variable region of the IL-6-1C7 monoclonal antibody is SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the IL-6-2E11 is SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is SEQ ID NO. 4. The enzyme-linked immunoassay kit of the application can effectively detect the soluble IL-6 content. It is found through the detection of the kit that the serum of healthy people contains a certain basic level of soluble IL-6 protein, and the soluble IL-6 content of the serum of patients with inflammatory and autoimmune diseases is obviously increased, which is related to the clinical stage and pathology of the patients, and the soluble IL-6 content detection in the body fluid of the patients with inflammation has clinical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical products, and particularly relates to an enzyme-linked immunosorbent assay kit for detecting the content of soluble IL-6 protein. BACKGROUND

[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, promoting immune response. Studies have shown that IL-6 is involved in the resistance of mice to Streptococcus pneumoniae infection, 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, detecting the content of IL-6 in the blood has important clinical significance for the diagnosis and treatment of certain diseases. SUMMARY

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

[0005] The present application first provides a monoclonal antibody against human IL-6 protein, wherein the first monoclonal antibody against human IL-6 is IL-6-1C7 antibody, which includes 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 IL-6-2E11 antibody, including heavy chain and 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, and is as follows:

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

[0013] The monoclonal antibody provided by the application is used for preparing a kit for detecting human IL-6 protein.

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

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

[0016] The monoclonal antibody screened by the application and the enzyme-linked immunoassay kit prepared by the application can effectively detect the content of soluble IL-6. It is found by using the kit of the application that the serum of healthy people contains a certain basic level of soluble IL-6 protein (~ 5 pg / ml), and the content of soluble IL-6 in the serum of patients with inflammatory and autoimmune diseases is significantly increased, indicating that it has relevance to the clinical stage and pathology of IL-6 patients. The detection of the content of soluble IL-6 in the body fluid of patients with inflammation has clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is a schematic diagram of the enzyme-linked immunoassay kit of the present application for detecting the content of soluble IL-6 protein in a sample. DETAILED DESCRIPTION

[0018] The present application will be described in detail below with reference to examples.

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

[0020] 1.1 Immunization of mice

[0021] The BALB / c mice were immunized with the IL-6 fusion recombinant protein for 4 times, and the interval between the first three times was 14 days. The antibody titer in the blood of the mice was determined 5-7 days after the third immunization. When the titer was good, the fourth booster immunization was performed.

[0022] 1.2 Culture of trophoblast cells

[0023] 1.2.1 The spleen of a 7-8 week old BALB / c mouse was taken out in a sterile clean bench. A single cell suspension was obtained by grinding in a 200 mesh sterile screen. 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 uniformly dropped into a 96-well culture plate using a sterile Pasteur glass pipette, and the volume of each well was about 80 ul. The cells were cultured overnight in a cell culture incubator.

[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 uniformly dropped into a 96-well culture plate using a sterile Pasteur glass pipette, and the volume of each well was about 80 ul. The cells were cultured overnight in a cell culture incubator.

[0025] 1.3 Cell fusion and screening

[0026] 1.3.1 The spleen of a 7-8 week old BALB / c mouse was taken out in a sterile clean bench. A single cell suspension was obtained by grinding in a 200 mesh sterile screen. The single cell suspension was washed twice with 37°C preheated 1640 basal medium by centrifugation at 1400 rpm / 5 min.

[0027] 1.3.2 The collected spleen cells and the SP2 / 0 cells in the logarithmic growth phase were mixed in a centrifuge tube at a ratio of about 5:1, and preheated RPMI-1640 basal medium was added and mixed thoroughly. Then, the mixture was centrifuged at 1400 rpm / 5 min once, and after the supernatant was discarded, the tube bottom was tapped with fingers to mix the two types of cells.

[0028] 1.3.3 1 ml of preheated PEG solution was added dropwise to the two types of mixed cells, and the centrifuge tube was gently shaken during the process to accelerate the fusion of the two types of cells.

[0029] 1.3.4 After the fusion was completed, 14 ml of RPMI-1640 basal medium was added dropwise to terminate the fusion process. The cells were centrifuged, and the supernatant was removed.

[0030] 1.3.5 Resuspend the confluent cells in RPMI-1640 whole medium, then use a Parshall glass plate to transfer approximately 80 μl of the cells into each well of a 96-well culture plate containing the feeder cells. Incubate in an incubator and change the medium with HT medium every 3-4 days. Afterward, take a small amount of supernatant from each well for ELISA detection.

[0031] 1.3.6 Select wells with high antibody detection values ​​for subcloning. Perform ELISA detection after each subcloning. Usually, it takes 3-4 subclonings to obtain cells with a stable antibody-secreting phenotype. Cryopreserve the cells after the last subcloning.

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

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

[0034] 2.1 Obtaining cDNA from hybridoma cells

[0035] RNA was obtained from the target hybridoma cells and reverse transcribed into cDNA using reverse transcription technology. The heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cells were then 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 ligated to the pJET cloning vector, respectively. Subsequently, the ligation products were transformed into DH5a competent cells, and the transformed bacterial culture was evenly spread on LB solid medium.

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

[0038] 2.4 Based on the sequencing results, candidate light and heavy chain variable region sequences were retained. The variable region sequences of the light and heavy chains that could be ligated to the expression vector were cloned again by PCR. The variable region sequences were then ligated to the expression vector, and the ligation product was transformed into DH5α. The transformed bacterial culture was evenly spread on LB solid medium and incubated overnight.

[0039] 2.5 Select well-growing bacteria for sequencing and compare the two sequencing results to obtain transformed bacteria with the correct sequence. After expansion culture, plasmid extraction is performed.

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

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

[0042] 2.7 The supernatant containing the antibody of interest was detected by ELISA kit, and the result showed good.

[0043] The anti-human IL-6-1C7 monoclonal antibody heavy chain variable region 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 variable regions of the heavy chain and the light chain of the antibody are extracted from the hybridoma cells expressing the anti-human IL-6 monoclonal antibody, the candidate light / heavy chain variable region sequences are reserved according to the sequencing results, and the light / heavy chain variable region sequences matching the expression vector are amplified by PCR, and the PCR product is connected with the double enzyme pretreated expression vector. The expression vector connected with the light chain and the heavy chain variable region of the monoclonal antibody is co-transfected into the eukaryotic expression cell strain HEK293, and the supernatant after culture contains the target antibody, which indicates that the obtained heavy chain and light chain variable region sequences are correct.

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

[0053] 3.1 Composition of kit

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

[0055] 3.2 Collection and processing of samples

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

[0057] 3.3.2 Before detecting the serum sample, pre-equilibrate the sample at room temperature for half an hour, shake and mix, take an appropriate amount of patient serum, dilute with sample diluent for 2-fold dilution.

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

[0059] 3.3.1 Dilute the coating antibody IL-6-1C7 (1 μg / ml) with coating solution (1×PBS), then add the coating antibody to the 96-well enzyme-labeled plate (50 μl / well), and incubate at 4℃ overnight.

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

[0061] 3.3.3 Remove the blocking solution, use a pipette to add 100 μl of diluted serum sample to be tested and gradient-diluted soluble IL-6 protein standard (start with 100 pg / ml, dilute by ratio, a total of 7 concentrations, the last one is sample diluent as blank control, and 3 repeats are made for each gradient), and incubate at room temperature with shaking at 500 rpm for 1 h.

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

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

[0064] 3.3.5PBST wash the plate for 6 times, 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 stop the color reaction, and determine the OD450 and OD630 values of each well by an enzyme-labeled instrument.

[0065] 3.3.6Take the product of IL-6 standard concentration and the dilution multiple of the sample (2) as the abscissa, and the corresponding measured OD450 minus OD630 value as the ordinate to draw a standard curve (such as Figure 1 ), and obtain a calculation formula, and calculate the content of soluble IL-6 in the sample according to the OD difference value of the sample.

[0066] The kit of the present application can achieve the following indexes according to methodological identification.

[0067] The linear range of the standard curve is R 2 ≥0.99, and the minimum detection limit is ≤2.0 pg / ml.

[0068] The enzyme-linked immunoassay kit of the present application can effectively detect the content of soluble IL-6. It is found by using the kit for detection that the serum of healthy people contains a certain basic level of soluble IL-6 protein (~5 pg / ml), and the content of soluble IL-6 in the serum of patients with inflammatory and autoimmune diseases is significantly increased, which has correlation with the clinical stage and pathology of the patients, and the detection of the content of soluble IL-6 in the body fluid of patients with inflammation has clinical value.

Claims

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

2.

2. A monoclonal antibody against a human IL-6 protein, characterized in that, The monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region 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 of claim 1 or claim 2 in the preparation of a kit for detecting human IL-6 protein.

4. A kit for detecting human IL-6 protein, characterized by, The kit comprises the monoclonal antibody of claim 1 and / or claim 2.

5. The kit of claim 4, wherein The kit is used for detecting soluble IL-6 protein in body fluid.

Images