A monoclonal antibody against human IL-6 and its use in preparing a detection kit

Abstract

The application provides a kind of anti-human IL-6 monoclonal antibody and its application in preparation flow cytometry detection kit, the anti-human IL-6 monoclonal antibody, wherein the amino acid sequence of heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of light chain variable region is SEQ ID NO.2.The monoclonal antibody screened in the application and the flow fluorescence detection kit established can effectively detect soluble IL-6 protein.Using the kit to detect, it is found that the lymphocyte containing IL-6 protein in the whole blood of healthy people is limited, and the content of soluble IL-6 in the serum of patients with inflammatory, autoimmune diseases and the like is significantly increased and is grouped, so as to assist in judging the immune function state of the body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical products, and particularly relates to a monoclonal antibody against human IL-6 and application thereof in preparing a detection kit. 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 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.

[0004] Currently, the methods for detecting IL-6 in human blood in research are mainly immune response assays, including ELISA and flow cytometry methods. Compared with the traditional ELISA method, the flow cytometry method has the advantages of simple sample pretreatment, short detection time, and simple result interpretation. At the same time, as a commonly used detection method in clinical and scientific research, flow cytometry detects target proteins based on flow antibody conjugated fluorescence, so the antibody for detecting target proteins has high requirements. Ordinary antibodies have the problems of low potency, and the effect of lymphocyte subpopulation is not obvious. SUMMARY

[0005] In order to overcome the above-mentioned defects of the prior art, the purpose of the present application is to provide a monoclonal antibody against human IL-6 and its application in preparing a flow cytometry detection kit, that is, on the basis of screening a monoclonal antibody against human IL-6, a human IL-6 flow cytometry fluorescence detection kit with good antibody potency and lymphocyte subpopulation effect is established.

[0006] The present application first provides a monoclonal antibody IL-6-2A11 antibody against human IL-6, which comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain variable region (mVH) of the IL-6-2A11 is SEQ ID NO: 1, and the specific sequence is as follows:

[0007] EVKLEESGAELVKPGASVKLSCTASGFNIKDTYIHWMKQRPEQGLKWIGKID PANGNTQYDPKFQGKATITADTSSNTAYLQLNSLTSEDTAVYYCAREGDWGQGTTL TVSS;

[0008] The amino acid sequence of the IL-6-2A11 light chain variable region (mVL) is SEQ ID NO. 2, and is specifically as follows:

[0009] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIY AASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAASYYCQQNYEDPPTFGGGTKLEI K.

[0010] In another aspect of the present application, the monoclonal antibody IL-6-2A11 is used in the preparation of a product for detecting interleukin 6 (IL-6).

[0011] As a specific embodiment of the present application, the product is a flow cytometry fluorescence detection kit.

[0012] In another aspect of the present application, the flow cytometry fluorescence detection kit comprises the monoclonal antibody IL-6-2A11.

[0013] The present application also provides a method for detecting soluble IL-6 protein for non-disease treatment purposes, which uses the flow cytometry fluorescence detection kit described above.

[0014] The monoclonal antibody and the flow cytometry fluorescence detection kit established by the present application can effectively detect soluble IL-6 protein. By using the kit, it is found that the lymphocytes containing IL-6 protein in the whole blood of healthy people are limited, and the content of soluble IL-6 in the serum of patients with inflammatory and autoimmune diseases is significantly increased and grouped, thereby assisting in judging the immune function state of the body. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A comparison diagram of the flow cytometry fluorescence detection kit of the present application for detecting soluble IL-6 protein in human whole blood. DETAILED DESCRIPTION

[0016] The present application will be described in detail below in conjunction with examples and drawings.

[0017] Example 1: Preparation of anti-human IL-6 monoclonal antibody hybridoma cells

[0018] 1.1 Immunization of mice

[0019] BALB / c mice were immunized with IL-6 fusion recombinant protein four times, with an interval of 14 days between the first three times. The antibody titer of the mice was determined 5-7 days after the third immunization. When the titer was good, a fourth booster immunization was performed.

[0020] 1.2 Culture of trophoblast cells

[0021] 1.2.1 The spleen of a 7-8 week old BALB / c mouse was removed in a sterile clean bench. A single cell suspension was obtained by grinding in a 200 mesh sterile sieve.

[0022] 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, with a volume of about 80ul per well. The cells were cultured overnight in a cell culture incubator.

[0023] 1.3 Cell fusion and screening

[0024] 1.3.1 The spleen of a 7-8 week old BALB / c mouse was removed in a sterile clean bench. A single cell suspension was obtained by grinding in a 200 mesh sterile sieve. The resuspended cells were washed twice with 37°C preheated 1640 basal medium at 1400 rpm / 5 min.

[0025] 1.3.2 The collected spleen cells were mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of about 5:1 in a centrifuge tube, and preheated RPMI-1640 basal medium was added and mixed thoroughly. Then, the mixture was centrifuged at 1400 rpm / 5 min once, and the supernatant was discarded. The tube bottom was gently tapped with a finger to mix the two cells.

[0026] 1.3.3 1ml of preheated PEG solution was added to the mixed cells, and the centrifuge tube was gently shaken during the process to accelerate the fusion of the two cells.

[0027] 1.3.4 After the fusion was completed, 14ml of RPMI-1640 basal medium was added to terminate the fusion process. The cells were centrifuged and the supernatant was removed.

[0028] 1.3.5 RPMI-1640 complete medium was added to resuspend the fused cells, and then a Pasteur glass was used to transfer the trophoblast cells into a 96-well culture plate, with a volume of about 80ul per well. The plate was incubated in a cell culture incubator, and the medium was changed every 3-4 days with HT medium. Then, a small amount of supernatant was taken from each well for ELISA detection.

[0029] 1.3.6 The wells with higher antibody detection values are selected for subcloning, and ELISA detection is performed after each subcloning. Usually, 3-4 subclonings are required to obtain cells stably secreting the antibody phenotype, and the cells after the last subcloning are frozen.

[0030] Example 2: Determination of the sequences of the light and heavy chain variable regions of the anti-human IL-6 monoclonal antibody

[0031] The method for determining the heavy and light chain variable regions of the anti-human IL-6 monoclonal antibody comprises the following steps:

[0032] 2.1 Obtaining of cDNA from hybridoma cells

[0033] RNA is obtained from the target hybridoma cells, and the obtained RNA is reverse transcribed into cDNA by reverse transcription technology. The heavy chain variable region (mVH) and the light chain variable region (mVL) of the hybridoma cells are cloned by PCR using specifically designed upstream and downstream primers.

[0034] 2.2 The heavy chain variable region (mVH) and the light chain variable region (mVL) are respectively connected with the pJET cloning vector cloning carrier. Then, the connected product is transformed into DH5a competent bacteria, and the transformed bacterial solution is uniformly coated on LB solid medium.

[0035] 2.3 The colonies with clear edges and good growth on the LB solid medium are selected for sequencing identification.

[0036] 2.4 According to the sequencing results, the candidate light and heavy chain variable region sequences are retained, and the light and heavy chain variable region sequences that can be connected with the expression vector are cloned again by PCR, and the variable region sequences are connected with the expression vector, and then the connected product is transformed into DH5a. The transformed bacterial solution is uniformly coated on LB solid medium and cultured overnight.

[0037] 2.5 The well-grown bacteria are sequenced, and the two sequencing results are compared to obtain the transformed bacteria with correct sequences, which are then expanded and subjected to plasmid extraction.

[0038] 2.6 The expression vector connected with the cloned antibody heavy and light chain variable region genes is co-transfected into eukaryotic expression cells HEK293.

[0039] 2.7 The HEK293 cells are suspended in serum-free medium, and the medium is replaced with serum-free medium at the time of transfection.

[0040] 2.7 The supernatant containing the target antibody is detected by ELISA kit, and the result shows good.

[0041] 2.7.1 The purified antibody is coated on a 96-well plate at a concentration of about 2 μg / mL;

[0042] 2.7.2 Add 100 μL of diluted IL-6 standard to the coated plate, 8 standard concentration gradients (S1-S8 from high to low, unit pg / mL) as shown in Table 1 below.

[0043] Table 1: 8 standard concentration gradient table

[0044] S1 S2 S3 S4 S5 S6 S7 S8 1000 500 250 125 62.50 31.25 15.63 0

[0045] 2.7.3 Add 50 μL of biotin-conjugated anti-IL-6 antibody (MABTECH) diluted to 2 μg / mL per well;

[0046] 2.7.4 After incubation at room temperature for 2 h, wash the plate 4 times with 1 × PBS-T;

[0047] 2.7.5 Add 100 μL of SA-HRP solution with a concentration of 1 ug / mL per well;

[0048] 2.7.6 Incubate at room temperature for 1 h, and wash the plate 4 times with 1 × PBS-T;

[0049] 2.7.7 After washing the plate, add 100 ul of TMB per well, and after 10 min of color development, add 100 ul of 1M sulfuric acid solution;

[0050] 2.7.8 Read the results using an enzyme marker, and the results are shown in Table 2 below. The anti-human IL-6-2A11 monoclonal antibody has good linearity in the concentration range of 15.63-1000 pg / mL for IL-6 protein.

[0051] Table 2: Human IL-6-2A11 monoclonal antibody binding efficiency table for IL-6 protein

[0052] pg / mL 2A11 2A11 1000 2.619 2.759 500 1.248 1.285 250 0.577 0.547 125 0.241 0.237 62.5 0.142 0.137 31.25 0.088 0.085 15.63 0.068 0.064 0 0.057 0.060

[0053] The anti-human IL-6-2A11 monoclonal antibody heavy chain variable region is obtained by the above method:

[0054] EVQLQQSGAELAKPGASVKMSCKASGYAFSNYWMHWVKQRPGQGLEWIGY IIPTTGYADYNQKFKDKATLTADKSSSTAYMQLTSLTSEDSAVYFCAKLLYGTYDFW GQGTTLTVS;

[0055] IL-6-2A11 monoclonal antibody light chain variable region:

[0056] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAASYYCQQNYEDPPTFGGGTKLEI K.

[0057] The present application extracts the variable regions of the heavy chain and the light chain of the antibody from the hybridoma cells expressing the anti-human IL-6 monoclonal antibody, retains the candidate light and heavy chain variable region sequences according to the sequencing results, and amplifies the light and heavy chain variable region sequences matched with the expression body through PCR, and connects the PCR product 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.

[0058] Example 3: Establishment of flow cytometry fluorescence detection kit and detection method for detecting soluble IL-6 protein

[0059] 3.1 Composition of kit

[0060] The flow cytometry fluorescence detection kit of the present application comprises the FITC-labeled anti-IL-6 detection antibody (IL-6-2A11) prepared in Example 1, and a reagent storage solution.

[0061] 3.2 Collection and processing of samples

[0062] 3.2.1 Collect a batch of whole blood of inflammatory patients in a hospital, store in a-80℃ refrigerator after sub-packaging, and avoid repeated freezing and thawing.

[0063] 3.3.2 Take 100 μL of whole blood sample, treat with red cell lysis and membrane fixation agent, add 5 μL of FITC-labeled anti-IL-6 antibody reagent, shake and mix, and incubate at room temperature for 15 minutes.

[0064] 3.3 Detection method of soluble IL-6 protein

[0065] 3.3.1 Add 2 mL of flow cytometry staining buffer to each sample tube, centrifuge at 300xg for 10 minutes, and discard the supernatant.

[0066] 3.3.2 Resuspend with 500 μL of flow cytometry staining buffer, and detect using a flow cytometer.

[0067] 3.3.3 Collect the FITC fluorescence signal of anti-IL-6 protein according to the instrument parameter setting, and output the results.

[0068] 3.3.4 Analyze the proportion of anti-IL-6 lymphocytes in the lymphocyte circle gate.

[0069] The kit of the present application can achieve the following indexes according to the methodological identification:

[0070] The positive proportion of anti-IL-6 protein lymphocyte subpopulation is greater than or equal to 30% compared with the same type control group.

[0071] 3.3.7 The flow fluorescence detection kit of the present application can effectively detect and distinguish the content of soluble IL-6. It is found by using the kit for detection that the lymphocytes containing IL-6 protein in the whole blood of healthy people are limited, and the content of soluble IL-6 in the serum of patients with inflammatory and autoimmune diseases is significantly increased and grouped, so as to assist in judging the immune function state of the body.

Claims

1. A monoclonal antibody against human IL-6, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain, 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. Use of the monoclonal antibody of claim 1 in the preparation of a preparation for detecting interleukin 6.

3. Use according to claim 2, wherein the compound is ###0002### The preparation is a flow fluorescence detection kit.

4. A flow-through fluorescent detection kit, characterized by, The flow fluorescence detection kit comprises the monoclonal antibody of claim 1.

5. A method for detecting soluble IL-6 protein for non-therapeutic and diagnostic purposes, characterized by, The method is to detect soluble IL-6 protein by using the monoclonal antibody of claim 1.

6. The method of claim 5, wherein, The method is to detect by using the flow fluorescence detection kit of claim 4.

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