A monoclonal antibody against human interleukin-6 and its application

Abstract

The present invention relates to the field of biological detection, and specifically to an anti-human interleukin 6 antibody and its application. The present application provides an anti-human interleukin 6 monoclonal antibody 2F7, which comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2, as well as a CBA kit comprising the detection antibody 2F7 and a detection method thereof. The antibody 2F7 provided in the present application can realize the qualitative and quantitative detection of human interleukin 6, and has high specificity and detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a monoclonal antibody against human interleukin 6 and its application. Background Art

[0002] Human interleukin-6 (IL-6) is an important proinflammatory cytokine that plays a key regulatory role in inflammation and immune response. Abnormal expression of IL-6 is closely related to a variety of diseases, including rheumatoid arthritis, autoimmune diseases and various cancers. Therefore, accurate determination of IL-6 levels is of great significance for early diagnosis, progression monitoring and efficacy evaluation of the disease. However, the concentration of IL-6 in the blood is usually low, and due to its complex structure, traditional detection methods have certain limitations in sensitivity and specificity.

[0003] Anti-human interleukin-6 monoclonal antibody is an antibody that specifically targets and binds to IL-6, and can recognize and bind to IL-6 molecules with high specificity. The stability and specificity of monoclonal antibodies give them significant advantages in the field of IL-6 detection. At present, detection methods using monoclonal antibodies (such as enzyme-linked immunosorbent assay ELISA and cytokine bioactivity analysis CBA) are widely used in research and clinical applications. Despite this, conventional monoclonal antibody detection methods still face the need to further improve specificity and sensitivity to meet the precise requirements of low-concentration IL-6 detection.

[0004] In order to improve the sensitivity and specificity of the CBA method in IL-6 detection, improving the monoclonal antibody to enhance its recognition ability for IL-6 has become a research direction. Through engineering modification, such as mutation optimization of the variable region (CDR) of the antibody, the affinity and specificity of the antibody can be significantly improved, thereby improving the performance of the CBA detection method. This CBA detection method based on improved monoclonal antibodies can achieve a lower detection limit and higher detection accuracy for IL-6, and reduce nonspecific reactions, providing a more effective tool for the diagnosis and treatment of IL-6-related diseases. Summary of the invention

[0005] In order to solve the above problems, the present invention first provides a highly sensitive and highly specific IL-6 detection method.

[0006] In certain embodiments, the present invention provides an antibody against human interleukin 6 protein, characterized in that the antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region, wherein the heavy chain variable region comprises VH-CDR1 as shown in SEQ ID NO: 4, VH-CDR2 as shown in SEQ ID NO: 5, and VH-CDR3 as shown in SEQ ID NO: 6; the light chain variable region comprises VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10.

[0007] In another preferred embodiment, the antibody heavy chain variable region is shown as SEQ ID NO:3, and the light chain variable region is shown as SEQ ID NO:7.

[0008] In another preferred embodiment, the heavy chain of the antibody is shown as SEQ ID NO:1, and the light chain is shown as SEQ ID NO:2.

[0009] In certain embodiments, the present invention provides a polynucleotide encoding the above-mentioned antibody.

[0010] In certain embodiments, the present invention provides a vector comprising the above-mentioned nucleotides.

[0011] In certain embodiments, the present invention provides a genetically engineered host cell, wherein the host cell contains the above-mentioned vector or the above-mentioned polynucleotide.

[0012] In certain embodiments, the present invention provides a human interleukin-6 protein detection kit, wherein the kit comprises the above-mentioned antibody.

[0013] Finally, the present invention provides an application, which is the application of the above-mentioned antibody in the preparation of a reagent for detecting human interleukin-6 protein.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] The present invention provides for the first time an anti-human interleukin-6 monoclonal antibody 2F7, which comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2;

[0016] The present invention also proposes a CBA kit comprising the detection antibody 2F7 and a detection method thereof. The antibody 2F7 provided by the present invention can be used to achieve qualitative and quantitative detection of human interleukin 6, with high specificity and detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 SDS-PAGE analysis results of human interleukin-6 antigen fragment.

[0018] Figure 2 Ascites titer determination of monoclonal antibody 2F7.

[0019] Figure 3 SDS-PAGE analysis results of the light and heavy chains of monoclonal antibody 2F7.

[0020] Figure 4 Specificity analysis of anti-human interleukin-6 monoclonal antibody 2F7.

[0021] Figure 5 Sensitivity analysis of anti-human interleukin-6 monoclonal antibody 2F7. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 Preparation of human interleukin-6 antigen fragment

[0024] According to the full-length sequence of human interleukin-6 (interleukin-6 [Homo sapiens], GenBank: AAD13886.1) recorded in NCBI, the conserved region was selected to design the human interleukin-6 antigen fragment: FPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSS (SEQ ID NO: 11).

[0025] Primer-BLAST was used to design a primer pair for the human interleukin-6 antigen fragment (IL-6-FR) as shown in SEQ ID NO: 1, and the primer pair also contained NdeI and HindIII restriction endonuclease sites.

[0026] Taq enzyme was used for PCR amplification under the following conditions: initial denaturation: 95°C, 5 min; denaturation: 95°C, 30 sec; annealing: 55°C, 30 sec; extension: 72°C, 1 min; cycle: 30 times final extension; 72°C, 10 min.

[0027] The amplified product and pET-28a(+) vector were double-digested with NdeI and HindIII, and the amplified fragment and the pET-28a(+) vector were connected with DNA ligase to obtain the pET-28a(+)-IL-6-FR recombinant plasmid, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3), and the positive clones were picked for culture. The positive clones were inoculated in LB medium and cultured at 37°C until OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.5mM and continued to be cultured at 37°C for 4-6 hours to improve solubility. The cells were collected by centrifugation at 4°C, 6000rpm, and 10 minutes. The cells were resuspended in lysis buffer (PBS+1% Triton X-100+protease inhibitor), incubated on ice for 30 minutes, and then lysed by ultrasonic treatment. Centrifuged at 12000rpm for 20 minutes, and the supernatant was collected as the soluble protein fraction. The supernatant was purified by a nickel ion affinity column (Ni-NTA), and the affinity column was washed with a washing buffer (PBS + 20 mM imidazole) to remove the unbound recombinant protein IL-6-FR. The bound recombinant protein IL-6-FR was eluted with an elution buffer (PBS + 250 mM imidazole), and the eluate was collected. The purity of the purified product was analyzed by SDS-PAGE, as shown in FIG. Figure 1 shown.

[0028] Example 2 Screening, preparation and identification of anti-human interleukin-6 monoclonal antibodies

[0029] The purified IL-6-FR recombinant protein was used as an antigen, and 7-week-old BALB / c female mice were immunized at a dose of 50 μg / mouse. The corresponding dose of protein was emulsified with an equal volume of Freund's adjuvant. Each immunization was performed 14 days apart, and a total of 4 immunizations were performed. Blood was collected from the tail vein on the 7th day after the 3 immunizations, and the serum titer was detected by indirect ELISA. When the antibody titer in the serum reached 1:12800 or above, the antigen without adjuvant was injected intraperitoneally for booster immunization once 3 days before fusion (double the dose, 100 μg / mouse).

[0030] SP2 / 0 cells were fused with spleen cells of immunized mice to generate hybridoma cells. After 5 days of cell fusion, the growth status of the fused cells was observed. When the cells grew to 1 / 10 of the size of the bottom of the well, the monoclonal antibodies were screened by indirect ELISA. At the same time, the best dilution multiples of positive and negative serum were set as negative and positive controls, and PBS was used as a blank control. The test was performed again after an interval of 2 days, and the 10 wells with the highest positive values ​​in both tests were taken for the next round of subcloning. Finally, a hybridoma cell line that stably secreted anti-IL-6-FR protein was obtained and named 2F7. The ascites of the prepared positive hybridoma cell line 2F7 was tested for antibody titer. The ascites titer was determined by indirect ELISA to be 1:3276800. Figure 2 shown.

[0031] The ELISA method (enzyme-linked immunosorbent assay) was used to determine the subtype of monoclonal antibody 2F7. By using subtype-specific secondary antibodies (such as anti-IgG1, anti-IgG2a, etc.), ELISA was used to detect antibodies of different subtypes. After the antibody in the sample binds to the antigen on the plate, a subtype-specific enzyme-labeled secondary antibody is added, and the subtype is determined by the absorbance value (OD value) after color development, as shown in Table 1.

[0032] Table 1 Identification of subtypes of monoclonal antibody 2F7

[0033]

[0034] The results in Table 1 show that the heavy chain of mAb 2F7 is of IgG3 subtype and the light chain is Lambda chain.

[0035] Identification of amino acid sequence of monoclonal antibody 2F7: Monoclonal antibody 2F7 is purified by protein A / G affinity chromatography, and then the antibody 2F7 is enzymatically hydrolyzed into peptides using trypsin or Lys-C specific protease. The peptide solution is injected into a mass spectrometer, usually using electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) method, and mass-to-charge ratio (m / z) data is obtained by LC-MS / MS scanning, and the fragment ions of the peptides are analyzed. Mascot or Sequest database software is used for peptide sequence alignment and protein identification, and the amino acid sequence of the heavy chain or light chain is matched. The amino acid sequence of the heavy chain or light chain is compared with the IMGT antibody database, and the FR and CDR regions of VH and VL are identified, and their CDR sequences are defined according to the Kabat rules.

[0036] Table 2 Amino acid sequence identification of monoclonal antibody 2F7

[0037]

[0038]

[0039] SDS-PAGE technology was used to separate and identify the heavy and light chains of monoclonal antibody 2F7: Take an appropriate amount of monoclonal antibody 2F7 sample 10μg, add 4X SDS sample buffer, add β-mercaptoethanol to the buffer to ensure that the antibody is fully reduced and the heavy chain and light chain are separated. Heat the sample at 95℃ for 5-10 minutes to ensure that the protein is completely denatured, prepare 10%-12% separation gel to separate the heavy chain and light chain, and pour 4%-5% concentration gel on it. After the gel is polymerized, inject electrophoresis buffer above the gel to prevent the formation of bubbles, and load the monoclonal antibody 2F7 and molecular weight standards into the gel wells, 10μL per well. Run the concentration gel part under 80V conditions. After the sample reaches the separation gel, adjust the voltage to 120V and continue running for 1.5-2 hours. After the electrophoresis is completed, place the gel in Coomassie Brilliant Blue staining solution and stain at room temperature for 30-60 minutes. Decolorize with decolorizing solution until the protein bands are clearly visible and the background becomes lighter. Compare the position and size of the bands by molecular weight standards, see Figure 3 .

[0040] Figure 3 The results showed that the light chain of monoclonal antibody 2F7 was 23.61 kDa, and the heavy chain was 48.62 kDa. The band size was similar to the molecular weight, which was in line with expectations.

[0041] Example 3 Specificity Identification of Anti-Human Interleukin-6 Monoclonal Antibody 2F7

[0042] The purified human IL-6 antigen and other non-target proteins (IL-1, IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-24, IL-25, IL-31) were added to 96-well plates, and 100 ng / mL of the same concentration of antigen protein was added to each well, and coated overnight at 4°C. The well plate was blocked with 5% BSA solution, incubated at room temperature for 1 hour and then washed. Anti-human IL-6 monoclonal antibody 2F7 (1 μg / mL) was added, incubated at 37°C for 1 hour, and washed again to remove unbound antibodies. HRP-labeled anti-mouse IgG secondary antibody was added, incubated at room temperature for 1 hour and then washed. The color developer (TMB) was added, incubated until the color development was complete, the stop solution was added and the OD value was read at 450nm, such as Figure 4 .

[0043] Figure 4The results showed that the binding of antibody 2F7 presented a significant signal response with a high OD value, indicating that 2F7 could specifically recognize the IL-6 antigen; in the non-target protein wells, the OD value was close to the background level, indicating that there was no obvious binding between antibody 2F7 and other cytokines, indicating that antibody 2F7 has a high specificity for IL-6.

[0044] Example 4: Sensitivity analysis of monoclonal antibody 2F7 to human IL-6

[0045] Resuspend the capture beads in assay buffer according to the CBA kit instructions. Mix the capture beads thoroughly before each experiment. Use the IL-6 standard and dilute it step by step according to the dilution ratio recommended by the kit to generate the different concentrations required for the standard curve (5000pg / mL, 2000pg / mL, 850pg / mL, 40pg / mL, 8pg / mL, 1.6pg / mL, and 0pg / mL). Add approximately 50μL of IL-6 capture beads to each flow tube, including the standard and blank control tubes. Add 50μL of the standard dilution to each flow tube to ensure that each sample is operated independently. Add 50μL of the fluorescently labeled IL-6 detection antibody 2F7 to each tube and mix gently. Incubate the samples at room temperature for 2 hours in the dark or at 4°C overnight to ensure that the capture beads are fully bound to IL-6 and the detection antibody 2F7. After the incubation, wash each flow tube once with 500 μL of analysis buffer (centrifuge at 300 × g for 5 minutes), carefully remove the supernatant, and resuspend the captured beads in 300 μL of analysis buffer. According to the instructions of the CBA kit and the channels equipped with the flow cytometer, adjust the appropriate detection parameters, detect the standards one by one, and collect at least 2000 capture bead events to ensure data reliability. Use the flow cytometer software for data analysis, generate a standard curve and calculate the linear range detection data of the human IL-6 standard, see Figure 5 .

[0046] Figure 5 The results showed that the detection curve of human IL-6 could be detected in the concentration range of 1.6 to 5000 pg / mL.

[0047] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibody against human interleukin-6 protein, characterized in that The antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region, wherein the heavy chain variable region comprises VH-CDR1 as shown in SEQ ID NO: 4, VH-CDR2 as shown in SEQ ID NO: 5, and VH-CDR3 as shown in SEQ ID NO: 6; the light chain variable region comprises VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO:

10.

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

7.

3. The antibody according to claim 1, characterized in that The heavy chain of the antibody is shown in SEQ ID NO:1, and the light chain is shown in SEQ ID NO:

2.

4. A polynucleotide encoding the antibody according to any one of claims 1 to 3. A vector comprising the polynucleotide according to claim 4.

6. A genetically engineered host cell, comprising the vector of claim 5, or an exogenous polynucleotide of claim 4 integrated into its genome.

7. A human interleukin-6 protein detection kit, comprising the antibody according to any one of claims 1 to 3.

8. Use of the antibody according to any one of claims 1 to 3 in the preparation of a reagent for detecting human interleukin-6 protein.

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