Antibody for recognizing interleukin-8 and its application

Abstract

The present invention relates to the field of antibodies. Specifically, the present invention provides an antibody that recognizes interleukin-8 and its application. This application obtains a monoclonal antibody against human interleukin-8 through screening, and based on this antibody, develops a quantitative detection kit for human interleukin-8. This kit can achieve quantitative detection of human interleukin-8 with high specificity and sensitivity, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular, to an antibody that recognizes interleukin-8 and applications thereof. Background Art

[0002] Interleukin-8 (IL-8), also known as the chemokine CXCL8, is a cytokine secreted by macrophages and epithelial cells. IL-8 binds to the chemokine receptors interleukin-8 receptor α (IL8RA, also known as CXCR1) and interleukin-8 receptor β (IL8RB, also known as CXCR2), acting as a chemoattractant for neutrophils and thereby regulating inflammatory responses. IL-8 also has a potent pro-angiogenic effect. IL-8 plays a key role in the pathogenesis of bronchiolitis and cystic fibrosis. A cytokine belonging to the chemokine family, IL-8 has been shown in recent studies to be involved in virtually all mammalian reproductive processes, including ovulation, corpus luteum formation, endometrial proliferation, decidualization, embryo implantation, and growth and development. Numerous clinical studies have also demonstrated that IL-8 is associated with certain reproductive pathologies, such as endometriosis and unexplained spontaneous abortion. The mechanism by which IL-8 participates in and regulates the physiological and pathological processes of human reproduction has attracted much attention from researchers at home and abroad. A more certain view is that IL-8 exerts its effect by binding to its specific receptor, triggering a series of biological events.

[0003] IL-8 is primarily produced by monocytes and macrophages; other cells, such as fibroblasts, epithelial cells, endothelial cells, and hepatocytes, can also produce IL-8 under appropriate stimulation. IL-8 has a molecular weight of approximately 8 kDa, and its primary active form consists of 72 amino acids. The amino acid sequence of IL-8 shares high homology with many inflammatory factors, indicating that it belongs to the same family. Preliminary confirmation suggests that the IL-8 family (also known as the PF4 family) contains at least 12 members. IL-8 can be divided into α and β subgroups, with the α subgroup gene located on chromosome 4 and the β subgroup gene on chromosome 17. There are two types of IL-8 receptors: one that binds exclusively to IL-8 and the other that can also bind to other chemokines. Neutrophils and basophils both express abundant IL-8 receptors on their surfaces.

[0004] Existing interleukin-8 quantitative detection kits on the market generally have low sensitivity and a narrow linear range. Therefore, there is an urgent need to develop highly sensitive antibodies that can meet detection needs. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the present invention utilizes hybridoma technology to screen for an antibody that recognizes interleukin-8 with high sensitivity and a wide linear range. This antibody has the characteristics of high affinity and strong specificity, and is used to prepare a chemiluminescence kit that can be used for clinical auxiliary diagnosis, risk assessment, and prognosis of various inflammatory diseases. To achieve the above technical effects, the present invention provides the following technical solutions:

[0006] In its first aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises CDRs selected from at least one of the following: light chain variable region CDR sequences: SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and heavy chain variable region CDR sequences: SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. The antibody or antigen-binding fragment according to an embodiment of the present invention has binding affinity for interleukin-8 and can be used in the preparation of an interleukin-8 antibody luminescent detection kit.

[0007] In a second aspect of the present invention, a recombinant protein is provided. According to an embodiment of the present invention, the recombinant protein comprises the antibody or antigen-binding fragment described in the first aspect of the present invention. The recombinant protein according to an embodiment of the present invention can be used to specifically detect interleukin-8.

[0008] In a third aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect of the present invention, or the recombinant protein described in the second aspect of the present invention. According to an embodiment of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can be used to specifically detect interleukin-8.

[0009] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect of the present invention. According to an embodiment of the present invention, the expression vector can efficiently express the antibody or antigen-binding fragment thereof in a suitable host cell for specific detection of interleukin-8 or preparation of a drug related to interleukin-8 diseases.

[0010] In its fifth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: a nucleic acid molecule according to the third aspect of the present invention, or an expression vector according to the fourth aspect of the present invention; or expresses the antibody or antigen-binding fragment according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention. According to an embodiment of the present invention, the recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under appropriate conditions, and the antibody or antigen-binding fragment can specifically bind to interleukin-8.

[0011] In a sixth aspect, the present invention provides a method for producing antibodies or antigen fragments that recognize interleukin-8. According to an embodiment of the present invention, the method comprises culturing the recombinant cell described in the fifth aspect of the present invention. The method according to an embodiment of the present invention can produce large quantities of antibodies or antigen fragments that bind to interleukin-8 with high specificity.

[0012] In a seventh aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: the antibody or antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, and the recombinant cell described in the fifth aspect of the present invention. Compositions according to embodiments of the present invention, including the aforementioned antibodies or antigen-binding fragments thereof, such as compositions, food compositions, and pharmaceutical compositions of the aforementioned antibodies or antigen-binding fragments and a buffer, can also bind to interleukin-8 with high specificity.

[0013] In an eighth aspect, the present invention provides use of the antibody or antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression vector of the fourth aspect of the present invention, the recombinant cell of the fifth aspect of the present invention, or the composition of the seventh aspect of the present invention in preparing a kit. According to an embodiment of the present invention, the kit is used to detect interleukin-8.

[0014] In the ninth aspect of the present invention, a kit is proposed. According to an embodiment of the present invention, the kit comprises: the antibody or antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant cell described in the fifth aspect of the present invention, and at least one of the compositions described in the seventh aspect of the present invention. According to an embodiment of the present invention, the aforementioned antibody or antigen-binding fragment thereof can bind to interleukin 8 with high specificity. Therefore, the kit comprising the aforementioned antibody or antigen-binding fragment thereof can be used for high-specificity and high-sensitivity detection of interleukin 8. In some scientific studies, the kit of the embodiment of the present invention can be used for qualitative or quantitative detection of interleukin 8 protein in biological samples, and can also be used to judge the status of an individual, such as after obtaining the interleukin 8 level of the individual, judging whether the interleukin 8 level is higher or lower than the normal level.

[0015] In a tenth aspect of the present invention, the present invention proposes use of the kit described in the ninth aspect of the present invention in preparing an interleukin-8 detection product.

[0016] In the eleventh aspect of the present invention, a method for detecting interleukin-8 is proposed. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant cell described in the fifth aspect of the present invention, or the kit described in the ninth aspect of the present invention to perform detection and treatment on the sample to be tested. The method according to the embodiment of the present invention can be used for high-sensitivity detection of interleukin-8, and has strong specificity and simple operation. In practical clinical applications, large quantities of samples can be detected quickly and with high throughput using a chemiluminescence instrument.

[0017] The twelfth aspect of the present invention is the use of the antibody or antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the recombinant cell described in the fifth aspect of the present invention, and the composition described in the seventh aspect of the present invention in the preparation of a medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 This is the result of chemiluminescence immunoassay using magnetic microspheres to separate interleukin-8. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0021] The amino acid sequences involved in this embodiment are shown in Table 1:

[0022] Table 1

[0023]

[0024]

[0025] Example 1: Synthesis of full-length interleukin-8 antigen

[0026] The full-length interleukin-8 antigen (amino acid sequence shown in SEQ ID NO: 11) produced by Beijing Sesvid Biotechnology Co., Ltd. was used.

[0027] Example 2: Immunization of BALB / c mice with interleukin-8 protein

[0028] In this example, BALB / c mice were immunized with the interleukin-8 protein synthesized in Example 1, and the antibody serum titer of the mice was detected. The specific steps are as follows:

[0029] BALB / c mice aged 6 to 8 weeks were immunized as follows: For the primary immunization, 25 μg of interleukin-8 was emulsified in an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Fourteen days after the primary immunization, a booster immunization was performed with 12.5 μg of interleukin-8 emulsified in Freund's incomplete adjuvant. Fourteen days after the secondary immunization, a booster immunization was performed with 12.5 μg of interleukin-8 emulsified in Freund's incomplete adjuvant. Fourteen days after the third immunization, blood was collected and serum was separated. Serum titers were determined by indirect ELISA using an ELISA plate coated with 1 μg / mL interleukin-8. The titer of the prepared mouse antiserum was 1:72,900. Typical results of serum testing against immunized mice are shown in Table 2.

[0030] Table 2

[0031]

[0032] Example 3: Cell fusion

[0033] In Example 2, cell fusion was performed 3 days after the mice were boosted with immunization. After removing the eyeballs of the mice and collecting blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied to find the spleen, which was removed with tweezers and gently ground into a 200-mesh stainless steel filter membrane. The cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and set aside. When preparing feeder cells, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied, and PBS was drawn with a syringe and gently injected into the peritoneum. The liquid containing the feeder cells was washed out from the other side, and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and set aside. 2.0×10 7 FO myeloma cells and 2.0×10 8The spleen cells were mixed, centrifuged at 200g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 mL of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, followed by 20 mL of DMEM medium. The mixture was centrifuged at 200g for 10 minutes, the supernatant was discarded, and the washing was repeated. The mixture was centrifuged at 200g for 10 minutes, the supernatant was discarded, and the hybridoma cells were obtained. The cells were plated into 10 96-well culture plates with 150 μL per well. Add 10,000 feeder cells / well to the 10 96-well cell culture plates described above, adding 100 μL per well. Label the plates and culture in a 37°C incubator with 5% CO₂. Add HAT selection medium (Merk, H0262) the next day. Within 1-2 days of HAT selection, a significant number of tumor cells will die. After 3-4 days, the tumor cells disappear, and the hybrid cells form small colonies. After 7-10 days in HAT selection medium, switch to HT medium (Merk, H0137) and continue culturing for an additional 2 weeks. Then, switch to DMEM medium supplemented with 20% FBS (ExCell, FSP500) to continue culturing. During this selection period, when hybridoma cells cover 1 / 10 of the well bottom, testing for specific antibodies can be initiated to identify the desired hybridoma cell line. During the selection period, half of the medium should be changed every 2-3 days.

[0034] Example 4: Screening and subcloning of positive hybridoma cell lines

[0035] First, an orthogonal experiment was used to determine the optimal coating amount of interleukin-8 as an antigen. A 96-well plate was coated with 0.5, 1.0, 2.0, and 4.0 μg of interleukin-8, with 6 wells for each concentration set as 3 positive and 3 negative. Founder titration was performed using positive serum from mice immunized with interleukin-8 at different dilutions, while negative serum from unimmunized mice was used as a negative control. A 96-well ELISA plate was coated with 0.5 μg of purified interleukin-8 per well and incubated at 4°C overnight. The plate was washed twice with PBST. 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then patted dry on trifold paper. Sample addition: 0.1 mL of the sample to be tested was added to the reaction well, incubated at 37°C for 1 hour, and then washed. Blank wells (no sample added), negative control wells, and positive control wells were also prepared. 0.1 mL of freshly diluted antibody was added to each reaction well, incubated at 37°C for 1 hour, and then washed three times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, then wash three times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1 M H₂SO₄ to each reaction well. Measure the OD value to determine the result. Use a microplate reader to measure the absorbance (A₄50) at 450 nm. A value 2.1 times or greater of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select hybridoma cell monoclonal clones expressing anti-IL-8 antibodies.

[0036] According to the above method, the positive hybridoma cells obtained by screening were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution and then redistributed into 96-well culture plates. The morphology and number of cells were then observed. The cells were adjusted to 3-10 cells / mL. Take the cell culture plate with the feeder cell layer prepared the day before and add 100 μL of diluted cells to each well. Culture statically in a 37°C, 5% CO2 incubator. Change the medium on the 7th day and every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity can be detected in time. The cells in the positive wells were transferred to 24-well plates for expansion culture. Each clone should be frozen as soon as possible, and the hybridoma cell line with clone number SS002 was finally selected for antibody production.

[0037] Example 5: Mass preparation of monoclonal antibodies and determination of antibody titer

[0038] (1) Large-scale preparation of monoclonal antibodies

[0039] 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1×10 6SS002 hybridoma cells should produce ascites 7–10 days after inoculation. Closely monitor the animal's health and signs of ascites. Once ascites is as abundant as possible and before the mouse dies, sacrifice the mouse and aspirate the ascites into a test tube using a dropper. 5–10 mL of ascites should be obtained from each mouse. Alternatively, ascites can be extracted using a syringe, which can be collected repeatedly. The ascites should be centrifuged at 3000 g for 10 minutes. Discard the supernatant and store in aliquots at -20°C. Thaw the supernatant and equilibrate to room temperature. Add 1 / 10 volume of 1M Tris-HCl, pH 8.0, to adjust the sample pH to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100mM Tris-HCl at pH 8.0, and the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column. The antibody was then washed with 20 column volumes of 100mM Tris-HCl at pH 8.0, and finally eluted with 100mM Glycine-HCl at pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged in a centrifuge (Xiangyi, L550) at room temperature for 20 minutes at 3000 × g. The solution was centrifuged in batches until the volume reached 1 mL / concentrator tube (2 tubes), 4 mL of 10mM PBS pH 7.4 buffer was added, and the solution was centrifuged at 3000 × g for 20 minutes at room temperature. The centrifugation was repeated 3 times to make the antibody buffer 10mM PBS pH 7.4, and 10mM PBS pH 7.4 was added to a total volume of 10 mL. Finally, 2 mL / tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. Antibody concentration was determined using a BCA kit (Solarbio, PC0020), and the concentration of the purified monoclonal antibody was 2.4 mg / mL.

[0040] (2) Antibody titer determination

[0041] The titer of the interleukin-8 antibody SS002 was determined using an indirect ELISA. Interleukin-8 was diluted in PBS and coated at 0.2 μg / mL at 100 μL / well in a 96-well microtiter plate. After overnight at 4°C, the plate was washed twice with PBST (300 μL / well per wash) and patted dry. The plate was blocked with 1% BSA in PBS (200 μL / well) for 2 hours at room temperature and patted dry. Interleukin-8 antibody SS002 diluted to 20 ng / mL in PTB was added and incubated at 37°C for 1 hour. The plate was washed twice with PBST (300 μL / well per wash) and patted dry. HRP-conjugated goat anti-mouse antibody diluted 5000-fold in PTB was added and incubated at 37°C for 1 hour. The plate was washed twice with PBST (300 μL / well per wash) and patted dry. TMB substrate solution was added, 100 μL / well, and incubated at room temperature for 10 minutes. The reaction was terminated by adding 100 μL / well of 1 M H₂SO₄. The absorbance (A₄50) was measured at 450 nm using a microplate reader. The results are shown in Table 3, indicating a titer of 1:800,000 for the purified antibody.

[0042] Table 3

[0043]

[0044] Example 6: Sequence analysis of monoclonal antibodies

[0045] (1) Monoclonal antibody subtype identification

[0046] Hybridoma cell line SS002 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO2) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube and counted with a hemocytometer. 4 × 10 6 The cells were centrifuged at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN Company.

[0047] Antibody subtypes were determined by PCR using subtype-specific primers. The synthesized cDNA described above was used as a template for the PCR reaction. The PCR reaction solution system included: 0.25 μL of TAKARA Ex Taq (5 U / μL, TAKARA, RR001B); 5 μL of 10× Ex Taq Buffer; 4 μL of dNTP mix (2.5 mM each); 1 μL of template cDNA; 1 μL of upstream primer (100 μM); 1 μL of downstream primer (100 μM); and double-distilled water to a total volume of 50 μL. The PCR reaction temperature program was: 94°C for 5 minutes for initial denaturation, 30 cycles of temperature cycling (94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute), and 10 minutes for extension at 72°C. After the reaction, 10 μL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. The order of samples added to each lane is shown in Table 5. The subtype of the SS002 antibody can be inferred from the PCR product results (Table 4). The results show that the monoclonal antibody SS002 obtained in the present invention has an IgG1 heavy chain and a kappa light chain.

[0048] Table 4 PCR primer information

[0049]

[0050] where S = C or G, M = A or C, R = A or G, and W = A or T.

[0051] Table 5: Antibody information

[0052]

[0053] (2) Sequencing of the variable region (V region) of the hybridoma cell line SS002

[0054] The fragments obtained after PCR amplification of the V region of the antibody of cell line SS002 (see above) were cut from an agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragments were linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB medium and cultured overnight before DNA sequencing. The nucleic acid sequence of the light chain V region of the antibody (SS002) that recognizes interleukin-8 provided by the present invention is shown in SEQ ID NO:9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO:10.

[0055] Example 7: Application of the ELISA kit prepared using antibody SS002 in the detection of interleukin-8 in human serum

[0056] (1) Horseradish peroxidase (HRP) labeling of antibody SS002 and identification of the labeled product

[0057] 0.5 mL of antibody SS002 (20 nmol, 3 mg) was added to a dialysis bag (10 kDa, 1 cm width) and dialyzed against 2 L of 10 mM PBS (pH 7.4) at 4°C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyzed at room temperature for 2 hours with stirring in preparation for conjugation with activated HRP.

[0058] At the same time, use an analytical balance to accurately weigh 1 mg of HRP, add 0.2 mL of ultrapure water to dissolve it, and make the HRP concentration be 5 mg / mL. Add 40 μL of 0.1 M NaIO4 to the above HRP solution, place it on a horizontal shaker, and react (activate) at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10 KDa, width 1 cm) and dialyze it in 2 L of 1 mM sodium acetate buffer (pH 4.4) at 4 ° C overnight. Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube, add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0-9.5.

[0059] Mix the above antibodies and HRP (for coupling reaction), place on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is complete, add 10 μL of freshly prepared NaBH4 (pre-chilled ultrapure water) and incubate at 4°C in the dark overnight to terminate the reaction. Transfer the antibody solution after the coupling reaction to a dialysis bag (10 kDa, 1 cm width) and dialyze at room temperature for 2 hours with stirring. Finally, transfer the solution to a brown centrifuge tube and store at 4°C.

[0060] (2) Detection of interleukin-8 in human serum using an enzyme-linked immunosorbent assay kit prepared using antibody SS002

[0061] In this experiment, a commercial interleukin-8 antibody (abcam, cat. no. ab289967) was diluted to 1 μg / mL in PBS and coated in a 96-well microtiter plate (overnight at 4°C) at 100 μL / well. The plate was washed twice with 300 μL / well PBST. The plate was patted dry and blocked with 1% BSA in PBS (200 μL / well) for 2 hours at room temperature. After patting dry, a 1000-fold diluted HRP-conjugated antibody SS002 (diluted in PTB) solution was added at 25 μL / well. Then, human serum samples (sera from patients with clinically confirmed inflammatory diseases were used as test samples, and normal serum was used as negative samples; all enrolled patients provided informed consent) at 75 μL / well were added and incubated at 37°C for 45 minutes. The plate was washed three times with 300 μL / well PBST. After patting dry, the plate was added with 100 μL / well TMB substrate solution and incubated at room temperature for 10 minutes. The reaction was then terminated by adding 100 μL / well of 1 M H2SO4, and the absorbance (A450) was measured at 450 nm using a microplate reader. The results are shown in Table 6. The test results for normal subjects were significantly different from those for positive patient samples, indicating that the ELISA kit prepared using the antibody SS002 of the present invention can efficiently detect interleukin-8 in human serum.

[0062] Table 6 Patient serum samples

[0063]

[0064] Example 8: Detection of interleukin-8 based on chemiluminescence

[0065] This example uses the interleukin-8 antibody SS002 prepared in the previous example to design a detection kit. Using a magnetic microsphere separation chemiluminescent immunoassay as an example, the detection principle is a double-antibody sandwich assay, demonstrating the specific steps of the kit for detecting interleukin-8 in a sample.

[0066] Instrument: The company's self-developed chemiluminescence detector;

[0067] Reagents: Labeled alkaline phosphatase (ALP) was purchased from BBI, UK, model: ALPI12G; SMCC was purchased from Thermo Fisher Scientific, item number: 22360; 2-IT was purchased from Thermo Fisher Scientific, CAS: 4781-83-3; item number: 26101; carboxyl-modified microparticles were purchased from Thermo Fisher Scientific; EDC was purchased from SIMGA, CAS: 25952-53-8, item number: E7750; the antibodies were the SS002 antibody prepared in the above example and the commercial interleukin-8 antibody, purchased from abcam, item number ab289967; the luminescent substrate came from the company's self-developed reagent; the remaining reagents were conventional products purchased commercially.

[0068] Experiment 1: Magnetic microspheres labeled with interleukin-8 antibody

[0069] Take 20 mg of carboxyl-modified microparticle solution, and sediment (magnetic separation) the magnetic microspheres with superparamagnetism, uniform particle size, and carboxyl (COOH-) active groups on the surface under the action of a magnetic field for 10 minutes. The supernatant is removed, and the sedimented magnetic microspheres are washed three times with an activation buffer having a molar concentration of 0.05 M (2-(N-morpholino)ethanesulfonic acid) MES, pH 6.0 buffer, each time using 2 mL.

[0070] The washed magnetic microspheres were fully suspended in 1.0 mL of activation buffer (2-(N-morpholinoline)ethanesulfonic acid) MES, pH 6.0, with a molar concentration of 0.05 M. 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was then added, and the suspension was reacted at room temperature for 30 minutes. The EDC reaction molar concentration was 7.5 mM.

[0071] Take 1.0 mg of interleukin-8 antibody SS002 and concentrate it to 2.5 mg / mL.

[0072] Interleukin-8 antibody SS002 concentrated to 2.5 mg / mL was added to 20 mg of the activated magnetic microsphere solution according to 1.0 mg of interleukin-8 coupled carrier protein antigen, and the mixture was gently shaken to mix. The mixture was reacted at 4°C under suspension conditions for 6.5 hours to covalently couple the interleukin-8 antibody to the surface of the magnetic microspheres. This reagent was named Reagent A.

[0073] Experiment 2: Alkaline phosphatase-labeled commercial interleukin-8 antibody

[0074] 1.0 mg of commercial interleukin-8 antibody was concentrated to 2.5 mg / mL. 5 μL of 13.76 mg / mL activator 2-Iminothiolane hydrochloride (2-IT) was added and allowed to react at room temperature for 15 minutes. The activated antibody was collected after desalting using a Sephadex G25 gel column.

[0075] 1.2 mg of alkaline phosphatase was concentrated to 2.5 mg / mL. 12 μL of 6.69 mg / mL activator, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), was added. The mixture was allowed to react at room temperature for 15 minutes. The activated alkaline phosphatase was collected after desalting using a Sephadex G25 column.

[0076] The activated commercial interleukin-8 antibody and alkaline phosphatase were mixed at a ratio of 1.0 mg of commercial interleukin-8 antibody to 1.0 mg of alkaline phosphatase, and the mixture was allowed to react at 4° C. for 18 hours.

[0077] The unconnected commercial interleukin-8 antibody and alkaline phosphatase were removed by separation and purification using a Supperdex200 gel chromatography column. The connection product was stored at 4°C and named as reagent B.

[0078] Experiment 3: Interleukin-8 magnetic microsphere separation and chemiluminescence immunoassay

[0079] (1) Immunoreaction: 50 μL of the calibrator or quality control sample was added to the reaction tubes, along with 50 μL of reagent A prepared in Experiment 1 and 50 μL of reagent B prepared in Experiment 2, and the mixture was incubated at 37°C for 10 min.

[0080] (2) Magnetic separation: allow the magnetic microspheres to settle in a magnetic field, remove the supernatant, add 200-500 μL of washing solution, remove the magnetic field, and then allow the magnetic microspheres to settle in a magnetic field again, and remove the supernatant; repeat this process 2-4 times to remove unbound antibodies and impurities;

[0081] (3) Reading: Add 200 μL of luminescent substrate solution, and after alkaline phosphatase catalyzes the substrate to emit light, use a self-developed chemiluminescence detector to measure the relative luminescence intensity (RLU);

[0082] (4) A four-parameter equation was used to fit the detected values ​​to obtain a standard curve of interleukin-8 concentration-luminescence value.

[0083] (5) Add 50 μL of the sample to be tested, 50 μL of reagent A prepared in Experiment 1, and 50 μL of reagent B prepared in Experiment 2 to the reaction tube in sequence, mix and incubate at 37°C for 15 min;

[0084] (6) Magnetic separation: allow the magnetic microspheres to settle in a magnetic field, remove the supernatant, add 200-500 μL of washing solution, remove the magnetic field, and then allow the magnetic microspheres to settle in a magnetic field again, and remove the supernatant; repeat this 2-4 times to remove unbound antibodies and impurities;

[0085] (7) Reading: Add 200 μL of luminescent substrate solution. After ALP catalyzes the substrate to emit light, the relative luminescence intensity (RLU) is measured using a self-developed chemiluminescence detector. The luminescence intensity of the sample to be tested is compared with the standard curve in step (4). The content of interleukin-8 in the sample to be tested can be calculated using a four-parameter equation fitting.

[0086] (8) Test interleukin-8 protein standard. Interleukin-8 protein was prepared with sample diluent to concentrations of 20,000 pg / mL, 10,000 pg / mL, 5,000 pg / mL, 1,000 pg / mL, and 500 pg / mL, respectively. Test the luminescence value results and use four-parameter fitting. The results are shown in Table 7 and Figure 1 As shown in the figure, the correlation coefficient r can reach 0.9999, and the upper limit of the linear range can reach 20000 pg / mL.

[0087] Table 7

[0088]

[0089] (9) Test the sample dilutions 20 times. The sensitivity calculation method is: calculate the mean value M and standard deviation SD of the measurement results, and substitute the value of the mean value M plus 2 times the standard deviation SD into the standard curve in (8). The calculated result is the sensitivity. The analysis results are shown in Table 8. The sensitivity test result can reach 0.021 pg / mL.

[0090] Table 8

[0091]

[0092] In summary, the monoclonal antibody against interleukin-8 prepared in this application has the characteristics of high affinity and specificity. The interleukin-8 detection kit prepared based on this antibody can be used to rapidly detect the interleukin-8 content in serum or plasma samples, with high sensitivity, wide linear range, simple operation, and low sample pretreatment requirements. By combining it with a fully automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and detection throughput are increased, the detection efficiency is improved, and the errors caused by human operation are avoided.

[0093] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An antibody or antigen-binding fragment thereof against interleukin-8, characterized in that: The light chain and heavy chain of the antibody or antigen-binding fragment thereof include the following CDR sequences: Light chain variable region CDR sequences: SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; Heavy chain variable region CDR sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: The light chain variable region is shown in the amino acid sequence of SEQ ID NO: 1, and the heavy chain variable region is shown in the amino acid sequence of SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that Further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region or the light chain constant region is derived from a mammalian antibody; the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is a full-length monoclonal antibody; or The antigen-binding fragment is selected from at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, and a scFv-Fv fusion protein.

5. A fusion protein, characterized in that include: The antibody or antigen-binding fragment thereof and a protein tag according to any one of claims 1 to 4.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the fusion protein according to claim 5.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleic acid molecule comprises: The nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:9; and / or The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO:

10.

8. An expression vector, characterized in that The expression vector carries the nucleic acid molecule according to claim 6 or 7.

9. A recombinant cell, characterized in that The recombinant cell comprises: The nucleic acid molecule according to claim 6 or 7, or the expression vector according to claim 8.

10. A method for preparing an antibody or an antigen-binding fragment thereof that recognizes interleukin-8, characterized in that: The method comprises: culturing the recombinant cell according to claim 9.

11. A composition, characterized in that The composition comprises: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to claim 8, or the recombinant cell according to claim 9.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to claim 8, the recombinant cell according to claim 9, or the composition according to claim 11 in preparing a kit, characterized in that: The kit is used for detecting interleukin-8.

13. A kit, characterized in that include: At least one of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to claim 8, the recombinant cell according to claim 9, and the composition according to claim 11.

14. The kit according to claim 13, characterized in that The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, magnetic particles, commercial interleukin-8 antibody, and a luminescent substrate; wherein the magnetic particles are coupled to the antibody or antigen-binding fragment thereof; The commercial interleukin-8 antibody has a marker modification; The label is used to catalyze the luminescent substrate to emit light.

15. The kit according to claim 14, characterized in that The kit further comprises a calibrator of interleukin-8 protein and / or a quality control product of interleukin-8 protein.

16. A method for detecting interleukin-8 for non-diagnostic purposes, characterized in that: include: The sample to be tested is detected using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to claim 8, the recombinant cell according to claim 9, or the kit according to any one of claims 13 to 15.

17. The method according to claim 16, characterized in that The detection process refers to detecting the sample to be tested by chemiluminescence, enzyme-linked immunosorbent assay or protein immunoblotting.

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