Antibody for recognizing interleukin 8 and application thereof

Abstract

The invention relates to the field of antibodies, in particular to an antibody for recognizing interleukin 8 and application of the antibody. The anti-human interleukin 8 monoclonal antibody is obtained through screening, the quantitative detection kit for the human interleukin 8 is developed on the basis of the anti-human interleukin 8 monoclonal antibody, quantitative detection of the human interleukin 8 can be achieved, and the monoclonal antibody has high specificity, high sensitivity and wide application prospects.

Description

Technical Field

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

[0002] Interleukin-8 (IL-8), also known as chemokine CXCL8, is a cytokine secreted by macrophages and epithelial cells, etc. Interleukin-8 binds to chemokine receptors interleukin-8 receptor alpha (IL8RA, also called CXCR1) and interleukin-8 receptor beta (IL8RB, also called CXCR2) to have chemotactic effects on neutrophils, thereby regulating the inflammatory response. Interleukin-8 also has a strong angiogenesis-promoting effect. Interleukin-8 plays an important role in the pathogenesis of bronchiolitis and cystic fibrosis. It is a cytokine belonging to the chemokine family. In recent years, it has been found in research that IL-8 is almost involved in the entire reproductive process of mammals, such as ovulation, corpus luteum formation, endometrial proliferation, decidualization, embryo implantation and its growth and development, etc. Many clinical studies have shown that IL-8 is also related to some reproductive pathological processes, such as endometriosis, unexplained spontaneous abortion, etc. How 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 relatively certain view is that IL-8 exerts its function by binding to its specific receptor and triggering a series of biological events.

[0003] IL-8 is mainly produced by monocytes - macrophages; others such as fibroblasts, epithelial cells, endothelial cells, hepatocytes, etc. can also produce IL-8 under appropriate stimulation conditions. The molecular weight of IL-8 is about 8KD, and the main active form is 72 amino acids. The amino acid sequence of IL-8 has a high homology with many inflammatory factors and belongs to the same family; it has been preliminarily confirmed that the IL-8 family (also known as the pf4 family) has at least 12 members. IL-8 can be divided into alpha and beta subgroups. The gene of the alpha subgroup is located on chromosome 4, and the gene of the beta subgroup is located on chromosome 17. There are also two types of IL-8 receptors: one can only bind to IL-8, while the other can also bind to other chemokines; both neutrophils and basophils express abundant IL-8 receptors on their surfaces.

[0004] The sensitivity of existing commercially available quantitative detection kits for interleukin-8 is generally not high, and the linear range is not wide enough. Therefore, there is an urgent need to develop high-sensitivity antibodies that can meet the detection requirements. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention uses hybridoma technology to screen out 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 a chemiluminescence kit is prepared therefrom, which can be used for clinical auxiliary diagnosis, risk assessment, and prognosis judgment of various inflammatory diseases. To achieve the above technical effects, the present invention provides the following technical solutions: In the first aspect of the present invention, 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; 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 the embodiment of the present invention has interleukin-8 binding affinity and can be used for the preparation of an interleukin-8 antibody luminescence detection kit.

[0006] In the second aspect of the present invention, the present invention provides a recombinant protein. 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 the embodiment of the present invention can be used for specifically detecting interleukin-8.

[0007] In the third aspect of the present invention, 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 its antigen-binding fragment encoded by the nucleic acid molecule can be used for specifically detecting interleukin-8.

[0008] In the fourth aspect of the present invention, 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 highly express the antibody or its antigen-binding fragment in a suitable host cell for specifically detecting interleukin-8 or for preparing drugs related to interleukin-8 diseases.

[0009] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: carrying the nucleic acid molecule described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention; or expressing 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 recombinant cell can highly express the above antibody or its antigen-binding fragment under suitable conditions, and the antibody or its antigen-binding fragment can specifically bind to interleukin-8.

[0010] In the sixth aspect of the present invention, a method for preparing an antibody or antigen fragment that recognizes interleukin-8 is proposed. According to an embodiment of the present invention, the method includes: culturing the recombinant cell described in the fifth aspect of the present invention. The method according to the embodiment of the present invention can prepare a large amount of an antibody or antigen fragment that specifically binds to interleukin-8 with high specificity.

[0011] In the seventh aspect of the present invention, a composition is proposed. According to an embodiment of the present invention, the composition includes: 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. The composition according to the embodiment of the present invention, which contains the aforementioned antibody or its antigen-binding fragment, such as a composition of the aforementioned antibody or its antigen-binding fragment and a buffer, a food composition, a pharmaceutical composition, etc., can also specifically bind to interleukin-8 with high specificity.

[0012] In the eighth aspect of the present invention, 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, or the composition described in the seventh aspect of the present invention in the preparation of a kit is proposed. According to an embodiment of the present invention, the kit is used for detecting interleukin-8.

[0013] In the ninth aspect of the present invention, a kit is proposed. According to an embodiment of the present invention, the kit includes at least one 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. According to an embodiment of the present invention, the aforementioned antibody or its antigen-binding fragment can specifically bind to interleukin-8 with high specificity. Therefore, the kit containing the aforementioned antibody or its antigen-binding fragment can be used for highly specific and highly sensitive detection of interleukin-8. In some scientific research, the kit according to the embodiment of the present invention can be used for qualitative or quantitative detection of interleukin-8 protein in a biological sample, and can also be used to judge the individual's status. For example, after obtaining the interleukin-8 level of the individual, it can be judged whether the interleukin-8 level is higher or lower than the normal level.

[0014] In the tenth aspect of the present invention, the use of the kit described in the ninth aspect of the present invention in the preparation of an interleukin-8 detection product is proposed.

[0015] The eleventh aspect of the present invention provides a method for detecting interleukin-8. According to an embodiment of the present invention, the method includes: detecting and processing a sample to be tested by 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. The method according to the embodiment of the present invention can be used for highly sensitive detection of interleukin-8, with strong specificity and simple operation. In clinical practical applications, a large number of samples can be quickly and highly throughput detected by means of a chemiluminescence analyzer.

[0016] The twelfth aspect of the present invention relates to 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 drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, but do not constitute a limitation to the present invention. In the drawings: Figure 1 It is a diagram of the chemiluminescence immunoassay results for the separation of interleukin-8 magnetic microspheres. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The following describes the preferred embodiments of the present invention with reference to the 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.

[0019] The amino acid sequences involved in this embodiment are shown in Table 1: Table 1

[0020]

[0021] Example 1: Synthesis of the full-length antigen of interleukin-8 The full-length antigen of interleukin-8 (the amino acid sequence is as shown in SEQ ID NO: 11) produced by Beijing Saisiweide Biotechnology Co., Ltd. was used.

[0022] Example 2: Immunization of BALB / c mice with interleukin-8 protein In this example, the interleukin-8 protein synthesized in Example 1 was used to immunize BALB / c mice, and the antibody serum titer of the mice was detected. The specific steps are as follows: BALB / c mice aged 6 - 8 weeks were selected for the following immunization protocol: During the primary immunization, 25 μg of interleukin-8 was mixed with an equal amount of Freund's complete adjuvant, emulsified, and then subcutaneously injected at multiple sites. Fourteen days after the first immunization, 12.5 μg of interleukin-8 was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. Fourteen days after the second immunization, 12.5 μg of interleukin-8 was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. Fourteen days after the third immunization, blood was collected and serum was separated. The ELISA plate was coated with 1 μg / mL of interleukin-8 for indirect ELISA assay to determine the serum titer. The results showed that the titer of the prepared mouse anti-serum was 1:72900. The typical serum test results of immunized mice are shown in Table 2.

[0023] Table 2

[0024] Example 3: Cell fusion Three days after boosting the immunized mice in Example 2, cell fusion was performed. After collecting blood by removing the eyeballs of the mice, the mice were sacrificed by cervical dislocation, placed in a 70% alcohol bottle for 2 minutes, then fixed on a foam board in a biosafety cabinet. The abdominal skin was cut open to find the spleen, which was removed with forceps and placed in a 200-mesh stainless steel filter membrane and gently ground. The cells were gently rinsed with DMEM medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. After discarding the supernatant, it was reserved for use. When preparing feeder cells, the mice were sacrificed by cervical dislocation, placed in a 70% alcohol bottle for 2 minutes, then fixed on a foam board in a biosafety cabinet. The abdominal skin was cut open, and PBS was aspirated with a syringe and gently injected subcutaneously into the peritoneum. The liquid containing feeder cells was washed out from the other side, and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. After discarding the supernatant, it was reserved for use. 2.0×10 7 cells of FO myeloma cells were mixed with 2.0×10 8Mix the spleen cells, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant, gently shake to mix, and within 90 seconds at 37 °C in a water bath, add 1 mL of an aqueous solution of PEG-1450 (Merk, P1458) with a volume concentration of 50%, then add 20 mL of DMEM medium, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant, repeat the washing once, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant to obtain hybridoma cells, and seed the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder layer cells per well to the above 10 96-well cell culture plates, 100 μL per well. After labeling the culture plates, place them in a cell culture incubator at 37 °C containing 5% CO2 for culture. On the second day, add HAT screening medium (Merk, H0262), and then use HAT selection culture. Within 1 - 2 days, a large number of tumor cells will die, and after 3 - 4 days, the tumor cells will disappear, and hybrid cells will form small colonies. After maintaining the HAT screening culture medium for 7 - 10 days, it should be replaced with HT medium (Merk, H0137), and then maintained for another 2 weeks, and then switched to DMEM medium containing 20% FBS (ExCell, FSP500) for continued culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom area of the well, the specific antibody can be detected to screen out the required hybridoma cell line. During the selection culture period, generally, half of the culture medium is changed every 2 - 3 days.

[0025] Example 4: Screening and subcloning culture of positive hybridoma cell lines First, determine the optimal coating amount of interleukin-8 as an antigen through orthogonal experiments. Coat a 96-well plate with 0.5, 1.0, 2.0, and 4.0 μg of interleukin-8, with 6 wells for each concentration, and set 3 wells as positive and 3 wells as negative respectively. Perform checkerboard titration with positive serum from mice immunized with interleukin-8 at different dilution multiples, and use negative serum from non-immunized mice as a negative control. Coat a 96-well ELISA plate with 0.5 μg of purified interleukin-8 per well and incubate overnight at 4°C; wash twice with PBST; add 200 μL of 1% BSA in PBS to each well, block at room temperature for 2 hours, and then pat dry on filter paper; add samples: add 0.1 mL of the sample to be tested to the reaction wells, incubate at 37°C for 1 hour, then wash, and at the same time, set up blank wells (without adding samples), negative control wells, and positive control wells. Add 0.1 mL of the newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: add 0.1 mL of the newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: add 0.1 mL of TMB substrate solution to each reaction well and let it stand at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure the OD value to judge the result, and use an enzyme-linked immunosorbent assay reader to measure the absorbance value (A450) at 450 nm. A value more than 2.1 times the OD value of the negative control is considered positive (calculated after zeroing with the blank control well). Select monoclonal hybridoma cells against interleukin-8.

[0026] According to the above method, perform subcloning on the selected positive hybridoma cells. Dilute the original wells with HAT selection medium by the limiting dilution method and then re-distribute them into a 96-well culture plate, and then observe the cell morphology and quantity. Adjust the cells to 3 - 10 cells / mL. Take the cell culture plate of the feeder cell layer prepared the previous day, and add 100 μL of the diluted cells to each well. Incubate statically in a 37°C, 5% CO2 incubator. Change the medium on the 7th day, and then change the medium once every 2 - 3 days. Cell clones can be seen on the 8th - 9th day, and detect the antibody activity in a timely manner. Transfer the cells in the positive wells to a 24-well plate for expansion culture. Each clone should be cryopreserved as soon as possible, and finally select the hybridoma cell line with the clone number SS002 for antibody production.

[0027] Example 5: Large-scale preparation of monoclonal antibody and determination of antibody titer (1) Large-scale preparation of monoclonal antibody Inject 0.5 mL of Freund's incomplete adjuvant intraperitoneally into 8-week-old BALB / C mice. Two weeks later, inject 1×10 6For SS002 hybridoma cells, ascites can be produced 7 to 10 days after inoculating the cells. Closely observe the health status of the animals and ascites signs. Before the ascites is as much as possible and the mice are on the verge of death, sacrifice the mice. Use a dropper to suck the ascites into a test tube. One mouse can obtain 5 to 10 mL of ascites. Ascites can also be extracted with a syringe and collected repeatedly several times. Centrifuge the obtained ascites at 3000g for 10 minutes, discard the upper layer of grease and the bottom precipitate, and collect the supernatant. Aliquot the supernatant and store it at -20°C. After thawing and equilibrating the ascites supernatant to room temperature, add 1 / 10 volume of 1M Tris-HCl pH 8.0 to adjust the pH of the sample to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100 mM pH 8.0 Tris-HCl. Load the ascites supernatant with adjusted pH to 8.0 onto the column, then wash it with 20 column volumes of 100 mM pH 8.0 Tris-HCl, and finally elute the antibody with 100 mM Glycine-HCL pH 2.5. Add the antibody eluate to a concentrator tube (Millipore, UFC801008, 10K) and centrifuge it at 3000×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifuge in batches until the solution volume reaches 1 mL / concentrator tube (2 tubes), add 4 mL of 10 mM PBS pH 7.4 buffer, and continue to centrifuge at 3000×g for 20 minutes at room temperature. Repeat centrifugation 3 times to make the buffer of the antibody 10 mM PBS pH 7.4, and add 10 mM PBS pH 7.4 to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution at 2 mL / tube into centrifuge tubes and store it at -80°C. Use a BCA kit (Solarbio, PC0020) to measure the antibody concentration, and the concentration of the purified monoclonal antibody is measured to be 2.4 mg / mL.

[0028] (2) Antibody titer determination The titer of interleukin-8 antibody SS002 was detected by the indirect ELISA method. Interleukin-8 was diluted with PBS and coated on a 96-well ELISA plate at 0.2 μg / mL, 100 μL / well. After overnight incubation at 4 °C, the ELISA plate was washed twice with PBST solution at 300 μL / well each time, and patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA at 200 μL / well for 2 hours at room temperature and patted dry after blocking; interleukin-8 antibody SS002 diluted to 20 ng / mL with PTB was added and reacted in an incubator at 37 °C for 1 hour. The ELISA plate was washed twice with PBST solution at 300 μL / well each time and patted dry after washing. Goat anti-mouse antibody labeled with HRP diluted 5000 times with PTB was added and reacted in an incubator at 37 °C for 1 hour. The ELISA plate was washed twice with PBST solution at 300 μL / well each time and patted dry after washing. TMB substrate solution was added at 100 μL / well and reacted at room temperature for 10 minutes, and 100 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 3, and it can be seen that the titer of the purified antibody is 1:800000.

[0029] Table 3

[0030] Example 6: Sequence analysis of monoclonal antibody (1) Identification of monoclonal antibody subtype The hybridoma cell line SS002 was cultured in a 10-cm diameter cell culture dish (37 °C, 5% CO2) with DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15-mL centrifuge tube. After counting with a hemocytometer, 4×10 6 cells were taken out, 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 used to synthesize cDNA using the reverse transcription kit (Qiagen, 74134) from QIAGEN.

[0031] Antibody subtypes were determined by performing PCR with subtype-specific primers for antibodies. The synthesized cDNA described above was used as the template for the PCR reaction. PCR reaction solution system: TAKARA Ex Taq (5 U / μL, TAKARA, RR001B), 0.25 μL; 10× Ex Taq Buffer, 5 μL; dNTP mixture (each 2.5 mM), 4 μL; template cDNA, 1 μL; upstream primer (100 μM), 1 μL; downstream primer (100 μM), 1 μL; double-distilled water was added to a total volume of 50 μL. PCR reaction temperature program: pre-denaturation at 94 °C for 5 minutes, 30 temperature cycles (94 °C for 1 minute, 57 °C for 1 minute, 72 °C for 1 minute), extension at 72 °C for 10 minutes. After the reaction, 10 μL of each PCR product was loaded onto a 1% agarose gel for electrophoresis, and the order of samples added to each lane is shown in Table 5. The subtype of the SS002 antibody can be inferred from the results of the PCR products (Table 4). The results show that the monoclonal antibody SS002 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0032] Table 4 PCR primer information

[0033] Where S = C or G, M = A or C, R = A or G, and W = A or T.

[0034] Table 5: Antibody information

[0035] (2) Sequencing of the variable region (V region) of the antibody of the hybridoma cell line SS002 The fragment obtained after PCR amplification of the V region of the antibody of cell line SS002 (see the above item) was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). Transformed bacterial colonies were picked into LB medium and cultured overnight for 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.

[0036] Example 7: Application of the enzyme immunoassay kit prepared with antibody SS002 in detecting interleukin 8 in human serum (1) Horseradish peroxidase (HRP) labeling of antibody SS002 and identification of the labeled product Take 0.5 mL of antibody SS002 (20 nmol, 3 mg) and add it to a dialysis bag (10 KDa, width 1 cm), and dialyze overnight at 4 °C in 2 L of 10 mM PBS solution (pH 7.4). The next day, place the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze with stirring at room temperature for 2 hours to prepare for coupling with the activated HRP.

[0037] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance, add 0.2 mL of ultrapure water to dissolve it, so that the concentration of HRP is 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 overnight at 4 °C in 2 L of 1 mM sodium acetate buffer (pH 4.4). Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube, and add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0 - 9.5.

[0038] Mix the above antibody and HRP (for the coupling reaction), place it on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is completed, add 10 μL of freshly prepared NaBH4 (using pre-cooled ultrapure water), and terminate the reaction at 4 °C in the dark overnight. Transfer the antibody solution after the coupling reaction is terminated to a dialysis bag (10 KDa, width 1 cm), and dialyze with stirring at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store it at 4 °C.

[0039] (2) Detect interleukin-8 in human serum using the enzyme immunoassay kit prepared with antibody SS002 In this experiment, a commercial interleukin-8 antibody (abcam, catalog number: ab289967) was diluted to 1 μg / mL with PBS and then coated in a 96-well ELISA plate at 100 μL / well (overnight at 4°C). The plate was washed twice with 300 μL / well of PBST. After patting dry, the ELISA plate was blocked with a PBS solution containing 1% BSA at 200 μL / well for 2 hours at room temperature. After patting dry, 25 μL / well of a 1000-fold diluted solution of HRP-labeled antibody SS002 (diluted with PTB) was added to the ELISA plate, and then different concentrations of human serum samples were added (serum from patients clinically diagnosed with inflammation-related diseases was selected as the test sample, and normal human serum was used as the negative sample. All enrolled patients signed informed consent), 75 μL / well, and the reaction was carried out in an incubator at 37°C for 45 minutes. The plate was washed 3 times with PBST, 300 μL / well each time. After patting dry, 100 μL / well of TMB substrate solution was added and the reaction was carried out at room temperature for 10 minutes. Then 100 μL / well of 1 M H2SO4 was added to terminate the reaction, and the absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 6. The test results of normal people were significantly different from those of 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.

[0040] Table 6 Patient serum samples

[0041] Example 8: Detection of interleukin-8 based on chemiluminescence method In this example, the interleukin-8 antibody SS002 prepared in the previous example was used to design a detection kit. Taking the magnetic microsphere separation chemiluminescence immunoassay method as an example, the detection principle is a double antibody sandwich, and the specific steps for detecting the content of interleukin-8 in the test sample using this kit are shown.

[0042] Instrument: A self-developed chemiluminescence detector of the company; Reagents: Alkaline phosphatase (ALP) labeled was purchased from BBI Company in the UK, model: ALPI12G; SMCC was purchased from thermofisher scientific company, catalog number: 22360; 2-IT was purchased from thermo fisher scientific company, CAS: 4781-83-3, catalog number: 26101; carboxyl-modified microparticles were purchased from thermo fisher scientific company; EDC was purchased from SIMGA company, CAS: 25952-53-8, catalog number: E7750; the antibodies were the SS002 antibody prepared in the above example and a commercial interleukin-8 antibody, purchased from abcam, catalog number ab289967; the chemiluminescent substrate was from a self-developed reagent of the company; the remaining reagents were conventional products obtained through commercial purchase.

[0043] Experiment 1: Magnetic microspheres labeled with interleukin-8 antibody Take 20 mg of carboxyl-modified particle solution. The magnetic microspheres with superparamagnetism, uniform particle size, and carboxyl (COOH-) active groups on the surface are sedimented (magnetic separation) for 10 minutes under the action of a magnetic field. Remove the supernatant. The sedimented magnetic microspheres are washed 3 times with a buffer solution of (2-(N-morpholino)ethanesulfonic acid) MES with a molar concentration of 0.05 M and pH 6.0, and the amount used each time is 2 mL.

[0044] The washed magnetic microspheres are fully suspended with 1.0 mL of a buffer solution of (2-(N-morpholino)ethanesulfonic acid) MES with a molar concentration of 0.05 M and pH 6.0, and then the activator 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) is added. The mixture is suspended and reacted at room temperature for 30 minutes, and the reaction molar concentration of EDC is 7.5 mM.

[0045] Take 1.0 mg of interleukin-8 antibody SS002 and concentrate it to 2.5 mg / mL.

[0046] The interleukin-8 antibody SS002 concentrated to 2.5 mg / mL is added to 20 mg of the activated magnetic microsphere solution at a ratio of 1.0 mg of interleukin-8 conjugated carrier protein antigen. The mixture is gently shaken and mixed evenly, and reacted under suspension conditions at 4°C for 6.5 hours, so that the interleukin-8 antibody is covalently conjugated to the surface of the magnetic microspheres, and it is named Reagent A.

[0047] Experiment 2: Alkaline phosphatase labeled with commercial interleukin-8 antibody Take 1.0 mg of commercial interleukin-8 antibody and concentrate it to 2.5 mg / mL. Add 5 μL of a solution of the activator 2-Iminothiolane hydrochloride (2-IT) with a concentration of 13.76 mg / mL and react at room temperature for 15 minutes. Use a Sephadex G25 gel column for desalting and collect the activated antibody.

[0048] Take 1.2 mg of alkaline phosphatase and concentrate it to 2.5 mg / mL. Add 12 μL of a solution of the activator Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) with a concentration of 6.69 mg / mL and react at room temperature for 15 minutes. Use a Sephadex G25 gel column for desalting and collect the activated alkaline phosphatase.

[0049] Mix the activated commercial interleukin-8 antibody and alkaline phosphatase at a ratio of 1.0 mg of commercial interleukin-8 antibody to 1.0 mg of alkaline phosphatase, and place it at 4 °C for 18 hours of reaction.

[0050] Separate and purify using a Superdex 200 gel chromatography column to remove the unlinked commercial interleukin-8 antibody and alkaline phosphatase. Store the conjugate at 4 °C and name it Reagent B.

[0051] Experiment 3: Chemiluminescent immunoassay method for interleukin-8 magnetic microsphere separation (1) Immune reaction: Take 50 μL of the calibrator or quality control product and add 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, and mix and incubate at 37 °C for 10 min; (2) Magnetic separation: Allow the magnetic microspheres to settle in a magnetic field, discard the supernatant, add 200 - 500 μL of washing solution, remove the magnetic field, and then allow the magnetic microspheres to settle in the magnetic field again to remove the supernatant; repeat this 2 - 4 times to remove the unbound antibodies and impurities; (3) Reading: Add 200 μL of the luminescent substrate solution, and measure the relative luminescence intensity (RLU) using a self-developed chemiluminescent detector after the alkaline phosphatase catalyzes the substrate to emit light; (4) Fit using a four-parameter equation based on the measured values to obtain the interleukin-8 concentration - luminescence value standard curve.

[0052] (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, and mix and incubate at 37 °C for 15 min; (6) Magnetic separation: Allow the magnetic microspheres to settle in a magnetic field, discard the supernatant, add 200 - 500 μL of washing solution, remove the magnetic field, and then allow the magnetic microspheres to settle in the magnetic field again to remove the supernatant; repeat this 2 - 4 times to remove the unbound antibodies and impurities; (7) Reading: Add 200 μL of the luminescent substrate solution, measure the relative luminescence intensity (RLU) using a self-developed chemiluminescent detector after the ALP catalyzes the substrate to emit light, compare the luminescence intensity of the sample to be tested with the standard curve in step (4), and use a four-parameter equation to fit to calculate the content of interleukin-8 in the sample to be tested.

[0053] (8) Test the interleukin-8 protein standard product, and prepare the interleukin-8 protein into concentrations of 20000 pg / mL, 10000 pg / mL, 5000 pg / mL, 1000 pg / mL, and 500 pg / mL in sequence using the sample diluent. Test the luminescence value results and use four-parameter fitting. The results are shown in Table 7 and Figure 1As shown, the correlation coefficient r can reach 0.9999, and the upper limit of the linear range can reach 20,000 pg / mL.

[0054] Table 7

[0055] (9) Detect the sample diluent 20 times. The sensitivity calculation method is as follows: calculate the average value M and the standard deviation SD of the measurement results, substitute the value obtained by adding 2 times the standard deviation SD to the average value M into the standard curve in (8), and the result obtained is the sensitivity. The analysis results are shown in Table 8, and the sensitivity test results can reach 0.021 pg / mL.

[0056] Table 8

[0057] In summary, the monoclonal antibody of 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 quickly detect the content of interleukin-8 in serum or plasma samples, with high sensitivity, wide linear range, simple operation, and low requirements for sample pretreatment. By being combined with an automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and detection throughput are increased, the detection efficiency is improved, and at the same time, the errors caused by manual operation are avoided.

[0058] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An antibody or antigen-binding fragment, characterized in that, The light chain and heavy chain of the antibody or antigen-binding fragment comprise 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 according to claim 1, wherein, The antibody or antigen-binding fragment comprises: A light chain variable region as shown by the amino acid sequence of SEQ ID NO:1, and a heavy chain variable region as shown by the amino acid sequence of SEQ ID NO:

2.

3. The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, Further comprising a heavy chain constant region and a light chain constant region, at least a part of the heavy chain constant region and the light chain constant region being from mammalian source antibodies; Optionally, the heavy chain constant region and the light chain constant region are derived from at least one of murine antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy cow antibodies, porcine antibodies, ovine antibodies, caprine antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies or mutants thereof; Optionally, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from kappa type or lambda type; Optionally, both the heavy chain constant region and the light chain constant region are derived from murine antibodies or mutants thereof; Optionally, the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region.

4. The antibody or antigen-binding fragment according to claim 1, wherein The antibody comprises at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab’ antibodies, F(ab’)2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units; or The antigen-binding fragment comprises at least one selected from F(ab’)2 fragments, Fab’ fragments, Fab fragments, F(ab)2 fragments, Fv fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, and minimal recognition units.

5. A recombinant protein, characterized in that, Comprises: The antibody or antigen-binding fragment according to any one of claims 1 to 4; Optionally, further comprising at least one selected from bioactive proteins or fragments thereof, bioactive polypeptides or fragments thereof; Optionally, the bioactive protein or fragment thereof comprises at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factors or fragments thereof, interferons or fragments thereof, biological response modifiers or fragments thereof, and Fc fragments.

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

7. The nucleic acid molecule according to claim 6, wherein, The nucleic acid molecule comprises: A nucleic acid sequence encoding a light chain variable region as shown by SEQ ID NO:9; and / or A nucleic acid sequence encoding a heavy chain variable region as shown by 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: Carrying the nucleic acid molecule according to claim 6 or 7, or the expression vector according to claim 8; or Expressing the antibody or antigen-binding fragment according to any one of claims 1 to 4, or the recombinant protein according to claim 5.

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

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

12. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 4, the recombinant 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 the preparation of a kit, characterized in that, The kit is used for detecting interleukin-8.

13. A kit, characterized in that, It includes: At least one of the antibody or antigen-binding fragment according to any one of claims 1 to 4, the recombinant 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, wherein The kit includes the antibody or antigen-binding fragment according to any one of claims 1 to 4, magnetic particles, a commercial interleukin-8 antibody, and a luminescent substrate; Wherein, the magnetic particles are conjugated with the antibody or antigen-binding fragment; The commercial interleukin-8 antibody is modified with a label; The label is used to catalyze the luminescence of the luminescent substrate.

15. The kit according to claim 14, wherein The particle size of the magnetic particles is not greater than 10 μm; Optionally, the particle size of the magnetic particles is 0.5 - 10 μm; Optionally, the label includes at least one of a bio-enzyme, a fluorophore, and a chemiluminescent label; Optionally, the bio-enzyme includes at least one of horseradish peroxidase and alkaline phosphatase; Optionally, the commercial interleukin-8 antibody specifically recognizes the conserved region of interleukin-8; Optionally, the luminescent substrate is selected from at least one of o-phenylenediamine, luminol, and acridinium ester; Optionally, the luminescent substrate is provided in a liquid form; Optionally, the luminescent substrate liquid further includes a buffer solution; Optionally, the buffer solution includes at least one selected from phosphate buffer solution, carbonate buffer solution, tris buffer solution, and citrate buffer solution; Optionally, the kit further includes a calibrator for interleukin-8 protein and / or a quality control product for interleukin-8 protein.

16. Use of the kit according to any one of claims 13 to 15 in the preparation of an interleukin-8 detection product.

17. A method for detecting interleukin-8, characterized in that, It includes: Using the antibody or antigen-binding fragment according to any one of claims 1 to 4, the recombinant 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 to perform detection processing on a sample to be tested.

18. The method according to claim 17, wherein Chemiluminescence method, enzyme-linked immunosorbent assay, WB.

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