A monoclonal antibody combination for detecting human IL-8 protein and its application

Abstract

This invention relates to the field of biological detection, and more particularly to a monoclonal antibody combination for detecting human IL-8 protein and its application. The combination comprises monoclonal antibodies 2G3 and 3H8. The heavy and light chain variable regions of monoclonal antibody 2G3 include the complementarity-determining regions shown in SEQ ID NO. 1-6; the heavy and light chain variable regions of monoclonal antibody 3H8 include the CDRs shown in SEQ ID NO. 7-12. The antibody combination of this invention exhibits high specificity and effectively avoids cross-reactivity with other CXC chemokine family members, thereby significantly reducing non-specific background interference and the risk of false positives during detection. This invention provides a high-performance, quality-controllable key tool for biological detection for non-diagnostic purposes, research on inflammatory mechanisms, and the development of related scientific reagents.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a combination of monoclonal antibodies for detecting human IL-8 protein and its application. Background Technology

[0002] Human interleukin-8 (IL-8), also known as chemokine CXCL8 (CXC motifchemokine ligand 8), is a small secreted protein belonging to the CXC chemokine family, along with other cytokines such as CXCL1 / 2 / 3 / 13, and plays a crucial role in inflammatory responses. IL-8 expression typically increases significantly under inflammatory stimuli, such as when cells are stimulated by inflammatory signals like bacterial lipopolysaccharide (LPS), IL-1β, or TNF-α, leading to the rapid synthesis and secretion of IL-8, which then participates in regulatory functions in the early stages of inflammation. In healthy individuals, however, IL-8 levels in peripheral blood circulation are usually low.

[0003] In terms of its mechanism of action, IL-8 primarily binds to CXCR1 and CXCR2 receptors on the cell surface, activating downstream signaling pathways such as PI3K, MAPK, and PLC, thereby inducing cytoskeleton rearrangement, enhanced expression of adhesion molecules, and improved cell migration. Unlike cytokines that mainly regulate the intensity of immune responses (such as IL-6), the core function of IL-8 is to induce the directed migration of inflammatory cells, such as neutrophils, to the site of inflammation and enhance their degranulation and oxidative burst responses. Therefore, IL-8 plays a more significant role in regulating "inflammatory cell recruitment and local inflammatory spread" during inflammation, and is considered an important molecule connecting inflammatory signals with the actual effects of inflammatory cells.

[0004] In clinical applications, elevated IL-8 levels typically indicate significant neutrophil infiltration in locally inflamed tissues. In diseases such as respiratory infections, acute lung injury, and acute respiratory distress syndrome (ARDS), IL-8 is considered a crucial chemokine driving the aggregation of inflammatory cells in the lungs. It also participates in the sustained recruitment of inflammatory cells in gastrointestinal inflammation, skin inflammation, and other tissue injuries. Furthermore, in various tumor microenvironments, IL-8 not only participates in inflammatory cell infiltration but may also influence tumor angiogenesis and local immune regulation. Therefore, IL-8 levels can serve as an important biomarker reflecting the degree of local inflammatory activity and the intensity of inflammatory cell infiltration in tissues.

[0005] Currently, IL-8 protein detection is mostly performed using immunological methods, with common sample types including serum, plasma, cell culture supernatant, and tissue homogenate. Among these, enzyme-linked immunosorbent assay (ELISA) is widely used in the assessment of inflammatory disease markers due to its high sensitivity, good specificity, mature operation, and ease of standardization.

[0006] Although various immunoassay kits for IL-8 detection are available on the market, the quality of the core antibody raw materials used varies, and some products use core antibody raw materials with insufficient characterization, affecting the accuracy of the test results. Furthermore, due to the structural similarities among members of the chemokine family, insufficient antibody specificity may lead to cross-reactions with other CXC family members, thereby reducing detection accuracy. In addition, low antibody affinity or inappropriate pairing may affect the detection of low-abundance samples, making it difficult to meet the requirements of high-sensitivity detection. Therefore, obtaining high-affinity, high-specificity, and sequence-defined human IL-8 monoclonal antibodies, and screening for stable and complementary pairings, is crucial for constructing a highly sensitive and standardized ELISA detection system. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a combination of monoclonal antibodies for detecting human IL-8 protein and its application, which solves the technical problems of low sensitivity and false positives caused by cross-reactivity in the existing detection technology.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0011] In a first aspect, this application provides a monoclonal antibody combination for detecting human IL-8 protein, the monoclonal antibody combination comprising monoclonal antibody 2G3 and monoclonal antibody 3H8;

[0012] The heavy chain variable region of the monoclonal antibody 2G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0013] The light chain variable region of the monoclonal antibody 2G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0014] The heavy chain variable region of the monoclonal antibody 3H8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0015] The light chain variable region of the monoclonal antibody 3H8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0016] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO. 14.

[0017] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO. 16.

[0018] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.18.

[0019] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.20.

[0020] In a further embodiment, the monoclonal antibody combination specifically recognizes recombinant human IL-8 protein and natural human IL-8 protein.

[0021] Secondly, this application provides the application of the combination of the monoclonal antibodies in the preparation of a tool for detecting human IL-8 protein; the tool is used to detect human IL-8 protein in a sample selected from any one of serum, plasma, cell culture supernatant and tissue homogenate, and the detection is not used for the diagnosis of disease.

[0022] In a further embodiment, the tool includes reagents, kits, test strips, and antibody chips.

[0023] In a further embodiment, the kit includes a double-antibody sandwich ELISA kit.

[0024] In a further embodiment, the ELISA kit uses monoclonal antibody 2G3 as the coating antibody and monoclonal antibody 3H8 as the labeling antibody.

[0025] (III) Beneficial Effects

[0026] This invention provides a monoclonal antibody combination for detecting human IL-8 protein, comprising monoclonal antibody 2G3 and monoclonal antibody 3H8. The heavy and light chain variable regions of monoclonal antibody 2G3 respectively contain complementarity-determining regions (CDRs) defined by SEQ ID NO. 1-3 and SEQ ID NO. 4-6, while the heavy and light chain variable regions of monoclonal antibody 3H8 respectively contain complementarity-determining regions defined by SEQ ID NO. 7-9 and SEQ ID NO. 10-12.

[0027] The antibody combination of this invention exhibits high specificity, effectively avoiding cross-reactions with other CXC chemokine family members, thereby significantly reducing non-specific background interference and the risk of false positives during detection. This monoclonal antibody combination demonstrates excellent affinity and sensitivity, lowering the detection limit of the detection system and enabling precise quantification of low concentrations of IL-8 protein in samples, meeting the high-precision requirements for trace detection in scientific experiments. In summary, this invention provides a high-performance, quality-controllable key tool for non-diagnostic biological detection, inflammatory mechanism research, and the development of related scientific reagents. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Image showing the results of SDS-PAGE protein identification;

[0030] Figure 2 The image shows the identification results of recombinant IL-8 protein;

[0031] Figure 3 The image shows the identification results of the purified monoclonal antibody;

[0032] Figure 4 The results of specificity and sensitivity of double-antibody sandwich ELISA for recombinant IL-8 protein are shown in the figure.

[0033] Figure 5 This is a diagram illustrating the activity of paired antibodies. Detailed Implementation

[0034] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0035] This invention utilizes hybridoma technology to successfully prepare monoclonal antibodies capable of specifically recognizing human interleukin-8 (IL-8) protein. Based on this, complementary antibody pairings with different binding sites were screened and obtained. These monoclonal antibodies can be used to construct ELISA detection systems, suitable for the quantitative detection of IL-8 protein in samples such as serum, plasma, and cell culture supernatants. Validation has shown that this monoclonal antibody pairing system exhibits good detection sensitivity and specificity for human IL-8, stable repeatability, and low background signal, meeting the accuracy and consistency requirements of scientific research and clinical testing. This provides a well-defined and reliable antibody raw material basis for the development of human IL-8-related immunodiagnostic reagents.

[0036] The double-antibody sandwich ELISA detection method described in this application is not intended for disease diagnosis and treatment.

[0037] Example 1

[0038] 1. Expression of recombinant IL-8 protein

[0039] Referring to the IL-8 gene sequence, the gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is SEQ ID NO.21:

[0040] ATGGAAGGCGCCGTGCTGCCGCGCAGCGCTAAAGAACTGCGCTGCCAGTGCATTAAGACCTATAGCAAACCGTTTCATCCGAAATTCATTAAGGAACTGCGTGTGATTGAAAGTGGCCCGCATTGTGCAAATACCGAAATTATTGTGAAACTGAGCGATGGTCGTGAACTGTGTCTGGACCCTAAAGAAAATTGGGTTCAGCGCGTGGTGGAAAAATTTCTGAAACGCGCAGAAAATAGC.

[0041] The amino acid sequence is SEQ ID NO.22:

[0042] MEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

[0043] The recombinant plasmid pET32a-IL-8 was transformed into BL21(DE3) competent cells and induced to express the gene using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600 The concentration of the sample was 0.6-0.9, preferably 0.7. IPTG was added to a final concentration of 0.1 mM, and the cells were collected after induction at 30°C and 200 rpm for 4 hours to obtain the recombinant IL-8 protein.

[0044] 2. Purification of recombinant IL-8 protein

[0045] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TM Purification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second sonication intervals. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 The protein eluted with 500mM imidazole showed a relatively single band around 33kDa, consistent with the expected size (estimated molecular weight of IL-8 recombinant protein: 31.8 kDa), indicating that the protein is relatively pure and can be used for downstream experiments.

[0046] Figure 1 In the diagram, M represents the protein marker, 1 represents the precipitate of the lysed bacterial cells, 2 represents the supernatant after centrifugation of the lysed bacterial cells, 3 represents the flow-through of the sample, 4 represents the recombinant IL-8 protein eluted with 100 mM imidazole, and 5 represents the recombinant IL-8 protein eluted with 500 mM imidazole.

[0047] 3. Identification of recombinant IL-8 protein

[0048] The purified recombinant human IL-8 protein was coated onto an ELISA plate, and its reaction with the IL-8 positive antibody was identified by indirect ELISA. The IL-8 antibody was a commercially available mouse monoclonal antibody (Sino-Pharmaceutical, 10098-MM05). The recombinant protein was first coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. IL-8 positive clonal antibody was diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to a microplate coated with the antigen. A negative control was prepared by diluting another mouse monoclonal antibody (Sino-Pharmaceutical IL-17A monoclonal antibody: 10247-M237) at the same concentration. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-mouse IgG secondary antibody (Solepro, diluted 5000 times with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 . Figure 2 The purified recombinant IL-8 protein showed a significant positive reaction with the commercial IL-8 monoclonal antibody, and this antigen can be used for subsequent mouse immunization and antibody screening.

[0049] 4. Screening of IL-8 monoclonal antibodies

[0050] Immunization in mice.

[0051] Mice were immunized with high-purity human IL-8 recombinant protein. Human CXCL13 recombinant protein expressed using the pET32a vector (expressed by the applicant using *E. coli*) was used as a reverse screening antigen for monoclonal antibody selection. Specifically, purified IL-8 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 15 μg / mouse. At weeks 4, 8, and 12, booster immunizations were administered subcutaneously at multiple sites with an equal volume of Freund's incomplete adjuvant at the same dose. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for intraperitoneal booster immunization with 15 μg of IL-8 recombinant protein. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0052] The nucleotide sequence of CXCL13 is SEQ ID NO.23: GTTCTGGAGGTCTATTACACAAGCTTGAGGTGTAGATGTGTCCAAGAGAGCTCAGTCTTTATCCCTAGACGCTTCATTGATCGAATTCAAATCTTGCCCCGTGGGAATGGTTGTCCAAGAAAAGAAAT CATAGTCTGGAAGAAGAACAAGTCAATTGTGTGTGTGGACCCTCAAGCTGAATGGATACAAAGAATGATGGAAGTATTGAGAAAAAGAAGTTCTTCAACTCTACCAGTTCCAGTGTTTAAGAGAAAGATTCCC.

[0053] The amino acid sequence of CXCL13 is SEQ ID NO.24: VLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP.

[0054] Screening of hybridoma cells.

[0055] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones that reacted positively with recombinant IL-8 protein were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET32a vector containing a His tag, background components needed to be screened to select specific cell lines targeting IL-8 protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0056] Positive clones were screened using indirect ELISA.

[0057] Recombinant IL-8 protein and other recombinant proteins of the pET32a vector (pET32a-CXCL13, His tag) were coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water), at a coating concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 2% sucrose + 3% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. 50 μL of cell culture supernatant was added, and a negative control was prepared by diluting another mouse monoclonal antibody (Sino-Pharmaceutical IL-17A monoclonal antibody: 10247-M237) to 1 μg / mL, and reacted at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer. Incubate at room temperature for 10 min, and finally add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to stop the reaction. Measure the OD450nm value using a microplate reader. Select positive cell lines that react with IL-8 recombinant protein but not with the control recombinant protein for subsequent experiments. The screening results are shown in Table 1.

[0058] Table 1: Screening results of IL-8 monoclonal antibodies

[0059]

[0060] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0061] Purification and identification of monoclonal antibodies.

[0062] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, preferably 4°C, and collect the supernatant. Filter the supernatant through defatted cotton, add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, incubate the precipitate overnight at 2–8°C, and centrifuge at 12000 rpm for 30 minutes at 2–8°C to collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl (pH 9) and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS (pH 7.4) for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0063] The purified monoclonal antibody was diluted to 1 μg / ml, and another mouse monoclonal antibody (Sino Biologics IL-17A monoclonal antibody: 10247-M237) was diluted to the same concentration as a negative control. The binding activity of the antibody to recombinant human IL-8 protein and control antigens CXCL13 and CXCL1 (Sino Biologics, 10877-HNCE, expressed in E. coli) recombinant proteins was detected using an indirect ELISA method. Specific results are shown below. Figure 3 The results showed that the selected monoclonal antibody specifically binds to human IL-8 recombinant protein and does not react with control antigens CXCL13 and CXCL1 recombinant protein, and can be used for subsequent testing.

[0064] 5. Screening of paired monoclonal antibodies

[0065] 5.1-HRP-labeled monoclonal antibody.

[0066] The selected antibody was diluted to a final concentration of 2 mg / mL using carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). 2 mg of HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 1 mg of the diluted antibody solution was added to the matching tube containing HRP and mixed by pipetting. The tube was incubated at room temperature for 1 hour, with regular mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0067] 5.2 Establishment of the double-antibody sandwich method.

[0068] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was then patted dry and blocked with 3% sucrose and 2% BSA at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. Dilute the test antigen IL-8 protein and the control antigen proteins CXCL1 and CXCL13 mixed protein to 10 ng / ml with PBS and add 50 μL / well to an ELISA plate. Incubate at 37°C for 35 min. Wash the plate four times with PBST wash buffer, blot dry, add 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS, incubate at 37°C for 35 min, wash four more times, blot dry, add 50 μL / well of TMB chromogenic buffer, and develop at room temperature for 10 min. Finally, add 50 μL of TMB stop solution to stop the reaction. Measure the OD using an ELISA reader. 450 nm value. Using IL-8 as the positive antigen (i.e., IL-8 protein) and the CXCL1 & CXCL13 mixed antigen (i.e., CXCL1 & CXCL13 recombinant protein) as the negative antigen, a sandwich assay was performed. The P / N ratio was calculated, and the monoclonal antibody combination with the highest P / N ratio (highest positive detection value and low negative detection value) was selected as the optimal pairing. The results are shown in Table 2. 2G3 as the coating antibody and 3H8 as the labeling antibody resulted in the highest P / N ratio when detecting recombinant protein.

[0069] Table 2: OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value analysis

[0070]

[0071] “ " " indicates the dilution factor."

[0072] 6. Optimization of the double-antibody sandwich ELISA method

[0073] The purified monoclonal antibody 2G3 was diluted with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, and then diluted to 50 μL / well. Coating was performed overnight at 4°C. The coating buffer was discarded the next day, and the wells were blocked with 150 μL of 3% sucrose and 2% BSA. The wells were incubated at 37°C for 2 hours, and the blocking buffer was discarded. The test antigen (IL-8 recombinant protein) and control antigen (CXCL1) were then added. The CXCL13 mixed antigen was diluted with PBS at 10 ng / ml and added to the microplate at 50 μL / well. The plate was incubated at 37°C for 35 min. The plate was washed four times with PBST wash buffer. Then, 50 μL / well of HRP-labeled monoclonal antibody 3H8 (diluted 1000, 2000, 3000, and 4000 times with PBS) was added and incubated at 37°C for 35 min. The plate was washed four more times, patted dry, and then 50 μL / well of TMB chromogenic buffer was added. The plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD was measured using a microplate reader. 450 nm value. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The screening results are shown in Table 3.

[0074] Table 3: Results of Optimization of Dual Antibody ELISA Conditions

[0075]

[0076] Table 3 shows that the P / N ratio was highest when the HRP-labeled antibody was coated at 1 μg / ml and diluted 2000 times, making it the optimal combination.

[0077] 7. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting recombinant IL-8 protein

[0078] After determining the optimal reaction conditions, including a coating monoclonal antibody concentration of 1 μg / mL and a 2000-fold dilution of the HRP-labeled monoclonal antibody, and following the above detection steps, the recombinant human IL-8 protein was first serially diluted with PBS buffer to concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL. Simultaneously, CXCL1 (Sinochem Bioscience, 10877-HNCE, expressed in *E. coli*), CXCL13 (expressed by the applicant using *E. coli*), and recombinant human IL-1β protein (Chongqing Tansheng, FAP-BC006, 293 cells) were also tested. The following recombinant proteins were expressed: human IL-2 (nearshore, GMP-CD66, 293 cells), human IL-6 (expressed by the applicant using E. coli), human IL-7 (Kaikai Biotechnology, IL7-HE001, expressed in E. coli), human IL-10 (expressed by the applicant using E. coli), human IL-17A (nearshore, C774, 293 cells), and human IL-33 (Yiqiao Shenzhou, 10368-HNAE, expressed in E. coli). 50 μL of each protein was added to each well at the same concentration for detection to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 4 It can be seen that the double-antibody sandwich ELISA composed of this group of paired antibodies still showed a weak positive reaction when the recombinant human IL-8 protein was diluted to 10 pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0079] Referring to the human IL-6 amino acid sequence (NP_000591.1), Val30-Met212 was selected for recombinant expression, with Met added at the N-terminus. The gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is SEQ ID NO. 25:

[0080] ATGGTACCCCCAGGAGAAGATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAACAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAACAAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAACCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAAATG。

[0081] The amino acid sequence is SEQ ID NO.26:

[0082] MVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM。

[0083] The recombinant plasmid pET32a-IL-6 was transformed into BL21(DE3) competent cells and induced to express the plasmid using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600 The concentration was 0.6-0.9, preferably 0.7. After adding IPTG to a final concentration of 0.1 mM, the cells were collected at 37°C and 200 rpm for 4 hours to obtain recombinant IL-6 protein.

[0084] Referring to the IL-10 gene sequence, the gene was synthesized by Anhui General Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is shown in SEQ ID NO.27:

[0085] ATGAGTCCGGGTCAGGGCACCCAGAGTGAAAATAGTTGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGATGCATTTTCACGTGATGCATTTTCACGTGTGAAAACCTTTTTCCAGATGAAAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGTCTGCTGGAAGATTTTAAAGGCTATCTGGGTTGTCAGGCCCTGAGCGAAATGATTCAGTTTTATCTGGAAGAAGTGATGCCGCAGGC AGAAAATCAGGACCCTGATATTAAGGCACATGTTAATAGCCTGGGTGAAAATCTGAAAACCCTGCGCCTGCGTCTGCGTCGCTGTCATCGTTTTCTGCCGTGCGAAAATAAGAGTAAAGCCGTTGAACAGGTTAAAAATGCCTTTAATAAGCTGCAGGAAAAAGGTATCTATAAAGCAATGAGCGAATTTGATATCTTCATTAATTACATCGAGGCCTATATGACCATGAAAATTCGTAAT.

[0086] The amino acid sequence of recombinant human IL-10 protein is shown in SEQ ID NO.28:

[0087] MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0088] The recombinant plasmid pET32a-IL-10 was transformed into BL21(DE3) competent cells and induced to express the gene using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 4 hours until OD500 was reached. 600 The concentration of the solution is 0.6-0.9, preferably 0.7. IPTG is added to a final concentration of 0.1 mM, and the cells are collected after induction at 16°C and 180 rpm for 16 hours.

[0089] 8. Identification of binding activity of paired monoclonal antibodies

[0090] Following the aforementioned indirect ELISA method, paired monoclonal antibodies were serially diluted to 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Another murine-derived unrelated monoclonal antibody, Yiqiao Shenzhou IL-17A monoclonal antibody (10247-M237), was used as a negative control. The binding activity of the antibodies to recombinant human IL-8 protein was measured. Results are as follows... Figure 5 The results showed that it still reacted positively with IL-8 at a dilution of 1 ng / ml, indicating high antibody activity.

[0091] 9. Light and heavy chain variable region sequences of paired monoclonal antibodies

[0092] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by 3 rounds of PCR. The PCR products were purified by gel excision and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the variable region gene sequences of the light and heavy chains of the monoclonal antibody.

[0093] Encapsulated monoclonal antibody 2G3:

[0094] Light chain variable region nucleotide sequence:

[0095] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.18:

[0096] GACATCCAGATGACACAATCTCCAACCACCATGGCTGCATCTCCCGGGGAAAAGATCACTGTCACCTGCAGTGCCAGCTCAAGTATAACTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGGCATCCAATCTG GCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0097] Light chain variable region amino acid sequence:

[0098] The amino acid sequence of the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.14:

[0099] DIQMTQSPTTMAASPGEKITVTCSASSSITSNYLHWYQQKPGFSPKLLIYRASNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIYTFGGGTKLEIKRTV.

[0100] CDR area annotation:

[0101] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO.4:

[0102] CDR-L1: SASSITSNYLH;

[0103] The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO. 5:

[0104] CDR-L2: RASNLAS;

[0105] The amino acid sequence of the complementarity-determining region (CDR-L3) of the light chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO. 6:

[0106] CDR-L3: QQGSSIYT.

[0107] Heavy chain variable region nucleotide sequence:

[0108] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.17:

[0109] GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTAGGTA CAATGACAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCCCGTCTACTGAGGGGTTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0110] Heavy chain variable region amino acid sequence:

[0111] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.13:

[0112] EFQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTRYNDKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARLLRGYYAMDYWGQGTSVTVSS.

[0113] CDR area annotation:

[0114] The amino acid sequence of CDR-H1 in the complementarity-determining region of the heavy chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO. 1:

[0115] CDR-H1: SYVMH;

[0116] The amino acid sequence of the CDR-H2 complementarity-determining region of the heavy chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO. 2:

[0117] CDR-H2: YINPYNDGTRYNDKFKG;

[0118] The amino acid sequence of the CDR-H3 complementarity-determining region of the heavy chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO. 3:

[0119] CDR-H3: LLRGYYAMDY.

[0120] Labeled monoclonal antibody 3H8:

[0121] Light chain variable region nucleotide sequence:

[0122] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.20:

[0123] GACATTTGTGCTCACCCAATCTCCAGCCTCCCTATCTGCATCTTTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTTTTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCTATGTTGCAAAAACCTTAGCA GAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCTGAAGATCAACAGCCTGCAGCCTGAAGATTTAGGGACTTATTACTGTCAACATCATTATGGTCATCCGCTCACGTTCGGTGCTGGGACAAAGTTGGAAAATAAAACGTACGGTG.

[0124] Light chain variable region amino acid sequence:

[0125] The amino acid sequence of the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.16:

[0126] DIVLTQSPASLSASLGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYVAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDLGTYYCQHHYGHPLTFGAGTKLEIKRTV.

[0127] CDR area annotation:

[0128] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO.10:

[0129] CDR-L1: RASENIYSFLA;

[0130] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO.11:

[0131] CDR-L2: VAKTLAE;

[0132] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO.12:

[0133] CDR-L3: QHHYGHPLT.

[0134] Heavy chain variable region nucleotide sequence:

[0135] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.19:

[0136] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCATTTTCAGTGACTATCCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGGATGGGTCGCAACCATTAGTAATGGTGGTGGTAAAACCTACTAT CCAGACACTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCATATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTACTTCTGTGCAAGATGGGATGACTACGGTAGTAGCCCCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0137] Heavy chain variable region amino acid sequence:

[0138] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.15;

[0139] EVQLVESGGGLVKPGGSLKLSCAASGFIFSDYPMSWVRQTPEKRLGWVATISNGGGKTYYPDTVKGRFTISRDNAKNNLYLHMSSLRSEDTALYFCARWDDYGSSPFAYWGQGTLVTVSA.

[0140] CDR area annotation:

[0141] The amino acid sequence of the CDR-H1 in the complementarity-determining region of the heavy chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO. 7:

[0142] CDR-H1: DYPMS;

[0143] The amino acid sequence of the CDR-H2 of the complementarity-determining region of the heavy chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO. 8:

[0144] CDR-H2: TISNGGGKTYYPDTVKG;

[0145] The amino acid sequence of CDR-H3 in the complementarity-determining region of the heavy chain variable region of monoclonal antibody 3H8 is shown in SEQ ID NO. 9:

[0146] CDR-H3: WDDYGSSPFAY.

[0147] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any manner.

Claims

1. A monoclonal antibody combination for detecting human IL-8 protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 2G3 and monoclonal antibody 3H8; The heavy chain variable region of the monoclonal antibody 2G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 2G3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 3H8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 3H8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting human IL-8 protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting human IL-8 protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting human IL-8 protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2G3 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting human IL-8 protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H8 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting human IL-8 protein according to claim 5, characterized in that, The monoclonal antibody combination specifically recognizes recombinant human IL-8 protein and native human IL-8 protein.

7. The use of the combination of monoclonal antibodies according to claim 1 in the preparation of a tool for detecting human IL-8 protein; the tool is used to detect human IL-8 protein in a sample selected from any one of serum, plasma, cell culture supernatant and tissue homogenate, and the detection is not used for the diagnosis of disease.

8. The application according to claim 7, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

9. The application according to claim 8, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The ELISA kit uses monoclonal antibody 2G3 as the coating antibody and monoclonal antibody 3H8 as the labeling antibody.

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