Anti-IP10 recombinant rabbit monoclonal antibody pair and application thereof

Abstract

The invention provides an anti-IP10 recombinant rabbit monoclonal antibody pair and application thereof, and relates to the technical field of biology, the anti-IP10 recombinant rabbit monoclonal antibody pair comprises an antibody 1 and an antibody 2, each of the antibody 1 and the antibody 2 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of a heavy chain variable region of the antibody pair 1 is as shown in SEQ ID NO: 1, and the amino acid sequence of a light chain variable region of the antibody pair 1 is as shown in SEQ ID NO: 2; the amino acid sequence of a heavy chain variable region of the antibody pair 2 is as shown in SEQ ID NO: 3, and the amino acid sequence of a light chain variable region of the antibody pair 2 is as shown in SEQ ID NO: 4; the anti-IP10 recombinant rabbit monoclonal antibody pair provided by the invention can specifically recognize and detect the IP10 protein level of a human body, has high specificity and sensitivity, and can accurately distinguish the IP10 protein from other similar proteins.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-IP10 recombinant rabbit monoclonal antibody pair and applications thereof. Background Art

[0002] The chemokine IP-10 (interferon inducible protein-10), also known as CXCL10, is classified as a CXC chemokine. Its receptor is CXCR3, and its molecular weight is 10 kDa. IP-10 is derived from over 30 cell types, including activated fibroblasts, monocytes and macrophages, endothelial cells, and various lymphocytes. It has multiple biological effects, including potent recruitment of neutrophils, promotion of multiple cytokine secretion, and inhibition of some tumor growth.

[0003] Non-invasive diagnosis. Inflammatory responses are a major pathological phenomenon in the development of NAFLD, involving a range of cytokines and chemokines. CXC chemokine 10 (CXCL10, also known as IP10) is a proinflammatory chemokine that plays a crucial role in the development and progression of MAFLD. In the liver, CXCL10 is primarily secreted by hepatocytes and sinusoidal endothelial cells. CXCL10 binds to its specific receptor, CXCR3, recruiting activated CXCR3+ T lymphocytes and macrophages to the liver parenchyma, promoting inflammation, apoptosis, and fibrosis. Activated inflammation can further induce liver fibrosis, a common scarring response in most chronic liver injuries. It can be triggered by a variety of factors, including infection, excessive alcohol consumption, chemical toxins, autoimmune liver disease, and can also be a side effect of metabolic syndrome. Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) proteins and a shift in ECM type from a normal basement membrane-like matrix to a fibrillar collagen-rich matrix. Abnormal ECM composition can lead to persistent disruption and abnormal differentiation of normal hepatocytes, further triggering cirrhosis and portal hypertension, and ultimately liver failure. CXCL10 levels in serum and hepatocytes correlate with the extent and severity of fibrosis in liver biopsies. Data suggest that CXCL10 plays a role in promoting fibrosis in steatohepatitis. Another important mechanism of liver damage in NAFLD is apoptosis, and increased apoptosis is closely associated with the severity of MAFLD. Concanavalin A (Con A) can induce tissue damage and hepatocyte apoptosis, and neutralizing CXCL10 can ameliorate Con A-induced tissue damage, indicating that CXCL10 is also closely linked to hepatocyte apoptosis and can serve as a biomarker for monitoring the progression of NAFLD.

[0004] The disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) to liver fibrosis / cirrhosis and finally liver cancer is often insidious. Liver biopsy has long been considered the "gold standard" for diagnosing NAFLD. However, the invasive nature of liver biopsy, coupled with its high cost and low patient acceptance, limits its clinical application. Therefore, there is an urgent need for highly sensitive and specific non-invasive diagnostic methods to reduce reliance on liver biopsy and identify patients requiring further treatment. IP10 levels are a hallmark of hepatocellular damage. Developing a highly sensitive monoclonal antibody pair that specifically recognizes IP10 and using it to develop a non-invasive diagnostic kit for diagnosing high-risk patients with NAFLD and NASH is crucial for identifying patients requiring drug intervention and monitoring clinical drug efficacy. Currently, there are no clinical products for detecting serum IP10 protein levels, so the development of a kit for detecting human IP10 protein levels is urgently needed. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of the prior art, the present invention provides a recombinant rabbit monoclonal antibody pair with wide application and the ability to accurately identify IP10 protein levels. This antibody pair can effectively detect IP10 protein levels in serum and can be applied in sandwich ELISA detection and screening. The present invention also relates to the nucleotide sequence, amino acid sequence, recombinant plasmid encoding this antibody pair, preparation methods, and the application of this antibody pair in IP10 protein detection, as follows: In a first aspect, the present invention provides an anti-IP10 recombinant rabbit monoclonal antibody pair, comprising an antibody pair 1 and an antibody pair 2, wherein both antibody pair 1 and antibody pair 2 comprise a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of antibody pair 1 being as shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of antibody pair 1 being as shown in SEQ ID NO: 2; The amino acid sequence of the heavy chain variable region of the antibody pair 2 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of the antibody pair 2 is shown in SEQ ID NO: 4.

[0006] Moreover, the anti-IP10 recombinant rabbit monoclonal antibody pair is obtained by recombinant expression in mammalian cells and can specifically recognize and detect the level of human IP10 protein.

[0007] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the antibody pair 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of the antibody pair 1 is shown in SEQ ID NO: 6; The nucleotide sequence encoding the heavy chain variable region of the antibody pair 2 is shown in SEQ ID NO: 7; The nucleotide sequence encoding the light chain variable region of the antibody pair 2 is shown in SEQ ID NO:8.

[0008] In a second aspect, the present invention provides a method for preparing an anti-IP10 recombinant rabbit monoclonal antibody pair, the preparation method being as follows: S1. constructing the nucleotide sequence encoding the antibody pair into an expression vector; S2, transforming the expression vector into a host cell; S3, culturing the host cells and collecting the cell supernatant; S4. Purify the cell supernatant to obtain the anti-IP10 recombinant rabbit monoclonal antibody pair.

[0009] In a third aspect, the present invention provides an application of an anti-IP10 recombinant rabbit monoclonal antibody pair for preparing a kit for detecting IP10 protein levels.

[0010] Furthermore, the kit is used for non-invasive diagnosis of NAFLD accompanied by non-alcoholic steatohepatitis (NASH).

[0011] In a fourth aspect, the present invention provides an application of an anti-IP10 recombinant rabbit monoclonal antibody pair for sandwich ELISA detection and screening.

[0012] In a fifth aspect, the present invention provides a kit for detecting IP10 protein levels, comprising an anti-IP10 recombinant rabbit monoclonal antibody pair.

[0013] Furthermore, the kit is used for non-invasive diagnosis of NAFLD accompanied by non-alcoholic steatohepatitis (NASH).

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The anti-IP10 recombinant rabbit monoclonal antibody pair provided by the present invention can specifically recognize and detect the level of human IP10 protein with high specificity and sensitivity. This antibody pair can accurately distinguish IP10 protein from other similar proteins, thereby accurately detecting the presence and amount of IP10 in complex biological samples. This antibody can be applied to detection and screening fields such as ELISA, which is conducive to obtaining accurate evaluation and detection results.

[0015] 2. The anti-IP10 recombinant rabbit monoclonal antibody pair provided by the present invention provides a non-invasive diagnostic method for high-risk patients with NAFLD accompanied by non-alcoholic steatohepatitis (NASH). Traditional liver biopsy is invasive, costly, and has low patient acceptance. The antibody pair of the present invention can be used to develop non-invasive diagnostic kits to assess the patient's disease status by detecting the IP10 protein level in the serum, greatly reducing the patient's pain and medical costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a standard curve diagram of the IP10 protein sandwich ELISA test and blank samples in Example 2-3; Figure 2 This is the interference experiment standard curve of the high-value IP10 sample in Example 4; Figure 3 This is the interference experiment standard curve of the low-value IP10 sample in Example 4. DETAILED DESCRIPTION

[0017] To further illustrate the technical means and effects adopted by the present invention, the present invention is further described below in conjunction with embodiments and drawings. It can be understood that the specific implementation methods described here are only used to explain the present invention, rather than to limit the present invention.

[0018] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0019] Example 1 This example is the preparation and screening of anti-IP10 recombinant rabbit monoclonal antibodies, the steps include: 1. Animal immunization 1.1 Immunization regimen The primary immunization antigen was mixed and emulsified with an equal volume of Freund's complete adjuvant, and the secondary, tertiary, quadruple, and quadruple immunization antigens were mixed and emulsified with an equal volume of Freund's incomplete adjuvant.

[0020] 1.2 Serum titer detection (1) Immunogen coating: Dilute the immunogen to 2 μg / ml with 10 mM PBS buffer and add 100 μl per well to a 96-well ELISA plate. Incubate at 4°C overnight. Wash the coated ELISA plate twice with a plate washer and tap dry. Add 200 μl of blocking solution per well and incubate in a 37°C incubator for 2 h. Then wash the blocked ELISA plate twice with a plate washer and tap dry.

[0021] (2) Incubation with primary antibody: Dilute immune rabbit serum with PBS buffer, with PBS as the negative control. The dilution ratios are 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000. Add 100ul of diluted serum to the corresponding wells and incubate at 37°C for 1.5h. After incubation, wash the plate in a plate washer three times and then dry it.

[0022] (3) Incubation with secondary antibody: Add 100 μl of HRP-labeled goat anti-rabbit secondary antibody to the corresponding wells, incubate at 37°C for 45 min, wash the ELISA plate 5 times in a plate washer and dry.

[0023] (4) Color development: Add 100 μl of color development solution to each well of a 96-well plate and incubate at 37°C for 15 min.

[0024] (5) Stop and read: Add stop solution to the 96-well plate, 50ul per well, and then place it in a microplate reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control is considered positive.

[0025] 2. B cell sorting Initial screening is performed using B cell surface markers. The immune protein and anti-IgG secondary antibody are fluorescently labeled. If the MBC surface displays IgG antibodies that specifically bind to the antigen, the cell will be labeled with two fluorescent markers. Target MBCs can be individually isolated from the cell suspension using flow cytometry and placed in a 96-well plate.

[0026] 3. High-throughput expression (1) Set up the PCR instrument program, lyse B cells to release RNA, and use reverse transcription reaction to obtain cDNA.

[0027] (2) Set the PCR instrument program and use light and heavy chain PCR specific primers to obtain the variable region sequence.

[0028] (3) Monoclonal bacteria were obtained by ligation and transformation, and colony PCR was used to screen candidate antibody clones with paired light and heavy chains.

[0029] (4) Clone the paired light and heavy chain candidate antibodies into bacteria and extract the plasmids.

[0030] 4. High-throughput expression and cell supernatant detection (1) Paired light and heavy chain plasmids are synchronously transfected into cells in a single well of a deep-well plate for cell culture.

[0031] (2) After the cells were grown for 5 days, the cell supernatant was collected.

[0032] (3) ELISA test of cell supernatant, the method is the same as serum ELISA test.

[0033] 5. Antibody expression and detection The expression plasmids corresponding to the 10 selected clones were transiently transfected and expressed, and finally 9 antibodies were obtained.

[0034] 6. Antibody pairing ELISA detection and sensitivity test 7. Antibody variable region sequencing Sequencing primers were designed and the base sequences of the light and heavy chain variable regions were obtained by first-generation sequencing.

[0035] Finally, the nucleotide sequence encoding the heavy chain variable region of the anti-IP10 recombinant rabbit monoclonal antibody of the antibody pair 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of the antibody pair 1 is shown in SEQ ID NO: 6; The nucleotide sequence encoding the heavy chain variable region of the antibody pair 2 is shown in SEQ ID NO: 7; The nucleotide sequence encoding the light chain variable region of the antibody pair 2 is shown in SEQ ID NO:8.

[0036] Example 2 This example is the development of a kit for producing anti-IP10 recombinant rabbit monoclonal antibodies, and the steps include: (1) Capture antibody coating: dilute (antibody pair 1) to 8 μg / mL with PBS buffer, add 100 μl per well, and incubate at 4°C overnight; (2) Washing: Add 300 μl of washing solution to each well, wash twice, and soak for 1 minute; (3) Blocking: Prepare 5% BSA (Sigma) as blocking solution, add 200 μl to each well, and incubate in a constant temperature incubator at 37°C for 2 h; (4) Washing: 300 μl of washing solution per well, 3 times, soaking for 1 minute; (5) Protein incubation: Dilute the IP10 protein to 6000 pg / mL using the sample and antibody diluent, and then dilute it 1.5 times in sequence. The negative control is the sample and antibody diluent. Each well has 100 μL of the protein and incubate at 37°C for 1.5 hours. (6) Washing: Add 300 μl of washing solution to each well, wash 5 times, and soak for 1 minute; (7) Detection antibody incubation: HRP (horseradish peroxidase) labeled IP10 detection antibody (antibody pair 2) was diluted to 0.1 μg / mL using sample and antibody diluent, 100 μL was added to each well, and incubated at 37°C for 1 hour; (8) Washing: Add 300 μl of washing solution to each well, wash 5 times, and soak for 1 minute; (9) Color development: Add 100 μl of color development solution to each well (keep away from light) and incubate at room temperature for 15 minutes. (10) Stop: Add 100 μl of stop solution to each well and shake the microplate for 5–10 seconds to mix the color development solution and the stop solution. (11) Reading: Measure OD value at 450nm.

[0037] Table 1 IP10 protein standard curve detection data

[0038] Standard curve Figure 1 As shown. Figure 1 It can be seen that the anti-IP10 antibody curve presents a typical "linear" feature, showing good response to both low and high concentration samples; this shows that this method has a wide detection range, reflecting the stability and reliability of the detection system, and can meet the needs of quantitative detection of IP10 protein in clinical samples.

[0039] Example 3 This example is a performance sensitivity verification of an anti-IP10 recombinant rabbit monoclonal antibody pair. The experimental steps are as follows: (1) Capture antibody coating: dilute (antibody pair 1) to 8 μg / mL with PBS buffer, add 100 μl per well, and incubate at 4°C overnight; (2) Washing: Add 300 μl of washing solution to each well, wash twice, and soak for 1 minute; (3) Blocking: Prepare 5% BSA (Sigma) as blocking solution, add 200 μl to each well, and incubate in a constant temperature incubator at 37°C for 2 h; (4) Washing: 300 μl of washing solution per well, 3 times, soaking for 1 minute; (5) Protein incubation: Dilute the Detai IP10 protein to 6000 pg / mL using sample and antibody diluent, and then dilute it in a 1.5-fold ratio. The negative control is the sample and antibody diluent, with 100 μl per well. Add 100 μl of antibody diluent to 24 blank wells in turn and incubate at 37°C for 1.5 hours. (6) Washing: Add 300 μl of washing solution to each well, wash 5 times, and soak for 1 minute; (7) Detection antibody incubation: Dilute the HRP-labeled IP10 detection antibody (antibody pair 2) to 0.1 μg / mL using the sample and antibody diluent, add 100 μL to each well, and incubate at 37°C for 1 hour. (8) Washing: Add 300 μl of washing solution to each well, wash 5 times, and soak for 1 minute; (9) Color development: Add 100 μl of color development solution to each well (keep away from light) and incubate at room temperature for 15 minutes. (10) Stop: Add 100 μl of stop solution to each well and shake the microplate for 5–10 seconds to mix the color development solution and the stop solution. (11) Reading: Measure OD value at 450nm.

[0040] Table 2 Detection limit analysis data of blank samples

[0041] Repeat the blank sample measurement 24 times, calculate the mean and standard deviation of the 24 reactions, and calculate the corresponding concentration as the mean + 2SD as the analytical sensitivity. The minimum detection can reach 86pg / ml. Figure 1 ) showed a good linear relationship (R²>0.99).

[0042] Example 4 This example is to verify the performance specificity of the antibody pair of anti-IP10 recombinant rabbit monoclonal antibodies. The experimental steps are as follows: (1) Prepare 1×PBST washing solution, sample and antibody diluent (1×PBST (Solybol) + 1% BSA + 0.1% ProClin 950, filtered) as required; (2) Antigen coating: Dilute IP10 monoclonal antibody (antibody pair 1) to 8.0 μg / ml with PBS buffer, add 100 μl to each well, and incubate at 4°C overnight; (3) Washing: Set the plate washer to 300 μl of washing solution per well, wash twice, and soak for 1 min; (4) Blocking: Prepare 5% BSA blocking solution, add 200 μl to each well, and incubate at 37°C for 2 h; (5) Washing: Set the plate washer to 300 μl of washing solution per well, wash three times, and soak for 1 min; (6) Sample incubation: Use antibody and sample diluent to dilute IP10 protein standard (starting at 6 ng / mL, 1.5-fold dilution 10+0 concentration), add the diluted protein standard to the corresponding wells in turn at 100 μl per well, and incubate at 37 °C for 1.5 h; add a certain concentration of influencing factors: CXCL9, CXCL11 and CXCL13 to the high-value samples and low-value samples, 100 μl per well, and incubate at 37 °C for 1.5 h; (7) Washing: Set the plate washer to 300 μl of washing solution per well, wash 5 times, and soak for 1 min; (8) Detection antibody incubation: dilute (antibody pair 2)-HRP (concentration 0.1 μg / mL) with antibody and sample diluent, add 100 μl to each well, and incubate at 37 °C for 1 h; (9) Washing: Set the plate washer to 300 μl of washing solution per well, wash 5 times, and soak for 1 min; (10) Color development: Add 100 μl of color development solution to each well (keep away from light) and incubate at 37°C in the dark for 15 min. (11) Stop: Add 100 μl of stop solution to each well and shake the microplate for 5–10 s to mix the color development solution and the stop solution. (12) Reading: Measure the OD value at 450 nm.

[0043] Table 3 High-value sample interference experimental data

[0044] Table 4 Specificity analysis of high-value samples

[0045] Table 5 Low-value sample interference experimental data

[0046] Table 6 Specificity analysis of low-value samples

[0047] The standard curve of high value samples is as follows Figure 1 As shown, the standard curve of low value samples is as follows Figure 3 As shown, the experimental results showed that when the three influencing factors of CXCL9, CXCL11 and CXCL13 were added to the low- and high-value samples, the protein concentration was calculated based on the OD value results substituted into the standard curve equation, and the final result was not significantly different from the high- and low-value sample controls (difference ≤ 10%). After adding CXCL9, CXCL11 and CXCL13 to the high- and low-value IP10 samples respectively, the detected IP10 concentration was ≤ 10% different from the control group without interference, proving that the antibody pair can specifically recognize IP10 protein and has almost no cross-reaction with the structurally similar CXCL9 / 11 / 13, fully meeting the specificity requirements of clinical testing.

Claims

1. An anti-IP10 recombinant rabbit monoclonal antibody pair, comprising antibody pair 1 and antibody pair 2, characterized in that: Both antibody pair 1 and antibody pair 2 include a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of antibody pair 1 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of antibody pair 1 is shown in SEQ ID NO:

2. The amino acid sequence of the heavy chain variable region of the antibody pair 2 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of the antibody pair 2 is shown in SEQ ID NO:

4.

2. An anti-IP10 recombinant rabbit monoclonal antibody pair according to claim 1, characterized in that The anti-IP10 recombinant rabbit monoclonal antibody pair is obtained by recombinant expression in mammalian cells and can specifically recognize and detect the level of human IP10 protein.

3. An anti-IP10 recombinant rabbit monoclonal antibody pair according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the antibody pair 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of the antibody pair 1 is shown in SEQ ID NO: 6; The nucleotide sequence encoding the heavy chain variable region of the antibody pair 2 is shown in SEQ ID NO: 7; The nucleotide sequence encoding the light chain variable region of the antibody pair 2 is shown in SEQ ID NO:

8.

4. A method for preparing an anti-IP10 recombinant rabbit monoclonal antibody pair according to any one of claims 1 to 3, characterized in that: The preparation method is as follows: S1. Constructing a nucleotide sequence encoding the antibody pair according to claim 1 into an expression vector; S2, transforming the expression vector into a host cell; S3, culturing the host cells and collecting the cell supernatant; S4. Purify the cell supernatant to obtain the anti-IP10 recombinant rabbit monoclonal antibody pair.

5. The use of an anti-IP10 recombinant rabbit monoclonal antibody pair as claimed in claim 1, characterized in that: Used to prepare a kit for detecting IP10 protein levels.

6. The use of an anti-IP10 recombinant rabbit monoclonal antibody pair as claimed in claim 5, characterized in that: The kit is used for non-invasive diagnosis of NAFLD accompanied by non-alcoholic steatohepatitis.

7. The use of an anti-IP10 recombinant rabbit monoclonal antibody pair according to claim 1, characterized in that: Used for sandwich ELISA detection and screening.

8. A kit for detecting IP10 protein levels, characterized in that: The invention comprises the anti-IP10 recombinant rabbit monoclonal antibody pair according to claim 1.

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