A Recombinant Rabbit Monoclonal Antibody Pair Against IP10 and Its Application

Abstract

This invention provides a recombinant rabbit monoclonal antibody pair against IP10 and its application, relating to the field of biotechnology. The anti-IP10 recombinant rabbit monoclonal antibody pair comprises antibody 1 and antibody 2. Both antibody 1 and antibody 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, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The amino acid sequence of the heavy chain variable region of antibody pair 2 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. The anti-IP10 recombinant rabbit monoclonal antibody pair provided by this invention can specifically recognize and detect the level of human IP10 protein, exhibiting high specificity and sensitivity. This antibody pair can accurately distinguish IP10 protein from other similar proteins.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant rabbit monoclonal antibody against IP10 and its applications. Background Technology

[0002] 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 more than 30 cell types, including activated fibroblasts, monocytes / macrophages, endothelial cells, and various lymphocytes. It possesses a variety of biological functions, including strongly recruiting neutrophils, promoting the secretion of various cytokines, and inhibiting the growth of some tumors.

[0003] In the non-invasive diagnosis of NAFLD, inflammation is one of the main pathological phenomena, involving a series of cytokines and chemokines. CXC chemokine 10 (CXCL10, also known as IP10) is a pro-inflammatory chemokine that plays an important role in the occurrence and development of MAFLD. Intrahepatic CXCL10 is mainly secreted by hepatocytes and sinusoidal endothelial cells. CXCL10 binds to its specific receptor CXCR3, recruiting activated CXCR3+ T lymphocytes and macrophages to aggregate in the liver parenchyma, promoting inflammation, apoptosis, and fibrosis. Activated inflammation can further induce liver fibrosis, a common scarring reaction in most chronic liver injuries. It can be caused by various factors such as infection, excessive alcohol consumption, chemical toxins, and autoimmune liver diseases, and can also be a side effect of metabolic syndrome. Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) proteins and a change in ECM type from normal basement membrane-like matrix to a fibrillary collagen-rich matrix. Abnormal ECM components lead to persistent disruption and abnormal differentiation of hepatocytes, further causing cirrhosis and portal hypertension, and ultimately liver failure. Serum and intracellular CXCL10 levels are correlated with the extent and severity of fibrosis in liver biopsies. Data suggests that CXCL10 promotes fibrosis in fatty liver disease. Another important mechanism of liver injury in NAFLD is apoptosis, and increased apoptosis is closely related to the severity of MAFLD. Concanavalin A (Con A) can induce tissue damage and hepatocyte apoptosis, while neutralizing CXCL10 can improve Con A-induced tissue damage, indicating that CXCL10 is also closely related to hepatocyte apoptosis and can serve as a biomarker for monitoring the progression of NAFLD.

[0004] The progression of NAFLD—from simple steatosis to non-alcoholic steatohepatitis (NASH) to liver fibrosis / cirrhosis to liver cancer—is often insidious. Liver biopsy has long been considered the "gold standard" for diagnosing NAFLD; however, its invasive nature, high cost, and low patient acceptance limit its clinical application. Therefore, there is an urgent need to develop highly sensitive and specific non-invasive diagnostic methods to reduce reliance on liver biopsy and to differentiate patients requiring further treatment. IP10 levels are a marker of hepatocellular damage. Developing a pair of highly sensitive and specific monoclonal antibodies that recognize IP10 and using them 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 the efficacy of clinical drugs. Currently, there are no products for clinically detecting serum IP10 protein levels; therefore, there is an urgent need to develop a kit for detecting human IP10 protein levels. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of existing technologies, this invention provides a recombinant rabbit monoclonal antibody pair that is widely applicable and can 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. This invention also relates to the nucleotide sequence, amino acid sequence, recombinant plasmid, preparation method, and application of this antibody pair in IP10 protein detection, as detailed below:

[0006] In a first aspect, the present invention provides a recombinant rabbit monoclonal antibody pair against IP10, the recombinant rabbit monoclonal antibody pair against IP10 comprising antibody pair 1 and antibody pair 2, both antibody pair 1 and antibody pair 2 comprising 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;

[0007] The amino acid sequence of the heavy chain variable region of antibody pair 2 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of antibody pair 2 is shown in SEQ ID NO: 4.

[0008] Moreover, the anti-IP10 recombinant rabbit monoclonal antibody is obtained by recombinant expression in mammalian cells and can specifically recognize and detect the level of human IP10 protein.

[0009] Furthermore, the nucleotide sequence encoding the heavy chain variable region of antibody pair 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of antibody pair 1 is shown in SEQ ID NO: 6;

[0010] The nucleotide sequence encoding the heavy chain variable region of antibody pair 2 is shown in SEQ ID NO: 7;

[0011] The nucleotide sequence encoding the light chain variable region of antibody pair 2 is shown in SEQ ID NO: 8.

[0012] Secondly, the present invention provides a method for preparing a recombinant rabbit monoclonal antibody pair against IP10, the preparation method being as follows:

[0013] S1. Construct the nucleotide sequence encoding the antibody pair into an expression vector;

[0014] S2. Transform the expression vector into host cells;

[0015] S3. Culture the host cells and collect the cell supernatant;

[0016] S4. Purify the cell supernatant to obtain the anti-IP10 recombinant rabbit monoclonal antibody pair.

[0017] Thirdly, the present invention provides an application of an anti-IP10 recombinant rabbit monoclonal antibody pair for preparing a kit to detect IP10 protein levels.

[0018] Furthermore, the kit is used for the non-invasive diagnosis of NAFLD with non-alcoholic steatohepatitis (NASH).

[0019] Fourthly, this invention provides an application of an anti-IP10 recombinant rabbit monoclonal antibody pair for sandwich ELISA detection and screening.

[0020] Fifthly, the present invention provides a kit for detecting IP10 protein levels, comprising a pair of recombinant rabbit monoclonal antibodies against IP10.

[0021] Furthermore, the kit is used for the non-invasive diagnosis of NAFLD with non-alcoholic steatohepatitis (NASH).

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The anti-IP10 recombinant rabbit monoclonal antibody pair provided by the present invention can specifically identify and detect the level of human IP10 protein, with high specificity and sensitivity. The antibody pair can accurately distinguish IP10 protein from other similar proteins, thereby accurately detecting the presence and amount of IP10 in complex biological samples. The antibody can be applied to detection and screening fields such as ELISA, which is beneficial to obtaining accurate assessment and detection results.

[0024] 2. The anti-IP10 recombinant rabbit monoclonal antibody pair provided by this invention offers a non-invasive diagnostic method for high-risk patients with NAFLD and non-alcoholic steatohepatitis (NASH). Traditional liver biopsy is invasive, costly, and has low patient acceptance. The antibody pair of this invention can be used to develop non-invasive diagnostic kits to assess the patient's disease status by detecting the level of IP10 protein in serum, greatly reducing patient suffering and medical costs. Attached Figure Description

[0025] Figure 1 The standard curves for IP10 protein sandwich ELISA detection and blank samples in Examples 2-3 are shown.

[0026] Figure 2 This is the standard curve of the interference experiment for the high-value IP10 sample in Example 4;

[0027] Figure 3 This is the standard curve for the interference experiment of the low-value IP10 sample in Example 4. Detailed Implementation

[0028] To further illustrate the technical means and effects of the present invention, the present invention will be further described below in conjunction with embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Example 1

[0031] This embodiment describes the preparation and screening of recombinant rabbit monoclonal antibodies against IP10, including the following steps:

[0032] 1. Animal immunization

[0033] 1.1 Immunization regimen

[0034] The primary immunization antigen was emulsified with an equal volume of Freund's complete adjuvant, while the secondary, tertiary, quaternary, and tertiary immunization shock antigens were emulsified with an equal volume of Freund's incomplete adjuvant.

[0035] 1.2 Serum titer detection

[0036] (1) Immunogen coating: Dilute the immunogen to 2ug / ml with 10 mM PBS buffer, add 100ul to each well of a 96-well microplate, and incubate overnight at 4°C. Wash the coated microplate twice with a plate washer and dry it. Add 200ul of blocking buffer to each well and incubate at 37°C for 2 hours. Then wash the blocked microplate twice with a plate washer and dry it.

[0037] (2) Incubation of primary antibody: Dilute rabbit serum with PBS buffer. PBS is used as 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 100 μL of the diluted serum to each well and incubate at 37°C for 1.5 h. After incubation, wash the microplate three times in a plate washer and then dry it.

[0038] (3) Incubation of secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody to each well and incubate at 37°C for 45 min. Then, wash the plate 5 times in a plate washer and dry it.

[0039] (4) Color development: Add 100 μL of color development solution to each well of the 96-well plate and incubate at 37°C for 15 min.

[0040] (5) Termination and reading: Add 50 μL of stop solution to each well of the 96-well plate, and then place it in an ELISA reader to read the value. Set the detection wavelength to 450 nm and read the detection result. An OD value greater than 2.1 times that of the negative control is considered positive.

[0041] 2. B cell sorting

[0042] Preliminary screening was performed using B cell surface markers. Secondary antibodies against immunoglobulins and anti-IgG were labeled with different fluorescent markers. If the surface of the mesoblast cells (MBCs) showed IgG antibodies that specifically bind to the antigen, the cells would carry two fluorescent markers. Target MBCs could then be individually sorted from the cell suspension using flow cytometry and placed in 96-well plates.

[0043] 3. High-throughput expression

[0044] (1) Set the PCR instrument program to lyse B cells to release RNA, and obtain cDNA by reverse transcription.

[0045] (2) Set the PCR instrument program and use light and heavy chain PCR specific primers to obtain the variable region sequence.

[0046] (3) Ligation and transformation were performed to obtain monoclonal bacteria, and colony PCR was used to screen for candidate antibody clones with light and heavy chains paired.

[0047] (4) Shake culture and extract plasmids of light and heavy chain paired candidate antibody clones.

[0048] 4. High-throughput expression and cell supernatant detection

[0049] (1) Pairs of light and heavy chain plasmids were simultaneously transfected into single-well cells of deep well plates for cell culture.

[0050] (2) After 5 days of cell growth, the cell supernatant was collected.

[0051] (3) ELISA detection of cell supernatant, the method is the same as serum ELISA detection.

[0052] 5. Antibody expression and detection

[0053] Ten selected clones were transiently transduced with their corresponding expression plasmids, resulting in nine antibody strains.

[0054] 6. Antibody-paired ELISA detection and sensitivity testing

[0055] 7. Antibody variable region sequencing

[0056] Design sequencing primers and obtain the base sequences of the light and heavy chain variable regions through first-generation sequencing.

[0057] Finally, the nucleotide sequence encoding the heavy chain variable region of the anti-IP10 recombinant rabbit monoclonal antibody 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.

[0058] The nucleotide sequence encoding the heavy chain variable region of antibody pair 2 is shown in SEQ ID NO: 7;

[0059] The nucleotide sequence encoding the light chain variable region of antibody pair 2 is shown in SEQ ID NO: 8.

[0060] Example 2

[0061] This embodiment describes the development of a kit for anti-IP10 recombinant rabbit monoclonal antibody, including the following steps:

[0062] (1) Capture antibody coating: Dilute (antibody pair 1) with PBS buffer to 8ug / mL, 100ul per well, and incubate overnight at 4°C;

[0063] (2) Washing: 300 μL of washing solution per well, wash twice, soak for 1 min;

[0064] (3) Blocking: Prepare 5% BSA (Sigma) as blocking solution, 200 μL per well, and incubate at 37°C for 2 h in a constant temperature incubator;

[0065] (4) Washing: 300 μL washing solution per well, 3 times, soaking for 1 min;

[0066] (5) Protein incubation: Dilute the Detai IP10 protein to 6000 pg / mL using sample and antibody diluent, and then perform serial dilutions of 1.5 times. The negative control is sample and antibody diluent. 100 μL of each corresponding well is used. Incubate at 37°C for 1.5 hours.

[0067] (6) Washing: 300 μL of washing solution per well, wash 5 times, soak for 1 min;

[0068] (7) Antibody incubation: The HRP (horseradish peroxidase) labeled IP10 detection antibody (antibody pair 2) was diluted to 0.1ug / mL with the sample and antibody diluent, 100ul per well, and incubated at 37°C for 1 hour;

[0069] (8) Washing: 300 μL of washing solution per well, wash 5 times, soak for 1 min;

[0070] (9) Color development: 100 μL of color development solution per well (avoid light) and incubate at room temperature in the dark for 15 min;

[0071] (10) Termination: Add 100 μL of stop solution to each well and shake the microplate for 5-10 seconds to mix the colorimetric solution with the stop solution.

[0072] (11) Reading: OD value is measured at 450nm.

[0073] Table 1. Detection data of IP10 protein standard curve

[0074]

[0075] Standard curve such as Figure 1 As shown. From Figure 1 As can be seen, the anti-IP10 antibody curve exhibits a typical "linear" shape, showing good response capability to both low and high concentration samples; this indicates that the method has a wide detection range, reflecting the stability and reliability of the detection system, and can meet the quantitative detection needs of IP10 protein in clinical samples.

[0076] Example 3

[0077] This example demonstrates the performance and sensitivity verification of the antibody pair against the recombinant rabbit monoclonal antibody against IP10. The experimental steps are as follows:

[0078] (1) Capture antibody coating: Dilute (antibody pair 1) with PBS buffer to 8ug / mL, 100ul per well, and incubate overnight at 4°C;

[0079] (2) Washing: 300 μL of washing solution per well, wash twice, soak for 1 min;

[0080] (3) Blocking: Prepare 5% BSA (Sigma) as blocking solution, 200 μL per well, and incubate at 37°C for 2 h in a constant temperature incubator;

[0081] (4) Washing: 300 μL washing solution per well, 3 times, soaking for 1 min;

[0082] (5) Protein incubation: Dilute the Detai IP10 protein to 6000 pg / mL using sample and antibody diluent, and then perform serial dilutions of 1.5 times. The negative control is sample and antibody diluent, 100 μL for each corresponding well. Add 100 μL of antibody diluent to each of the 24 blank wells and incubate at 37°C for 1.5 hours.

[0083] (6) Washing: 300 μL of washing solution per well, wash 5 times, soak for 1 min;

[0084] (7) Antibody incubation: Dilute the HRP-labeled IP10 detection antibody (antibody pair 2) to 0.1ug / mL with sample and antibody diluent, 100ul per well, and incubate at 37°C for 1 hour;

[0085] (8) Washing: 300 μL of washing solution per well, wash 5 times, soak for 1 min;

[0086] (9) Color development: 100 μL of color development solution per well (avoid light) and incubate at room temperature in the dark for 15 min;

[0087] (10) Termination: Add 100 μL of stop solution to each well and shake the microplate for 5-10 seconds to mix the colorimetric solution with the stop solution.

[0088] (11) Reading: OD value is measured at 450nm.

[0089] Table 2. Detection limit analysis data for blank samples

[0090]

[0091] The blank sample was measured 24 times repeatedly. The mean and standard deviation of the 24 reactions were calculated. The corresponding concentration was calculated as the analytical sensitivity using the mean + 2SD. The lowest detectable concentration was 86 pg / ml. Standard curve ( Figure 1The results show a good linear relationship (R²>0.99).

[0092] Example 4

[0093] This example demonstrates the performance and specificity verification of the antibody pair against the recombinant rabbit monoclonal antibody against IP10. The experimental steps are as follows:

[0094] (1) Prepare 1×PBST wash solution, sample and antibody diluent as required (1×PBST (Solepro) + 1% BSA + 0.1% ProClin950, filtered);

[0095] (2) Antigen coating: Dilute IP10 monoclonal antibody (antibody pair 1) with PBS buffer to 8.0 μg / ml, 100 μl per well, and incubate overnight at 4 ℃;

[0096] (3) Washing: Set the plate washer to 300 μl of washing solution per well, wash twice, and soak for 1 min;

[0097] (4) Blocking: Prepare 5% BSA blocking solution, 200 μl per well, and incubate at 37 °C for 2 h;

[0098] (5) Washing: Set the plate washer to 300 μl of washing solution per well, wash 3 times, and soak for 1 min;

[0099] (6) Sample incubation: Dilute IP10 protein standard with antibody and sample diluent (starting concentration of 6 ng / mL, serially diluted 1.5 times to 10+0 concentrations), add 100 μl of the diluted protein standard to each well, and incubate at 37 ℃ for 1.5 h; add certain concentrations of influencing factors: CXCL9, CXCL11 and CXCL13 to high-value and low-value samples, 100 μl per well, and incubate at 37 ℃ for 1.5 h;

[0100] (7) Washing: Set the plate washer to 300 μl of washing solution per well, wash 5 times, and soak for 1 min;

[0101] (8) Antibody incubation: Dilute the antibody with sample diluent (antibody-2)-HRP (concentration of 0.1 μg / mL), 100 μl per well, and incubate at 37 °C for 1 h;

[0102] (9) Washing: Set the plate washer to 300 μl of washing solution per well, wash 5 times, and soak for 1 min;

[0103] (10) Color development: 100 μl of color development solution per well (avoid light) and incubate at 37°C for 15 min in the dark;

[0104] (11) Termination: Add 100 μl of stop solution to each well and shake the microplate for 5-10 s to mix the color development solution with the stop solution.

[0105] (12) Reading: OD value is measured at 450 nm.

[0106] Table 3. Experimental data on high-value sample interference

[0107]

[0108] Table 4. Specificity analysis of high-value samples

[0109]

[0110] Table 5. Experimental data on interference from low-value samples

[0111]

[0112] Table 6. Specificity analysis of low-value samples

[0113]

[0114] High-value sample standard curve as follows Figure 1 As shown, the standard curve for low-value samples is as follows: Figure 3 As shown, the experimental results indicate that when CXCL9, CXCL11, and CXCL13, three influencing factors, are added to low- and high-value samples, the protein concentration is calculated by substituting the OD value results into the standard curve equation. The final results show no significant difference (≤10%) compared with the high- and low-value sample controls. After adding CXCL9, CXCL11, and CXCL13 to high- and low-value IP10 samples, respectively, the detected IP10 concentrations are ≤10% different from the control group without interfering substances. This demonstrates that the antibody can specifically recognize the IP10 protein and has almost no cross-reactivity with the structurally similar CXCL9 / 11 / 13, fully meeting the specificity requirements for clinical testing.

Claims

1. A pair of anti-IP10 recombinant rabbit monoclonal antibodies, wherein the anti-IP10 recombinant rabbit monoclonal antibody pair comprises 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 antibody pair 2 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of antibody pair 2 is shown in SEQ ID NO:

4.

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

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

8.

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

5. The application of the anti-IP10 recombinant rabbit monoclonal antibody pair as described in claim 1, characterized in that, This kit is used to prepare a reagent for detecting IP10 protein levels.

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