A recombinant rabbit monoclonal antibody against the CK18 M30 fragment and its application

Abstract

The application discloses an anti-CK18 M30 fragment recombinant rabbit monoclonal antibody pair and application thereof, relates to the field of biotechnology, and the antibody pair comprises antibody 1 and antibody 2; the heavy chain variable region amino acid sequence of the antibody 1 is shown as SEQ ID NO:1, and the light chain variable region amino acid sequence is shown as SEQ ID NO:2; the heavy chain variable region amino acid sequence of the antibody 2 is shown as SEQ ID NO:3, and the light chain variable region amino acid sequence is shown as SEQ ID NO:4; the antibody pair is obtained by mammalian cell recombinant expression; the anti-CK18 M30 fragment recombinant rabbit monoclonal antibody pair provided by the application can specifically recognize and combine CK18 fragment M30 in human serum, has high specificity and sensitivity, and the antibody pair can accurately detect the level of CK18 fragment M30 in serum.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant rabbit monoclonal antibody against the CK18 M30 fragment and its application. Background Technology

[0002] CK18 is an important component of intermediate filament proteins (IFPs) in the hepatocyte cytoskeleton system and is an acidic protein. In patients with liver disease, due to the induction of hepatocyte apoptosis, CK18 is hydrolyzed by caspases and released into the bloodstream. The level of CK18 fragments in the bloodstream is related to the three death processes of epithelial cells: apoptosis, autophagy, and necrosis. During apoptosis, CK18 is targeted by caspases at two sites (Asp238 and Asp396). CK18 at the Asp396 site is broken down, exposing a new epitope (Asp-387-396-NE) ​​that can be recognized by the M-30 antibody. This broken-down fragment is stable at physiological concentrations in the blood and has high specificity for detecting apoptosis.

[0003] Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity, type 2 diabetes, and metabolic syndrome. Due to sedentary lifestyles and the prevalence of high-calorie diets, the incidence of NAFLD is increasing year by year, making it the most common liver disease worldwide. Its average prevalence in Asia is 15%-40%, and in urban areas of my country, it reaches as high as 43.3%. The pathological progression of NAFLD is from simple steatosis to non-alcoholic steatohepatitis (NASH), leading to progressive liver fibrosis / cirrhosis, and ultimately liver cancer. 12%-40% of NAFLD patients will progress to NASH, 15%-33% of NASH patients will develop cirrhosis, and 15%-27% of cirrhosis patients will progress to liver cancer. Due to the increasing prevalence of obesity and type 2 diabetes, the incidence of NASH is expected to increase in the next decade. The proportion of NASH patients progressing to cirrhosis and liver cancer is significantly higher than that of patients with simple steatosis. Recent studies have shown that there are no effective treatments for NASH-related liver cancer compared to liver cancer caused by other factors. Differentiating between simple steatosis and NASH is crucial for preventing disease progression.

[0004] The progression of simple steatosis → NASH → liver fibrosis / cirrhosis → liver cancer is often insidious. Liver biopsy has long been considered the "gold standard" for NASH diagnosis. However, due to its invasive nature, high cost, and low patient acceptance, its clinical application is limited. Therefore, there is an urgent need to develop highly sensitive and specific non-invasive diagnostic methods to reduce reliance on liver biopsy and differentiate patients requiring further treatment. CK18 fragment M30 level is a marker of hepatocyte apoptosis and the most widely used biomarker for assessing fatty liver. Developing a pair of highly sensitive and specific monoclonal antibodies that recognize CK18 fragment M30 and using them to develop a non-invasive diagnostic kit for diagnosing high-risk patients with NASH and liver fibrosis is of great significance for identifying patients requiring drug intervention and monitoring the efficacy of clinical drugs. Currently, there are few commercially available products for clinically detecting serum CK18M30 protein, and their performance is generally average. 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 the CK18 fragment M30 level. This antibody pair can effectively detect the fragment of CK18 that is broken down during hepatocyte apoptosis in serum and can be applied to 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 the detection of the CK18 fragment M30, as detailed below:

[0006] A pair of recombinant rabbit monoclonal antibodies against the CK18 M30 fragment, the antibody pair comprising antibody 1 and antibody 2;

[0007] The amino acid sequence of the heavy chain variable region of antibody 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;

[0008] The amino acid sequence of the heavy chain variable region of antibody 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.

[0009] Moreover, the antibody pair is obtained through recombinant expression in mammalian cells and is able to specifically recognize and bind to the CK18 fragment M30 in human serum.

[0010] Furthermore, the nucleotide sequence encoding the heavy chain variable region of antibody 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of antibody 1 is shown in SEQ ID NO: 6;

[0011] The nucleotide sequence encoding the heavy chain variable region of antibody 2 is shown in SEQ ID NO: 7; the nucleotide sequence encoding the light chain variable region of antibody 2 is shown in SEQ ID NO: 8.

[0012] Furthermore, the method for preparing the antibody pair includes the following:

[0013] Two New Zealand white rabbits were immunized. After final immunization, serum titers were measured by indirect ELISA. Once the titer was greater than 1:100K, rabbit blood was collected to prepare PBMCs (peripheral blood mononuclear cells). Utilizing the surface markers and antigen-specific binding characteristics of memory B cells (MBCs), antigen-specific memory B cells were sorted from PBMCs by flow cytometry and transferred to 96-well plates. The sorted B cells were subjected to programmed lysis in a PCR instrument to release intracellular RNA, which was then reverse transcribed to obtain nucleic acid templates. The variable regions of the antibody light and heavy chains were amplified using a specific amplification system. The amplified products were ligated and transformed to obtain monoclonal bacteria. Candidate antibody clones with paired light and heavy chains were screened and plasmids were extracted. The paired light and heavy chain plasmids were simultaneously transfected into mammalian cells for expression, and the cell supernatant was collected. The cell supernatant was detected by ELISA, and 8 positive clones were selected for sequencing. Based on the results, 6 clones were selected for amplification, expression, and purification. After obtaining antibodies, antibody pairing was performed, and finally, one pair of paired antibodies was selected.

[0014] Secondly, the present invention provides an application of a recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment for preparing a kit to detect the level of the CK18 fragment M30 in human serum.

[0015] Furthermore, the kit is used for sandwich ELISA detection.

[0016] Thirdly, the present invention provides an application of a recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment for sandwich ELISA detection to detect the level of the CK18 fragment M30 in human serum.

[0017] Fourthly, the present invention provides an application of a recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment, characterized in that it is used to prepare a kit for diagnosing non-alcoholic fatty liver disease.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The recombinant rabbit monoclonal antibody against the CK18 M30 fragment provided by this invention can specifically recognize and bind to the CK18 fragment M30 in human serum, exhibiting high specificity and sensitivity. This antibody can accurately detect the level of the CK18 fragment M30 in serum, is suitable for sandwich ELISA detection, and can effectively distinguish different forms of cell death such as hepatocyte apoptosis, autophagy, and necrosis.

[0020] 2. Traditional liver biopsies are invasive, expensive, and have low patient acceptance. The antibody pairs provided by this invention can be used to develop non-invasive diagnostic kits, reducing reliance on liver biopsies and providing a more convenient and safer diagnostic method.

[0021] 3. As the incidence of non-alcoholic fatty liver disease (NAFLD) and its progressive form NASH increases year by year, and the treatment options for NASH-related liver cancer are limited, the antibody pair provided by this invention can help identify high-risk patients in the early stage, provide timely drug intervention, and monitor the treatment effect, thereby effectively preventing the occurrence of cirrhosis and liver cancer.

[0022] 4. The detection method established using the antibody pair provided by this invention has a detection limit as low as 97.4 pg / mL and high detection sensitivity, which can meet the detection requirements of low concentrations of M30 antigen. The detection method has a good linear relationship in the M30 antigen concentration range of 97.4-2000 pg / mL (R²>0.99, the closer R² is to 1, the better the linear relationship), proving that its detection results are accurate and reliable, and the actual concentration of M30 antigen can be accurately quantified by absorbance value. Attached Figure Description

[0023] Figure 1 : Standard curve of CK18 M30 protein in Example 2;

[0024] Figure 2 Example 3: Detection limit analysis chart of blank sample;

[0025] Figure 3 Standard curve of high-value samples in Example 4;

[0026] Figure 4 Example 4: Standard curve of low-value samples. Detailed Implementation

[0027] Example 1

[0028] This embodiment describes the preparation and screening of recombinant rabbit monoclonal antibodies against the CK18 M30 fragment. The steps include:

[0029] (a) Animal Immunization

[0030] 1.1 Immunization regimen

[0031] Primary immunization: Mix the antigen with an equal volume of Freund's complete adjuvant and emulsify.

[0032] Secondary, tertiary, and quaternary immunizations, as well as shock immunization: The antigen is mixed with an equal volume of Freund's incomplete adjuvant and emulsified.

[0033] 1.2 Serum titer detection

[0034] (1) Antigen coating:

[0035] Dilute the immunogen to 2 µg / mL with 10 mM PBS buffer, add 100 µL to each well of a 96-well microplate, incubate overnight at 4°C, wash the microplate twice with a plate washer and dry, add 200 µL of blocking buffer to each well, incubate at 37°C for 2 hours, wash the microplate twice more with a plate washer and dry.

[0036] (2) Incubation of primary antibody:

[0037] Dilute the immunized rabbit serum with PBS buffer at dilution ratios from 1:1000 to 1:512000. Add 100 µL of the diluted serum to each well and incubate at 37°C for 1.5 hours. After incubation, wash the ELISA plate three times with a plate washer and then dry it.

[0038] (3) Incubation of secondary antibody:

[0039] Add 100 µL of HRP-labeled goat anti-rabbit secondary antibody to each well and incubate at 37°C for 45 minutes. After incubation, wash the microplate 5 times with a plate washer and then dry it.

[0040] (4) Color development: Add 100 µL of color development solution to each well and incubate at 37°C for 15 minutes.

[0041] (5) Termination and reading: Add 50 µL of stop solution to each well and read the OD value at 450 nm wavelength using an ELISA reader. The OD value is considered positive if it is greater than 2.1 times that of the negative control.

[0042] (II) Isolation of rabbit peripheral blood mononuclear cells (PBMCs)

[0043] On day 7 after the last immunization, peripheral blood was collected from immunized rabbits, and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation for subsequent B cell sorting.

[0044] (III) B cell sorting

[0045] Preliminary screening was performed using B cell surface markers. Secondary antibodies against immunoproteins and anti-IgG were labeled with different fluorescent markers. Memory B cells (MBCs) with two fluorescent markers were sorted from the PBMCs obtained in step (II) by flow cytometry and then sorted into 96-well plates.

[0046] (iv) High-throughput expression

[0047] (1) Set the PCR instrument program, place the sorted B cells in the PCR instrument for programmed lysis, release intracellular RNA, and then perform reverse transcription to obtain nucleic acid templates that can be used for subsequent amplification.

[0048] (2) The PCR instrument is programmed to use the continuously amplified nucleic acid template to perform programmed amplification of the variable regions of the antibody light chain and heavy chain in the PCR instrument, and the variable region sequences of the light chain and heavy chain are obtained respectively.

[0049] (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.

[0050] (4) Shake culture and extract plasmids of light and heavy chain paired candidate antibody clones.

[0051] (v) High-throughput expression and cell supernatant detection

[0052] (1) Pairs of light and heavy chain plasmids were simultaneously transfected into single-well cells of deep well plates for cell culture.

[0053] (2) After 5 days of cell growth, the cell supernatant was collected.

[0054] (3) ELISA detection of cell supernatant, the method is the same as serum ELISA detection.

[0055] (vi) Antibody expression and detection

[0056] The expression plasmids corresponding to the six selected clones were transiently transduced to obtain six antibodies. Antibody-paired ELISA detection and sensitivity testing were performed, sequencing primers were designed, and the base sequences of the light and heavy chain variable regions were obtained through first-generation sequencing.

[0057] The final sequence of the recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment is as follows:

[0058] The amino acid sequence of the heavy chain variable region of antibody 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;

[0059] The amino acid sequence of the heavy chain variable region of antibody 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.

[0060] Moreover, the antibody pair is obtained through recombinant expression in mammalian cells and is able to specifically recognize and bind to the CK18 fragment M30 in human serum.

[0061] Furthermore, the nucleotide sequence encoding the heavy chain variable region of antibody 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of antibody 1 is shown in SEQ ID NO: 6;

[0062] The nucleotide sequence encoding the heavy chain variable region of antibody 2 is shown in SEQ ID NO: 7; the nucleotide sequence encoding the light chain variable region of antibody 2 is shown in SEQ ID NO: 8.

[0063] Example 2

[0064] This example describes the development of a kit for an anti-CK18 M30 recombinant rabbit monoclonal antibody, including the following steps:

[0065] (1) Capture antibody coating: Dilute (antibody 1) with 1×PBS buffer to 1 μg / mL, corresponding to 100 μL per well, and incubate overnight at 4 ℃;

[0066] (2) Plate washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0067] (3) Blocking: Prepare 200 μL of 5% BSA blocking solution for each well and incubate at 37 ℃ for 2 h;

[0068] (4) Washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0069] (5) Protein incubation: Dilute CK18 M30 recombinant protein with diluent (starting concentration of 2000 pg / mL, serially diluted 7+0 times), add 100 μL of antigen to each well, and incubate at 37 ℃ for 1.5 h;

[0070] (6) Washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min.

[0071] (7) Antibody incubation detection: HRP (horseradish peroxidase) labeled (antibody 2) was diluted to 0.1 μg / mL with diluent, 100 μL per well, and incubated at 37 °C for 1.5 h;

[0072] (8) Plate washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min.

[0073] (9) Color development: Add 100 µL of TMB color development solution to each well and incubate at 37 °C in the dark for 15 min;

[0074] (10) Termination: Add 100 µL of termination solution to each well and shake for 5-10 seconds;

[0075] (11) Reading: Turn on the microplate reader and preheat for 10 minutes. Set the wavelength of the microplate reader to 450 nm. Read the absorbance value (OD value) of each well within 30 minutes and record the data.

[0076] Table 1. Detection data of CK18 M30 protein standard curve

[0077]

[0078] Standard curve such as Figure 1 As shown. From Figure 1 As can be seen, the curve of the anti-CK18 M30 antibody exhibits typical linear characteristics, 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 CK18 M30 protein in clinical samples.

[0079] Example 3

[0080] This example demonstrates the performance and sensitivity verification of the antibody pair against the recombinant rabbit monoclonal antibody against CK18 M30. The experimental steps are as follows:

[0081] (1) Capture antibody coating: Dilute (antibody 1) with 1×PBS buffer to 1 μg / mL, corresponding to 100 μL per well, and incubate overnight at 4 ℃;

[0082] (2) Plate washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0083] (3) Blocking: Prepare 200 μL of 5% BSA blocking solution for each well and incubate at 37 ℃ for 2 h;

[0084] (4) Washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0085] (5) Protein incubation: Dilute CK18 M30 recombinant protein with diluent (starting concentration of 2000 pg / mL, serially diluted 7+0 times). Use blank control as diluent. Add 100 μL of antigen to each well and incubate at 37 °C for 1.5 h.

[0086] (6) Washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min.

[0087] (7) Antibody incubation detection: HRP (horseradish peroxidase) labeled (antibody 2) was diluted to 0.1 μg / mL with diluent, 100 μL per well, and incubated at 37 ℃ for 1.5 h;

[0088] (8) Plate washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min.

[0089] (9) Color development: Add 100 µL of TMB color development solution to each well and incubate at 37 °C in the dark for 15 min;

[0090] (10) Termination: Add 100 µL of termination solution to each well and shake for 5-10 seconds;

[0091] (11) Reading: Preheat the microplate reader for 10 minutes in advance. Set the wavelength to 450 nm. Read the absorbance (OD value) of each well within 30 minutes and record the data. Repeat the blank sample test 24 times. Calculate the mean and standard deviation of the 24 reactions. Calculate the OD value as mean + 2SD, which is 0.0696. Substitute this into y = 0.0014x - 0.0668 to calculate the concentration of X as 97.4 pg / ml. Figure 2 The detection method established in this invention demonstrates that the lowest detection limit is 97.4 pg / mL, exhibiting high detection sensitivity and meeting the detection requirements for low concentrations of M30 antigen. The method also demonstrates good linearity (R²>0.99, with R² closer to 1 indicating better linearity) within the CK18 M30 antigen concentration range of 97.4-2000 pg / mL, proving its accuracy and reliability. The actual concentration of CK18 M30 antigen can be precisely quantified through absorbance values.

[0092] Table 2 Standard Curve and Blank Sample Data

[0093]

[0094] Example 4

[0095] This example demonstrates the performance and specificity verification of the antibody pair against the recombinant rabbit monoclonal antibody against CK18 M30. The experimental steps are as follows:

[0096] (1) Capture antibody coating: Dilute (antibody 1) with 1×PBS buffer to 1 μg / mL, corresponding to 100 μL per well, and incubate overnight at 4 ℃;

[0097] (2) Plate washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0098] (3) Blocking: Prepare 200 μL of 5% BSA blocking solution for each well and incubate at 37 ℃ for 2 h;

[0099] (4) Washing: Set the plate washer to 300 μL of washing solution per well, wash 3 times, and soak for 1 min;

[0100] (5) Protein incubation: Dilute CK18 M30 recombinant protein with diluent (starting concentration of 2000 pg / mL, serially diluted 7+0 times). Add a certain concentration of ten influencing factors to high-value and low-value samples respectively, 100 μL per well, and incubate at 37 ℃ for 1.5 h. Add 100 μL of antigen to the corresponding wells respectively, and incubate at 37 ℃ for 1.5 h.

[0101] (6) Washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min.

[0102] (7) Antibody incubation detection: HRP (horseradish peroxidase) labeled (antibody 2) was diluted to 0.1 μg / mL with diluent, 100 μL per well, and incubated at 37 ℃ for 1.5 h;

[0103] (8) Washing: Set the plate washer to 300 μL of washing solution per well, wash 5 times, and soak for 1 min;

[0104] (9) Color development: Add 100 µL of TMB color development solution to each well and incubate at 37 °C in the dark for 15 min;

[0105] (10) Termination: Add 100 µL of termination solution to each well and shake for 5-10 seconds;

[0106] (11) Reading: Turn on the microplate reader and preheat for 10 minutes. Set the wavelength of the microplate reader to 450 nm. Read the absorbance value (OD value) of each well within 30 minutes and record the data.

[0107] Table 3. Experimental data on high-value sample interference

[0108]

[0109] Table 4. Specificity analysis of high-value samples

[0110]

[0111] Table 5. Experimental data on interference from low-value samples

[0112]

[0113] Table 6. Specificity analysis of low-value samples

[0114]

[0115] High-value sample standard curve as follows Figure 3 As shown, the standard curve for low-value samples is as follows: Figure 4As shown, the experimental results indicate that when seven influencing factors—CXCL11, IL-6, INF-γ, IL-33, IL-2, IL-10, and TIMP-1—were added to low- and high-value samples, the protein concentration was calculated by substituting the OD values ​​into the standard curve equation. The final results showed no significant difference (≤10%) compared to the high- and low-value sample controls. Specific data are shown in Tables 4 and 6. After adding CXCL11, IL-6, INF-γ, IL-33, IL-2, IL-10, and TIMP-1 to high- and low-value CK18 M30 samples, respectively, the detected CK18 M30 concentrations were ≤10% different from the control group without interfering substances. This demonstrates that the antibody can specifically recognize the CK18 M30 protein and has almost no cross-reactivity with structurally similar CXCL11, IL-6, INF-γ, IL-33, IL-2, IL-10, and TIMP-1, fully meeting the specificity requirements for clinical testing.

Claims

1. A recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment, characterized in that, The antibody pair includes antibody 1 and antibody 2; The amino acid sequence of the heavy chain variable region of antibody 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 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.

2. The anti-CK18 M30 fragment recombinant rabbit monoclonal antibody pair as described in claim 1, characterized in that, The antibody pair was obtained through recombinant expression in mammalian cells and is capable of specifically recognizing and binding to the CK18 fragment M30 in human serum.

3. The anti-CK18 M30 fragment recombinant rabbit monoclonal antibody pair as described in claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of antibody 1 is shown in SEQ ID NO: 5; the nucleotide sequence encoding the light chain variable region of antibody 1 is shown in SEQ ID NO: 6; The nucleotide sequence encoding the heavy chain variable region of antibody 2 is shown in SEQ ID NO: 7; the nucleotide sequence encoding the light chain variable region of antibody 2 is shown in SEQ ID NO:

8.

4. The application of the recombinant rabbit monoclonal antibody pair against the CK18 M30 fragment as described in any one of claims 1-3, characterized in that, This kit is used to prepare a reagent for detecting the level of CK18 fragment M30 in human serum.

5. The application of the recombinant rabbit monoclonal antibody against the CK18 M30 fragment as described in claim 4, characterized in that, The kit is used for sandwich ELISA detection.

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