Free nucleic acid ALU115 and ALU247 complete antigen, monoclonal antibody and application thereof
Producing protein conjugates and hybridoma technology through EDC/NHS method, monoclonal antibodies against ALU115 and ALU247 were developed, which solved the problems of low sensitivity and high cost of existing detection methods, and achieved efficient and accurate detection results. Clinical application provides an important basis.
Patent Information
- Application Number
- CN202411360355.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2024-12-27
AI Technical Summary
The existing methods for detecting ALU115 and ALU247 gene sequences have low sensitivity and high cost, and the traditional methods are cumbersome to operate. Since the ALU sequence is a hapten substance, it is difficult to stimulate the body to produce corresponding antibodies, affecting the accuracy of the detection results.
Protein conjugates were prepared by EDC/NHS method, and highly efficient specific antibodies against ALU115 and ALU247 were developed in combination with hybridoma technology. Monoclonal antibodies were obtained using complete antigens to improve the accuracy and reliability of the detection.
The high sensitivity detection of the existence of ALU115 and ALU247 provides a reliable diagnostic basis, provides accurate antibodies for clinical applications, and ensures the accuracy and reliability of the detection results.
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Figure CN119192335A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and more specifically to a free ALU115 and ALU247 complete antigen, a monoclonal antibody and applications thereof. Technical Background
[0002] Free nucleic acid DNA (cfDNA) consists of nuclear DNA fragments in the blood and originates from various cells in the body. DNA fragments released from apoptotic cells are usually 185 to 200 base pairs (bp) in length. In contrast, cfDNA fragments from necrotic cells or tumor cells are longer, indicating more disordered decomposition of DNA. Therefore, high levels of long DNA fragments can serve as potential markers of disease. The ratio of long DNA fragments from necrosis (ALU247) to short apoptotic fragments (ALU115) representing total cfDNA represents the level of cfDNA integrity. Therefore, the higher the ratio, the more conducive it is to the occurrence and development of the disease, making it a suitable candidate biomarker that can be used in clinical applications (an emerging field of non-invasive assessment), such as prenatal diagnosis, cancer, diabetes, cardiovascular and neurological diseases.
[0003] The cfDNA sequence contains a large number of ALU sequences, which are highly abundant primate-specific short interspersed nuclear elements, accounting for about 10% of the human genome. Because they are preferentially located in gene-rich regions, especially in introns and 3'UTRs, ALU elements can regulate the expression of host genes and neighboring genes, among which AUL115 and ALU247 are conducive to becoming effective marker genes for the occurrence and development of diseases. ALU sequences do not directly encode proteins, but play a key role in life processes such as gene expression regulation, cell proliferation, differentiation, and apoptosis. In recent years, with the rapid development of high-throughput sequencing technology, more and more ALU sequences have been discovered and confirmed to be closely related to the occurrence and development of various diseases. Therefore, it is particularly important to develop efficient research tools for ALU sequences, among which non-coding ALU sequence-specific antibodies are an indispensable part.
[0004] At present, the gene sequences of AUL115 and ALU247 are generally detected by traditional ultraviolet spectrophotometer and gel electrophoresis, which has low sensitivity and complicated operation. In addition, fluorescence PCR reaction is a means commonly used in disease detection, which has high sensitivity, but the test cost is also high. Immunological detection method is widely used in the detection of small molecule content as a rapid, specific and sensitive detection technology. Since the ALU sequence belongs to a hapten substance, it cannot stimulate the body to produce corresponding antibodies. Only after coupling with a macromolecular protein to prepare an ALU complete antigen can the animal be stimulated to produce an immune response, and it has immunogenicity. Therefore, the synthesis of ALU with strong immunogenicity is the key to obtaining anti-ALU monoclonal antibodies.
[0005] Currently, commonly used protein coupling technologies include biotin / streptavidin, DCC / NHS (dicyclohexylcarbodiimide / N-hydroxysuccinimide) and EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide). The following is a comparison of the advantages and disadvantages of these methods:
[0006] DCC method: DCC is a powerful carboxyl activator that can efficiently promote the formation of amide bonds. DCC has good solubility in organic solvents and is suitable for reactions in the organic phase. Although DCU (dicyclohexylurea) generated after the DCC reaction precipitates in some solvents, it has a high solubility and is difficult to completely remove, which may affect subsequent steps. DCC is insoluble in water, which limits its application in aqueous environments.
[0007] The biotin / streptavidin chemical binding system is widely used in biochemistry and molecular biology. The binding between biotin and streptavidin is highly specific, with a very low dissociation constant (approximately 10^-14 mol / L). This tight and specific binding is rapid and can withstand the extreme effects of pH, temperature, organic solvents and denaturants. However, it is expensive, and high concentrations of biotin in samples may interfere with detection methods based on the biotin / streptavidin system and interfere with the generation of immune responses, affecting the accuracy of the test results.
[0008] The present invention prepares a protein conjugate by screening the conditions of the EDC / NHS method. First, EDC is easily soluble in water and organic solvents, thereby avoiding the problem of low coupling efficiency caused by poor water solubility of DCC. Byproducts generated after the EDC reaction are relatively easy to remove by washing with water, thereby obtaining a relatively pure product, thereby avoiding contamination caused by poor water solubility of DCC and interference with the results caused by biotin / streptavidin. The presence of NHS can stabilize the intermediate generated after EDC activates the carboxyl group, thereby improving the coupling efficiency.
[0009] The present invention develops highly efficient and specific antibodies against specific non-coding ALU115 and ALU247 through hybridoma technology. The successful preparation of AUL115 and ALU247 antibodies is expected to be of great significance in technologies such as immunoluminescence, ELISA (enzyme-linked immunosorbent assay) and immunocolloidal gold. High-quality antibody preparation can ensure the accuracy and reliability of test results and provide an important basis for the diagnosis, prevention and control of diseases. Summary of the invention
[0010] The purpose of the present invention is to provide a free nucleic acid ALU115 and ALU247 complete antigen, obtain ALU115 and ALU247 monoclonal antibodies using the complete antigen and their application, so as to provide accurate antibodies for clinical prenatal diagnosis, cancer, diabetes, cardiovascular and nervous system diseases.
[0011] The specific technical solutions for achieving the above-mentioned invention objectives are as follows:
[0012] In one aspect, the present invention provides a complete antigen of free nucleic acid ALU115 and ALU247, wherein the complete antigen is formed by coupling ALU hapten with a protein carrier, and the ALU hapten is an ALU115 and ALU247 nucleic acid fragment containing an amino group label. The structure of the complete antigen is as follows Figure 1 As shown, the template sequence of ALU is shown in SEQ ID NO.1.
[0013] The method for preparing the complete antigens of ALU115 and ALU247 comprises the following steps:
[0014] (1) Add SYBR Green I Buffer Amix containing amino-labeled dUTP (the components include:
[0015] 20mM tris-SO4, 100mM KCL, 0.3mM betaine, 20% glycerol, 0.2mM dNTP, SYBR Green I and 0.5mM MgCL2) were introduced into the ALU115 gene sequence through primer amplification. The amplification system of ALU115 is as follows:
[0016]
[0017] The amplification procedure is as follows:
[0018]
[0019] Preferably, the specific primer sequences of the AUL115 gene are shown in SEQ ID No. 2-3.
[0020] SYBR Green Ⅰ Buffer Amix containing amino-labeled dUTP was introduced into the ALU247 gene sequence through primer amplification. The amplification system and amplification procedure of ALU247 were the same as those of ALU115.
[0021] Preferably, the specific primer sequences of the AUL247 gene are shown in SEQ ID No. 4-5.
[0022] (2) Purify the ALU115 and ALU247 nucleotide sequences using a gel recovery kit. After detection by a UV spectrophotometer, the DNA concentration is required to be no less than 40 ng / μL and the OD260 / OD280 ratio is required to be no less than 1.80.
[0023] (3) The bovine serum albumin was coupled to the ALU115 and ALU247 nucleic acid fragments using the EDC / NHS method. 20 mg BSA and 40 mg EDC were weighed and fully dissolved in 2 mL 0.1 M MES pH 5.0 buffer. 20 mg NHS was added. The mixture was stirred at 4°C in the dark for 15 min and then adjusted to pH 8 to obtain a reaction solution A. 4 mg of the purified ALU115 and ALU247 amplification products were added dropwise to the A solution (stirring while adding), sealed, protected from light, and stirred at 4°C overnight. After the above reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed with PBS at 4°C for 2 days. After the dialysis was completed, it was stored for future use.
[0024] In another aspect, the present invention provides monoclonal antibodies obtained by immunization with the complete antigen.
[0025] The preparation method of the ALU115 and ALU247 monoclonal antibodies comprises the following specific steps:
[0026] (1) Five BALB / c mice aged 4-6 weeks were selected for immunization. ALU115-BSA and ALU247-BSA were used as immunization antigens. 100 μg of antigen was weighed and emulsified with an equal volume of Freund's complete adjuvant (antigen concentration was 1 mg / ml) and then injected subcutaneously on the back. Two weeks later, an equal amount of antigen was emulsified with Freund's incomplete adjuvant and then injected subcutaneously on the back. Three immunizations were performed after an interval of 2 weeks, 100 μg / mouse, also emulsified with incomplete adjuvant.
[0027] (2) Two weeks after the third immunization, blood was collected from the eye sockets to separate the serum, and the titer of the mouse immune serum was measured by indirect ELISA, and mice with high titers were selected.
[0028] Preferably, unimmunized / immunized SPF-grade BALB / mice are bled out of the eye sockets and blood is collected. The killed mice are placed in 75% alcohol and soaked in a clean bench. The spleen of the mouse is removed aseptically, and the spleen is first ground with RPMI 1640 incomplete culture medium and then the excess tissue is filtered with a cell filter. After centrifugation at 1000rpm / min for 10min, the supernatant is discarded, and the cell pellet is resuspended with RPMI1640HAT culture medium, and 100μL is added to each well of a 96-well plate and cultured in a cell culture incubator.
[0029] (3) Fusion of high titer mouse spleen cells with SP2 / 0 myeloma cells. Add SP / 20 to spleen cells according to the ratio, centrifuge for 4 minutes, and discard the supernatant. Suspend the cell pellet and add 1 ml of PEG1500 fusion agent. Let it stand for 90 seconds to allow the fusion agent to take effect. Add RPMI 1640 to the centrifuge tube to terminate the fusion. Resuspend the cells with HAT medium, take out the 96-well plate, add 200 ml of the above cell solution to each well, and observe the growth of hybridoma cells every day.
[0030] (4) After 5 days of fusion, the medium is replaced with HAT / HT medium, and the replaced medium is used for indirect ELISA experiment to screen positive clones. Screen at least twice, and the hybridoma cells that are positive after two tests are transferred to 24-well plates for culture, and then transferred to T25cm cell flasks for expansion. After testing, the cells are frozen and called positive clone cells.
[0031] (5) Select the positive clone cells in the initial screening. After the cell count, aspirate 100 cells into 20 mL of HT culture medium. After mixing, take out the 96-well plate and add 100 μL to each well for culture. On the 8th to 10th day, test the tumor cells and select the positive clones for subcloning until all wells are tested positive.
[0032] (6) The selected hybridoma cell lines were transferred to cell culture flasks and expanded in RPMI 1640 / 10 complete medium. When the cells reached logarithmic growth, 1×10 50 μg / ml was injected into the peritoneal cavity of paraffin-sensitized multiparous mice. 6 After about 10 days, the abdomen of the mouse swelled, and the ascites was collected and the supernatant was collected by centrifugation. The ascites was purified according to the instructions of Protein G SefinoseTM Resin (Settled Resin) of Bioengineering.
[0033] The beneficial effects of the present invention are as follows: the present invention discloses for the first time a complete antigen of ALU115 and ALU247 and a monoclonal antibody obtained by immunization thereof; the complete antigen has a good immunization effect, and the obtained monoclonal antibody has a strong specificity, which provides a reliable method for promoting the clinical detection of ALU115 and ALU247; the monoclonal antibody of the present invention can detect the presence of ALU115 and ALU247 with high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The structural diagram of ALU15 and ALU247 binding to BSA.
[0035] Figure 2 This is a protein gel image of ALU15-BSA and ALU247-BSA coupling.
[0036] Figure 3 This is the inhibition rate curve of ALU115 and ALU247.
[0037] Figure 4 The amplification effects of ALU115 and ALU247 in different reaction systems; AF represents ALU115 reaction system 1, ALU115 reaction system 2, ALU115 reaction system 3, ALU247 reaction system 1, ALU247 reaction system 2, and ALU247 reaction system 3, respectively. DETAILED DESCRIPTION
[0038] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0039] Example 1 Preparation of ALU115 and ALU247 Complete Antigens
[0040] (1) Extraction of cfDNA from peripheral blood samples
[0041] A healthy human serum / plasma circulating DNA extraction kit (Novozyme, catalog number N903-01) was used to extract human peripheral blood DNA. After the peripheral blood DNA was tested by UV spectrophotometer, the DNA concentration was required to be no less than 40 ng / μL and the OD260 / OD280 ratio was required to be no less than 1.80;
[0042] (2) Preparation of amino-labeled ALU115 and ALU247 nucleic acid fragments
[0043] SYBR Green I Buffer Amix containing amino-labeled dUTP (Thermo: Product No. R1101) was used to introduce amino-labeled dUTP into the ALU115 gene sequence through primer amplification. The amplification system and amplification procedure of ALU115 are as follows:
[0044]
[0045]
[0046] The SYBR Green I Buffer Amix containing amino-labeled dUTP was used to introduce the amino-labeled dUTP into the ALU247 gene sequence through primer amplification. The amplification system and amplification procedure of ALU247 were the same as above. (3) Gel recovery of amino-labeled ALU115 and ALU247 nucleic acid sequences
[0047] The ALU115 and ALU247 nucleotide sequences were purified by gel recovery kit (Tian Gen, catalog number: DP209). After detection by UV spectrophotometer, the DNA concentration was required to be no less than 40 ng / μL and the OD260 / OD280 ratio was required to be no less than 1.80.
[0048] (4) ALU115 and ALU247 conjugated to BSA
[0049] The bovine serum albumin was coupled to the ALU115 and ALU247 nucleic acid fragments by the EDC / NHS method. 20 mg BSA and 40 mg EDC were weighed and fully dissolved in 2 mL 0.1 M MES pH 5.0 buffer. 20 mg NHS was added, and the mixture was stirred at 4°C in the dark for 15 min, and then the pH was adjusted to 8 to obtain a reaction solution A. 4 mg of the purified ALU115 and ALU247 amplification products were added dropwise to the A solution (stirring while adding), and the solution was sealed, protected from light, and stirred at 4°C overnight. After the above reaction was completed, the reaction solution was transferred to a dialysis bag, dialyzed with PBS at 4°C for 2 days, and stored for later use after the dialysis was completed.
[0050] (5) Gel electrophoresis results and content detection of ALU115 and ALU247 whole antigens
[0051] Identify the prepared concentrated gel with a concentration of 5% and the separation gel with a concentration of 12% by SDS-PAGE gel electrophoresis. Weigh a certain amount of BSA, ALU115-BSA, and ALU247-BSA, dilute and mix with 5 times the concentration of sample buffer respectively. Heat the mixed sample and protein marker in boiling water for 5 to 8 minutes, centrifuge at 12000r / min in a centrifuge for 3-5 minutes, and use it as the sample to be tested. Load 10μL per well, use 90V voltage, and use 120V separation gel voltage. After electrophoresis, stain with Coomassie Brilliant Blue staining solution. Refer to the instructions of the Coomassie Brilliant Blue protein content test kit to detect the protein content of the prepared complete antigen ALU115-BSA and ALU247-BSA.
[0052] The results showed that: SDS-PAGE gel electrophoresis identification results were as follows: Figure 2 As shown in the figure, it can be seen that the electrophoresis bands of the artificial coupling products ALU115-BSA and ALU247-BSA are somewhat lagging compared with the bands of the carrier protein BSA, which preliminarily indicates that the hapten small molecule is successfully coupled to the carrier protein. The protein concentrations of the prepared complete antigens ALU115-BSA and ALU247-BSA were measured by the Coomassie Brilliant Blue protein content test kit to be 5.2 mg / ml and 4.3 mg / ml, respectively.
[0053] Example 2 Preparation of ALU115 and ALU247 Antibodies
[0054] (1) Immunization of animals
[0055] ① Five BALB / c mice aged 4-6 weeks were selected for immunization. ALU115-BSA and ALU247-BSA were used as immunization antigens. 100 μg of antigen was weighed respectively and emulsified with an equal volume of Freund's complete adjuvant (antigen concentration was 1 mg / ml), and then injected subcutaneously in the back. After 2 weeks, an equal amount of antigen was emulsified with Freund's incomplete adjuvant and then injected subcutaneously in the back. After an interval of 2 weeks, three immunizations were performed, 100 μg / mouse, also emulsified with incomplete adjuvant.
[0056] ② Two weeks after the three immunizations, isolate the serum by collecting blood from the eye sockets, determine the titer of the mouse immune serum by indirect ELISA, and select mice with high titer.
[0057] (2) Fusion and screening of hybridoma cells
[0058] ① Take unimmunized / immunized SPF-grade BALB / mice and bleed them from their orbits to collect blood. Soak the killed mice in 75% alcohol and move them to a clean bench. Remove the spleen of the mice aseptically, grind it with 1640 incomplete culture medium, and filter the excess tissue with a cell filter. Centrifuge at 1000rpm / min for 10min and discard the supernatant. Resuspend the cell pellet with 1640HAT culture medium and spread it on a 96-well plate, 100μL per well. Culture the cells in a 37℃, 5% CO2 cell culture incubator.
[0059] ③ One week before fusion, resuscitate SP2 / 0 cells and passage. When the cells are in good condition and grow to 80% to 90%, discard the culture medium, wash the cells twice with incomplete RPMI 1640, centrifuge for 10 minutes, resuspend the precipitate in 5mL incomplete RPMI 1640, and count.
[0060] ④ Fusion of high titer mouse spleen cells with SP2 / 0 myeloma cells at a cell number ratio of 8:1, adding SP / 20 to spleen cells, centrifuging at 1000r for 4min, and discarding the supernatant. Suspend the cell pellet and add 1ml PEG1500 fusion agent. Add to cells within 1min, and let stand for 90s after addition to allow the fusion agent to take effect. Add RPMI 1640 to the centrifuge tube to terminate the fusion. Resuspend the cells with 3ml HAT medium, add to 40ml HAT medium respectively, take out the 96-well plate, and add 200mL to each well.
[0061] ⑤ After 5 days of fusion, replace the medium with HAT / HT medium, and use the replaced medium for indirect ELISA experiment to screen positive clones. Screen at least twice, and transfer the hybridoma cells that are positive after two tests to 24-well plates for culture, and then transfer to T25cm cell flasks for expansion. After testing, the cells are frozen and called positive clone cells.
[0062] ⑥ Select the positive clone cells in the initial screening. After the cell count, aspirate 100 cells into 20mL HT culture medium. After mixing, take out the 96-well plate, add 100μL cell solution to each well and culture in a 37℃, 5% CO2 cell culture incubator. On the 8th-10th day, test the tumor cells, select the positive clones and continue subcloning until all the wells are tested positive.
[0063] ⑦ The selected hybridoma cell lines were transferred to cell culture flasks and expanded in RPMI 1640 / 10 complete medium. When the cells reached logarithmic growth, 1×10 / ml of the culture medium was injected into the peritoneal cavity of paraffin-sensitized multiparous mice. 6 After about 10 days, the abdomen of the mouse swelled, and the ascites was collected and the supernatant was collected by centrifugation. The ascites was purified according to the instructions of Protein G SefinoseTM Resin (Settled Resin) of Bioengineering.
[0064] The results showed that the titer of polyclonal antibody serum was determined by indirect ELISA. The ratio of the OD450nm of the antibody to be tested to the blank control (Positive / negative, P / N) was calculated. When P / N was greater than 2, it was positive, and when P / N was less than 2, it was negative. The positive result with the largest dilution multiple was taken as the measured antibody titer. As shown in Table 1, the titers of polyclonal antibody serum of 5 mice immunized with ALU115 and ALU247 reached 1:64000 and 1:32000, respectively, indicating that the prepared artificial antigens ALU115-BSA and ALU247-BSA have good immunogenicity.
[0065] Table 1 ALU115 and ALU247 antibody titers
[0066]
[0067]
[0068] Example 3 The immune effects of ALU115-BSA and ALU247-BSA under different coupling conditions
[0069] After purification of amino-labeled ALU115 and ALU247 using a gel recovery kit, protein coupling was performed using the following method:
[0070] Method 1: Couple bovine serum albumin to ALU115 and ALU247 nucleic acid fragments, weigh 20 mg BSA and 40 mg EDC, fully dissolve them in 2 mL 0.1 M MES pH 5.0 buffer, add 20 mg NHS, stir and react at 4°C in the dark for 15 minutes, then adjust to pH 8 to obtain the reaction solution A; add 4 mg of purified ALU115 and ALU247 amplification products dropwise to solution A (stirring while adding), seal, protect from light, and stir overnight at 4°C; after the above reaction is completed, transfer the reaction solution to a dialysis bag, dialyze with PBS at 4°C for 2 days, and store for later use after dialysis is completed.
[0071] Method 2: Couple bovine serum albumin to ALU115 and ALU247 nucleic acid fragments, weigh 20 mg BSA and 40 mg EDC, fully dissolve them in 2 mL 0.1 M MES pH 6.0 buffer, add 20 mg NHS, stir and react at 4°C in the dark for 15 minutes, then adjust to pH 7 to obtain a reaction solution A; add 4 mg of purified ALU115 and ALU247 amplification products dropwise to solution A (stirring while adding), seal, protect from light, and stir overnight at 4°C; after the above reaction is completed, transfer the reaction solution to a dialysis bag, dialyze with PBS at 4°C for 2 days, and store for later use after dialysis is completed.
[0072] Method three: Couple bovine serum albumin to ALU115 and ALU247 nucleic acid fragments, weigh 20 mg BSA and 40 mg EDC, fully dissolve them in 2 mL 0.1 M MES pH 5.0 buffer, add 10 mg NHS, stir and react at 4°C in the dark for 15 minutes, then adjust to pH 8 to obtain the reaction solution A; add 4 mg of purified ALU115 and ALU247 amplification products dropwise to solution A (stirring while adding), seal, protect from light, and stir overnight at 4°C; after the above reaction is completed, transfer the reaction solution to a dialysis bag, dialyze with PBS at 4°C for 2 days, and store for later use after dialysis is completed.
[0073] The above-obtained antigens were used for animal immunization. Five BALB / c mice aged 4-6 weeks were selected for immunization. ALU115-BSA and ALU247-BSA were used as immunization antigens. 100 μg of antigen was weighed and emulsified with an equal volume of Freund's complete adjuvant (antigen concentration was 1 mg / ml) and injected subcutaneously on the back; after 2 weeks, an equal amount of Freund's incomplete adjuvant was used to emulsify the antigen and inject it subcutaneously on the back; after an interval of 2 weeks, three immunizations were performed, 100 μg / mouse. After 2 weeks of the three immunizations, serum was separated by orbital blood sampling, and the titer of mouse immune serum was determined by indirect ELISA, and mice with high titers were detected.
[0074] As shown in Table 2, the antibody titers produced by the antigens of ALU115 and ALU247 prepared under the conditions of method 1 were better than those of methods 2 and 3. The antigens of ALU115 and ALU247 produced under the conditions of method 1 produced titers of 1:64000 and 1:32000 in 5 mice, respectively. The antigens of ALU115 produced under the conditions of method 2 produced titers of 1:8000 in mice No. 1 and 2, and 1:16000 in mice No. 3, 4 and 5, respectively. The antigens of ALU247 produced under the conditions of method 2 produced titers of 1:800 in mice No. 1, 2, 4 and 5, and 1:16000 in mice No. 3. The antigen produced by ALU115 under the conditions of method 3 produced a titer of 1:4000 in mice 1 and 3, and a titer of 1:8000 in mice 2, 4 and 5. The antigen produced by method 3 produced a titer of 1:2000 in mice 1, 2 and 4, and a titer of 1:4000 in mice 3 and 5. The above results show that the whole antigen prepared by method 1 has a better immune effect.
[0075] Table 2 ALU115 and ALU247 antibody titers under different coupling conditions
[0076]
[0077]
[0078] Example 4 Amplification effects of ALU115 and ALU247 in different amplification reaction systems and the immune effects of their antigens
[0079] (1) Extraction of cfDNA from peripheral blood samples
[0080] A healthy human serum / plasma circulating DNA extraction kit (Novozyme, catalog number N903-01) was used to extract human peripheral blood DNA. After the peripheral blood DNA was tested by UV spectrophotometer, the DNA concentration was required to be no less than 40 ng / μL and the OD260 / OD280 ratio was required to be no less than 1.80;
[0081] (2) Preparation of amino-labeled ALU115 and ALU247 nucleic acid fragments
[0082] SYBR GreenⅠBuffer A mix, SYBR GreenⅠBuffer B mix and SYBR GreenⅠBuffer C mix containing amino-labeled dUTP were prepared, wherein the components of SYBR GreenⅠBuffer B Mix are: 20mM tris-SO4, 100mM KCL, 0.2mM betaine, 20% glycerol, 0.2mM dNTP and SYBR GreenⅠ and 0.5mM MgCL2. The components of SYBR GreenⅠBuffer C Mix are: 20mM tris-SO4, 150mM KCL, 0.2mM betaine, 20% glycerol, 0.2mM dNTP and SYBR GreenⅠ, 15mM TMAC and 0.5mM MgCL2. The amino-labeled dUTP was introduced into the ALU115 gene sequence according to the following different reaction systems. The amplification system and amplification procedure of ALU115 are as follows:
[0083]
[0084]
[0085] SYBR GreenⅠBuffer A mix, SYBR GreenⅠBuffer B mix and SYBR GreenⅠBuffer C mix containing amino-labeled dUTP were used to introduce amino-labeled dUTP into the ALU247 gene sequence according to the following different reaction systems. The amplification system and amplification procedure of ALU247 are as follows:
[0086]
[0087]
[0088] The amplified ALU115 and ALU247 were purified by a gel recovery kit and then protein coupled by the following method: bovine serum albumin was coupled to the ALU115 and ALU247 nucleic acid fragments, 20 mg BSA and 40 mg EDC were weighed and fully dissolved in 2 mL 0.1 M MES pH 5.0 buffer, 20 mg NHS was added, and the mixture was stirred at 4°C in the dark for 15 min, and then the pH was adjusted to 8 to obtain a reaction solution A; 4 mg of the purified ALU115 and ALU247 amplification products were added dropwise to the A solution (stirring while adding), sealed, protected from light, and stirred at 4°C overnight; after the above reaction was completed, the reaction solution was transferred to a dialysis bag, dialyzed with PBS at 4°C for 2 days, and stored for later use after the dialysis was completed.
[0089] The antigens obtained above were used for animal immunization: 5 BALB / c mice aged 4-6 weeks were selected for immunization, ALU115-BSA and ALU247-BSA were used as immunization antigens, 100 μg of antigens were weighed and emulsified with an equal volume of Freund's complete adjuvant (antigen concentration was 1 mg / ml), and then injected subcutaneously on the back; after 2 weeks, an equal amount of antigens were emulsified with Freund's incomplete adjuvant and injected subcutaneously on the back; after an interval of 2 weeks, three immunizations were performed, 100 μg / mouse. After 2 weeks of the three immunizations, serum was separated by orbital blood sampling, the titer of mouse immune serum was determined by indirect ELISA, and mice with high titers were selected.
[0090] The results are as follows Figure 4 As shown in Table 3, the fluorescence intensity of the amplification effect of ALU115 and ALU247 under the conditions of reaction system 1 is stronger and the Ct value is smaller, indicating that the amplification effect of PCR reaction system 1 is better than that of reaction system 2 and reaction system 3. After ALU115 and ALU247 produced in different reaction systems were coupled by the above-mentioned protein coupling method, the immune effects of ALU115 and ALU247 produced under different reaction system conditions were detected. The results are shown in Table 3. The antibody titers produced by the antigens prepared under the conditions of reaction system 1 are better than those of reaction system 2 and reaction system 3. The antigens produced under the conditions of reaction system 1 produce titers of 1:64000 and 1:32000 in 5 mice, respectively. The antigens of ALU115 and ALU247 produced under the conditions of reaction system 2 produced titers of 1:16000 and 1:16000 in 5 mice, respectively; the antigens of ALU115 and ALU247 produced under the conditions of reaction system 2 produced titers of 1:8000 and 1:4000 in 5 mice, respectively. The above results show that the full antigens obtained under the amplification conditions of reaction system 1 have a better immune effect.
[0091] Table 3 The titers of ALU115 and ALU247 antibodies under different amplification systems
[0092]
[0093]
[0094] Example 5 Immune response test of ALU115 and ALU247 antibodies
[0095] The sensitivity of the antibody was detected by indirect competitive ELISA. ALU115-BSA and ALU247-BSA were used as coating antigens, with a coating amount of 300 ng / well, incubated overnight at 4°C, washed 6 times with PBST, blocked with blocking solution at 37°C for 2h, and washed 6 times with PBST. The gradient dilution concentrations of ALU115-BSA and ALU247-BSA antigens were 16ug / mL, 8ug / mL, 4ug / mL, 2ug / mL, 1ug / mL, 0.5ug / mL, 0.25ug / mL, and 0.125ug / mL, respectively, and mixed with equal volumes of ALU115 and ALU247 monoclonal antibodies diluted 1:1000, and 100uL of the mixture was added to each well, and a negative control (NC) was set at the same time. Add 100uL 1:4000 diluted goat anti-mouse IgG secondary antibody and incubate at 37℃ for 30min, develop color with TMB for 8min, terminate the reaction with 2mmol / LH2SO4, and measure the absorbance at OD450nm.
[0096] The results are shown in Table 4. The sensitivity of the antibody was determined by indirect competitive ELISA. The addition of ALU115 and ALU115 inhibited the binding of ALU115 and antibody ALU247 to the coated antigen. The inhibition rate curve was drawn with the logarithmic value of the mass concentration of added ALU115 and ALU247 as the horizontal axis and the inhibition rate as the vertical axis. Figure 3 As shown, the linear equations of the inhibition rate curves of ALU115 and ALU247 are: y = -39.394x + 54.393 and y = -44.803x + 60.758, respectively. The inhibition rate is the OD450 nm value B0 of the control well, and the OD450 nm value of the well with added antigen is B. The ratio of B / B0 is called the inhibition rate. When the inhibition rate of ALU115 and ALU247 is 50%, the corresponding mass concentration is IC50, which is 1.271ug / mL and 1.806ug / mL respectively. The half inhibition of IC50 is used to measure the sensitivity of the antibody. The lower the half inhibition, the higher the sensitivity of the antibody. This result further proves that the sensitivity of ALU115 and ALU247 antibodies in this test is good.
[0097] Table 4 ALU115 and ALU247 antibody sensitivity test
[0098]
[0099] The cross-reaction of the antibody was detected by indirect competitive ELISA method, and human gene structural analogs ACE, CYP2C9, CYP2D6 and ADRB1 were selected for cross-reaction. The IC50 of this gene was determined by the same indirect competitive ELISA method, and the IC50 of ALU115 and ALU247 was calculated and compared with the IC50 values of other genes to calculate the cross-reaction rate. According to the formula CR = IC50 (ALU115 or ALU247) / IC50 (others) * 100%.
[0100] According to the results shown in Table 5, the cross-reaction rates of ALU115 and ALU247 monoclonal antibodies for ACE, CYP2C9, CYP2D6 and ADRB1 genes were all less than 0.01, indicating that the antibodies have good specificity.
[0101] Table 5 Cross-test of ALU115 and ALU247 antibodies
[0102]
Claims
1. A free nucleic acid ALU115 and ALU247 complete antigen, characterized in that: The complete antigen is formed by coupling the ALU hapten with a protein carrier, and the ALU hapten is respectively an ALU115 nucleic acid fragment and an ALU247 nucleic acid fragment containing an amino group label.
2. The free nucleic acid ALU115 and ALU247 complete antigen according to claim 1, characterized in that: The protein carrier is bovine serum albumin (BSA).
3. The method for preparing the free nucleic acid ALU115 and ALU247 complete antigens as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) Introducing amino-modified dUTP into the ALU115 and ALU247 gene sequences respectively through primer amplification; (2) purifying ALU115 and ALU247 nucleotide sequences using a gel recovery kit; (3) The purified ALU115 and ALU247 nucleic acid fragments were coupled to bovine serum albumin using the EDC / NHS method to obtain ALU115-BSA and ALU247-BSA.
4. The preparation method according to claim 3, characterized in that: In step (1), the PCR reaction system is: 12.5 μL SYBR Green I Buffer Amix; 0.5 μL taq enzyme; 0.8 μL each of the upstream and downstream primers of ALU115 and AUL247; 2 μL of genomic DNA sample; and sterile water is added to 25 μL.
5. The preparation method according to claim 4, characterized in that: The composition of the SYBR Green Ⅰ Buffer Amix includes: 20mM tris-SO4, 100mM KCL, 0.3mM betaine, 20% glycerol, 0.2mM dNTP, SYBR Green Ⅰ and 0.5mM MgCL2.
6. The preparation method according to claim 3, characterized in that: In step (1), the specific primer sequences for amplifying the AUL115 gene are shown in SEQ ID Nos. 2 to 3, and the specific primer sequences for amplifying the AUL247 gene are shown in SEQ ID Nos. 4 to 5.
7. The preparation method according to claim 3, characterized in that: In step (3), the specific coupling method is: weigh 20 mg BSA and 40 mg EDC, dissolve them in 2 mL 0.1 M MES pH 5.0 buffer, add 20 mg NHS, stir and react at 4 ° C in the dark for 15 minutes, and then adjust the pH to 8 to obtain a reaction solution A; add 4 mg of the purified amplification products of ALU115 and ALU247 obtained in step (2) dropwise to the A solution, seal, protect from light, and stir overnight at 4 ° C; after the above reaction is completed, transfer the reaction solution to a dialysis bag, dialyze with PBS at 4 ° C for 2 days, and store it for future use after the dialysis is completed.
8. The monoclonal antibody obtained by immunization with the complete antigen as claimed in claim 1 or 2.
9. The monoclonal antibody according to claim 8, characterized in that: The method for preparing the monoclonal antibody comprises the following steps: ①Animal Immunity Test Five BALB / c mice aged 4-6 weeks were selected for immunization. ALU115-BSA and ALU247-BSA were used as immunization antigens. 100 μg of antigens with an antigen concentration of 1 mg / ml were weighed and emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously on the back. After 2 weeks, an equal amount of antigens were emulsified with Freund's incomplete adjuvant and injected subcutaneously on the back. Three immunizations were performed after an interval of 2 weeks, 100 μg / mouse. After 2 weeks of the three immunizations, blood was collected from the eye sockets to separate serum. ② Screening of hybridoma cells and preparation of antibodies The titer of mouse immune serum was determined by indirect ELISA, and mice with high titer were selected. The spleen cells were fused with SP2 / 0 myeloma cells. The positive hybridoma cell lines were screened by three rounds of subcloning. The screened hybridoma cell lines were transferred to cell culture flasks and expanded in RPMI1640 / 10 complete medium in the cell culture flasks. When the cells reached the logarithmic growth state, 1×10 per ml of paraffin-sensitized multiparous mice were injected into the peritoneal cavity. 6 cells, and wait until the mouse abdomen swells, collect the ascites, and collect the supernatant by centrifugation.
10. Use of the complete antigen according to claim 1 and the monoclonal antibody according to claim 8 in the preparation of products for detecting ALU115 and ALU247.