Immune encephalopathy early detection method based on serum marker detection

Abstract

The invention discloses an early detection method for immune encephalopathy based on serum marker detection, and relates to the technical field of medical laboratory science, and the early detection method comprises the following steps: screening antigen epitopes by using technologies such as proteomics, constructing a phage antibody library, carrying out panning and mutation to obtain a specific antibody, constructing a nucleic acid library, screening and treating nucleic acid aptamers, and carrying out quantitative analysis on the nucleic acid aptamers. The preparation method comprises the following steps: synthesizing nanogold and magnetic nanoparticles by adopting a reduction method and a coprecipitation method, modifying a nanomaterial to obtain a compound, mixing the compound to form a probe, and optimizing a detection system to complete preparation. A bacteriophage antibody library is constructed, and an antibody with strong specificity and high affinity is prepared through multiple rounds of panning and site-directed mutagenesis optimization, so that the problems of poor specificity and poor sample enrichment effect of a detection reagent in the prior art are effectively solved, the detection sensitivity and specificity are remarkably improved, and early low-concentration markers of immune encephalopathy can be accurately detected.

Description

Technical Field

[0001] The present invention relates to the technical field of medical laboratory testing, and in particular to an early detection method for immune encephalopathy based on serum marker detection. Background Art

[0002] Immune encephalopathy is a type of disease caused by a dysfunctional immune system that mistakenly attacks the central nervous system. The pathogenesis of these diseases is complex, involving abnormal activity of multiple immune cells and molecules, leading to pathological changes in the central nervous system, such as inflammation and damage. With the deepening of medical research, the incidence of immune encephalopathy has gradually attracted attention. Because it can cause severe neurological dysfunction, such as cognitive impairment, epileptic seizures, and psychiatric symptoms, which seriously affect patients' quality of life and prognosis, early detection and intervention are particularly important.

[0003] There are certain defects in the existing technology. The first is the problem of poor specificity. The current reagents used to detect serum markers have cross-reaction problems, and the specific recognition ability of immune encephalopathy-related markers is not strong enough. During the detection process, they are easily affected by other similar substances or interfering factors, resulting in false positive results. This will not only mislead clinical testing, but may also cause patients to undergo unnecessary further examinations and treatments, increase the burden on patients and waste medical resources. The second problem is the poor sample enrichment effect. For serum markers with extremely low content, the existing enrichment materials and reagents cannot achieve efficient enrichment, which makes it difficult to accurately detect the markers due to their low concentration. For example, some small molecule metabolites related to immune encephalopathy are rare in serum, and existing enrichment technologies cannot effectively enrich them to detectable levels, limiting the sensitivity of early detection. For this reason, we propose an early detection method for immune encephalopathy based on serum marker detection. Summary of the Invention

[0004] The purpose of the present invention is to provide an early detection method for immune encephalopathy based on serum marker detection.

[0005] To solve the problems raised in the above background technology, the present invention provides the following technical solution: an early detection method for immune encephalopathy based on serum marker detection, the detection method comprising the following specific steps:

[0006] Step 1: Use proteomics technology to analyze specific antigens of immune encephalopathy, and with the help of bioinformatics databases and computer simulation technology, screen out highly specific antigenic epitopes, then construct a phage antibody library, fix the antigenic epitopes on an ELISA plate, add the phage antibody library to each well of the ELISA plate, and perform multiple rounds of panning to screen out single-chain antibody fragments with highly specific recognition capabilities for immune encephalopathy-related markers. Then, use site-directed mutagenesis technology to mutate the amino acid sites of the screened single-chain antibody fragments to construct a mutant library, screen out antibody variants, and then clone the screened antibody variant genes into expression vectors, transform them into Escherichia coli competent cells, and induce expression to obtain specific antibodies;

[0007] Step 2: Construct a random single-stranded nucleic acid library, target immune encephalopathy markers, incubate with the nucleic acid library, and perform multiple rounds of screening through filtration, elution, and PCR amplification to enrich nucleic acid aptamers with high affinity and specificity for the target. The nucleic acid aptamers are then modified, purified, and sequence optimized, and stability tested to prepare nucleic acid aptamers.

[0008] Step 3: Add ultrapure water to a round-bottom flask, heat to boiling, quickly add chloroauric acid solution, stir evenly, then add sodium citrate solution, continue stirring and keep boiling, and then cool to room temperature to obtain nanogold solution, and then synthesize magnetic nanoparticles by chemical coprecipitation method to complete the preparation of nanomaterials;

[0009] Step 4: The gold nanoparticle solution is centrifuged and then redispersed, and then a specific antibody is added for incubation. After that, a bovine serum albumin solution is added to block the nonspecific binding sites. The modified gold nanoparticle antibody complex is then collected by centrifugation. The magnetic nanoparticles are amino-treated, activated, and then incubated with aptamers to obtain the modified magnetic nanoparticle-aptamer complex, thus completing the surface modification of the nanomaterial.

[0010] Step 5: Mix the modified gold nanoparticle antibody complex and the magnetic nanoparticle nucleic acid aptamer complex, add detection buffer to form a mixed detection probe, and then incubate the serum sample of the immune encephalopathy patient with the mixed detection probe to allow the probe to fully bind to the marker in the serum. Detect the marker and probe binding signal through fluorescence detection and magnetic detection methods, determine the optimal detection conditions, and complete the construction and optimization of the detection system.

[0011] As a further solution of the present invention: In step 1, 10-15 specific antigens of immune encephalopathy are analyzed using proteomics technology, and 5-7 highly specific antigen epitopes are screened out with the help of bioinformatics database and computer simulation technology, and a capacity of 1.0×10 12 -3.0×10 12The phage antibody library was prepared, and the antigen epitope with a concentration of 8μg / mL-12μg / mL was fixed to the ELISA plate. After adding 100μL-150μL of phage antibody library to each well, the plates were incubated at 35℃-38℃ for 1.5h-2.5h. After 3-5 rounds of panning, the unbound phages were eluted with 0.08mol / L-0.12mol / L glycine-HCl buffer with a pH value of 2-2.5 after each round of panning, and then neutralized with 0.8mol / L-1.2mol / L Tris-HCl buffer with a pH value of 9-9.5 to screen out single-chain antibody fragments with highly specific recognition ability for immune encephalopathy-related markers.

[0012] As a further solution of the present invention: In the step 1, the amino acid sites of the screened single-chain antibody fragments were mutated by site-directed mutagenesis technology, and the mutations were introduced by PCR. The amount of template DNA used in the PCR reaction system was 40ng-60ng, 8pmol-12pmol of upstream and downstream primers, 180μM-220μM of dNTPs, 1IU-2IU of TaqDNA polymerase, 4μL-6μL of 10×PCR buffer, and ddH2O was added to 40μL-60 μL, PCR reaction conditions were: 94℃-96℃ pre-denaturation for 4min-6min, 94℃-96℃ denaturation for 25s-35s, 54℃-56℃ annealing for 25s-35s, 71℃-73℃ extension for 0.8min-1.2min, for a total of 28-32 cycles, and then 71℃-73℃ final extension for 8min-12min. Then, a mutant library was constructed and the binding affinity of the markers related to immune encephalopathy was screened by enzyme-linked immunosorbent assay to reach a Kd value of 10 -9 M-10 -11 The optimized antibody variant gene was cloned into the expression vector pET-28a, transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in LB medium containing 40 μg / mL-60 μg / mL kanamycin at 36°C-38°C and 180r / min-220r / min with shaking until the OD 600 When the RT-PCR reaction temperature is 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.4 mM-0.6 mM, induce expression at 16° C.-20° C. for 14 h-18 h, and complete the preparation of specific antibodies.

[0013] As a further solution of the present invention: In the step 2, the development of nucleic acid aptamers is to construct a 1.0×10 15 -1.5×10 15Random single-stranded nucleic acid libraries of different sequences were prepared, targeting immune encephalopathy markers at a concentration of 0.8 μM-1.2 μM, and dissolved in binding buffer. The binding buffer had a pH of 7.3-7.7 and included 20 mM-25 mM Tris-HCl, 90 mM-110 mM NaCl, and 1.5 mM-2.5 mM MgCl2. The libraries were incubated with the nucleic acid library at 35°C-38°C for 25 min-35 min, and unbound nucleic acids were removed by filtration through a nitrocellulose membrane. The bound nucleic acids were eluted and then amplified by PCR. The PCR reaction system included 8 μL-12 μL template nucleic acid, 8 pmol-1 of each of the upstream and downstream primers. 2 pmol, dNTPs concentration 180 μM-220 μM, Taq DNA polymerase 1 IU-2 IU, 10× PCR buffer 4 μL-6 μL, add ddH2O to 40 μL-60 μL, PCR reaction conditions: 94℃-96℃ pre-denaturation 4 min-6 min, 94-96℃ denaturation 25s-35s, 56℃-60℃ annealing 25s-35s, 71℃-73℃ extension 0.8min-1.2min, a total of 28-32 cycles, 71℃-73℃ final extension 8min-12min, after 8-10 rounds of screening, nucleic acid aptamers with high affinity and specificity for the target were enriched.

[0014] As a further embodiment of the present invention: In step 2, a carboxyfluorescein marker is introduced at the 5' end of the screened nucleic acid aptamer, and the nucleic acid aptamer is reacted at a molar ratio of 1:4-6 in a buffer containing 90mM-110mM 4-hydroxyethylpiperazineethanesulfonic acid, 8mM-12mM dithiothreitol, and 0.8-1.2mM ethylenediaminetetraacetic acid at 35°C-38°C for 1.5h-2.5h. The reaction is purified by polyacrylamide gel electrophoresis, the sequence is optimized by computer-aided design, and a stability test is performed in a 40mM-60mM sodium chloride, 8mM-12mM Tris-HCl buffer at 23°C-27°C to ensure stability within 20h-28h, thereby completing the preparation of the nucleic acid aptamer.

[0015] As a further embodiment of the present invention: in the step three, nanogold is synthesized by a sodium citrate reduction method, 80 mL to 100 mL of ultrapure water is added to a 100 mL to 120 mL round-bottom flask, the mixture is heated to boiling, 0.8 mL to 1.2 mL of a 0.8% to 1.2% chloroauric acid solution is added, the mixture is stirred evenly, and 2.5 mL to 3.5 mL of a 0.8% to 1.2% sodium citrate solution is added dropwise, the mixture is stirred continuously and kept boiling for 12 min to 18 min until the color of the solution turns wine red, and the mixture is then cooled to room temperature to obtain a nanogold solution.

[0016] As a further scheme of the present invention: in the step three, a chemical coprecipitation method is subsequently used to synthesize magnetic nanoparticles Fe3O4, 0.04-0.06 mol of FeCl2·4H2O and 0.08 mol-0.12 mol of FeCl3·6H2O are dissolved in 80-120 mL of deionized water, heated to 75°C-85°C under nitrogen protection and stirred evenly, 20 mL-30 mL of 20%-30% ammonia water is slowly added dropwise, and after the addition is complete, the stirring reaction is continued for 0.8 h-1.2 h, and then the magnetic nanoparticles are separated by a magnet, washed with deionized water and anhydrous ethanol for 3-5 times respectively until the pH value of the washing liquid is 6.8-7.2, and then dried in a vacuum drying oven at 55°C-65°C for 10 h-14 h to obtain magnetic nanoparticles, thereby completing the preparation of the nanomaterial.

[0017] As a further solution of the present invention: in the step 4, the prepared nanogold solution is centrifuged at 7000r / min-9000r / min for 8min-12min, the supernatant is discarded, and the precipitate is redispersed in a potassium carbonate buffer solution with a pH value of 8.8-9.2 and containing 0.08M-0.12M, and then the specific antibody is added, the antibody and nanogold molar ratio is 1:40-60, and the mixture is stirred and incubated at 4°C-5°C for 10h-14h, and then 8%-12% bovine serum albumin solution is added, and the stirring and incubation is continued for 1.5h-2.5h to block the non-specific binding sites, and then the modified nanogold antibody complex is collected by centrifugation at 7000r / min-9000r / min for 8min-12min, and the complex is washed three times with PBS buffer containing 0.04%-0.06% Tween-20, and then the magnetic The magnetic nanoparticles were dispersed in an ethanol solution containing 0.8%-1.2% 3-aminopropyltriethoxysilane, stirred and reacted at 60°C-65°C for 2.5h-3.5h to amino-amino the surface of the nanoparticles. The nanoparticles were separated by a magnet and washed three times with ethanol. The amino-modified magnetic nanoparticles were dispersed in a PBS buffer containing 8mM-12mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 8mM-12mM N-hydroxysuccinimide and activated for 25min-35min. Then, the aptamer was added at a molar ratio of aptamer to magnetic nanoparticles of 1:80-120. The mixture was stirred and incubated at 35°C-38°C for 5h-7h. The mixture was then separated by a magnet and washed three times with a PBS buffer containing 0.04%-0.06% Tween-20 to obtain a modified magnetic nanoparticle-aptamer complex, thereby completing the surface modification of the nanomaterial.

[0018] As a further solution of the present invention: in the step 5, the modified nanogold antibody complex and the magnetic nanoparticle nucleic acid aptamer complex are mixed in a volume ratio of 0.8:1-1.1, and added to a detection buffer containing 8mM-12mMTris-HCl, 90mM-110mMNaCl and 0.8mM-1.2mMMgCl2 to form a mixed detection probe, and the serum sample of the immune encephalopathy patient and the mixed detection probe are incubated at 35℃-38℃ for 0.8h-1.2h to allow the probe to fully bind to the marker in the serum, and the immune encephalopathy related marker standard is used as the detection object, and the marker and probe binding signal is detected by fluorescence detection and magnetic detection methods. The optimal detection conditions are determined to be: ionic strength 80mM-120mMNaCl, pH 7.3-7.7, and incubation temperature 35℃-38℃. Under these conditions, the detection sensitivity of the detection system for immune encephalopathy related markers can reach 10 -9 M level, the construction and optimization of the detection system is completed. At this point, the complete preparation process of the early detection method of immune encephalopathy based on serum marker detection is completed.

[0019] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. The present invention uses proteomics and bioinformatics to screen for antigenic epitopes related to immune encephalopathy, constructs a phage antibody library, and through multiple rounds of panning and site-directed mutagenesis optimization, prepares antibodies with strong specificity and high affinity. Then, SELEX technology is used to screen and modify nucleic acid aptamers to improve their stability and specificity. Then, gold nanoparticles and magnetic nanoparticles with good biocompatibility and stability are designed and synthesized. Antibodies and nucleic acid aptamers are separately modified onto the surface of nanomaterials to construct specific nanoprobes. Finally, a hybrid detection probe is formed and the detection conditions are optimized. This effectively solves the problems of poor specificity of detection reagents and poor sample enrichment effect in the prior art, significantly improves detection sensitivity and specificity, and can accurately detect low-concentration markers of early immune encephalopathy, providing an efficient method for early detection, facilitating early intervention, and improving patient prognosis.

[0021] 2. The present invention uses glycine-HCl buffer and Tris-HCl buffer in the preparation of specific antibodies to precisely control the elution and neutralization of phages during the panning process, ensuring the screening of antibodies with extremely high specificity for markers related to immune encephalopathy. The isopropyl-β-D-thiogalactopyranoside reagent plays a key role in the induced expression of antibody variants, ensuring the large-scale and stable production of high-affinity antibodies. In the modification of nucleic acid aptamers, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide reagents activate the surface of magnetic nanoparticles, promoting their strong binding to nucleic acid aptamers, greatly enhancing the stability and reliability of the detection system. When synthesizing nanomaterials, sodium citrate is used as a reducing agent to synthesize gold nanoparticles, providing a stable and high-performance basic material for the subsequent construction of multifunctional detection probes. These materials and reagents work synergistically to fundamentally overcome the problem of poor specificity of detection reagents in the prior art and significantly improve the specificity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the method steps in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention will be further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention.

[0024] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Please see the attached Figure 1 The present invention provides an early detection method for immune encephalopathy based on serum marker detection, and the detection method comprises the following specific steps:

[0026] Step 1: Use proteomics technology to analyze specific antigens of immune encephalopathy, and with the help of bioinformatics databases and computer simulation technology, screen out highly specific antigenic epitopes, then construct a phage antibody library, fix the antigenic epitopes on an ELISA plate, add the phage antibody library to each well of the ELISA plate, and perform multiple rounds of panning to screen out single-chain antibody fragments with highly specific recognition capabilities for immune encephalopathy-related markers. Then, use site-directed mutagenesis technology to mutate the amino acid sites of the screened single-chain antibody fragments to construct a mutant library, screen out antibody variants, and then clone the screened antibody variant genes into expression vectors, transform them into Escherichia coli competent cells, and induce expression to obtain specific antibodies;

[0027] Step 2: Construct a random single-stranded nucleic acid library, target immune encephalopathy markers, incubate with the nucleic acid library, and perform multiple rounds of screening through filtration, elution, and PCR amplification to enrich nucleic acid aptamers with high affinity and specificity for the target. The nucleic acid aptamers are then modified, purified, and sequence optimized, and stability tested to prepare nucleic acid aptamers.

[0028] Step 3: Add ultrapure water to a round-bottom flask, heat to boiling, quickly add chloroauric acid solution, stir evenly, then add sodium citrate solution, continue stirring and keep boiling, and then cool to room temperature to obtain nanogold solution, and then synthesize magnetic nanoparticles by chemical coprecipitation method to complete the preparation of nanomaterials;

[0029] Step 4: The gold nanoparticle solution is centrifuged and then redispersed, and then a specific antibody is added for incubation. After that, a bovine serum albumin solution is added to block the nonspecific binding sites. The modified gold nanoparticle antibody complex is then collected by centrifugation. The magnetic nanoparticles are amino-treated, activated, and then incubated with aptamers to obtain the modified magnetic nanoparticle-aptamer complex, thus completing the surface modification of the nanomaterial.

[0030] Step 5: Mix the modified gold nanoparticle antibody complex and the magnetic nanoparticle nucleic acid aptamer complex, add detection buffer to form a mixed detection probe, and then incubate the serum sample of the immune encephalopathy patient with the mixed detection probe to allow the probe to fully bind to the marker in the serum. Detect the marker and probe binding signal through fluorescence detection and magnetic detection methods, determine the optimal detection conditions, and complete the construction and optimization of the detection system.

[0031] In one embodiment of the present invention, in step 1, 10-15 specific antigens of immune encephalopathy are analyzed using proteomics technology, and 5-7 highly specific antigen epitopes are screened out with the help of bioinformatics database and computer simulation technology, and a capacity of 1.0×10 12 -3.0×10 12 The phage antibody library was prepared, and the antigen epitope with a concentration of 8μg / mL-12μg / mL was fixed to the ELISA plate. After adding 100μL-150μL of phage antibody library to each well, the plates were incubated at 35℃-38℃ for 1.5h-2.5h. After 3-5 rounds of panning, the unbound phages were eluted with 0.08mol / L-0.12mol / L glycine-HCl buffer with a pH value of 2-2.5 after each round of panning, and then neutralized with 0.8mol / L-1.2mol / L Tris-HCl buffer with a pH value of 9-9.5 to screen out single-chain antibody fragments with highly specific recognition ability for immune encephalopathy-related markers.

[0032] In one embodiment of the present invention: in step 1, the amino acid sites of the screened single-chain antibody fragments are mutated by site-directed mutagenesis technology, and mutations are introduced by PCR. The amount of template DNA used in the PCR reaction system is 40ng-60ng, 8pmol-12pmol of upstream and downstream primers, 180μM-220μM of dNTPs, 1IU-2IU of TaqDNA polymerase, 4μL-6μL of 10×PCR buffer, and ddH2O is added to 40μL-60 μL, PCR reaction conditions were: 94℃-96℃ pre-denaturation for 4min-6min, 94℃-96℃ denaturation for 25s-35s, 54℃-56℃ annealing for 25s-35s, 71℃-73℃ extension for 0.8min-1.2min, for a total of 28-32 cycles, and then 71℃-73℃ final extension for 8min-12min. Then, a mutant library was constructed and the binding affinity of the markers related to immune encephalopathy was screened by enzyme-linked immunosorbent assay to reach a Kd value of 10 -9 M-10 -11 The optimized antibody variant gene was cloned into the expression vector pET-28a, transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in LB medium containing 40 μg / mL-60 μg / mL kanamycin at 36°C-38°C and 180r / min-220r / min with shaking until the OD 600 When the RT-PCR reaction temperature is 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.4 mM-0.6 mM, induce expression at 16° C.-20° C. for 14 h-18 h, and complete the preparation of specific antibodies.

[0033] In one embodiment of the present invention: In step 2, the development of nucleic acid aptamers is to construct a nucleic acid aptamer containing 1.0×10 15 -1.5×10 15Random single-stranded nucleic acid libraries of different sequences were prepared, targeting immune encephalopathy markers at a concentration of 0.8 μM-1.2 μM, and dissolved in binding buffer. The binding buffer had a pH of 7.3-7.7 and included 20 mM-25 mM Tris-HCl, 90 mM-110 mM NaCl, and 1.5 mM-2.5 mM MgCl2. The libraries were incubated with the nucleic acid library at 35°C-38°C for 25 min-35 min, and unbound nucleic acids were removed by filtration through a nitrocellulose membrane. The bound nucleic acids were eluted and then amplified by PCR. The PCR reaction system included 8 μL-12 μL template nucleic acid, 8 pmol-1 of each of the upstream and downstream primers. 2 pmol, dNTPs concentration 180 μM-220 μM, Taq DNA polymerase 1 IU-2 IU, 10× PCR buffer 4 μL-6 μL, add ddH2O to 40 μL-60 μL, PCR reaction conditions: 94℃-96℃ pre-denaturation 4 min-6 min, 94-96℃ denaturation 25s-35s, 56℃-60℃ annealing 25s-35s, 71℃-73℃ extension 0.8min-1.2min, a total of 28-32 cycles, 71℃-73℃ final extension 8min-12min, after 8-10 rounds of screening, nucleic acid aptamers with high affinity and specificity for the target were enriched.

[0034] In one embodiment of the present invention: In step 2, a carboxyfluorescein marker is introduced at the 5' end of the screened nucleic acid aptamer, and the nucleic acid aptamer and the marker are reacted at a molar ratio of 1:4-6 in a buffer containing 90mM-110mM 4-hydroxyethylpiperazineethanesulfonic acid, 8mM-12mM dithiothreitol and 0.8-1.2mM ethylenediaminetetraacetic acid at 35°C-38°C for 1.5h-2.5h. The reaction is purified by polyacrylamide gel electrophoresis, the sequence is optimized by computer-aided design, and the stability test is performed in 40mM-60mM sodium chloride, 8mM-12mM Tris-HCl buffer at 23°C-27°C to ensure stability within 20h-28h, thereby completing the preparation of the nucleic acid aptamer.

[0035] In one embodiment of the present invention: In step three, nanogold is synthesized by sodium citrate reduction method, 80mL-100mL of ultrapure water is added to a 100mL-120mL round-bottom flask, heated to boiling, 0.8mL-1.2mL of 0.8%-1.2% chloroauric acid solution is added, stirred evenly, 2.5mL-3.5mL of 0.8%-1.2% sodium citrate solution is added dropwise, stirring is continued and boiling is maintained for 12min-18min, the solution color turns wine red, and then cooled to room temperature to obtain a nanogold solution.

[0036] In one embodiment of the present invention: in step three, magnetic nanoparticles Fe3O4 are subsequently synthesized by a chemical coprecipitation method, 0.04-0.06 mol of FeCl2·4H2O and 0.08 mol-0.12 mol of FeCl3·6H2O are dissolved in 80-120 mL of deionized water, heated to 75°C-85°C under nitrogen protection and stirred evenly, 20 mL-30 mL of 20%-30% ammonia water is slowly added dropwise, and after the addition is complete, the stirring reaction is continued for 0.8 h-1.2 h, and then the magnetic nanoparticles are separated by a magnet, washed with deionized water and anhydrous ethanol for 3-5 times respectively until the pH value of the washing solution is 6.8-7.2, and then dried in a vacuum drying oven at 55°C-65°C for 10 h-14 h to obtain magnetic nanoparticles, thereby completing the preparation of the nanomaterial.

[0037] In one embodiment of the present invention: in step 4, the prepared nanogold solution is centrifuged at 7000r / min-9000r / min for 8min-12min, the supernatant is discarded, and the precipitate is redispersed in a potassium carbonate buffer solution with a pH value of 8.8-9.2 and containing 0.08M-0.12M, and then the specific antibody is added, the antibody and nanogold molar ratio is 1:40-60, and the mixture is stirred and incubated at 4°C-5°C for 10h-14h, and then 8%-12% bovine serum albumin solution is added, and the stirring and incubation is continued for 1.5h-2.5h to block the nonspecific binding sites, and then the modified nanogold antibody complex is collected by centrifugation at 7000r / min-9000r / min for 8min-12min, and washed three times with PBS buffer containing 0.04%-0.06% Tween-20, and then the magnetic The nanoparticles are dispersed in an ethanol solution containing 0.8%-1.2% 3-aminopropyltriethoxysilane, stirred and reacted at 60°C-65°C for 2.5h-3.5h to aminate the surface, separated by a magnet and washed three times with ethanol, and the aminated magnetic nanoparticles are dispersed in a PBS buffer containing 8mM-12mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 8mM-12mM N-hydroxysuccinimide for activation for 25min-35min, and then the nucleic acid aptamer is added with a molar ratio of nucleic acid aptamer to magnetic nanoparticles of 1:80-120. The mixture is stirred and incubated at 35°C-38°C for 5h-7h, separated by a magnet, and washed three times with a PBS buffer containing 0.04%-0.06% Tween-20 to obtain a modified magnetic nanoparticle nucleic acid aptamer complex, thereby completing the surface modification of the nanomaterial.

[0038] In one embodiment of the present invention: in step five, the modified nanogold antibody complex and the magnetic nanoparticle nucleic acid aptamer complex are mixed in a volume ratio of 0.8:1-1.1, and added to a detection buffer containing 8mM-12mMTris-HCl, 90mM-110mMNaCl and 0.8mM-1.2mMMgCl2 to form a mixed detection probe, and the serum sample of the immune encephalopathy patient and the mixed detection probe are incubated at 35°C-38°C for 0.8h-1.2h to allow the probe to fully bind to the marker in the serum, and the immune encephalopathy related marker standard is used as the detection object, and the marker and probe binding signal is detected by fluorescence detection and magnetic detection methods. The optimal detection conditions are determined to be: ionic strength 80mM-120mMNaCl, pH 7.3-7.7, and incubation temperature 35°C-38°C. Under these conditions, the detection sensitivity of the detection system for immune encephalopathy related markers can reach 10 -9 M level, the construction and optimization of the detection system is completed. At this point, the complete preparation process of the early detection method of immune encephalopathy based on serum marker detection is completed.

[0039] In one embodiment of the present invention: In step one, when using proteomics technology to analyze the specific antigens of immune encephalopathy, proteomics technology uses two-dimensional gel electrophoresis and mass spectrometry technology, which can more comprehensively and accurately analyze the protein composition and expression of immune encephalopathy specific antigens, and thus more accurately screen out antigen epitopes with high specificity.

[0040] In one embodiment of the present invention: in step 1, when screening antigen epitopes with the help of bioinformatics databases and computer simulation technology, at least three different bioinformatics databases are used for comparison analysis, including but not limited to UniProt, NCBI and OMIM databases, to ensure that the screened antigen epitopes are highly specific and reliable.

[0041] In one embodiment of the present invention, in step 1, when constructing the phage antibody library, the recombinant phage DNA is introduced into Escherichia coli cells by electroporation to improve the construction efficiency and quality of the phage antibody library, and the conversion efficiency is not less than 1×10 8 transformants / μgDNA.

[0042] In one embodiment of the present invention: in step 1, when fixing the antigen epitope on the ELISA plate, glutaraldehyde cross-linking method is used for fixation, so that the antigen epitope can be firmly bound to the surface of the ELISA plate, and the activity retention rate of the antigen epitope after fixation is not less than 80%, so as to ensure the accuracy of subsequent rounds of panning.

[0043] In one embodiment of the present invention: when the eluted phage is amplified and cultured after each round of panning in step 1, the culture medium used is LB medium containing 1% glucose, 0.5% yeast extract and 1% peptone, the culture temperature is 37°C, and the shaker speed is 220 r / min to promote the large-scale reproduction of phages to meet subsequent screening requirements.

[0044] Example 1, please refer to the attached Figure 1 The specific method for early detection of immune encephalopathy based on serum marker detection is as follows: 15 specific antigens of immune encephalopathy were analyzed using proteomics technology. With the help of bioinformatics database and computer simulation technology, 7 highly specific antigen epitopes were screened. These epitopes were specifically expressed in the serum of patients with immune encephalopathy and were lowly expressed or not expressed in the serum of normal people and patients with common diseases. The construction capacity was 3.0×10 12 The phage antibody library was prepared, and the antigen epitope with a concentration of 12 μg / mL was fixed to the ELISA plate. After adding 150 μL of phage antibody library to each well, it was incubated at 38°C for 2.5 hours. After 5 rounds of panning, the unbound phage was eluted with 0.12 mol / L glycine-HCl buffer with a pH value of 2.5 after each round of panning, and then neutralized with 1.2 mol / L Tris-HCl buffer with a pH value of 9.5. The single-chain antibody fragment with highly specific recognition ability for immune encephalopathy-related markers was screened out, and the amino acid sites of the single-chain antibody fragment were mutated by site-directed mutagenesis technology. Mutations were introduced. The template DNA used in the PCR reaction system was 60 ng, 12 pmol of upstream and downstream primers, 220 μM of dNTPs, 2 IU of Taq DNA polymerase, 6 μL of 10× PCR buffer, and ddH2O was added to 60 μL. The PCR reaction conditions were as follows: pre-denaturation at 96°C for 6 min, denaturation at 96°C for 35 s, annealing at 56°C for 35 s, extension at 73°C for 1.2 min, for a total of 32 cycles, and final extension at 73°C for 12 min. A mutant library was constructed and the binding affinity of the mutants to the markers related to immune encephalopathy was screened by enzyme-linked immunosorbent assay (ELISA) to reach a Kd value of 10 -11 The optimized antibody variant gene was cloned into the expression vector pET-28a, transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in LB medium containing 60 μg / mL kanamycin at 38°C and 220 rpm until the OD 600 When the concentration of isopropyl-β-D-thiogalactopyranoside was 0.7, the final concentration of 0.6 mM was added and the expression was induced at 20 ° C for 18 h to complete the preparation of specific antibodies. 15A random single-stranded nucleic acid library with different sequences was prepared, targeting immune encephalopathy markers at a concentration of 1.2 μM. The library was dissolved in a binding buffer with a pH of 7.7 and containing 25 mM Tris-HCl, 110 mM NaCl, and 2.5 mM MgCl2. The library was incubated with the nucleic acid library at 38°C for 35 min. Unbound nucleic acids were removed by filtration through a nitrocellulose membrane. The bound nucleic acids were eluted and then amplified by PCR. The PCR reaction system included 12 μL of template nucleic acid, 12 pmol of upstream and downstream primers, 220 μM of dNTPs, 2 IU of Taq DNA polymerase, 6 μL of 10× PCR buffer, and ddH2O was added to 60 μL. The PCR reaction conditions were: 9 Pre-denaturation at 6°C for 6 min, denaturation at 96°C for 35 s, annealing at 60°C for 35 s, extension at 73°C for 1.2 min, for a total of 32 cycles, and final extension at 73°C for 12 min. After 10 rounds of screening, nucleic acid aptamers with high affinity and specificity for the target were enriched. A carboxyfluorescein marker was introduced at the 5' end of the screened nucleic acid aptamers. The aptamer-to-marker molar ratio was 1:6 in a buffer containing 110 mM 4-hydroxyethylpiperazineethanesulfonic acid, 12 mM dithiothreitol, and 1.2 mM ethylenediaminetetraacetic acid, and the reaction was carried out at 38°C for 2.5 h. The aptamer was purified by polyacrylamide gel electrophoresis, and the sequence was optimized by computer-aided design. Cl buffer, stability test was performed at 27 ° C to ensure stability within 28 h, and the preparation of nucleic acid aptamers was completed. 100 mL of ultrapure water was added to a 120 mL round-bottom flask, heated to boiling, and then 1.2 mL of 1.2% chloroauric acid solution was added and stirred evenly. 3.5 mL of 1.2% sodium citrate solution was added dropwise, and stirring was continued and boiling was maintained for 18 min. The color of the solution turned to wine red and then cooled to room temperature to obtain nanogold solution. 0.06 mol of FeCl2·4H2O and 0.12 mol of FeCl3·6H2O were dissolved in 120 mL of deionized water, heated to 85 ° C under nitrogen protection and stirred evenly, and 30 m L of 30% ammonia water was added dropwise. After the addition was complete, the reaction was stirred for 1.2 hours. The magnetic nanoparticles were then separated using a magnet and washed five times with deionized water and anhydrous ethanol until the pH of the washing solution reached 7.2. The solution was then dried in a 65°C vacuum drying oven for 14 hours to obtain the magnetic nanoparticles, completing the preparation of the nanomaterial. The prepared nanogold solution was centrifuged at 9000 rpm for 12 minutes, the supernatant was discarded, and the precipitate was redispersed in a pH 9.2 buffer solution containing 0.12M potassium carbonate. Specific antibodies were then added at a molar ratio of 1:60 between the antibody and nanogold. The solution was stirred and incubated at 5°C for 14 hours. A 12% bovine serum albumin solution was then added and the solution was stirred and incubated for 2 hours.5h, block nonspecific binding sites, and then centrifuge at 9000r / min for 12min to collect the modified nanogold antibody complex, wash it three times with PBS buffer containing 0.06% Tween-20, and then disperse the magnetic nanoparticles in an ethanol solution containing 1.2% 3-aminopropyltriethoxysilane, stir the reaction at 65°C for 3.5h to aminoate the surface, separate it with a magnet and wash it three times with ethanol, disperse the amino magnetic nanoparticles in a PBS buffer containing 12mM1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 12mM N-hydroxysuccinimide and activate it for 35min, then add nucleic acid aptamers, the molar ratio of nucleic acid aptamers to magnetic nanoparticles is 1:120, incubate it with stirring at 38°C for 7h, then separate it with a magnet and use PBS buffer containing 0.06% Tween-20. The modified magnetic nanoparticle-aptamer complex was obtained by washing three times, completing the surface modification of the nanomaterial. The modified gold nanoparticle-antibody complex and the magnetic nanoparticle-aptamer complex were mixed in a volume ratio of 1:1 and added to a detection buffer containing 12mM Tris-HCl, 110mM NaCl, and 1.2mM MgCl2 to form a mixed detection probe. Serum samples from patients with immune encephalopathy were incubated with the mixed detection probe at 38°C for 1.2 hours to allow the probe to fully bind to the marker in the serum. Standard samples of immune encephalopathy-related markers were used as detection objects, and the marker-probe binding signals were detected by fluorescence detection and magnetic detection methods. The optimal detection conditions were determined to be: ionic strength 120mM NaCl, pH 7.7, and incubation temperature 38°C. Under these conditions, the detection system had a detection sensitivity of 10 for immune encephalopathy-related markers. -9 M level, the construction and optimization of the detection system is completed. At this point, the complete preparation process of the early detection method of immune encephalopathy based on serum marker detection is completed.

[0045] Example 2, please refer to the attached Figure 1 The specific method for early detection of immune encephalopathy based on serum marker detection is as follows: proteomics technology is used to analyze the specific antigens of 10 immune encephalopathy. With the help of bioinformatics database and computer simulation technology, 5 highly specific antigen epitopes are screened. These epitopes are specifically expressed in the serum of patients with immune encephalopathy and are lowly expressed or not expressed in the serum of normal people and patients with common diseases. The capacity of the constructed epitope is 1.0×10 12The phage antibody library was prepared, and the antigen epitope with a concentration of 8 μg / mL was fixed to the ELISA plate. After adding 100 μL of phage antibody library to each well, it was incubated at 35°C for 1.5 hours. After three rounds of panning, the unbound phage was eluted with 0.08 mol / L glycine-HCl buffer with a pH value of 2 and then neutralized with 0.8 mol / L Tris-HCl buffer with a pH value of 9. The single-chain antibody fragment with highly specific recognition ability for immune encephalopathy-related markers was screened out, and the amino acid sites of the single-chain antibody fragment were mutated by site-directed mutagenesis technology, and the PCR was used to introduce Mutation, the amount of template DNA used in the PCR reaction system was 40 ng, 8 pmol of upstream and downstream primers each, 180 μM of dNTPs, 1 IU of Taq DNA polymerase, 4 μL of 10× PCR buffer, and ddH2O was added to 40 μL. The PCR reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, extension at 71°C for 0.8 min, for a total of 28 cycles, and final extension at 71°C for 8 min. A mutant library was constructed and the binding affinity of the mutants to the markers related to immune encephalopathy was screened by enzyme-linked immunosorbent assay technology to reach a Kd value of 10 -9 The optimized antibody variant gene was cloned into the expression vector pET-28a, transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in LB medium containing 40 μg / mL kanamycin at 36°C and 180 rpm until the OD 600 When the concentration of isopropyl-β-D-thiogalactopyranoside was 0.5, the final concentration of 0.4 mM isopropyl-β-D-thiogalactopyranoside was added and the expression was induced at 16 °C for 14 h to complete the preparation of specific antibodies. 15A random single-stranded nucleic acid library with different sequences was prepared, targeting immune encephalopathy markers at a concentration of 0.8 μM. The library was dissolved in a binding buffer with a pH of 7.3 and containing 20 mM Tris-HCl, 90 mM NaCl, and 1.5 mM MgCl2. The library was incubated with the nucleic acid library at 35°C for 25 min. Unbound nucleic acids were removed by filtration through a nitrocellulose membrane. The bound nucleic acids were eluted and then amplified by PCR. The PCR reaction system included 8 μL of template nucleic acid, 8 pmol of upstream and downstream primers, 180 μM dNTPs, 1 IU of Taq DNA polymerase, 4 μL of 10× PCR buffer, and ddH2O was added to 40 μL. The PCR reaction conditions were: pre-denaturation at 94°C for 4 min, incubation at 94°C for 5 min, and PCR amplification. Denaturation for 25s, annealing at 56°C for 25s, extension at 71°C for 0.8min, for a total of 28 cycles, and final extension at 71°C for 8min. After 8 rounds of screening, nucleic acid aptamers with high affinity and specificity for the target were enriched. A carboxyfluorescein marker was introduced at the 5' end of the screened nucleic acid aptamers. The molar ratio of nucleic acid aptamer to marker was 1:4 in a buffer containing 90mM 4-hydroxyethylpiperazineethanesulfonic acid, 8mM dithiothreitol and 0.8mM ethylenediaminetetraacetic acid. The reaction was carried out at 35°C for 1.5h, purified by polyacrylamide gel electrophoresis, and the sequence was optimized by computer-aided design. The stability test was carried out at 23°C in 40mM sodium chloride, 8mMTris-HCl buffer to ensure stability within 20h. To complete the preparation of nucleic acid aptamers, 80 mL of ultrapure water was added to a 100 mL round-bottom flask, heated to boiling, and then 0.8 mL of 0.8% chloroauric acid solution was added and stirred evenly. 2.5 mL of 0.8% sodium citrate solution was added dropwise, and stirring was continued and boiling was maintained for 12 minutes. The color of the solution turned wine red and then cooled to room temperature to obtain a nanogold solution. 0.04 mol of FeCl2·4H2O and 0.08 mol of FeCl3·6H2O were dissolved in 80 mL of deionized water, heated to 75°C under nitrogen protection and stirred evenly. 20 mL of 20% ammonia water was slowly added dropwise. After the addition was complete, the reaction was continued with stirring for 0.8 h, and then the magnetic nanoparticles were separated using a magnet. , washed three times with deionized water and anhydrous ethanol respectively until the pH value of the washing solution reached 6.8, and then dried in a vacuum drying oven at 55°C for 10 hours to obtain magnetic nanoparticles, completing the preparation of the nanomaterial. The prepared nanogold solution was centrifuged at 7000r / min for 8 minutes, the supernatant was discarded, and the precipitate was redispersed in a potassium carbonate buffer solution with a pH value of 8.8 and containing 0.08M. Then, specific antibodies were added, and the molar ratio of antibody to nanogold was 1:40. The mixture was stirred and incubated at 4°C for 10 hours, and then 8% bovine serum albumin solution was added and stirred and incubated for 1.5 hours to block nonspecific binding sites. The modified nanogold antibody complex was then collected by centrifugation at 7000r / min for 8 minutes and 0.The magnetic nanoparticles were washed three times with PBS buffer containing 0.04% Tween-20, and then dispersed in an ethanol solution containing 0.8% 3-aminopropyltriethoxysilane, stirred at 60°C for 2.5h to amino-aminoate the surface, separated with a magnet and washed three times with ethanol, and the amino-modified magnetic nanoparticles were dispersed in a PBS buffer containing 8mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 8mM N-hydroxysuccinimide and activated for 25min, and then nucleic acid aptamers were added, with a molar ratio of nucleic acid aptamers to magnetic nanoparticles of 1:80, stirred and incubated at 35°C for 5h, and then separated with a magnet and washed three times with PBS buffer containing 0.04% Tween-20 to obtain the modified magnetic nanoparticle nucleic acid aptamer complex. The surface of the nanomaterial was modified. The modified gold nanoparticle antibody complex and magnetic nanoparticle nucleic acid aptamer complex were mixed at a volume ratio of 0.8:1 and added to a detection buffer containing 8mM Tris-HCl, 90mM NaCl and 0.8mM MgCl2 to form a mixed detection probe. The serum samples of patients with immune encephalopathy were incubated with the mixed detection probe at 35°C for 0.8h to allow the probe to fully bind to the markers in the serum. The standard samples of immune encephalopathy-related markers were used as the detection objects. The marker-probe binding signals were detected by fluorescence detection and magnetic detection methods. The optimal detection conditions were determined to be: ionic strength 80mM NaCl, pH 7.3, and incubation temperature 35°C. Under these conditions, the detection sensitivity of the detection system for immune encephalopathy-related markers can reach 10. -9 M level, the construction and optimization of the detection system is completed. At this point, the complete preparation process of the early detection method of immune encephalopathy based on serum marker detection is completed.

[0046] According to the contents of the above two groups of embodiments, it can be concluded that by using proteomics and bioinformatics to screen antigen epitopes, constructing and optimizing phage antibody libraries, using SELEX technology to screen and modify nucleic acid aptamers, designing and synthesizing nanogold and magnetic nanoparticles and performing surface modification, and forming a mixed detection probe and optimizing detection conditions, a series of operations, while at the same time with the help of glycine-hydrochloric acid buffer, Tris-HCl buffer, isopropyl-β-D-thiogalactopyranoside, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, sodium citrate and other reagents, the problems of poor specificity of detection reagents and poor sample enrichment effect in the prior art are effectively solved, the detection sensitivity and specificity are significantly improved, and the early low-concentration markers of immune encephalopathy can be accurately detected, which provides an efficient method for the early detection of immune encephalopathy, helps early intervention, improves patient prognosis, and shows broad application prospects in the field of early detection of immune encephalopathy.

[0047] Although the present invention is disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any person skilled in the art may make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent variations, and modifications made to the above embodiments in accordance with the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for early detection of immune encephalopathy based on serum marker detection, characterized in that: The detection method comprises the following specific steps: Step 1: Use proteomics technology to analyze specific antigens of immune encephalopathy, and with the help of bioinformatics databases and computer simulation technology, screen out highly specific antigenic epitopes, then construct a phage antibody library, fix the antigenic epitopes on an ELISA plate, add the phage antibody library to each well of the ELISA plate, and perform multiple rounds of panning to screen out single-chain antibody fragments with highly specific recognition capabilities for immune encephalopathy-related markers. Then, use site-directed mutagenesis technology to mutate the amino acid sites of the screened single-chain antibody fragments to construct a mutant library, screen out antibody variants, and then clone the screened antibody variant genes into expression vectors, transform them into Escherichia coli competent cells, and induce expression to obtain specific antibodies; Step 2: Construct a random single-stranded nucleic acid library, target immune encephalopathy markers, incubate with the nucleic acid library, and perform multiple rounds of screening through filtration, elution, and PCR amplification to enrich nucleic acid aptamers with high affinity and specificity for the target. The nucleic acid aptamers are then modified, purified, and sequence optimized, and stability tested to prepare nucleic acid aptamers. Step 3: Add ultrapure water to a round-bottom flask, heat to boiling, quickly add chloroauric acid solution, stir evenly, then add sodium citrate solution, continue stirring and keep boiling, and then cool to room temperature to obtain nanogold solution, and then synthesize magnetic nanoparticles by chemical coprecipitation method to complete the preparation of nanomaterials; Step 4: The gold nanoparticle solution is centrifuged and then redispersed, and then a specific antibody is added for incubation. After that, a bovine serum albumin solution is added to block the nonspecific binding sites. The modified gold nanoparticle antibody complex is then collected by centrifugation. The magnetic nanoparticles are amino-treated, activated, and then incubated with aptamers to obtain the modified magnetic nanoparticle-aptamer complex, thus completing the surface modification of the nanomaterial. Step 5: Mix the modified gold nanoparticle antibody complex and the magnetic nanoparticle nucleic acid aptamer complex, add detection buffer to form a mixed detection probe, and then incubate the serum sample of the immune encephalopathy patient with the mixed detection probe to allow the probe to fully bind to the marker in the serum. Detect the marker and probe binding signal through fluorescence detection and magnetic detection methods, determine the optimal detection conditions, and complete the construction and optimization of the detection system.

2. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the first step, proteomics technology is used to analyze 10-15 specific antigens of immune encephalopathy, and 5-7 highly specific antigen epitopes are screened with the help of bioinformatics database and computer simulation technology, and a capacity of 1.0×10 12 -3.0×10 12 The phage antibody library was prepared, and the antigen epitope with a concentration of 8μg / mL-12μg / mL was fixed to the ELISA plate. After adding 100μL-150μL of phage antibody library to each well, the plates were incubated at 35℃-38℃ for 1.5h-2.5h. After 3-5 rounds of panning, the unbound phages were eluted with 0.08mol / L-0.12mol / L glycine-HCl buffer with a pH value of 2-2.5 after each round of panning, and then neutralized with 0.8mol / L-1.2mol / L Tris-HCl buffer with a pH value of 9-9.5 to screen out single-chain antibody fragments with highly specific recognition ability for immune encephalopathy-related markers.

3. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step 1, the amino acid sites of the screened single-chain antibody fragments were mutated by site-directed mutagenesis technology, and mutations were introduced by PCR. The amount of template DNA in the PCR reaction system was 40 ng-60 ng, 8 pmol-12 pmol of upstream and downstream primers, 180 μM-220 μM of dNTPs, 1 IU-2 IU of Taq DNA polymerase, 4 μL-6 μL of 10× PCR buffer, and ddH2O was added to 40 μL-60 μL. PCR The reaction conditions were as follows: pre-denaturation at 94°C-96°C for 4 min-6 min, denaturation at 94°C-96°C for 25s-35s, annealing at 54°C-56°C for 25s-35s, extension at 71°C-73°C for 0.8 min-1.2 min, for a total of 28-32 cycles, and a final extension at 71°C-73°C for 8 min-12 min. A mutant library was then constructed, and the binding affinity of the mutants to immune encephalopathy-related markers was screened by enzyme-linked immunosorbent assay (ELISA) to achieve a Kd value of 10 -9 M-10 -11 The optimized antibody variant gene was cloned into the expression vector pET-28a, transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in LB medium containing 40 μg / mL-60 μg / mL kanamycin at 36°C-38°C and 180r / min-220r / min with shaking until the OD 600 When the RT-PCR reaction temperature is 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.4 mM-0.6 mM, induce expression at 16° C.-20° C. for 14 h-18 h, and complete the preparation of specific antibodies.

4. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the second step, the development of nucleic acid aptamers was carried out by constructing a 1.0×10 15 -1.5×10 15 Random single-stranded nucleic acid libraries of different sequences were prepared, targeting immune encephalopathy markers at a concentration of 0.8 μM-1.2 μM, and dissolved in binding buffer. The binding buffer had a pH of 7.3-7.7 and included 20 mM-25 mM Tris-HCl, 90 mM-110 mM NaCl, and 1.5 mM-2.5 mM MgCl2. The libraries were incubated with the nucleic acid library at 35°C-38°C for 25 min-35 min, and unbound nucleic acids were removed by filtration through a nitrocellulose membrane. The bound nucleic acids were eluted and then amplified by PCR. The PCR reaction system included 8 μL-12 μL template nucleic acid, 8 pmol-1 of each of the upstream and downstream primers. 2 pmol, dNTPs concentration 180 μM-220 μM, Taq DNA polymerase 1 IU-2 IU, 10× PCR buffer 4 μL-6 μL, add ddH2O to 40 μL-60 μL, PCR reaction conditions: 94℃-96℃ pre-denaturation 4 min-6 min, 94-96℃ denaturation 25s-35s, 56℃-60℃ annealing 25s-35s, 71℃-73℃ extension 0.8min-1.2min, a total of 28-32 cycles, 71℃-73℃ final extension 8min-12min, after 8-10 rounds of screening, nucleic acid aptamers with high affinity and specificity for the target were enriched.

5. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step 2, a carboxyfluorescein marker is introduced into the 5' end of the screened nucleic acid aptamer, and the nucleic acid aptamer and the marker are reacted at a molar ratio of 1:4-6 in a buffer containing 90mM-110mM 4-hydroxyethylpiperazineethanesulfonic acid, 8mM-12mM dithiothreitol, and 0.8-1.2mM ethylenediaminetetraacetic acid at 35°C-38°C for 1.5h-2.5h. The aptamer is purified by polyacrylamide gel electrophoresis, the sequence is optimized by computer-aided design, and a stability test is performed in a 40mM-60mM sodium chloride, 8mM-12mM Tris-HCl buffer at 23°C-27°C to ensure stability within 20h-28h, thereby completing the preparation of the nucleic acid aptamer.

6. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step 3, a sodium citrate reduction method is used to synthesize nanogold. 80-100 mL of ultrapure water is added to a 100-120 mL round-bottom flask. After heating to boiling, 0.8-1.2 mL of a 0.8%-1.2% chloroauric acid solution is added. The mixture is stirred evenly, and 2.5-3.5 mL of a 0.8%-1.2% sodium citrate solution is added dropwise. The mixture is stirred continuously and kept boiling for 12-18 minutes, and then cooled to room temperature to obtain a nanogold solution.

7. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step three, a chemical coprecipitation method is subsequently used to synthesize magnetic nanoparticles Fe3O4, where 0.04-0.06 mol of FeCl2·4H2O and 0.08 mol-0.12 mol of FeCl3·6H2O are dissolved in 80-120 mL of deionized water, heated to 75°C-85°C under nitrogen protection and stirred evenly, 20 mL-30 mL of 20%-30% ammonia water is slowly added dropwise, and after the addition is complete, the stirring reaction is continued for 0.8 h-1.2 h, and then the magnetic nanoparticles are separated by a magnet, washed with deionized water and anhydrous ethanol for 3-5 times respectively until the pH value of the washing solution is 6.8-7.2, and then dried in a vacuum drying oven at 55°C-65°C for 10 h-14 h to obtain magnetic nanoparticles, thereby completing the preparation of the nanomaterial.

8. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step 4, the prepared nanogold solution is centrifuged at 7000r / min-9000r / min for 8min-12min, the supernatant is discarded, and the precipitate is redispersed in a potassium carbonate buffer solution with a pH value of 8.8-9.2 and containing 0.08M-0.12M, and then the specific antibody is added, the antibody and nanogold molar ratio is 1:40-60, and the mixture is stirred and incubated at 4°C-5°C for 10h-14h, and then 8%-12% bovine serum albumin solution is added, and the mixture is stirred and incubated for 1.5h-2.5h to block non-specific binding sites, and then the modified nanogold antibody complex is collected by centrifugation at 7000r / min-9000r / min for 8min-12min, and the magnetic nanoparticles are separated. The particles were dispersed in an ethanol solution containing 0.8%-1.2% 3-aminopropyltriethoxysilane, stirred at 60°C-65°C for 2.5h-3.5h to amino-amino the surface of the particles. The particles were separated by a magnet and washed three times with ethanol. The amino-amino magnetic nanoparticles were dispersed in a PBS buffer containing 8mM-12mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 8mM-12mM N-hydroxysuccinimide and activated for 25min-35min. The aptamer was then added at a molar ratio of aptamer to magnetic nanoparticles of 1:80-120. The particles were stirred and incubated at 35°C-38°C for 5h-7h. The particles were then separated by a magnet and washed three times with a PBS buffer containing 0.04%-0.06% Tween-20 to obtain a modified magnetic nanoparticle-aptamer complex, thereby completing the surface modification of the nanomaterial.

9. The method for early detection of immune encephalopathy based on serum marker detection according to claim 1, characterized in that: In the step 5, the modified gold nanoparticle antibody complex and the magnetic nanoparticle nucleic acid aptamer complex are mixed in a volume ratio of 0.8:1-1.1, and added to a detection buffer containing 8mM-12mMTris-HCl, 90mM-110mMNaCl and 0.8mM-1.2mMMgCl2 to form a mixed detection probe. The serum sample of the immune encephalopathy patient and the mixed detection probe are incubated at 35°C-38°C for 0.8h-1.2h to allow the probe to fully bind to the marker in the serum. The standard of immune encephalopathy-related markers is used as the detection object, and the marker and probe binding signal is detected by fluorescence detection and magnetic detection methods. The optimal detection conditions are determined to be: ionic strength 80mM-120mMNaCl, pH 7.3-7.7, and incubation temperature 35°C-38°C. Under these conditions, the detection sensitivity of the detection system for immune encephalopathy-related markers can reach 10 -9 M level, the construction and optimization of the detection system is completed. At this point, the complete preparation process of the early detection method of immune encephalopathy based on serum marker detection is completed.

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