A method and kit for isolating human immunoglobulin E based on nucleic acid aptamers

Abstract

The application discloses a human immunoglobulin E separation method and kit based on nucleic acid aptamer and belongs to the field of biomedical technology, and comprises the following steps: combining nucleic acid aptamer capable of specifically recognizing human immunoglobulin E with a magnetic carrier, and selectively combining and separating human immunoglobulin E in a sample containing human immunoglobulin E. The application utilizes the ion-reversible conformation of the nucleic acid aptamer, realizes high-specificity capture and mild non-damaging dissociation of IgE, effectively avoids the interference of other immunoglobulins, and maintains the biological activity of IgE.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method and kit for isolating human immunoglobulin E based on nucleic acid aptamers. Background Technology

[0002] Allergic diseases are a group of diseases caused by abnormal immune system responses, including allergic asthma, allergic rhinitis, and atopic dermatitis, and their incidence has been rising continuously in recent years. Studies have shown that mast cells play a key role in the occurrence and development of allergic diseases. Mast cells are widely distributed in tissues that come into direct contact with the external environment, such as the skin, respiratory tract, and digestive tract. They can be rapidly activated under specific stimuli and release a variety of inflammatory mediators, thereby mediating allergic reactions. In classic type I hypersensitivity reactions, immunoglobulin E (IgE) and its mediated signaling pathways are the core mechanisms of mast cell activation. IgE can specifically bind to the high-affinity Fcε receptor (Fc epsilon receptor I, FcεRI) on the surface of mast cells through its homeostasis region Fc fragment, sensitizing mast cells. When the body is exposed to the corresponding allergen again, the allergen binds to IgE on the cell surface, causing it to cross-link. This activates mast cells, leading to degranulation and the release of various bioactive mediators such as histamine and β-aminohexosidase, which in turn trigger allergic symptoms.

[0003] Human IgE is mainly produced by B cells after immunoglobulin class switching in a specific cytokine environment. Compared with other immunoglobulins, IgE has characteristics such as lower structural stability and stronger non-specific adsorption tendency, which increases the technical difficulty of its separation and purification to a certain extent. Therefore, a method for isolating IgE with high purity and good functional preservation is an important prerequisite for carrying out related basic and applied research.

[0004] Currently, the in vitro isolation and purification of IgE mainly relies on protein ligand affinity chromatography, with Protein L being a commonly used IgE binding ligand. Protein L can bind to some light chain regions of IgE, thereby enriching IgE. However, this method still has significant limitations in practical applications. On the one hand, Protein L has limited binding ability to λ-type IgE, which may lead to the loss of some IgE isoforms during purification; on the other hand, Protein L can also non-specifically bind to other types of immunoglobulins, easily causing co-purification of IgE with immunoglobulins such as IgG and IgM, thus affecting the purity of the final product and the accuracy of subsequent experiments.

[0005] In view of the above problems, there is an urgent need to establish a new method for IgE separation to achieve specific separation of IgE from human serum and minimize interference from other immunoglobulins. Summary of the Invention

[0006] To address the problems of low purity due to non-specific binding and impaired activity due to harsh elution conditions in existing technologies, this invention proposes a method and kit for separating human immunoglobulin E based on nucleic acid aptamers. By utilizing conformationally reversibly ion-dependent nucleic acid aptamers in conjunction with ion environment switching, highly specific capture and gentle, non-destructive dissociation of IgE can be achieved.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses a method for separating human immunoglobulin E based on nucleic acid aptamers, which utilizes a nucleic acid aptamer capable of specifically recognizing human immunoglobulin E to bind to a magnetic carrier, thereby selectively binding to and separating human immunoglobulin E from a sample containing human immunoglobulin E.

[0008] Preferably, the above method includes: Nucleic acid aptamers that specifically recognize human immunoglobulin E are contacted with samples containing human immunoglobulin E in a system in the presence of divalent metal ions to capture human immunoglobulin E. In a system containing an ion chelating agent, the captured human immunoglobulin E is dissociated from the nucleic acid aptamer to obtain isolated human immunoglobulin E.

[0009] The above scheme utilizes the conformation-dependent properties of nucleic acid aptamers to maintain the specific binding conformation of aptamers in a system containing conformation-maintaining ions to capture IgE with high specificity, and in a system containing ion chelating agents, it gently releases IgE by altering the conformation of aptamers through chelation of ions. This achieves highly specific separation of IgE and good preservation of its biological activity.

[0010] As a preferred embodiment, the nucleic acid aptamer is a single-stranded DNA molecule. Using a single-stranded DNA molecule as the aptamer provides a structural basis for conformational reversibility and ion dependence, ensuring the effective realization of the mechanism by which ion environment switching controls conformation.

[0011] More preferably, the sequence of the single-stranded DNA molecule is shown in SEQ ID NO.1. This single-stranded DNA molecule has been experimentally verified to ensure high affinity and high specificity for human immunoglobulin E, effectively avoiding interference from other immunoglobulins.

[0012] As a preferred embodiment, the ion chelating agent is ethylenediaminetetraacetic acid (EDTA) or its salt, with a concentration of 5-20 mM. The concentration of the divalent metal ion is 1-5 mM. This suitable range of ion and chelating agent concentrations ensures both the stable maintenance of the aptamer conformation during capture and the effective alteration of the aptamer conformation during dissociation, thus balancing separation efficiency and product activity.

[0013] As a preferred embodiment, the nucleic acid aptamer is immobilized on the surface of magnetic microspheres. By introducing magnetic microspheres as a solid-phase support, rapid separation of the complex is achieved with the aid of an external magnetic field, significantly simplifying the operation process and improving separation efficiency.

[0014] As a preferred embodiment, the surface of the magnetic microspheres is coated with streptavidin, and biotin is coupled to the ends of the nucleic acid aptamers. Utilizing the high affinity and specific binding between streptavidin and biotin, stable and oriented immobilization of the nucleic acid aptamers on the magnetic microspheres is achieved, further improving capture efficiency.

[0015] As a preferred approach, nonionic surfactants are added to both systems containing divalent metal ions and systems containing ion chelating agents. The addition of nonionic surfactants effectively reduces nonspecific adsorption, further improving the purity of the target product.

[0016] A second aspect of the present invention discloses a kit for isolating human immunoglobulin E, comprising: Nucleic acid aptamers that specifically recognize human immunoglobulin E; Buffer solution containing divalent metal ions; Buffer solutions containing ion chelating agents.

[0017] The nucleic acid aptamer in the kit is a single-stranded DNA molecule, preferably with a single-stranded DNA sequence as shown in SEQ ID NO.1; the ion chelating agent in the kit is ethylenediaminetetraacetic acid or ethylenediaminetetraacetic acid salt.

[0018] Compared with the prior art, the present invention has the following beneficial effects: First, this invention uses nucleic acid aptamers that specifically recognize human IgE instead of traditional protein ligands, fundamentally avoiding non-specific binding with other immunoglobulins such as IgG and IgM, and significantly improving the purity of the separated products. Second, this invention utilizes the conformational reversibility of nucleic acid aptamers to achieve highly specific capture in systems containing divalent metal ions. In systems containing ion chelating agents, the conformation of the aptamers is altered by chelating ions, thus achieving gentle dissociation. This avoids the damage to the IgE structure caused by harsh elution conditions such as strong acids and bases in traditional affinity chromatography, effectively preserving the biological activity of IgE. Finally, by combining magnetic microsphere carriers with a non-ionic surfactant system, rapid separation and washing can be achieved using a magnetic field, significantly shortening the operation time and reducing non-specific adsorption interference. The entire process is easy to standardize and widely apply. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the IgE separation process based on nucleic acid aptamer magnetic beads according to an embodiment of the present invention; Figure 2 This is a Coomassie brilliant blue stained electrophoresis image of the human serum IgE separation results according to an embodiment of the present invention; Figure 3 This is an immunoblot identification image of the human serum IgE separation results according to an embodiment of the present invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0021] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0022] The present invention will now be described in further detail with reference to the accompanying drawings: This invention provides a method for separating human immunoglobulin E based on nucleic acid aptamers. The core of this method lies in utilizing the conformation-dependent ion-dependent properties of nucleic acid aptamers to achieve highly specific capture in systems containing divalent metal ions, and to achieve gentle dissociation by chelating ions to alter the conformation of the aptamers in systems containing ion chelating agents. This results in both specific capture and gentle dissociation of human immunoglobulin E.

[0023] Specifically, the conformationally reversibly dependent ions described above in this invention refer to ionic substances that can provide positive charge shielding and structural support for nucleic acid aptamers, prompting the nucleic acid aptamers to fold into a stable three-dimensional spatial structure (i.e., a specific binding conformation). The ion chelating agent referred to in this embodiment refers to a chemical substance that can specifically bind to divalent metal ions, thereby stripping or removing the ions from the environment surrounding the nucleic acid aptamer, causing the nucleic acid aptamer to lose structural support and undergo conformational unfolding.

[0024] The fundamental reason for choosing single-stranded DNA molecules as the recognition ligand for reversible conformation-dependent ion mechanisms lies in the unique structural characteristics of single-stranded DNA. Unlike double-stranded DNA, which forms a stable, rigid double helix structure due to the strong hydrogen bonds between complementary strands, single-stranded DNA molecules, lacking the constraint of complementary strands, have a highly flexible and loosely coiled backbone in its natural state. This structural freedom makes the secondary and tertiary structures of single-stranded DNA highly dependent on the stabilizing effect of external ions. When conformation-maintaining ions (divalent metal ions) are present in the system, single-stranded DNA can rapidly fold into stable stem-loops, pseudo-knots, or G-quadruplexes through electrostatic neutralization and coordination bridging; conversely, when the ions are deprived of chelation, single-stranded DNA easily loses its support and reverts to a loose linear state. It is this highly sensitive structural plasticity to the ionic environment that constitutes the structural premise for controlling capture and dissociation through ion environment switching in this invention. If double-stranded DNA or other ligands with strong internal constraints are used, their conformation will be difficult to respond sensitively to the increase or decrease of external ions, and thus the mild and controllable conformational switching mechanism in this invention cannot be achieved.

[0025] Preferably, divalent metal ions play a universal and crucial role in stabilizing the secondary structure of nucleic acids. Specifically, when single-stranded DNA molecules fold to form specific binding conformations such as stem-loops or G-quadruplexes, their phosphate backbone carries a dense concentration of negative charges. The electrostatic repulsion between these negative charges severely hinders the approach and folding of the strands. Divalent metal ions, such as magnesium or calcium ions, can not only effectively neutralize these repulsive forces through a strong positive charge shielding effect, but also form bridging supports between specific spatial positions of the nucleic acid strands through coordination bonds. This acts like a rivet, anchoring loose strands in the correct folding position and providing the structural rigidity necessary for the aptamer to maintain the specific binding conformation. It should be understood that although magnesium and calcium ions are listed as preferred examples in this embodiment, other divalent metal ions that can provide similar electrostatic shielding and coordination bridging functions, such as manganese or zinc ions, can also be used in other embodiments, as long as they can effectively support the binding conformation of the human immunoglobulin E aptamer.

[0026] Accordingly, this invention selects ethylenediaminetetraacetic acid (EDTA) or its salts as ion chelating agents based on their extremely high specific chelating ability and extremely fast reaction kinetics for divalent metal ions. The multidentate structure of EDTA can tightly encapsulate divalent metal ions like claws, forming an extremely stable coordination complex. This rapidly reduces the concentration of free divalent metal ions in the system to extremely low levels, quickly and thoroughly depriving the aptamer of the ion support required to maintain its conformation, forcing the aptamer to undergo rapid and complete unfolding, and thus gently releasing human immunoglobulin E. This precise ion deprivation mechanism avoids the risk of using strong acids or bases to damage the conformation of the target protein.

[0027] See Figure 1 This study demonstrates a complete experimental procedure for enriching human immunoglobulin E (IgE) in human peripheral blood serum using biotin-streptavidin magnetic beads coupled with nucleic acid aptamers. The core materials used in the experiment were biotin-labeled nucleic acid aptamers specifically recognizing IgE, magnetic microspheres modified with streptavidin, and human peripheral blood serum containing various proteins and human IgE. Magnesium ions served as a cofactor to maintain the spatial conformation of the aptamers. First, the linear biotinylated nucleic acid aptamers folded into stem-loop structures with recognition activity under the action of magnesium ions. Then, relying on the strong specific interaction between biotin and streptavidin, the aptamers were immobilized on the surface of the magnetic beads, thus preparing targeted capture magnetic beads. Subsequently, the functionalized magnetic beads were mixed and incubated with human peripheral blood serum, with magnesium ions continuously stabilizing the aptamer structure. After incubation, the nucleic acid aptamers on the magnetic beads specifically bound to the target IgE in the serum, while the remaining proteins remained free in the liquid phase. This method relies on the convenient separation properties of magnetic beads and the high specificity of aptamer recognition to efficiently enrich IgE from serum samples with complex composition. It is suitable for clinical pre-immunoassay analysis such as IgE detection related to allergic diseases and allergen screening.

[0028] Example 1 A method for isolating human immunoglobulin E based on nucleic acid aptamers, specifically comprising the following steps: 1) Preparation of nucleic acid aptamers The nucleic acid aptamer sequence used in this embodiment is: 5′-GCGCGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCCGCGC-3′; (as shown in SEQ ID NO. 1).

[0029] During the synthesis process, a biotin group is coupled to its 3′ end to enable it to bind to streptavidin magnetic beads.

[0030] 2) Pretreatment of magnetic beads Take 20 μl of magnetic bead stock solution, wash 3 times with magnetic bead washing solution, and resuspend the magnetic beads in 500 μl of magnetic bead washing solution for later use. Take 20 μL of streptavidin magnetic bead stock solution, place it in a 1.5 mL centrifuge tube, add magnetic bead washing solution and mix thoroughly. Then, use a magnetic rack to separate the supernatant and discard the supernatant. Repeat the washing process 3 times in total.

[0031] 3) Refolding treatment of nucleic acid aptamers The nucleic acid aptamer was diluted with deionized water to a final concentration of 100 μM.

[0032] The diluted nucleic acid aptamer solution was heated in a 90 °C metal bath or water bath for 3 min to completely denature it; then the heating was stopped and the solution was allowed to cool slowly to room temperature to form a stable secondary structure for later use.

[0033] 4) Construction of magnetic bead-nucleic acid aptamer complex Take 1.6 μL of the nucleic acid aptamer solution processed in step 3) and add it to 500 μL of the magnetic bead suspension obtained in step 2. Incubate slowly in a rotary mixer at room temperature for 60 min to allow the biotin-labeled nucleic acid aptamers to fully bind to the streptavidin on the surface of the magnetic beads. After incubation, separate the magnetic beads on a magnetic rack and discard the supernatant. Wash the magnetic beads 5 times with magnetic bead washing buffer to remove unbound nucleic acid aptamers. The final magnetic bead-nucleic acid aptamer complex is obtained and ready for use.

[0034] 5) Preparation of serum samples Peripheral venous blood was collected from patients with fresh allergic diseases and placed in a coagulation-promoting tube. The blood was allowed to coagulate naturally at room temperature for 20 minutes. The blood was then centrifuged at 4000×g for 5 minutes, and the supernatant serum was collected. 500 μL of the serum was taken and mixed gently with 500 μL of serum diluent to obtain a diluted serum sample for later use.

[0035] 6) Specific capture of IgE Mix the magnetic bead-nucleic acid aptamer complex obtained in step 4) with the diluted serum sample obtained in step 5. Incubate slowly in a rotary mixer at 4 °C for 12 h to allow the IgE in the serum to fully bind to the nucleic acid aptamers on the surface of the magnetic beads. After incubation, place the reaction system on a magnetic rack to separate the magnetic beads and discard the supernatant; wash three times with complex washing buffer to remove unbound or non-specifically bound protein components.

[0036] After washing, the magnetic bead-nucleic acid aptamer-IgE complex was obtained and set aside for later use.

[0037] 7) IgE dissociation and purification Add IgE dissociation buffer to the magnetic bead-nucleic acid aptamer-IgE complex obtained in step 6) to fully resuspend the magnetic beads. Incubate at room temperature for 1 h to allow the IgE bound to the nucleic acid aptamer to dissociate into the solution. After incubation, centrifuge at 4000 × g for 10 min and collect the supernatant. The obtained supernatant is the purified IgE sample, which can be used for subsequent detection or functional analysis.

[0038] See results Figure 2 and Figure 3 , Figure 2 The image shows the Coomassie brilliant blue stained electrophoresis results of immunoglobulin E separation from human serum, including samples from patients with allergic rhinitis and healthy individuals. Under reducing conditions in the allergic rhinitis patient group, heavy and light chain bands were clearly visible, while no obvious bands were observed in the healthy group, consistent with the extremely low concentration of immunoglobulin E in the serum of healthy individuals. This result demonstrates that the present invention achieves extremely high separation purity through the specific recognition of single-stranded DNA aptamers and the synergistic anti-adsorption effect of nonionic surfactants. Figure 3 The image shows an immunoblot pattern of immunoglobulin E separation from human serum. The pattern includes samples from both patients with allergic rhinitis and healthy individuals. Under reducing conditions in the allergic rhinitis patient group, a complete immunoglobulin E band is visible at the top, along with a heavy chain band below, and the band edges are sharp without tailing. This indicates that the present invention's gentle dissociation mechanism, which uses ion chelators to deprive ions and cause aptamer conformational unfolding, does not damage the spatial structure of immunoglobulin E itself; the folded state of its heavy and light chains remains intact, and its biological activity is well preserved. This confirms that the mechanism of the present invention, which controls aptamer conformation through environmental switching, successfully achieves a synergistic effect of highly specific capture and gentle, non-destructive dissociation, solving the defect problem of elution damaging target activity in existing technologies.

[0039] Example 2 This embodiment provides a kit for isolating human immunoglobulin E, which includes: a nucleic acid aptamer that specifically recognizes human immunoglobulin E, a buffer containing divalent metal ions, and a buffer containing an ion chelating agent.

[0040] Specifically, based on the core innovations of the aforementioned method, the reagent combination included in this kit fundamentally solves two major problems commonly found in existing immunoglobulin E purification kits: non-specific binding and elution damage. Specifically, the first component is a nucleic acid aptamer that specifically recognizes human immunoglobulin E, effectively preventing non-specific binding to other immunoglobulins; the second component is a buffer containing divalent metal ions, providing conformational support for the aptamer during the capture phase to ensure high affinity and high specificity binding; the third component is a buffer containing ion chelating agents, gently stripping ions during the dissociation phase, causing the aptamer to unfold and release the target without damage.

[0041] As a preferred embodiment, the above-mentioned kit for isolating human immunoglobulin E can use streptavidin-coated magnetic microspheres as a solid-phase carrier, directly eliminating the tedious steps of coating and blocking microspheres by the user, and ensuring batch-to-batch consistency of aptamer immobilization efficiency; the buffer containing divalent metal ions and the buffer containing ethylenediaminetetraacetic acid or its salts use standardized reagents that can be used directly, preventing the adverse effects of ion concentration deviations or pH fluctuations caused by self-preparation on the aptamer conformation.

[0042] Specifically, this embodiment provides a formula that can be implemented. In actual production, the formula can be adjusted according to requirements.

[0043] Formula 1: Magnetic Bead Washing Solution. Weigh 58.44 g of sodium chloride, 1.21 g of Tris base, and 0.372 g of disodium ethylenediaminetetraacetate (EDTA), and dissolve them thoroughly in approximately 800 mL of deionized water. Adjust the pH to 7.5 with hydrochloric acid solution. Then add 0.5 mL of Tween 20 to bring the final concentration to 0.05%. Mix well and bring the volume to 1000 mL with deionized water. Filter through a 0.22 μm filter membrane for sterilization and store at 4°C for later use. The introduction of a low concentration of disodium ethylenediaminetetraacetate aims to pre-remove any interfering metal ions that may remain on the surface of the microspheres.

[0044] Formula 2: Serum Diluent. Weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, and dissolve them in approximately 800 mL of deionized water. Add 0.41 g of magnesium chloride to achieve a final concentration of 2 mM divalent metal ions. After thorough stirring and dissolution, adjust the pH to 7.2-7.4 with hydrochloric acid or sodium hydroxide solution. Finally, bring the volume to 1000 mL with deionized water. After sterilization by filtration through a 0.22 μm filter membrane, store at 4 °C for later use. This formula uses a phosphate buffer system and adds 2 mM magnesium chloride to ensure that the aptamers in the diluted sample receive sufficient conformational support, achieving highly specific capture.

[0045] Formula 3: Complex Washing Solution. This washing solution is mainly used in the post-capture washing step to maintain conformation and remove contaminating proteins. Weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, and dissolve them in approximately 800 mL of deionized water; add 0.20 g of magnesium chloride to achieve a final concentration of 1 mM divalent metal ions; then add 0.5 mL of Tween 20 to achieve a final concentration of 0.05%; mix thoroughly and adjust the pH to 7.2-7.4, then bring the volume to 1000 mL with deionized water. After sterilization by filtration through a 0.22 μm filter membrane, store at 4 °C for later use. Compared to serum diluent, the complex washing buffer lowers the concentration of divalent metal ions to the lower limit of 1 mM. This is because after capture, the aptamer and target have formed a stable complex. At this point, only the critical concentration of ions is needed to maintain the conformation and prevent collapse. Moderately reducing the ion concentration helps to weaken some non-specific electrostatic adsorption. At the same time, the addition of 0.05% Tween 20 further synergistically removes impurities such as immunoglobulin G or immunoglobulin M that may have attached to the surface of the microspheres during the capture stage.

[0046] Formula 4: IgE Dissociation Buffer. This dissociation buffer is mainly used for the gentle release of the target, providing a system environment containing ion chelating agents. Weigh 14.91 g of potassium chloride and 1.21 g of Tris base, and dissolve them in approximately 800 mL of deionized water; add 3.72 g of disodium ethylenediaminetetraacetate (EDTA) to a final concentration of 10 mM EDTA or its salt; after thorough dissolution, adjust the pH to 7.6 with hydrochloric acid; finally, bring the volume to 1000 mL with deionized water. After sterilization by filtration through a 0.22 μm filter membrane, store at 4 ℃ for later use. This formula uses a high osmotic pressure Tris-hydrochloric acid buffer system to provide a suitable salt environment for the dissociated naked nucleic acid aptamers and free targets to prevent protein precipitation. The 10 mM concentration of disodium EDTA can efficiently and thoroughly chelate residual divalent metal ions in the system, forcing the aptamer conformation to rapidly unfold, achieving a gentle and efficient release of human immunoglobulin E.

[0047] In summary, the method for isolating human immunoglobulin E based on nucleic acid aptamers disclosed in this invention has the following advantages: 1. By using a nucleic acid aptamer that specifically recognizes human IgE instead of a traditional protein ligand, non-specific binding with other immunoglobulins such as IgG and IgM is fundamentally avoided, significantly improving the purity of the separated products and resulting in high separation specificity. 2. Utilizing the conformation-dependent properties of nucleic acid aptamers, highly specific capture is achieved in systems containing conformation-maintaining ions, and mild dissociation is achieved by chelating ions to change the conformation of aptamers in systems containing ion chelating agents. This avoids the damage to the IgE structure caused by harsh elution conditions such as strong acids and strong bases in traditional affinity chromatography, and effectively maintains the biological activity of IgE. 3. By combining magnetic microsphere carriers with nonionic surfactant systems, rapid separation and washing can be achieved through a magnetic field, which significantly shortens the operation time and reduces non-specific adsorption interference, making the operation simple and quick.

[0048] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for isolating human immunoglobulin E based on nucleic acid aptamers, characterized in that, By utilizing nucleic acid aptamers that specifically recognize human immunoglobulin E to bind to magnetic carriers, human immunoglobulin E can be selectively bound to and separated from samples containing human immunoglobulin E.

2. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 1, characterized in that, include: Nucleic acid aptamers that specifically recognize human immunoglobulin E are contacted with samples containing human immunoglobulin E in a system in the presence of divalent metal ions to capture human immunoglobulin E. In a system containing an ion chelating agent, the captured human immunoglobulin E is dissociated from the nucleic acid aptamer to obtain isolated human immunoglobulin E.

3. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 1 or 2, characterized in that, The nucleic acid aptamer is a single-stranded DNA molecule.

4. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 3, characterized in that, The sequence of the single-stranded DNA molecule is shown in SEQ ID NO.

1.

5. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 2, characterized in that, The ion chelating agent is ethylenediaminetetraacetic acid or ethylenediaminetetraacetic acid salt, with a concentration of 5-20 mM.

6. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 2, characterized in that, The concentration of the divalent metal ions is 1-5 mM.

7. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 1 or 2, characterized in that, The nucleic acid aptamer is immobilized on the surface of the magnetic microspheres, and the surface of the magnetic microspheres is coated with streptavidin.

8. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 1 or 2, characterized in that, Biotin is coupled to the end of the nucleic acid aptamer.

9. The method for isolating human immunoglobulin E based on nucleic acid aptamers according to claim 2, characterized in that, Nonionic surfactants are added to both systems containing divalent metal ions and systems containing ion chelating agents.

10. A kit for isolating human immunoglobulin E, characterized in that, include: Nucleic acid aptamers that specifically recognize human immunoglobulin E; Buffer solution containing divalent metal ions; Buffer solutions containing ion chelating agents.

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