Method for improving stability of antigen, cross-linked antigen and application of cross-linked antigen

Abstract

The invention relates to a method for improving antigen stability, a cross-linked antigen and application of the cross-linked antigen. The method comprises the following steps: mixing a target antigen with a cross-linking agent to obtain a cross-linked antigen; the cross-linking agent is a hydrophilic cross-linking agent. The cross-linked antigen provided by the invention is relatively good in stability, and relatively good stability can be kept for a long time.

Description

Technical Field

[0001] The present application relates to the field of biological reagent technology, and in particular to a method for improving antigen stability, cross-linked antigens and applications thereof. Background Art

[0002] In the field of clinical medical analysis, the corresponding antibodies are usually obtained by immunizing animals with purified antigens, and then various types of antigen-antibody reaction models and detection methods are established. At present, there are two main ways to obtain antigens. One is to obtain them by natural methods, that is, natural antigens obtained after extraction and purification from the body. The antigens obtained in this way can maintain the structural characteristics of the antigen itself. The second is to obtain some antigens through cultivation, expression, recombination and other methods. In the process of obtaining such antigens, the effective information of the original antigen is expressed, but compared with natural antigens, there is still a gap in conformation, modification and protein activity or stability, which often becomes a more difficult problem faced in medical detection.

[0003] At present, in order to solve the problem of antigen stability, antigen protectants are generally added to antigen reagents, such as amino acids, proteins, polyhydroxyl groups, etc. However, this method is not suitable for all types of antigens. Even if protective agents are added to some antigen reagents, they still cannot effectively improve the stability of the antigen in the long term, or the improvement in stability cannot reach the expected level. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides a method for improving antigen stability, cross-linked antigens and their applications, which can improve the stability of antigens, are simple to prepare and have low cost.

[0005] The first aspect of the present application provides a method for improving antigen stability, comprising: mixing a target antigen with a cross-linking agent to obtain a cross-linked antigen; the cross-linking agent is a hydrophilic cross-linking agent.

[0006] In some embodiments, both ends of the cross-linking agent can bind to the target antigen; preferably, the cross-linking agent binds to the amino group on the surface of the target antigen.

[0007] In some embodiments, the cross-linking agent is bis-n polyethylene glycol-NHS lipid, wherein 6≤n≤60; preferably, 12≤n≤48.

[0008] In some embodiments, the mass ratio of the target antigen to the cross-linking agent is 1:(10-50).

[0009] In some embodiments, after mixing the target antigen with the cross-linking agent, the method further comprises:

[0010] The mixed solution is dialyzed to retain the monomeric cross-linked antigen.

[0011] The second aspect of the present application provides a cross-linked antigen, which is prepared according to the above method.

[0012] In some embodiments, there are multiple amino groups on the surface of the target antigen of the cross-linked antigen; preferably, the target antigen is hepatitis B virus e antigen.

[0013] The third aspect of the present application provides a neutralizing antigen reagent, comprising a buffer and the above-mentioned cross-linked antigen.

[0014] The fourth aspect of the present application provides an immunoassay kit, which comprises:

[0015] Reagent 1, comprising receptor particles coupled to a first partner antibody;

[0016] Reagent 2, comprising a biotinylated secondary partner antibody;

[0017] Reagent 3, the neutralizing antigen reagent as described in claim 7.

[0018] Preferably, the kit further comprises a reagent 4, wherein the reagent 4 comprises donor particles, wherein the donor particles are capable of generating active oxygen under an excited state, and avidin is bonded to the surface of the donor microspheres.

[0019] The fifth aspect of the present application provides a use of the cross-linked antigen as described in the second aspect of the present application, or the neutralization antigen reagent as described in the third aspect of the present application, or the kit as described in the fourth aspect of the present application in photochemiluminescence detection.

[0020] The technical solution provided by this application has the following beneficial effects:

[0021] The cross-linked antigen of the present application has good stability in the liquid phase and can maintain good stability for a long time; it is also simple to prepare and low in cost.

[0022] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. DETAILED DESCRIPTION

[0023] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0024] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials or equivalent methods and materials described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0026] I. Terminology

[0027] The term "particle" as used herein refers to a small, localized object that can be attributed to physical properties such as volume, mass or size. Particles can be symmetrical, spherical, or irregular, asymmetric shapes; they can be of any size. Particles suitable for use in the present application can be spherical, for example, particles with diameters in the nanometer or micrometer range. Particles can be solids (such as polymers, metals, glasses, organic or inorganic substances such as minerals, salts and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, siliceous fluids), vesicles (such as synthetic such as phospholipids, or natural such as cells and organelles). Particles can be organic or inorganic, such as latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silicon particles, metal sols, cells and microcrystalline dyes. Particles can be expandable or non-expandable, they can be porous or non-porous, they have any density, but preferably have a density close to that of water, preferably float in water, and are composed of transparent, partially transparent or non-transparent materials. The particles may or may not be charged, and when charged, they are preferably negatively charged. The particles are typically multifunctional or capable of binding to a donor or receptor through specific or non-specific covalent or non-covalent interactions. Typical functional groups include carboxyl, aldehyde, amino, cyano, vinyl, hydroxyl, thiol, and the like. A non-limiting example of particles suitable for the present application is polystyrene latex microspheres. The particles described herein have a diameter of 50 nm to 20 μm; preferably, the particles have a diameter of 100 nm to 10 μm; more preferably, the particles have a diameter of 200 nm to 5 μm; further preferably, the particles have a diameter of 750 nm to 1 μm.

[0028] The term "donor particles" used in the present invention refers to particles containing sensitizers that can generate active intermediates such as active oxygen that react with receptor particles after being activated by energy or active compounds. Donor particles can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In a specific embodiment, the donor particles are polymer microspheres filled with photosensitizers, and the photosensitizers can be photosensitizers known in the art, preferably compounds that are relatively stable to light and do not react effectively with singlet oxygen.

[0029] The term "acceptor particles" used in the present invention refers to particles containing compounds that can react with active oxygen to produce detectable signals. Donor particles are activated by energy or active compounds and release high-energy active oxygen, which is captured by nearby acceptor particles, thereby transferring energy to activate the acceptor particles.

[0030] The term "active oxygen" as used herein refers to a general term for substances in the body or in the natural environment that are composed of oxygen, contain oxygen and are active in nature, mainly an excited state of oxygen molecules, including the one-electron reduction product of oxygen, superoxide anion (O 2 ·-), the two-electron reduction product hydrogen peroxide (H 2 O 2 ), three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and reactive oxygen species ( 1 O 2 )wait.

[0031] The term "test sample" of the present invention refers to a mixture containing or suspected to contain the target molecule to be tested. The test sample that can be used in the present invention includes body fluids, such as blood (can be anticoagulated blood commonly seen in the collected blood sample), plasma, serum, urine, semen, saliva, cell culture, tissue extracts, etc. Other types of test samples include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The test sample can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, the test sample can be diluted with a diluent before being tested on the machine and then tested on the detection instrument.

[0032] The term "antibody" as used herein is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, for example, biotin or avidin (a member of a biotin-avidin specific binding pair member), etc.

[0033] The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0034] As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.

[0035] The term "specific binding" used in the present invention refers to the mutual recognition and selective binding reaction between two substances, which is the correspondence of the conformations between the corresponding reactants from the perspective of the three-dimensional structure. Under the technical concept disclosed in the present invention, the detection method of the specific binding reaction includes but is not limited to: double antibody sandwich method, competition method, neutralization competition method, indirect method or capture method.

[0036] II. Specific implementation plan

[0037] The embodiment of the present application provides a method for improving the stability of an antigen, which includes: mixing a target antigen with a cross-linking agent to obtain a cross-linked antigen; the cross-linking agent is a hydrophilic cross-linking agent. There are multiple amino groups on the surface of the target antigen, and each two amino groups cross-link a cross-linking agent molecule. The entire target antigen can cross-link multiple cross-linking agent molecules, thereby playing a binding effect similar to a rope, so that it binds the spatial structure of the target antigen and is not easily dispersed due to certain extreme conditions; and the use of a hydrophilic cross-linking agent enhances the hydrophilicity of the target antigen surface, which can prevent the target antigen from aggregating and settling, thereby obtaining an antigen with good stability in the liquid phase.

[0038] In some embodiments, there are multiple amino groups on the surface of the target antigen. In some embodiments, both ends of the cross-linking agent can bind to the target antigen. In some embodiments, the cross-linking agent binds to the amino groups on the surface of the target antigen. That is, after the cross-linking agent binds to the target antigen, the corresponding cross-linked antigen is generated.

[0039] In some embodiments, the crosslinking agent can be a bis-n polyethylene glycol-NHS lipid, for example, n represents the number of polyethylene glycols, i.e., PEG, 6≤n≤60; preferably, 12≤n≤48. For example, n can be 12, 18, 20, 24, 30, 35, 40, 43, 48, etc. Among them, bis-n polyethylene glycol-NHS lipid, i.e., Bis-PEGn-NHS, is a PEG linker containing two NHS ester groups. It can be understood that PEG has a good hydrophilic effect and can be used to generate a hydrophilic crosslinking agent, which then reacts with the target antigen to increase the hydrophilicity of the target antigen, thereby increasing the stability of the antigen.

[0040] In some embodiments, the mass ratio of the target antigen to the cross-linking agent is 1:(10-50). Preferably, for example, the mass ratio of the target antigen to the cross-linking agent can be 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60, etc., which are only exemplified here and are not limited. By controlling the mass ratio of the cross-linking agent to the target antigen, it is possible to avoid waste caused by adding too much cross-linking agent, and to avoid failure to achieve the expected effect due to too little cross-linking agent, so that the amino group on the target antigen has an appropriate amount of cross-linking agent to complete cross-linking, thereby achieving a better stability of the target antigen.

[0041] In some embodiments, after mixing the target antigen with the cross-linking agent, the method further comprises:

[0042] The mixed solution is dialyzed and purified to intercept and obtain monomeric cross-linked antigens. Preferably, cross-linked antigens with a molecular weight greater than 3500D are intercepted. After the target antigen is mixed with the cross-linking agent for reaction, the reacted solution is loaded into a dialysis bag with a preset molecular weight cutoff, and the dialysis bag is placed in a buffer solution with a volume greater than 100 times, and dialyzed within a preset temperature range. By replacing the buffer solution several times, each dialysis lasts for a preset time, thereby removing excess cross-linking agent, so that cross-linked antigens with a molecular weight greater than 3500D are intercepted in the dialysis bag. It should be noted that the molecular weight of the antigen is generally greater than 3500D, and the molecular weight of the cross-linking agent is less than 3500D. By using a dialysis bag with a cutoff of 3500D, the excess cross-linking agent free in the solution can be dialyzed and removed.

[0043] In some embodiments, after the mixed solution is dialyzed, the dialyzed cross-linked antigens can be purified to separate and obtain monomeric antigens. The monomeric antigen solution is fixed to a preset concentration and can be used to prepare an antigen reagent.

[0044] The embodiment of the present application also provides a cross-linked antigen, which includes the cross-linked antigen prepared by the above method. For example, the target antigen corresponding to the cross-linked antigen can be hepatitis B virus e antigen, hepatitis B virus surface antigen, etc., which is illustrated here by way of example and is not intended to be limiting.

[0045] The embodiments of the present application provide a neutralizing antigen reagent, including a buffer and the cross-linked antigens in the above embodiments. For example, the buffer may be a commonly used buffer such as a mixed buffer of PBS and BSA, a mixed buffer of HEPES and BSA, etc.; wherein the pH value of the buffer may be 7 to 8. For example, a buffer with 50mmol / mL PBS and 3% BSA as the main raw materials has a pH of 7.4. The above antigen is diluted to a working concentration by a buffer to obtain a neutralizing antigen reagent. Among them, the working concentration is designed according to the actual situation, for example, it can be 0.2μg / mL to 0.5μg / mL, such as 0.25μg / mL, etc., without limitation.

[0046] The embodiments of the present application also provide an immunoassay kit, which includes: reagent 1, reagent 2 and reagent 3. Among them, reagent 1 contains receptor particles coupled to a first partner antibody; reagent 2 contains a second partner antibody labeled with biotin; and reagent 3 is the above-mentioned neutralizing antigen reagent. The immunoassay kit of the present application can perform immunoassay by photochemiluminescence technology. It should be noted that reagent 3 refers to a neutralizing antigen reagent that can be detected by a neutralization competition method, and the neutralizing antigen in the reagent 3 refers to a cross-linked antigen in which the target antigen is combined with the above-mentioned cross-linking agent.

[0047] In some embodiments, the kit further comprises a reagent 4, wherein the reagent 4 comprises donor particles, wherein the donor particles are capable of generating reactive oxygen species under excitation, and the surface of the donor microspheres is bonded with avidin. In some specific embodiments of the present application, the avidin is selected from egg white avidin, streptavidin, egg yolk avidin, neutravidin and avidin-like, preferably selected from neutravidin and / or streptavidin.

[0048] Specifically, the basic principle of photoinduced chemiluminescence is a homogeneous immune reaction, which is based on the first partner antibody (i.e., reagent 1) coated on the surface of the receptor particle, the second partner antibody (i.e., reagent 2) labeled with biotin, and the neutralizing antigen reagent (i.e., reagent 3) containing the neutralizing antigen; when the sample to be tested does not contain the target molecule, for example, does not contain the target antibody, the three form an immune complex in the liquid phase, i.e., the receptor particle-first partner antibody-neutralizing antigen-second partner antibody-biotin. In addition, the surface of the donor particle is coated with avidin (i.e., reagent 4), and through the biotin-avidin reaction, the donor particle and the receptor particle are brought closer to generate an immune complex of receptor particle-first partner antibody-neutralizing antigen-second partner antibody-biotin-avidin-donor particle. Under the excitation of the laser, the transfer of ionized oxygen between the donor particle and the receptor particle occurs, thereby generating high-energy red light, and the corresponding chemiluminescent signal value is obtained through the single-photon counter on the chemiluminescent immunoassay. When the sample reagent to be tested contains target molecules, such as target antibodies, the target antibodies compete to neutralize the antigens, and the above-mentioned immune complexes cannot be formed between the donor particles and the acceptor particles, resulting in the distance between the two particles exceeding the propagation range of ionized oxygen. Ionized oxygen is quickly quenched in the liquid phase, and no high-energy red light is generated. The corresponding chemiluminescent signal value obtained during detection is reduced. The target antibody content in the sample to be tested can be calculated through the final measured chemiluminescent signal value.

[0049] III. Specific Examples

[0050] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0051] The present application example uses the detection of samples containing hepatitis B virus e antibodies as an example to further explain the present application in detail. After the corresponding neutralizing antigen reagent is prepared by combining the amino group of the neutralizing antigen with different cross-linking agents, the neutralizing antigen reagent prepared by the same cross-linking agent is divided into 2 parts, one of which is not heat-treated (i.e., 0-day heat treatment), stored at 2°C-8°C, and directly tested with the sample; the other is first heat-treated for 7 consecutive days (i.e., 7-day heat treatment) and then tested with the sample.

[0052] The detection principle is: On the fully automatic chemiluminescence immunoassay, the samples containing hepatitis B virus e antibodies were detected by the neutralization competition immunological method, and the corresponding chemiluminescence signal values ​​were obtained. The stability of the neutralization antigen reagent after cross-linking with different types of cross-linking agents was evaluated by comparing the signal deviations corresponding to 0-day heat treatment and 7-day heat treatment.

[0053] Example 1 Preparation method of reagents

[0054] (I) Coupling of the first partner antibody to the receptor particle

[0055] 1. The first partner antibody was dialyzed into 50 mmol / mL CB buffer with a pH value of 9.0, and the concentration was measured to be 1 mg / mL. The first partner antibody was an Anti-HBe antibody.

[0056] 2. Add 0.5 mL of receptor particles and 0.5 mL of the first partner antibody obtained in step 1 to a 2 mL centrifuge tube, mix well and add 100 μL of 10 mg / mL NaBH 4 Solution (prepared with 50 mmol / mL CB buffer) was reacted at 2°C to 8°C for 4 hours.

[0057] 3. After the reaction in step 2 is completed, add 0.5 mL of 100 mg / mL BSA solution (50 mmol / mL CB buffer) to the centrifuge tube and react at 2°C to 8°C for 2 hours.

[0058] 4. After the reaction in step 3 is completed, centrifuge the centrifuge tube at 30000G for 45 minutes, discard the supernatant, and resuspend with 50mmol / mL MES buffer. Repeat the centrifugation and washing four times, and dilute to a final concentration of 100μg / mL to obtain a receptor particle solution coupled to the first partner antibody.

[0059] (ii) Biotin-labeled secondary partner antibody

[0060] 1. The second partner antibody was dialyzed to 50 mmol / mL PBS buffer with a pH value of 7.4, and the concentration was measured to be 1 mg / mL. The second partner antibody is a specific Anti-HBe antibody that binds to a different antigen epitope than the first partner antibody.

[0061] 2. Take the 1 mg / mL second partner antibody prepared in step 1 and mix it with the prepared 1.997*10 -5 mol / mL Biotin-NHS (DMSO) solution was mixed at a volume ratio of 10000:116, and mixed quickly. The reaction was allowed to stand at 2°C to 8°C for 12 hours to 16 hours to obtain a biotin-labeled second partner antibody.

[0062] 3. Place the biotin-labeled second partner antibody in a dialysis bag with a molecular weight cutoff of 3500D, and then place it in a 50mmol / mL PBS buffer solution that is 100 times larger than the volume of the biotin-labeled second partner antibody solution, and dialyze at 2℃~8℃. Change the buffer twice, and dialyze for 4 hours to 5 hours each time.

[0063] 4. Aspirate the dialyzed biotin-labeled second partner antibody and transfer it to a clean centrifuge tube, take a sample to determine the concentration of the second partner antibody and the ratio of the second partner antibody to biotin. Adjust the concentration of the biotin-labeled second partner antibody that has passed the quality inspection to 0.5 mg / mL.

[0064] (III) Antigen cross-linking

[0065] 1. The recombinant hepatitis B virus HBeAg antigen was dialyzed into 50 mmol / mL PBS buffer with a pH value of 7.4, and the concentration was measured to be 1 mg / mL.

[0066] 2. Cross-linking: Take the 1 mg / mL neutralizing antigen treated in step 1 and mix them with the pre-prepared cross-linking agent solutions according to the preset mass ratio, mix them quickly, and let them react at 2°C to 8°C for 12 to 16 hours to obtain a neutralizing antigen solution combined with a cross-linking agent.

[0067] 3. Dialysis: The neutralized antigen solutions of each mass ratio reacted in step 2 were respectively placed in a dialysis bag with a molecular weight cut-off of 3500D, and then placed in a 50mmol / mL PBS buffer with a volume greater than 100 times for dialysis at 2℃~8℃. The buffer was replaced twice, and the dialysis was performed for 4 hours to 5 hours each time to remove the free excess cross-linking agent in the solution.

[0068] 4. Purification: Aspirate each cross-linked neutralized antigen dialyzed in step 3 and transfer it to a clean centrifuge tube, and use a purifier to separate monomer cross-linked antigens and polymer cross-linked antigens.

[0069] 5. Take the purified monomer cross-linked antigen solution or the uncross-linked antigen (recombinant hepatitis B virus HBeAg antigen) solution to determine the concentration and prepare the neutralized antigen solution. Adjust the concentration of each qualified neutralized antigen solution to 0.5 mg / mL.

[0070] (IV) Preparation of Reagent 1, Reagent 2 and Reagent 3

[0071] 1. Dilute the receptor particle solution coupled to the first partner antibody obtained in step (i) above to a working concentration of 25 μg / mL using MES buffer as reagent 1.

[0072] 2. Dilute the biotin-labeled second partner antibody solution obtained in the above step (ii) to a working concentration of 1 μg / mL with PBS buffer, and use them as the corresponding reagent 2.

[0073] 3. Dilute each neutralized antigen solution obtained in the above step (iii) to a working concentration of 0.25 μg / mL using a mixed buffer of PBS and BSA as reagent 3.

[0074] 4. Reagent 1, reagent 2 and reagent 3 are respectively packed into reagent bottle 1, reagent bottle 2 and reagent bottle 3 to assemble into a corresponding complete reagent set.

[0075] Example 2 Comparison of the presence and type of cross-linking agent

[0076] (I) Reagent handling

[0077] The reagent 3 prepared in Example 1 was divided into two parts, one of which was not heat treated and stored in an environment of 2°C to 8°C for 7 days (i.e., heat treatment time was 0 days), and the other was heat treated at 37°C for 7 consecutive days (i.e., heat treatment time was 7 days). Then, each sample was detected simultaneously according to the following detection steps to obtain the corresponding chemiluminescence signal value.

[0078] (II) Detection method

[0079] Each complete reagent kit is used to test each calibrator, negative sample, positive sample, or other sample to be tested according to the following test steps. The specific test steps are as follows:

[0080] 1. Add 25 μL of Reagent 1 to the reaction wells.

[0081] 2. Add 25 μL of reagent 2 to the reaction well.

[0082] 3. Add 50 μL of reagent 3 to the reaction well.

[0083] 4. Add 25 μL of the sample to be tested into the reaction well.

[0084] 5. Incubate the mixed solution in the reaction well for the first time at 37°C for 1020 seconds.

[0085] 6. Add 175 μL of universal solution containing 50 μg / mL donor particles, i.e., reagent 4, to the reaction wells.

[0086] 7. Incubate the mixed solution in the reaction well for the second time at 37°C for 900 seconds.

[0087] 8. Perform photoinduced chemiluminescence detection to obtain the corresponding chemiluminescence signal value.

[0088] 3. Data analysis methods

[0089] The deviation of the chemiluminescent signal values ​​of the same sample detected by reagent 3 heat-treated for 7 days and reagent 3 heat-treated for 0 days was calculated. The smaller the deviation, the more stable the neutralizing antigen reagent is, that is, the more stable the neutralizing antigen in the reagent is.

[0090] (IV) Experimental results

[0091] The stabilities of uncrosslinked antigens and crosslinked antigens crosslinked with crosslinkers Bis-LC-LC-NHS, Bis-Glu6-NHS and Bis-PEG6-NHS at a mass ratio of 1:30 (antigen: crosslinker) were compared.

[0092] The results (Table 1) show that regardless of the type of cross-linking agent, the deviation values ​​of the neutralization antigen reagent prepared by cross-linking antigens are all smaller than the deviation values ​​of the neutralization antigen reagent prepared by non-cross-linking antigens, and the stability is better.

[0093] Further comparison of the stabilities of neutralizing antigen reagents prepared by antigens cross-linked with different cross-linkers LC-LC and Glu and PEG revealed that the neutralizing antigen reagent prepared by antigens cross-linked with Glu and PEG had better stability, that is, the effect of cross-linking antigens with hydrophilic cross-linkers was better.

[0094] Table 1

[0095]

[0096] Example 3 Comparison of the length of cross-linking agents

[0097] According to the method in Example 1, the recombinant hepatitis B virus HBeAg antigen and Bis-PEG6-NHS, Bis-PEG12-NHS, Bis-PEG24-NHS, Bis-PEG48 and Bis-PEG60-NHS-NHS cross-linking agents with different numbers of PEG were cross-linked at a mass ratio of 1:30 to prepare a neutralizing antigen reagent. After the neutralizing antigen reagent was treated in the manner in Example 2, it was tested with each sample to compare the stability of the neutralizing antigen reagent prepared with cross-linking agents of different lengths. The results (Table 2) show that the neutralizing antigen reagent cross-linked Bis-PEG (12-48)-NHS has better stability.

[0098] Table 2

[0099]

[0100]

[0101] Example 4 Comparison of the mass ratio of antigen to cross-linking agent

[0102] According to the method in Example 1, the recombinant hepatitis B virus HBeAg antigen and the cross-linking agent Bis-PEG12-NHS were cross-linked at mass ratios of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 to prepare a neutralizing antigen reagent. After the neutralizing antigen reagent was treated in the manner in Example 2, it was tested with each sample to compare the stability of the neutralizing antigen reagent after cross-linking with different mass ratios of neutralizing antigen and cross-linking agent.

[0103] The results (Table 3) showed that the neutralization antigen reagent had better stability after cross-linking with the neutralization antigen and the cross-linking agent at a mass ratio of 1: (10-50).

[0104] Table 3

[0105]

[0106]

[0107] Comprehensive experimental results show that the neutralization antigen reagent prepared by cross-linking the neutralization antigen and Bis-PEG (12-48)-NHS cross-linker at a mass ratio of 1: (10-50) has the best stability, with the stability improved by about 30%.

[0108] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for improving antigen stability, characterized in that: The target antigen is mixed with a cross-linking agent to obtain a cross-linked antigen; the cross-linking agent is a hydrophilic cross-linking agent.

2. The method according to claim 1, characterized in that Both ends of the cross-linking agent can bind to the target antigen; preferably, the cross-linking agent binds to the amino group on the surface of the target antigen.

3. The method according to claim 1 or 2, characterized in that: The cross-linking agent is bis-n polyethylene glycol-NHS lipid, wherein 6≤n≤60; preferably, 12≤n≤48.

4. The method according to any one of claims 1 to 3, characterized in that: The mass ratio of the target antigen to the cross-linking agent is 1:(10-50).

5. The method according to claim 1, characterized in that After the target antigen is mixed with the cross-linking agent, the method further comprises: The mixed solution is dialyzed and purified to retain the monomeric cross-linked antigen.

6. A cross-linked antigen, characterized in that Prepared according to the method according to any one of claims 1 to 5.

7. The cross-linked antigen according to claim 6, characterized in that There are multiple amino groups on the surface of the target antigen of the cross-linked antigen; preferably, the target antigen is hepatitis B virus e antigen.

8. A reagent for neutralizing an antigen, comprising a buffer and the cross-linked antigen according to claim 6 or 7.

9. An immunoassay kit, characterized in that: include: Reagent 1, comprising receptor particles coupled to a first partner antibody; Reagent 2, comprising a biotinylated secondary partner antibody; Reagent 3, the neutralizing antigen reagent as described in claim 7. Preferably, the kit further comprises a reagent 4, wherein the reagent 4 comprises donor particles, wherein the donor particles are capable of generating active oxygen under an excited state, and avidin is bonded to the surface of the donor microspheres.

10. Use of the cross-linked antigen according to claim 6 or 7, the neutralizing antigen reagent according to claim 8, or the kit according to claim 9 in photoinduced chemiluminescence detection.

Images