Method for improving stability of labeled antigen, labeled antigen and application of labeled antigen

Abstract

The invention relates to a method for improving stability of a labeled antigen, the labeled antigen and application of the labeled antigen. The method comprises the step of combining the antigen and a marker through a hydrophilic molecular chain to obtain the labeled antigen. When being applied to light-excited chemiluminescence detection, the labeled antigen provided by the invention has better specificity, higher sensitivity and high detection accuracy.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a method for improving the stability of a labeled antigen, a labeled antigen and an application thereof. Background Art

[0002] Chemiluminescent immunoassay is widely used in clinical testing, drug analysis and other fields due to its high sensitivity and high accuracy. Common chemiluminescent immunoassays such as direct chemiluminescence, enzymatic chemiluminescence, electrochemiluminescence, luminescent oxygen channel and other methods all involve the use of labeled antibodies or labeled antigens in the detection process.

[0003] Taking the Luminescent Oxygen Channeling Immunoassay (LOCI) method for detecting antibodies in the sample to be tested as an example, the "sandwich structure" (antigen coated with luminescent microspheres - antibody to be tested - biotin-labeled antigen) combines with the photosensitive microspheres coated with avidin to generate the transfer of ion oxygen energy and emit a light signal to detect the sample to be tested. In the prior art, when the LOCI method is used for antibody detection, the labeled antibodies are easily aggregated in the reagent due to hydrophobic interaction, resulting in a decrease in the stability of the labeled antigen and affecting the sensitivity of the detection. 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 the stability of labeled antigens, labeled antigens and their applications. When the labeled antigens are applied to photochemiluminescence detection, the false positive and false negative rates are low, the specificity is good, the sensitivity is high, and the detection accuracy is high.

[0005] Based on this, the first aspect of the present application provides a method for improving the stability of a labeled antigen, wherein the antigen is combined with a marker via a hydrophilic molecular chain to obtain a labeled antigen.

[0006] In some embodiments of the present application, the hydrophilic molecule chain is selected from a sugar chain; preferably, the sugar chain is selected from one of dextran, glycogen, galactan, fructan, starch, mannan, agarose, polyribose, and a dextran derivative; preferably selected from one of dextran, starch, glycogen, polyribose, and a dextran derivative; more preferably selected from dextran or a dextran derivative.

[0007] In some embodiments of the present application, the hydrophilic molecular chain is formed by N monomer sugars connected in sequence, 2≤N≤15; preferably 2≤N≤12.

[0008] In some embodiments of the present application, the molar ratio of the hydrophilic molecular chain to the number of amino groups on the antigen surface is 1:(0.1-6); preferably 1:(1-6); more preferably 1:(1-3).

[0009] In some embodiments of the present application, the marker is selected from an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, an immunoglobulin binding protein, avidin, avidin derivatives, biotin or a biotin derivative.

[0010] The second aspect of the present application provides a labeled antigen prepared according to the method described in the first aspect of the present application.

[0011] The third aspect of the present application provides a labeled antigen reagent, comprising a buffer solution and the labeled antigen described in the second aspect of the present application, wherein the reagent does not contain a surfactant.

[0012] In some embodiments of the present application, the buffer does not contain sugar.

[0013] The fourth aspect of the present application provides an immunoassay kit, comprising:

[0014] Reagent a, the labeled antigen reagent as described in the third aspect of the present application;

[0015] Reagent b, a receptor reagent that binds to the antigen or its binding fragment and is capable of reacting with singlet oxygen to generate a detectable signal;

[0016] Reagent c is a donor reagent capable of generating singlet oxygen, which is bound to a ligand capable of specifically recognizing and binding to the label.

[0017] The fifth aspect of the present application provides a use of the labeled antigen described in the second aspect of the present application, the labeled antigen reagent described in the third aspect of the present application, or the kit described in the fourth aspect of the present application in photochemiluminescence detection.

[0018] The technical solution provided by the present application has the following beneficial effects: the labeled antigen of the present application has good stability in the liquid phase; when the labeled antigen is applied to photochemiluminescence detection, better photochemiluminescence detection results can be obtained, with low false positive and false negative rates, good specificity and high sensitivity, and high detection accuracy. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] 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.

[0022] I. Terminology

[0023] The term "homogeneous" as used in this application is defined as "homogeneous", which means that detection can be performed without separating the bound antigen-antibody complex and the remaining free antigen or antibody.

[0024] The term "test sample" used in this application refers to a mixture that may contain an analyte. Typical test samples that can be used in this application include body fluids, including blood, plasma, serum, urine, semen, saliva, etc.

[0025] The term "hydrophilicity" as used herein refers to molecules with polar groups that have a greater affinity for water, can attract water molecules, or are easily soluble in water. The term "hydrophobicity" is also known as water repellency, and refers to the property of non-polar molecules that have an affinity for water.

[0026] The term "antibody" as used herein is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to antigens, 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 can be further conjugated to other moieties, such as specific binding pair members, for example, biotin or avidin, etc.

[0027] The term "antigen" as used herein refers to a substance that can stimulate a matrix to produce an immune response and can bind to the immune response product antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect. The antigen may be a fusion antigen, and in any desired case, the antigen may be further conjugated with other parts, such as specific binding partner members, such as biotin or avidin.

[0028] As used herein, the term "binding" or "bonding" 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.

[0029] The term "specific binding" as used in this application refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the responding reactants from a three-dimensional structural perspective.

[0030] The term "specific binding pair member" as used herein refers to a pair of molecules that can specifically bind to each other, for example, enzyme-substrate, antigen-antibody, ligand-receptor. A specific example of a specific binding pair member is the biotin-avidin system, in which "biotin" is widely present in animal and plant tissues, and has two cyclic structures on its molecule, namely, an imidazolide ring and a thiophene ring, wherein the imidazolide ring is the main site for binding to avidin, and "avidin" is a protein secreted by Streptomyces with a molecular weight of 65kD.

[0031] The term "donor" as used herein refers to a sensitizer that can produce active intermediates such as reactive oxygen species that react with an acceptor after being activated by energy or an active compound. The donor can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In some embodiments of the present application, the donor is coated on microparticles through functional groups to form polymer microspheres filled with photosensitive compounds, which can produce reactive oxygen species under light excitation. At this time, the polymer microspheres can be called photosensitive microspheres or oxygen supply microspheres. The donor surface can have hydrophilic aldehyde dextran, and the interior is filled with a sensitizer. The sensitizer can be a sensitizer known in the art, preferably a compound that is relatively stable to light and does not react effectively with reactive oxygen species, and its non-limiting examples include compounds such as methylene blue, rose bengal, porphyrin, and phthalocyanine, and derivatives of these compounds with 1-50 atomic substituents, the substituents are used to make these compounds more lipophilic or more hydrophilic, and / or as a linking group connected to a specific binding pair. The donor surface may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into active oxygen and water. Other examples of donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or directly absorbing light will release active oxygen.

[0032] The term "acceptor" as used herein refers to a compound that can react with active oxygen to produce a detectable signal. The donor is activated by energy or an active compound and releases high-energy active oxygen, which is captured by the receptor in close proximity, thereby transferring energy to activate the receptor. In some embodiments of the present application, the receptor is filled in the microparticles through functional groups to form a polymer microsphere filled with a luminescent composition, which reacts with active oxygen to produce a light signal, and the polymer microsphere can be called a luminescent microsphere. The luminescent composition contains a chemiluminescent compound that can react with active oxygen. In some specific embodiments of the present application, the chemiluminescent compound undergoes a chemical reaction with active oxygen to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of these substances include, but are not limited to, enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridan, aryl ethyl ether ene, diethylene oxide, dimethylthiophene, aryl imidazole or luscin.

[0033] 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.

[0034] 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 microparticles may or may not be charged, and when charged, they are preferably negatively charged. The microparticles are generally multifunctional or can be bound to a donor or receptor by 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 microparticles suitable for the present application is polystyrene latex microspheres. The microparticles described in the present application have a diameter of 50nm-20μm; preferably, the microparticles have a diameter of 100nm-10μm; more preferably, the microparticles have a diameter of 200nm-5μm; further preferably, the microparticles have a diameter of 750nm-1μm.

[0035] The term "luminescent composition" as used herein refers to a compound, referred to as a marker, that can undergo a chemical reaction to cause luminescence, such as by being converted into another compound formed in an electronically excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state to emit light directly, or by transferring the excitation energy to an emitting energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particle will be transitioned to an excited state and emit light.

[0036] II. Specific implementation plan

[0037] The present application will be described in more detail below.

[0038] The technical principle of LOCI is as follows: the sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules under laser irradiation, and the singlet oxygen molecules can react with optical reaction substances about 200nm away to generate light signals of a certain wavelength; when the sample contains the antigen or antibody to be tested, the immune reaction of the antigen and antibody can make the photosensitive microspheres containing the sensitizer combine with the luminescent microspheres containing the optical reaction substances, thereby generating light signals of specific wavelengths. The antigen or antibody to be tested can be detected by detecting the light signal, thereby realizing "separation-free" homogeneous photochemiluminescence immunoassay.

[0039] In the photoinduced chemiluminescent immunoreaction process described above, it is inevitable to use antibody protein or antigen protein. The structure of antibody protein is more consistent with that of antigen protein, and the overall stability of antibody is better than that of antigen. The properties of antigen protein are affected by its own sequence. For example, the presence of highly hydrophobic amino acids together will cause some areas to be highly hydrophobic, and there will be more positively charged amino acids or more negatively charged amino acids. In different pH environments, the protein will carry different charges, which will affect the structure of the protein and the activity of surface sites, and thus have a certain impact on the stability of the protein.

[0040] For immunoassays, the activity of the antigen has a great influence on the test results. At present, there are generally two methods to improve the stability of liquid-phase antigen proteins. One is to add bovine serum albumin (BSA) to protect the target antigen protein; the other is to add protease inhibitors to prevent enzymes from decomposing proteins. These methods can resist the influence of exogenous factors on protein stability, but cannot solve the problem that the strong hydrophobicity of the antigen protein itself affects its stability. Proteins with strong hydrophobicity tend to aggregate with each other to form dimers, trimers, etc., and are prone to precipitation for a long time, which will still affect the activity of the antigen and thus affect the test results. Especially for labeled antigens, once the stability of the labeled antigen decreases, it will seriously affect the labeling effect of the labeled antigen, and thus affect the test results.

[0041] The present application increases the hydrophilicity of the surface of the labeled antigen protein by modifying the surface of the labeled antigen with a hydrophilic molecule chain, such as a sugar chain, thereby preventing the labeled antigen protein from aggregating and settling, thereby improving the long-term stability of the labeled antigen protein in the liquid phase, achieving a good labeling effect, and obtaining highly accurate immunoassay results.

[0042] To this end, the first aspect of the present application provides a method for improving the stability of a labeled antigen, wherein the antigen and the marker are combined through a hydrophilic molecular chain to obtain a labeled antigen. The labeled antigen modified by the hydrophilic molecular chain enhances the surface hydrophilicity of the labeled antigen protein, can improve the stability of the labeled antigen in the liquid phase, avoid the occurrence of nonspecific adsorption, and prevent the aggregation and sedimentation of the labeled antigen protein.

[0043] In some embodiments of the present application, the hydrophilic molecular chain is preferably a sugar chain. A sugar chain is a carbohydrate containing two or more unmodified or modified monosaccharide units. The stability of the labeled antigen modified by the sugar chain is higher, which is conducive to improving the long-term stability of the antigen protein in the liquid phase.

[0044] In some specific embodiments of the present application, the sugar chain is selected from one of glucan, glycogen, galactan, fructan, starch, mannan, agarose, polyribose, and glucan derivatives; preferably selected from one of glucan, starch, glycogen, polyribose, and glucan derivatives; more preferably selected from glucan or a glucan derivative.

[0045] In some embodiments of the present application, the hydrophilic molecule chain is formed by sequentially connecting N monomer sugars, 2≤N≤15; preferably 2≤N≤12. For example, N can be 2, 4, 6, 8, 10, 12, 15. The number of monomer sugars in the hydrophilic molecule chain, that is, the length of the sugar chain, will affect the stability of the labeled antigen protein conformation, and then affect the stability of the labeled antigen protein in the liquid phase. When the sugar chain length on the surface of the labeled antigen protein is 2 to 15, the stability of the labeled antigen protein is good, and the optimal chain length that has an effect on the stability of the labeled antigen protein is 2 to 12.

[0046] In some embodiments of the present application, the molar ratio of the number of amino groups on the surface of the antigen to the hydrophilic molecular chain is 1:(0.1-6); preferably 1:(1-6); more preferably 1:(1-3). For example, the molar ratio of the number of amino groups on the surface of the antigen to the hydrophilic molecular chain can be 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6. When the molar ratio of the number of amino groups on the surface of the antigen to the hydrophilic molecular chain is controlled within the above range, the stability of the labeled antigen in the liquid phase is better, and insufficient hydrophilic molecular chains do not have a good stabilizing effect; when the hydrophilic molecular chain is excessive, excessive labeling occurs, which will affect the activity of the antigen surface site, thereby causing a certain degree of decrease in the intensity of the photochemiluminescence detection signal, affecting the detection results.

[0047] In some embodiments of the present application, the specific binding pair member is selected from a pair of substances that can specifically bind to each other, consisting of antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, immunoglobulin binding proteins, avidin, avidin derivatives, biotin or biotin derivatives. The marker can be selected from one of the above-mentioned specific binding pair members.

[0048] In some specific embodiments of the present application, the marker is biotin or a biotin derivative; preferably, the marker is an NHS derivative of biotin. Both biotin and biotin derivatives can specifically bind to avidin and avidin derivatives, respectively, and have strong binding ability. Biotin, especially NHS derivatives of biotin, can be coupled to almost all known biological macromolecules, such as proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein crosslinkers; "avidin" molecules are composed of 4 identical peptide chains, each of which can bind to one biotin, and biological macromolecules such as antigens can bind to many biotins. Therefore, each antigen can be coupled to multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve analytical sensitivity.

[0049] In some specific embodiments of the present application, the avidin is selected from egg albumin avidin, streptavidin, egg yolk avidin, neutravidin and avidin-like, preferably selected from neutravidin and / or streptavidin.

[0050] The second aspect of the present application provides a labeled antigen, which is prepared according to the method described in the first aspect of the present application. That is, the labeled antigen of the present application is prepared by connecting the antigen and the marker with a hydrophilic molecular chain, which enhances the surface hydrophilicity of the labeled antigen and can prevent the labeled antigen from aggregating and settling, thereby obtaining a labeled antigen with good stability in the liquid phase.

[0051] In some embodiments of the present application, the preparation step of the labeled antigen includes: connecting the hydrophilic molecule chain with the marker, and then coupling it with the antigen to obtain the labeled antigen. Specifically, the sugar chain can be connected with the NHS derivative of biotin to obtain Biotin-GLU-NHS, and then coupled with the antigen to obtain the labeled antigen.

[0052] In some embodiments of the present application, Biotin-GLU-NHS is stored at ≤15°C.

[0053] The third aspect of the present application provides a labeled antigen reagent, comprising a buffer solution and the labeled antigen described in the second aspect of the present application.

[0054] In some embodiments of the present application, the buffer is selected from a biotin reagent buffer; preferably a HEPES buffer containing BSA; and more preferably a HEPES buffer containing BSA at pH 8.0.

[0055] In some embodiments of the present application, the buffer does not contain sugar.

[0056] In some embodiments of the present application, the labeled antigen reagent does not contain a surfactant.

[0057] Different from adding surfactants, sugars and other substances to reagents to increase the hydrophilicity of proteins in the liquid phase, the present application modifies the labeled antigen protein by hydrophilic molecular chains to change the conformation of the labeled antigen protein itself, thereby improving the stability of the labeled antigen protein.

[0058] In some embodiments of the present application, the steps of preparing the labeled antigen reagent include:

[0059] The antigen protein is mixed with biotin or a biotin derivative for labeling reaction, and diluted with a buffer to obtain a labeled antigen reagent.

[0060] In some embodiments of the present application, the molar ratio of the antigen protein to biotin or a biotin derivative during the labeling reaction is 1:(1-60), for example, it can be 1:1, 1:10, 1:20, 1:30, or 1:60.

[0061] The fourth aspect of the present application provides an immunoassay kit, comprising reagent a, reagent b and / or reagent c. Reagent a is the labeled antigen reagent as described in the third aspect of the present application; reagent b is a receptor reagent that can bind to the antigen or its binding fragment and can react with singlet oxygen to generate a detectable signal; and reagent c is a donor reagent that can generate singlet oxygen and bind to a ligand that can specifically recognize and bind to the marker.

[0062] In some embodiments of the present application, the reagent b comprises luminescent microspheres and luminescent reagent buffer. In some embodiments of the present application, the luminescent microspheres are coated antigens coated with luminescent microparticles.

[0063] In some embodiments of the present application, the luminescent particles are stored in a sealed state and away from light at 2 to 8°C.

[0064] In some embodiments of the present application, the luminescent reagent buffer is a CB buffer.

[0065] In some embodiments of the present application, the preparation step of reagent b includes: mixing the antigen protein and the luminescent particles for coating reaction, and diluting with a luminescent reagent buffer to obtain reagent b.

[0066] In some embodiments of the present application, the kit further comprises a sample diluent; preferably a calf serum solution.

[0067] In some embodiments of the present application, the kit further includes one or more of a negative control, a positive control, and a weak positive control.

[0068] The fifth aspect of the present application provides the use of the above-mentioned labeled antigen, labeled antigen reagent or the above-mentioned kit in photochemical detection.

[0069] III. Specific Examples

[0070] 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.

[0071] The present application is further described in detail in the examples of HIV (Human Immunodeficiency Virus) antibody detection.

[0072] When HIV antibodies are present in clinical samples, HIV antibodies specifically bind to receptors coated with HIV antigens and biotin-labeled antigens at the same time, and form a sandwich complex of "antigen-antibody-antigen" on the surface of the receptor; at this time, an avidin-modified donor is added, and biotin specifically binds to avidin to bring the receptor and the donor close to each other. Under the excitation of the excitation light source, the donor releases singlet oxygen, and chemiluminescence is generated after hitting the receptor in the system, and the intensity of the light signal is detected. When the biotin of the HIV marker antigen protein in this application adopts sugar chain-modified Biotin-GLU-NHS, the long-term stability of the HIV antigen protein in the liquid phase can be increased, while the labeling effect of the HIV marker antigen protein is not affected, and the sensitivity and specificity of the detection are improved. Further, when the number of monomer sugars in Biotin-GLU-NHS is 2-15 and the molar ratio of Biotin-GLU-NHS to the surface amino group of the HIV antigen protein reaches (1-6): 1, the stability of the HIV antigen protein in the liquid phase is optimal, and the sensitivity and specificity of the detection are optimal.

[0073] Example 1: Preparation of biotin reagent (reagent a)

[0074] 1. Raw material dialysis: Take 2 mg HIV antigen, place it in a 3.5KD dialysis bag, and dialyze it three times in 0.1M NaHCO3 buffer; transfer the protein recovered by dialysis to a clean centrifuge tube, and measure the concentration using the BCA method, which is 1.62 mg / ml;

[0075] 2. Labeling reaction: prepare 20 mg / ml of Biotin-NHS active ester and Biotin-GLU-NHS (Biotin-NHS active ester and Biotin-GLU-NHS are purchased from WuXi AppTec), respectively, and carry out labeling reaction according to the HIV antigen protein (with 10 oligoamines on the surface): biotin molar ratio of 1:20, and carry out labeling reaction by rotating mixing, react for 20 hours, and obtain labeled product;

[0076] 3. Product dialysis: Place the labeled product in a 3.5KD dialysis bag and dialyze it in 0.02M HEPES buffer for 3 times, 3 hours each time, at a dialysis temperature of 2-8°C; aspirate the HIV antigen protein recovered by dialysis and transfer it to a clean centrifuge tube, and use the BCA method to determine the protein concentration, which is 1.43 mg / m and 1.54 mg / ml to obtain a concentrated biotin solution.

[0077] 4. Use 50 ml of biotin reagent buffer (0.02 M HEPES buffer containing BSA, pH 8.0) to dilute the above prepared biotin concentrated solution to 0.5 ug / ml and equilibrate for 2 hours.

[0078] Example 3: Preparation of Luminescent Reagent (Reagent b)

[0079] 1. Luminescent microsphere treatment: Take 1 ml of 10 mg / ml luminescent microspheres in a centrifuge tube, centrifuge for 30 minutes, discard the supernatant, and resuspend with 0.05 MCB buffer by ultrasonication. Repeat the operation once and adjust the volume to 50 mg / ml for use;

[0080] 2. Raw material dialysis: Take 1 mg HIV antigen, place it in a 3.5KD dialysis bag, and dialyze it three times in 0.05M CB buffer (pH 9.6); aspirate the HIV antigen protein recovered by dialysis and transfer it to a clean centrifuge tube, and measure the concentration using the BCA method, which is 2.78 mg / ml;

[0081] 3. Coupling reaction: Mix the luminescent microspheres with HIV antigen protein at a mass ratio of (luminescent microspheres: HIV antigen protein) of (10:0.2), and perform the coupling reaction by rotating mixing for 20 hours; prepare 8 mg / mL NaBH4 solution using 0.05 M CB buffer and immediately add it to each reaction tube, and rotate for 2 hours;

[0082] 4. Volume adjustment: Repeat centrifugation and washing twice to remove excess protein, and finally adjust the volume to 10 mg / ml in the luminescent reagent buffer to obtain a luminescent concentrated solution;

[0083] 5. Use 50 ml of luminescent reagent buffer to dilute the luminescent concentrated solution prepared above to 50 ug / ml and equilibrate for 2 hours.

[0084] Example 3: Evaluation of HIV marker antigen stability

[0085] The kit is composed of reagent a (prepared in Example 1), reagent b (prepared in Example 2), reagent c (photosensitive reagent containing donor particles), Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control, Anti-HIV weak positive control, and Anti-HIV sample diluent. Each component is prepared separately, and the negative and positive controls, weak positive controls, and sample diluents are packaged separately and then assembled into a box.

[0086] Among them, Anti-HIV negative control: Anti-HIV qualitative reference dilution solution;

[0087] Anti-HIV-1 positive control: prepared by diluting HIV-1 positive serum with Anti-HIV qualitative reference diluent;

[0088] Anti-HIV-2 positive control: prepared by diluting HIV-2 polyclonal antibody with Anti-HIV qualitative reference diluent;

[0089] Anti-HIV weak positive control: prepared by diluting HIV-1 positive serum using Anti-HIV qualitative reference diluent.

[0090] This kit can be used for qualitative detection of human immunodeficiency virus HIV (type 1+2) antibodies. Each reagent must be equilibrated to ambient temperature before use.

[0091] Evaluation method:

[0092] 1. The biotin reagent (reagent a) prepared in Example 1 was stored at 4° C. and 37° C. (7 days) respectively;

[0093] 2. Combine the above biotin reagent and luminescent reagent and react with the sample to detect the luminescent signal. The results are shown in Table 1. HIV samples include negative samples, positive samples, and Conchestan quality control products. The specific steps for using the kit are as follows:

[0094] Step 1: Dilute the HIV sample with Anti-HIV sample diluent and mix thoroughly;

[0095] Step 2: Add 25 μL of diluted sample and Anti-HIV negative control, Anti-HIV-1 positive control, Anti-HIV-2 positive control, and Anti-HIV weak positive control to the reaction wells respectively;

[0096] Step 3: Add 25 μL of biotin reagent, 25 μL of luminescent reagent and 25 μL of photosensitizer reagent to the reaction wells in sequence;

[0097] Step 4: Place the sample into a chemiluminescence analyzer and let the instrument automatically operate to perform reaction and detection.

[0098] Table 1 Evaluation results (photochemiluminescence detection signal amount and signal deviation value)

[0099]

[0100] As can be seen from Table 1, compared with Biotin-NHS, the positive samples and Conchestan third-party quality control test signal deviations of the Biotin-GLU-NHS group were better than those of the Biotin-NHS group, with the deviation being as high as about 20%. It can be seen that HIV Ag labeled with Biotin-GLU-NHS has better stability.

[0101] Example 4: Effect of Sugar Chain Length in Biotin-GLU-NHS on Detection Results

[0102] Experimental steps:

[0103] The biotin reagents for labeling HIV antigen proteins with Biotin-GLU-NHS with different numbers of monomeric sugars (i.e. Biotin-GLU-NHS with different sugar chain lengths) were prepared with reference to the steps for preparing the concentrated biotin solution in Example 2. The above biotin reagents were combined with the luminescent reagents and reacted with the samples to detect the luminescent signals. The results are shown in Tables 2, 3 and 4. Among them, HIV samples include negative samples, positive samples, and Conchestan quality control products. The specific steps for using the kit refer to Example 3.

[0104] Table 2 HIV sample stability results (photochemiluminescence detection signal)

[0105]

[0106]

[0107] Table 3 HIV sample stability results (photochemiluminescence detection signal deviation value)

[0108]

[0109] Table 4 HIV sample signal deviation results (photochemiluminescence detection signal amount and signal deviation value)

[0110]

[0111]

[0112] From the HIV sample stability experimental data in Tables 2 and 3, it can be seen that the stability of HIV antigen proteins labeled with Biotin-GLU-NHS with a monomer sugar number of 2-15, i.e., a sugar chain length of 2-15, is not significantly different, and the positive mean deviation is basically around 5%, indicating that the stability of biotin-labeled HIV antigen proteins with a sugar chain length of 2-15 is improved.

[0113] From the experimental data of HIV sample signal deviation in Table 4, it can be seen that when the sugar chain length in Biotin-GLU-NHS reaches 15, the mean value of positive samples decreases by about 13% compared with the chain length of Biotin-GLU-NHS of 2, indicating that the excessive length of the sugar chain affects the activity of the HIV antigen protein's own site, thereby affecting the detection results of the photochemiluminescence detection signal.

[0114] In summary, the sugar chain length of Biotin-GLU-NHS that has the best effect on the stability of HIV marker antigen protein is 2-12, and the more preferred sugar chain length of Biotin-GLU-NHS is 2-6.

[0115] Example 5: Effects of different ratios of Biotin-GLU-NHS and HIV antigen protein labeling on test results

[0116] Experimental steps:

[0117] Using Biotin-GLU6-NHS as a biotin marker, the concentrated biotin solutions with different labeling ratios (hereinafter referred to as the molar ratio of Biotin-GLU6-NHS to the surface amino groups of HIV antigen protein) were prepared with reference to the preparation steps of the concentrated biotin solution in Example 1, and the above biotin reagents were combined with luminescent reagents to react with samples, and the luminescent signals were detected. The results are shown in Tables 5, 6 and 7. Among them, HIV samples include negative samples, positive samples, and quality control products.

[0118] Table 5 HIV sample stability results (photochemiluminescence detection signal)

[0119]

[0120] Table 6 HIV sample stability results (photochemiluminescence detection signal deviation value)

[0121]

[0122]

[0123] Table 7 HIV sample signal deviation results (photochemiluminescence detection signal amount and signal deviation value)

[0124]

[0125]

[0126] From the HIV sample stability and signal deviation experimental data in Tables 5, 6 and 7, we can see that:

[0127] 1. When Biotin-GLU-NHS is used to label HIV antigen protein, when the molar number of Biotin-GLU-NHS is 1-6 times the molar number of amino groups on the surface of HIV antigen protein, the photochemiluminescence detection signal is stronger and the stability of HIV labeled antigen protein is better;

[0128] 2. From the initial signal value, it can be seen that when the molar ratio of Biotin-GLU-NHS and the surface amino group of HIV antigen protein reached 6:1 and 0.1:1, the signal decreased.

[0129] Cause analysis: When the labeling ratio is 0.1:1, the number of Biotin-GLU-NHS on the actual HIV antigen protein label is insufficient, the stability is improved, but not saturated, and the surface hydrophilicity is not in the optimal state; when the labeling ratio reaches the range of 1:1 to 3:1, the signal reaches the highest, which may be that the surface hydrophilicity of the HIV labeled antigen reaches the most suitable state; when the ratio reaches 6:1, over-labeling occurs, affecting the site activity on the surface of the HIV labeled antigen, resulting in a certain degree of signal decline. Therefore, the optimal labeling ratio is between 1:1-3:1, that is, the molar number of Biotin-GLU-NHS is 1-3 times the molar number of amino groups on the surface of the HIV antigen protein.

[0130] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present application may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A method for improving the stability of labeled antigens, wherein the antigens are combined with markers via hydrophilic molecular chains to obtain labeled antigens.

2. The method according to claim 1, characterized in that: The hydrophilic molecule chain is selected from a sugar chain; preferably, the sugar chain is selected from one of dextran, glycogen, galactan, fructan, starch, mannan, agarose, polyribose, and a dextran derivative; preferably selected from one of dextran, starch, glycogen, polyribose, and a dextran derivative; more preferably selected from dextran or a dextran derivative.

3. The method according to claim 1, characterized in that: The hydrophilic molecular chain is formed by sequentially connecting N monomer sugars, 2≤N≤15; preferably 2≤N≤12.

4. The method according to any one of claims 1 to 3, characterized in that: The molar ratio of the hydrophilic molecular chain to the amino groups on the antigen surface is 1:(0.1-6); preferably 1:(1-6); more preferably 1:(1-3).

5. The method according to any one of claims 1 to 4, characterized in that: The label is selected from an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, an immunoglobulin binding protein, avidin, avidin, an avidin derivative, biotin or a biotin derivative.

6. A labeled antigen obtained by the method according to any one of claims 1 to 5.

7. A labeled antigen reagent, comprising a buffer solution and the labeled antigen according to claim 6, wherein the reagent does not contain a surfactant.

8. The labeled antigen reagent according to claim 7, characterized in that: The buffer contained no sugar.

9. An immunoassay kit comprising: Reagent a, a labeled antigen reagent as described in claim 7 or 8; Reagent b, a receptor reagent that binds to the antigen or its binding fragment and is capable of reacting with singlet oxygen to generate a detectable signal; Preferably, the kit further comprises reagent c, a donor reagent capable of generating singlet oxygen which is combined with a ligand capable of specifically recognizing and binding to the marker.

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

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