A homogeneous immunoassay kit without matrix effect and its analysis method and application

Abstract

The present invention relates to a homogeneous immunoassay kit without matrix effect, which comprises: a first composition, which comprises a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or a second antibody fragment bound thereto, wherein the second antibody or the second antibody fragment can specifically bind to a first epitope of an analyte in a sample to be tested; and a third composition, which comprises magnetic microspheres. The present invention also relates to a homogeneous immunoassay method and application of a homogeneous immunoassay kit without matrix effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a homogeneous immunoassay kit without matrix effect and an analysis method and application thereof. Background Art

[0002] Labeled immunoassay combines the high specificity of antigen-antibody binding and the high sensitivity of labeled (tracer) substance detection, and is widely used in the quantitative analysis of ultra-trace substances in clinical body fluid specimens. Depending on whether it is necessary to separate the bound marker (Bind, B) and the free marker (Free, F), labeled immunoassay is divided into homogeneous immunoassay and heterogeneous immunoassay. Homogeneous immunoassay does not need to separate and remove the free marker, and directly measures the signal of the bound marker to obtain a dose curve or a standard curve, such as fluorescence polarization immunoassay and photoinduced chemiluminescence assay (LICA). Heterogeneous immunoassay needs to separate and remove the free marker first, and then measure the signal of the bound marker to obtain a dose curve or a standard curve, such as chemiluminescence immunoassay (CLIA) and electrochemiluminescence immunoassay (ECLIA).

[0003] The methods of measuring antibodies by labeled immunoassay include: indirect method, double antigen sandwich method, competition method, neutralization method, etc. Among them, the indirect method can distinguish the immunoglobulin class of the antibody to be tested through anti-antibodies (anti-human-IgG or anti-human-IgM), which is of great value in judging the course of infectious diseases, so it is a commonly used analysis mode. The classic indirect method requires a two-step method, in which the known antigen binds to the antibody to be tested, and the labeled anti-antibody (second antibody) binds to the antibody to be tested. The non-specific (antigen-independent) antibodies need to be separated and removed between the two steps, otherwise the non-specific antibodies will also bind to the second antibody, consuming a large amount of the second antibody and increasing the blank signal value.

[0004] In heterogeneous labeling immunoassay technology, removing free markers that are not involved in binding through "separation and washing" is one of the key links in the analysis process. Whether it is done manually or automatically by the instrument, washing errors will occur. At the same time, the washing process will also cause cross-contamination between specimens, and strongly positive specimens will affect the test results of adjacent specimens. The characteristic of homogeneous labeling immunoassay technology is that there is no separation and washing process throughout the process, and there is no washing error.

[0005] Although there is no need for separation and washing steps in homogeneous immunoassay technology, some substances in serum or plasma can interfere with the detection of label signals or interfere with chemiluminescence reactions. In addition, in the field of chemiluminescence involved in homogeneous labeling immunoassay technology, some involve redox reactions. For example, some redox drugs (such as vitamin C) contained in blood samples also interfere with the generation process of light signals, seriously affecting the accuracy of the test results. Summary of the invention

[0006] The present invention provides a homogeneous immunoassay kit without matrix effect and its analysis method and application in view of the deficiencies of the prior art. The method of analyzing antigens or antibodies in a sample to be tested by using the kit can eliminate the matrix effect in homogeneous immunoassay, changing the current situation that homogeneous immunoassay cannot use indirect methods to measure antibodies and the hook effect occurs when detecting antigens.

[0007] To this end, the first aspect of the present invention provides a homogeneous detection kit without matrix effect, comprising:

[0008] A first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or a second antibody fragment bound thereto, wherein the second antibody or the second antibody fragment is capable of specifically binding to a first epitope of an analyte in a sample to be tested;

[0009] a second composition comprising a donor capable of generating singlet oxygen in an excited state;

[0010] The third composition comprises magnetic microspheres, and the magnetic microspheres are used to separate the matrix in the sample to be tested and other non-specific substances in the sample to be tested that are not specifically bound to the first antibody or the first antibody fragment.

[0011] The inventors of the present application surprisingly found that the method of analyzing antigens or antibodies in the sample to be tested by using the kit of the present invention adds a process of removing serum / plasma matrix, effectively avoiding the influence of serum / plasma matrix on the subsequent luminescence process in homogeneous detection, and changing the current situation that the homogeneous immunoassay technology cannot use the indirect method to measure antibodies and the hook effect when detecting antigens. In addition, the process of removing serum / plasma matrix of the present invention is different from the separation and washing of general heterogeneous immunoassay. Its purpose is not to remove free markers, but to remove possible interfering substances in serum / plasma. It only needs to remove most of the interfering substances (more than 80%), and there is no excessive requirement for washing accuracy, which will not affect the precision (repeatability). In addition, the kit of the present invention has strong versatility and can detect a variety of items.

[0012] In some embodiments of the present invention, the kit further comprises a fourth composition, wherein the fourth composition comprises a first antibody or a first antibody fragment that can specifically bind to a second epitope of the analyte.

[0013] According to the present invention, the magnetic microspheres and the donor are both coated with one member of the specific binding pair, and the first antibody is coated with the other member of the specific binding pair.

[0014] In other embodiments of the present invention, the kit further comprises a fourth composition comprising a known antigen that can specifically bind to a third epitope of the analyte.

[0015] According to the present invention, the magnetic microspheres and the donor are both coated with one member of the specific binding pair, and the surface of the known antigen is coated with the other member of the specific binding pair.

[0016] In some preferred embodiments of the present invention, the specific binding pair member is a biotin-streptavidin system.

[0017] In other preferred embodiments of the present invention, the magnetic microspheres are indirectly coated with streptavidin via biotin-labeled bovine serum albumin and / or biotin-labeled globulin.

[0018] According to the present invention, the particle size of the magnetic microspheres is selected to be 100 nm to 1 μm, preferably 200 nm to 800 nm, and more preferably 300 nm to 600 nm.

[0019] The second aspect of the present invention provides a homogeneous detection method for detecting whether an analyte is present in a sample to be tested using the kit according to the first aspect of the present invention, which comprises the following steps:

[0020] S1, separating the first complex formed by the magnetic microsphere-analyte from the matrix in the sample to be tested and other non-specific substances that are not specifically bound to the first antibody or the first antibody fragment by a magnetic field;

[0021] S2, contacting the first composition, the second composition and the first complex to form a second complex;

[0022] S3, contacting the second complex with energy or an active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the received singlet oxygen to generate a detectable chemiluminescent signal;

[0023] S4, analyzing the chemiluminescent signal to determine whether the analyte exists in the sample to be tested and the content or concentration of the analyte.

[0024] In some embodiments of the present invention, step S1 is preceded by step S0, which involves contacting the sample to be tested, the third composition and the fourth composition to generate the first complex.

[0025] According to the present invention, when the analyte is an antigen to be detected, the fourth composition includes a first antibody or a first antibody fragment that can specifically bind to the second epitope of the analyte, and the magnetic microspheres and the analyte in the first complex are combined through the first antibody or the first antibody fragment.

[0026] According to the present invention, when the analyte is an antibody to be detected, the fourth composition comprises a known antigen that can specifically bind to the third epitope of the analyte, and the magnetic microspheres in the first complex and the analyte are bound via the known antigen.

[0027] In some preferred embodiments of the present invention, the method comprises the following steps:

[0028] S0, mixing the sample to be tested, the third composition and the fourth composition to obtain a first mixture including a first complex formed by magnetic microspheres and analytes;

[0029] S1, using a magnetic field to adsorb the first complex and remove the liquid containing the matrix and other non-specific substances in the first mixture.

[0030] In some specific embodiments of the present invention, the volume of the liquid removed in step S1 accounts for 80%-95% of the volume of the liquid in the first mixture.

[0031] According to the present invention, there is no washing step between step S1 and step S2.

[0032] According to the present invention, there is no washing step between step S2 and step S3.

[0033] In some preferred embodiments of the present invention, in step S2, the magnetic field is removed first, and then the first composition and the second composition are added to the first complex to form the second complex.

[0034] According to the present invention, the analyte in the sample to be tested is selected from one or more of pathogen antibodies, autoantibodies, allergen-specific antibodies and allergen total antibodies in serum or plasma.

[0035] The inventors of the present application were surprised to find that the method of the present invention has changed the current situation that the photochemiluminescence analysis in homogeneous immunoassay cannot use the indirect method to determine antibodies. Since the proportion of the antibody to be tested in the non-specific antibody is relatively small, there is no separation and washing process in the existing photochemiluminescence analysis procedure, and the non-specific antibody cannot be removed. The non-specific antibody can also bind to the receptor microspheres coated with the anti-antibody, thereby interfering with the detection system. Therefore, conventional photochemiluminescence analysis cannot adopt the indirect analysis mode of "known antigen-antibody to be tested-labeled anti-antibody", and can only adopt the double antigen sandwich or competitive analysis mode to detect the antibody to be tested, and the double antigen sandwich or competitive analysis mode has high requirements for known antigens or competitive antibodies, and cannot distinguish the category of immunoglobulins of the antibody to be tested. The above-mentioned photochemiluminescence analysis in the new homogeneous detection adds a magnetic field separation link after the known antigen (antigen bound to the antibody to be analyzed) is combined with the antibody to be analyzed, which can effectively remove non-specific antibodies and other interferents, thereby eliminating the influence of non-specific antibodies and other interferents.

[0036] In addition, the above-mentioned process of separating and removing serum / plasma matrix by using magnetic field in the homogeneous immunoassay method is different from the separation and washing process of heterogeneous immunoassay, and will not affect the precision (repeatability). The "separation and washing" process of heterogeneous immunoassay is carried out after the labeled antibody and the corresponding antigen to be detected or the labeled antigen and the corresponding antibody to be detected occur, and its purpose is to remove the influence of free markers on signal detection, and also to remove interfering substances in serum / plasma. However, this separation and washing needs to be repeated 3-5 times to completely remove the free markers, which not only increases the detection time, but more importantly, due to the difficulty in standardizing the washing process, it will bring about a large error (washing error), thereby affecting the precision of the analytical method. The process of removing serum / plasma matrix of the present invention is carried out before adding donor microspheres and acceptor microspheres. The purpose of separation is not to remove free markers, but to remove possible interfering substances in serum / plasma. It is only necessary to remove most of the interfering substances, and there is no excessive requirement for washing accuracy.

[0037] The third aspect of the present invention provides a second complex involved in the method according to the second aspect of the present invention, comprising:

[0038] a centrosome portion selected from any one of the immune molecules;

[0039] a receptor portion capable of reacting with singlet oxygen to generate a detectable luminescent signal, which is bound to the centrosome via a second antibody or a second antibody fragment, which is capable of specifically binding to a fourth epitope of the centrosome;

[0040] a donor moiety capable of generating singlet oxygen in an excited state, which is bound to the centrosome via a bridging body, said bridging body specifically binding to a fifth epitope on the centrosome that does not overlap with said fourth epitope;

[0041] The magnetic part is bound to the centrosome via a bridge body, and the bridge body specifically binds to a sixth epitope on the centrosome that does not overlap with the fourth epitope.

[0042] In some embodiments of the present invention, the cross-body is selected from a known antigen, a first antibody or a first antibody fragment that can specifically bind to the centrosome.

[0043] In other embodiments of the present invention, the magnet is indirectly coated with streptavidin via biotin-labeled bovine serum albumin and / or biotin-labeled globulin, and the bridge body is coated with biotin.

[0044] According to the present invention, the centrosome is selected from one of pathogenic antibodies, autoantibodies or allergen-specific antibodies in human serum or plasma.

[0045] The fourth aspect of the present invention provides a use of the kit described in the first aspect of the present invention, the method described in the second aspect of the present invention, or the second complex described in the third aspect of the present invention in clinical detection of antigens or antibodies in serum or plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be described in detail below with reference to the accompanying drawings.

[0047] Figure 1 The schematic diagram of the principle of analyzing antibodies in a sample to be tested by the method of the present invention. The meanings of the reference numerals in the figure are as follows: 1 magnetic microspheres labeled with streptavidin; 2 known antigens labeled with biotin; 3 antibody to be analyzed; 4 second antibody bound to acceptor microspheres; 5 donor microspheres labeled with streptavidin.

[0048] Figure 2 The schematic diagram of the principle of analyzing antigens in a sample to be tested by the method of the present invention is as follows: 1 magnetic microspheres labeled with streptavidin; 2 first antibody labeled with biotin; 3 antigen to be analyzed; 4 second antibody bound to acceptor microspheres; 5 donor microspheres labeled with streptavidin.

[0049] Figure 3 The schematic diagram is a principle diagram of analyzing antigens in a sample to be tested using magnetic microspheres indirectly bound to streptavidin. The meanings of the reference numerals in the figure are as follows: 1 magnetic microspheres labeled with streptavidin; 1-1 magnetic microspheres; 1-2 bovine serum albumin or globulin labeled with biotin; 1-3 streptavidin; 2 first antibody bound to biotin; 3 antigen to be analyzed; 4 second antibody bound to acceptor microspheres; 5 donor microspheres labeled with streptavidin. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] 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 similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0053] I. Terminology

[0054] The term "sample to be tested" as used herein refers to a mixture that may contain an analyte, which includes but is not limited to proteins, hormones, antibodies or antigens. Typical samples to be tested that can be used in the method disclosed herein include body fluids, such as blood, plasma, serum, urine, semen, saliva, etc.

[0055] The terms "antibody" and "immunoglobulin" used in the present invention are used in the broadest sense, including antibodies or immunoglobulins 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. Where necessary, the antibody can be further conjugated to other moieties, such as specific binding pair members, for example, biotin or streptavidin (a member of a biotin-streptavidin specific binding pair member), etc.

[0056] The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted by a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

[0057] The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced by more than one B lymphocyte clones, which can be prepared by methods known to those skilled in the art.

[0058] 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.

[0059] 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.

[0060] The term "specific binding" used in the present invention refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the corresponding reactants from the perspective of three-dimensional structure.

[0061] The term "specific binding pair member" used in the present invention 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-streptavidin system, in which "biotin" is widely present in animal and plant tissues, and has two ring structures on its molecule, namely, the imidazolone ring and the thiophene ring, wherein the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled to almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces with a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which can bind to one biotin. Therefore, each antigen or antibody can be coupled to multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the sensitivity of the analysis.

[0062] In any desired case, any reagent used in the present invention, including antigen, antibody, receptor or donor, can be conjugated to any member of the biotin-streptavidin specific binding pair according to actual needs.

[0063] The term "donor" as used herein refers to a sensitizer that can produce an active intermediate such as singlet oxygen that reacts 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 specific embodiments of the present invention, the donor is a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively stable to light and does not effectively react with singlet oxygen, and its non-limiting examples include compounds such as methylene blue, rose red, porphyrin, phthalocyanine and chlorophyll disclosed in, for example, U.S. Pat. No. 5,709,994 (the entire text of this patent document is cited as a reference), and derivatives of these compounds having 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 member. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as the contents described in U.S. Pat. No. 6,406,913, which is incorporated herein by reference. In other specific embodiments of the present invention, the donor is other chemically activated sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water. Other examples of donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc., which release singlet oxygen when heated or directly absorb light.

[0064] The term "acceptor" as used herein refers to a compound that can react with singlet oxygen to produce a detectable signal. The donor is activated by energy or an active compound and releases high-energy singlet oxygen, which is captured by the receptor in close proximity, thereby transferring energy to activate the receptor. In some specific embodiments of the present invention, the receptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that 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 acridans, aryl vinyl ethers, diethylene oxides, dimethylthiophene, aromatic imidazoles or lucigenin. In other specific embodiments of the present invention, the receptor is an olefin capable of reacting with singlet oxygen to form hydroperoxides or dioxetanes that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetanes that can be decomposed by the action of light; an acetylene that can react with singlet oxygen to form diketones; a hydrazone or hydrazide that can form azo compounds or azocarbonyl compounds, such as luminol; and an aromatic compound that can form endoperoxides. Specific, non-limiting examples of receptors that can be used according to the present disclosure and claimed invention are described in U.S. Pat. No. US5340716 (which is hereby incorporated by reference in its entirety). In other specific embodiments of the present invention, the receptor comprises an olefin compound and a metal chelate that is non-particulated and soluble in an aqueous medium, such as PCT / US2010 / 025433 (which is hereby incorporated by reference in its entirety)

[0065] In the present invention, the "donor" and / or "acceptor" can be coated on a substrate through functional groups to form "donor microspheres" and / or "acceptor microspheres". The "substrate" described in the present invention is a microsphere or particle known to those skilled in the art, which can be of any size, can be organic or inorganic, can be expandable or non-expandable, can be porous or non-porous, has any density, but preferably has a density close to that of water, preferably can float in water, and is composed of transparent, partially transparent or opaque materials. The substrate may or may not have an electric charge, and when it has an electric charge, it is preferably a negative charge. The substrate can be a solid (such as a polymer, metal, glass, organic and inorganic substances such as minerals, salts and diatoms), a small oil droplet (such as hydrocarbons, fluorocarbons, siliceous fluids), a vesicle (such as synthetic such as phospholipids, or natural such as cells, and cell organs). The matrix can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silicon particles, metal sols, cells and microcrystalline dyes. The matrix is ​​usually multifunctional or can be bound to the donor or receptor through specific or non-specific covalent or non-covalent interactions. There are many functional groups that are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfhydryl, etc. A non-limiting example of a matrix suitable for the present invention is a carboxyl-modified latex particle. The details of this matrix can be found in U.S. Patents US5709994 and US5780646 (these two patent documents are cited in their entirety as reference).

[0066] The term "epitope" as used herein refers to any protein determinant that can specifically bind to an immunoglobulin or T cell receptor. In some embodiments of the present invention, an epitope is a region of an antigen surface that can be specifically bound by an antibody. Epitope determinants can generally include chemically active surface groups of a molecule, such as, but not limited to, amino acids, sugar side chains, phosphoryl and / or sulfonyl groups. In other embodiments of the present invention, an epitope can specifically specify three structural features and specific charge characteristics.

[0067] There are many types of "immune molecules" used in the present invention, some of which have structural and evolutionary homology, the main ones are the following: membrane surface antigen receptors, major histocompatibility complex antigens, leukocyte differentiation antigens, adhesion molecules, antibodies, complement, cytokines, antigens, etc.; in particular, the "immune molecules" mentioned in the present invention mainly refer to antigens and antibodies.

[0068] II. Examples

[0069] As mentioned above, the classical indirect method for detecting antibodies must include a separation and washing process to remove the influence of nonspecific antibodies and free antibodies. The characteristic of photoinduced chemiluminescence analysis (LICA) in homogeneous detection is that there is no separation and washing process throughout the process. Therefore, it cannot remove substances in serum or plasma that interfere with the detection of label signals or the chemiluminescence reaction. It cannot use the indirect method to detect antibodies, nor can it distinguish the types of immunoglobulins of the antibodies to be detected.

[0070] The inventors of the present application introduced magnetic microspheres during the detection process and removed most of the interfering substances in the serum / plasma by magnetic separation before adding the first composition containing the donor and the second composition containing the receptor, thereby effectively avoiding the influence of the interfering substances on the detection results.

[0071] To this end, the homogeneous detection kit without matrix effect involved in the first aspect of the present invention comprises the following reagents:

[0072] A first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or a second antibody fragment bound thereto, wherein the second antibody or the second antibody fragment is capable of specifically binding to a first epitope of an analyte in a sample to be tested;

[0073] a second composition comprising a donor capable of generating singlet oxygen in an excited state;

[0074] The third composition comprises magnetic microspheres, and the magnetic microspheres are used to separate the matrix in the sample to be tested and other non-specific substances in the sample to be tested that are not specifically bound to the first antibody or the first antibody fragment.

[0075] Specifically, the matrix-effect-free homogeneous detection kit of the present invention comprises the following reagents:

[0076] (a) a receptor solution bound to a second antibody or a second antibody fragment; the receptor is capable of reacting with singlet oxygen to generate a detectable signal; the second antibody or the second antibody fragment is capable of specifically binding to a first epitope of an analyte in a sample to be tested; the receptor solution is 0.25 mg / ml to 0.75 mg / ml;

[0077] (b) a donor solution, wherein the donor is capable of generating singlet oxygen in an excited state; the concentration of the donor solution is 35 mg / ml-45 mg / ml;

[0078] (c) a magnetic microsphere solution, wherein the magnetic microspheres are used to separate the matrix in the sample to be tested and other non-specific substances in the sample to be tested that are not specifically bound to the first antibody or the first antibody fragment; the concentration of the magnetic microsphere solution is 15 mg / ml-20 mg / ml;

[0079] (d) a first antibody or first antibody fragment solution that specifically binds to a second epitope of the analyte (the analyte is an antigen); or a known antigen solution that specifically binds to a third epitope of the analyte (the analyte is an antibody); the concentration of the first antibody or first antibody fragment solution and the concentration of the known antigen solution are both 0.25 mg / ml-0.75 mg / ml.

[0080] In the present invention, the second antibody is selected from anti-human monoclonal antibodies and / or polyclonal antibodies; in a specific embodiment, the second antibody is selected from anti-human IgE polyclonal antibodies.

[0081] In the present invention, the first antibody is selected from anti-human monoclonal antibodies and / or polyclonal antibodies. In a specific embodiment, the first antibody is selected from anti-human IgE monoclonal antibodies.

[0082] According to the reagent requirements, the magnetic microspheres and donors, the first antibody and the known antigen can be connected with streptavidin or biotin, so as to achieve the connection of the two molecules through the specific interaction between the "specific binding pair members". In addition, the donor and the receptor in the reagent can be coated on the matrix particles to form donor microspheres and receptor microspheres.

[0083] The streptavidin can be directly or indirectly bound to the magnetic microspheres; preferably, the streptavidin is indirectly bound to the magnetic microspheres. The streptavidin is indirectly bound to the magnetic microspheres in the following manner: first, biotin-labeled bovine serum albumin and / or biotin-labeled globulin are connected to the surface of the magnetic microspheres, and then the streptavidin is indirectly bound to the magnetic microspheres through biotin. This method will further expand the space on the surface of the magnetic microspheres to reduce the steric hindrance effect of biological molecule binding. The schematic diagram of the principle of using magnetic microspheres indirectly bound to streptavidin to analyze antigens in the sample to be tested is shown in the figure. Figure 3 shown.

[0084] More specifically, the matrix-effect-free homogeneous detection kit of the present invention comprises the following reagents:

[0085] (a) a solution of receptor microspheres bound to a second antibody or a second antibody fragment; the receptor microspheres are capable of reacting with singlet oxygen to generate a detectable signal; the second antibody or the second antibody fragment is capable of specifically binding to a first epitope of an analyte in a sample to be tested;

[0086] (b) a solution of biotin-labeled donor microspheres, wherein the donor microspheres are capable of generating singlet oxygen in an excited state;

[0087] (c) a streptavidin-labeled magnetic microsphere solution, wherein the magnetic microsphere is used to separate the matrix in the sample to be tested and other non-specific substances in the sample to be tested that are not specifically bound to the first antibody or the first antibody fragment;

[0088] (d) a solution of a first antibody or first antibody fragment labeled with streptavidin that specifically binds to a second epitope of the analyte (the analyte is an antigen); or a solution of a known antigen labeled with streptavidin that specifically binds to a third epitope of the analyte (the analyte is an antibody).

[0089] The second aspect of the present invention involves a homogeneous detection method for detecting whether an analyte is present in a sample to be tested using the kit as described in the first aspect of the present invention, which includes a method for detecting whether an antibody is present in a sample to be tested using the kit as described in the first aspect of the present invention and a method for detecting whether an antigen is present in a sample to be tested using the kit as described in the first aspect of the present invention.

[0090] Specifically, the method for detecting whether there is an antibody in a sample to be tested using the kit according to the first aspect of the present invention is as follows: Figure 1 As shown, the specific steps include:

[0091] S0, mixing the sample to be tested, reagent (c) and reagent (d) to obtain a first mixture containing a first complex formed by magnetic microspheres and the antibody to be analyzed; the reagent (d) is a known antigen solution labeled with streptavidin that specifically binds to the third epitope of the analyte.

[0092] S1, using a magnetic field to adsorb the first complex, removing the matrix and other non-specific liquid substances contained in the first mixture, so that the first complex is separated from the matrix in the sample to be tested and other non-specific substances that are not specifically bound to the first antibody or the first antibody fragment.

[0093] S2, removing the magnetic field, and contacting the reagent (a) and the reagent (b) with the first complex to form a second complex.

[0094] S3, contacting the second complex with energy or an active compound to excite the donor microsphere to generate singlet oxygen, and the acceptor microsphere reacts with the received singlet oxygen to generate a detectable chemiluminescent signal.

[0095] S4, analyzing the chemiluminescent signal to determine whether the antibody to be analyzed exists in the sample to be tested; and determining the content or concentration of the antibody to be analyzed based on the standard curve of the antibody to be analyzed.

[0096] In the present invention, the "standard curve of the antibody to be analyzed" refers to a mathematical function curve of "antibody concentration-signal value" obtained using a standard sample of the antibody to be analyzed.

[0097] The method for detecting whether an antigen exists in a sample to be tested using the kit according to the first aspect of the present invention is shown in the schematic diagram. Figure 2 As shown, the specific steps include:

[0098] S0, mixing the sample to be tested, reagent (c) and reagent (d) to obtain a first mixture containing a first complex formed by magnetic microspheres and the antibody to be analyzed; the reagent (d) is a first antibody or a first antibody fragment solution labeled with streptavidin that specifically binds to the second epitope of the analyte.

[0099] S1, using a magnetic field to adsorb the first complex, removing the matrix and other non-specific liquid substances contained in the first mixture, so that the first complex is separated from the matrix in the sample to be tested and other non-specific substances that are not specifically bound to the first antibody or the first antibody fragment.

[0100] S2, removing the magnetic field, and contacting the reagent (a) and the reagent (b) with the first complex to form a second complex.

[0101] S3, contacting the second complex with energy or an active compound to excite the donor microsphere to generate singlet oxygen, and the acceptor microsphere reacts with the received singlet oxygen to generate a detectable chemiluminescent signal.

[0102] S4, analyzing the chemiluminescent signal to determine whether the analyzed antigen exists in the sample to be tested; and determining the content or concentration of the analyzed antibody according to the standard curve of the analyzed antigen.

[0103] In the present invention, "the standard curve of the analyzed antigen" refers to a mathematical function curve of "antigen concentration-signal value" obtained using a standard sample of the analyzed antigen.

[0104] The above-mentioned homogeneous immunoassay analysis method for determining antigens using a double antibody sandwich introduces a magnetic separation process to remove irrelevant components in serum or plasma, thereby preventing these components from affecting the subsequent chemiluminescence process and effectively improving the ability of the analytical reagent to resist the "hook effect".

[0105] Example

[0106] 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.

[0107] Example 1 Indirect detection of sIgE antibodies using the method of the present invention

[0108] The detection system consists of a streptavidin-labeled magnetic microsphere (M-SA) solution, a streptavidin-labeled donor microsphere (GG-SA) solution, a biotin-labeled known antigen (egg white protein (Bio-Ag)) solution, and an anti-human IgE antibody (FG-anti-hIgE) solution bound to the acceptor microsphere.

[0109] Main reagents:

[0110] (1) Streptavidin-labeled magnetic microsphere solution: commercially available, concentration 17.5 mg / ml. The diluent is a 0.1 M Tris-HCl solution containing 3% (3 g / 100 ml) bovine serum albumin (BSA).

[0111] (2) Biotin-labeled egg white protein allergen solution: provided by Boyang Biotechnology (Shanghai) Co., Ltd., with a concentration of 0.50 mg / ml. The diluent is a 0.1 M Tris-HCl solution containing 3% (3 g / 100 ml) bovine serum albumin (BSA).

[0112] (3) Anti-human IgE antibody (monoclonal antibody) (FG-anti-hIgE) solution bound to receptor microspheres: provided by Boyang Biotechnology (Shanghai) Co., Ltd., with a concentration of 0.50 mg / ml. The diluent is a 0.1 M Tris-HCl solution containing 3% (3 g / 100 ml) bovine serum albumin (BSA).

[0113] (4) Streptavidin-labeled donor microspheres (GG-SA) solution was provided by Boyang Biotechnology (Shanghai) Co., Ltd. with a concentration of 40 mg / ml.

[0114] (5) Photochemiluminescence microplate (96-T): provided by Boyang Biotechnology (Shanghai) Co., Ltd.

[0115] (6) The preparation method, composition structure and content of the donor microspheres and acceptor microspheres used in the present invention can be found in Example 1 of Chinese Patent CN100429197C (the entire text of this patent document is incorporated herein by reference).

[0116] Testing steps:

[0117] (1) Add 25 ml of biotin-labeled egg white protein allergen solution, 25 ml of serum / plasma sample to be tested (diluted 40 times with saline), and 25 ml of streptavidin-labeled magnetic microsphere solution to the microplate, mix well, and incubate at 37°C for 20 minutes to obtain a first mixture containing a first complex. The egg white protein allergen captures the sIgE antibody to be tested to form an immune complex, which is captured by the magnetic microsphere via the biotin-streptavidin system.

[0118] (2) Place the microplate close to the magnet, use the magnetic field to suck the magnetic microspheres to one side, and carefully suck the liquid from the other side with a negative pressure pump, being careful not to suck away the magnetic microspheres. Keep the magnetic microspheres and remove interfering components in the system, especially nonspecific IgE antibodies.

[0119] (3) Remove the magnetic field, add 100 ml of anti-human IgE antibody (FG-anti-hIgE) solution bound to the acceptor microspheres, mix well, and incubate at 37°C for 20 minutes. Then add 175 ml of donor microsphere (GG-SA) solution labeled with streptavidin, mix well, and incubate at 37°C for 10 minutes to obtain a second mixture containing the second complex.

[0120] Anti-human IgE antibody binds to the sIgE antibody to be tested, and the streptavidin linked to the donor microspheres and the biotin linked to the egg white protein allergen bind to bring the donor microspheres and the acceptor microspheres close to each other. Figure 1 shown.

[0121] (4) The chemiluminescence signal value of the second mixture was read using a photochemiluminescence analyzer (excitation wavelength 680 nm, measurement wavelength 615 nm), and a standard curve was obtained based on the standard solution of sIgE antibody to calculate the content of sIgE antibody in the sample to be tested. The test results are shown in Table 1.

[0122] Table 1: Detection results of egg white protein sIgE antibodies in serum / plasma samples

[0123]

[0124] As can be seen from Table 1, the present invention can accurately detect the sIgE antibody to be detected in serum / plasma by an indirect method, which changes the current situation that photochemiluminescence analysis cannot use an indirect method to measure antibodies.

[0125] Example 2 Detection of serum total IgE (tIgE) antibodies using the method of the present invention

[0126] The detection system consists of a magnetic microsphere solution labeled with streptavidin, a donor solution labeled with streptavidin, an anti-human IgE polyclonal antibody solution labeled with biotin, and an anti-human IgE monoclonal antibody solution combined with a receptor.

[0127] Main reagents:

[0128] (1) Streptavidin-labeled magnetic microsphere solution: commercially available, concentration 17.5 mg / ml. The diluent is a 0.1 M Tris-HCl solution containing 3% (3 g / 100 ml) bovine serum albumin (BSA).

[0129] (2) Biotin-labeled anti-human IgE polyclonal antibody (Bio-anti-hIgE) solution: provided by Boyang Biotechnology (Shanghai) Co., Ltd., with a concentration of 0.5 mg / ml.

[0130] (3) Receptor-bound anti-human IgE monoclonal antibody solution: prepared according to the example described in patent PCT / US2010 / 025433. The structure of the receptor-bound anti-human IgE monoclonal antibody is: anti-human IgE monoclonal antibody-BSA-(dimethylthiophene)-(BHHCT), which is non-particulated and soluble in aqueous solution, and the concentration of the solution is 0.50 mg / ml.

[0131] (4) Streptavidin-labeled donor solution: According to the method described in the example of patent US5780646, 200 g of chlorophyll A was placed in 200 nm carboxyl-modified latex particles, and streptavidin was coated on the surface to form the streptavidin-labeled donor of the present invention. The concentration of the solution was 0.50 mg / ml.

[0132] Testing steps:

[0133] (1) Add 25 ml of biotin-labeled anti-human IgE polyclonal antibody solution, 25 ml of serum / plasma sample to be tested, and 25 ml of streptavidin-labeled magnetic microsphere solution to the microplate, mix well, and incubate at 37°C for 20 minutes to obtain a first mixture containing a first complex. The IgE in the sample forms an immune complex with the anti-human IgE polyclonal antibody, and the complex is captured by the magnetic microsphere via the biotin-streptavidin system.

[0134] (2) Place the microplate close to the magnet and use the magnetic field to suck the magnetic microspheres to one side. Carefully suck the liquid from the other side with a negative pressure pump, taking care not to suck away the magnetic microspheres. Keep the magnetic microspheres and remove the serum or plasma matrix in the system.

[0135] (3) Remove the magnetic field, add 100 ml of receptor-bound anti-human IgE monoclonal antibody solution, mix well, and incubate at 37°C for 20 minutes; then add 175 ml of streptavidin-labeled donor solution, mix well, and incubate at 37°C for 10 minutes to obtain a second mixture.

[0136] (4) The chemiluminescence signal value of the second mixture was read using a photoluminescence analyzer (excitation wavelength 680 nm, measurement wavelength 615 nm), and a standard curve was obtained based on the tIgE antibody standard solution to calculate the content of tIgE antibody in the sample to be tested. The test results are shown in Table 2.

[0137] Table 2: Detection results of tIgE antibodies in serum / plasma samples

[0138]

[0139] As can be seen from Table 2, the present invention can accurately detect tIgE antibodies in serum / plasma using the double antigen sandwich method, and effectively avoid the hook effect that occurs when detecting antigens.

[0140] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been 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 invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A homogeneous immunoassay kit without matrix effect, include: A first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or a second antibody fragment bound thereto, wherein the second antibody or the second antibody fragment is capable of specifically binding to a first epitope of an analyte in a sample to be tested; A third composition, comprising magnetic microspheres, wherein the magnetic microspheres are used to separate the matrix in the sample to be tested and other non-specific substances in the sample to be tested that are not specifically bound to the first antibody or the first antibody fragment; The homogeneous immunoassay method of the kit for detecting whether an analyte exists in a sample to be tested comprises the following steps: S1, separating the first complex formed by the magnetic microsphere-analyte from the matrix in the sample to be tested and other non-specific substances that are not specifically bound to the first antibody or the first antibody fragment by a magnetic field; S2, contacting the first composition, the second composition comprising donor microspheres and the first complex to form a second complex; S3, contacting the second complex with energy or an active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the received singlet oxygen to generate a detectable chemiluminescent signal; S4, analyzing the chemiluminescent signal to determine whether the analyte exists in the sample to be tested and the content or concentration of the analyte.

2. The kit according to claim 1, It is characterized in that The kit also includes a second composition comprising donor microspheres capable of generating singlet oxygen in an excited state.

3. The kit according to claim 1 or 2, It is characterized in that The kit further comprises a fourth composition comprising a first antibody or a first antibody fragment that is capable of specifically binding to a second epitope of the analyte.

4. The kit according to claim 1, It is characterized in that The magnetic microspheres and the donor microspheres are both coated with one member of the specific binding pair, and the first antibody is coated with the other member of the specific binding pair.

5. The kit according to claim 1, It is characterized in that The kit further includes a fourth composition comprising a known antigen that is capable of specifically binding to a third epitope of the analyte.

6. The kit according to claim 5, It is characterized in that The magnetic microspheres and the donor microspheres are both coated with one member of the specific binding pair, and the surface of the known antigen is coated with the other member of the specific binding pair.

7. The kit according to claim 4 or 6, It is characterized in that The specific binding pair member is a biotin-streptavidin system.

8. The kit according to claim 7, It is characterized in that The magnetic microspheres are indirectly coated with streptavidin via biotin-labeled bovine serum albumin and / or biotin-labeled globulin.

9. The kit according to any one of claim 1, It is characterized in that The particle size of the magnetic microspheres is 100nm-1μm.

10. The kit according to any one of claim 8, It is characterized in that The particle size of the magnetic microspheres is 200nm to 800nm.

11. The kit according to any one of claim 9, It is characterized in that The particle size of the magnetic microspheres is 300nm to 600nm.

12. The method according to claim 1, It is characterized in that Before step S1, there is step S0, which is to contact the sample to be tested, the third composition and the fourth composition to generate the first complex.

13. The method according to any one of claims 12, The characteristic is that When the analyte is an antigen to be detected, the fourth composition includes a first antibody or a first antibody fragment that can specifically bind to a second epitope of the analyte, and the magnetic microspheres and the analyte in the first complex are bound by the first antibody or the first antibody fragment.

14. The method according to any one of claims 12, The characteristic is that When the analyte is an antibody to be detected, the fourth composition comprises a known antigen that can specifically bind to the third epitope of the analyte, and the magnetic microspheres in the first complex and the analyte are bound via the known antigen.

15. The method according to any one of claims 1 or 12, It is characterized in that The following steps are involved: S0, mixing the sample to be tested, the third composition and the fourth composition to obtain a first mixture including a first complex formed by magnetic microspheres and analytes; S1, using a magnetic field to adsorb the first complex and remove the matrix and other non-specific liquid contained in the first mixture.

16. The method according to claim 15, It is characterized in that The volume of the liquid removed in step S1 accounts for 80%-95% of the volume of the liquid in the first mixture.

17. The method according to claim 1, It is characterized in that There is no washing step between step S1 and step S2.

18. The method according to claim 1, It is characterized in that There is no washing step between step S2 and step S3.

19. The method according to claim 1, It is characterized in that In step S2, the magnetic field is removed first, and then the first composition and the second composition are added to the first composite to form a second composite.

20. The method according to claim 1, It is characterized in that The analyte in the sample to be tested is selected from one or more of pathogen antibodies, autoantibodies, allergen-specific antibodies and allergen total antibodies in serum or plasma.

21. A second complex included in the method of claim 1, wherein include: a centrosome portion selected from any one of the immune molecules; a receptor portion capable of reacting with singlet oxygen to generate a detectable luminescent signal, which is bound to the centrosome via a second antibody or a second antibody fragment, which is capable of specifically binding to a fourth epitope of the centrosome; a donor moiety capable of generating singlet oxygen in an excited state, which is bound to the centrosome via a bridging body, said bridging body specifically binding to a fifth epitope on the centrosome that does not overlap with said fourth epitope; a magnet portion, which is bound to the centrosome via a bridge body, and the bridge body specifically binds to a sixth epitope on the centrosome that does not overlap with the fourth epitope; Wherein, the bridge body is selected from a known antigen, a first antibody or a first antibody fragment that can specifically bind to the centrosome.

22. The second composite according to claim 21, It is characterized in that The magnet is indirectly coated with streptavidin via biotin-labeled bovine serum albumin and / or biotin-labeled globulin, and the cross-body is coated with biotin.

23. The second composite according to any one of claims 21 or 22, It is characterized in that The centrosome is selected from one of pathogenic antibodies, autoantibodies or allergen-specific antibodies in human serum or plasma.

24. Use of the kit according to any one of claims 1 to 11, the method according to any one of claims 1, 12 to 20, or the second complex according to any one of claims 21 to 23 in clinical detection of antigens or antibodies in serum or plasma.

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