CN114910634A - Homogeneous phase immunoassay kit without matrix effect and analysis method and application thereof - Google Patents

Abstract

The present invention relates to a homogeneous immunoassay kit without matrix effect, comprising: 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 bound to it An antibody fragment, the second antibody or second antibody fragment can specifically bind to the first epitope of the analyte in the sample to be tested; and a third composition, which comprises magnetic microspheres. The invention also relates to a homogeneous immunodetection method and application of a homogeneous immunoassay kit without matrix effect.

Description

A homogeneous immunoassay kit with no matrix effect and its analytical method and application

technical field

The invention belongs to the technical field of biomedical detection, in particular to a homogeneous immunodetection kit without matrix effect, an analysis method and application thereof.

Background technique

Labeled immunoassay integrates 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 medical body fluid samples. According to whether it is necessary to separate bound label (Bind, B) and free label (Free, F), labeling immunoassay is divided into homogeneous immunoassay and heterogeneous immunoassay. Homogeneous immunoassays do not need to separate and remove free labels, and directly measure the signal of bound labels, that is, to obtain a dose curve or standard curve, such as fluorescence polarization immunoassay and light-activated chemiluminescence assay (LICA). Heterogeneous immunoassays, such as chemiluminescence immunoassays (CLIA) and electrochemiluminescence immunoassays (ECLIA), require separation and removal of free labels, followed by measurement of the signal of bound labels, in order to obtain a dose curve or standard curve.

The methods of detecting 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 type of the antibody to be detected by anti-antibody (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 classical indirect method needs to use a two-step method. The known antigen is combined with the antibody to be tested, and the labeled anti-antibody (second antibody) is combined with the antibody to be tested. Nonspecific antibodies will also bind to secondary antibodies, depleting large amounts of secondary antibodies and increasing the blank signal.

In the heterogeneous labeling immunoassay technology, the removal of free labels that are not involved in binding by "separation and washing" is one of the key links in the analysis process. Then, whether it is done manually or automatically by the instrument, it will lead to washing errors. At the same time, the washing process will also lead to cross-contamination between samples, and strong positive samples will affect the test results of adjacent samples. The characteristic of homogeneous labeling immunoassay technology is that there is no separation and washing process in the whole process, and there is no washing error.

Although separation washing steps are not required in homogeneous immunoassay techniques, some substances in serum or plasma can interfere with the detection of the labeled signal or interfere with the chemiluminescent reaction. In addition, the field of chemiluminescence is involved in the homogeneous labeling immunoassay technology, and some involve redox reactions. For example, blood samples containing some redox drugs (such as vitamin C) also interfere with the light signal generation process, which seriously affects the detection results. accuracy.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a homogeneous immunoassay kit with no matrix effect and its analysis method and application. The method for analyzing the antigen or antibody in the sample to be tested by the kit can eliminate the matrix effect in the homogeneous immunoassay, and change the current situation of the hook effect when the homogeneous immunoassay cannot use the indirect method to detect the antibody and detect the antigen.

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

A first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or second antibody fragment bound thereto, the second antibody or second antibody fragment capable of interacting with the sample to be tested. specifically binds to the first epitope of the analyte;

a second composition comprising a donor capable of producing singlet oxygen in an excited state;

A third composition comprising magnetic microspheres for separating 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 inventors of the present application have surprisingly found that the method for analyzing antigens or antibodies in a sample to be tested using the kit of the present invention increases the process of removing serum/plasma matrix, effectively avoiding the follow-up in the homogeneous detection of serum/plasma matrix The influence of the luminescence process has changed the current situation that the homogeneous immunoassay technology cannot use the indirect method to detect the antibody and the hook effect when the antigen is detected. In addition, the process of removing the serum/plasma matrix described in the present invention is different from the separation and washing of the general heterogeneous immunoassay, and its purpose is not to remove free labels, but to remove possible interfering substances in serum/plasma. Part of the interfering substances is sufficient (above 80%), there is no high requirement for washing precision, and it will not affect the precision (repeatability). In addition, the kit of the present invention has strong versatility and can detect various items.

In some embodiments of the invention, the kit further comprises a fourth composition comprising a first antibody or first antibody fragment capable of specifically binding to a second epitope of the analyte.

According to the present invention, both the magnet microsphere and the donor are coated with one member of the specific binding pair, and the first antibody is coated with the other member of the specific binding pair.

In other embodiments of the present invention, the kit further comprises a fourth composition comprising a known antigen capable of specifically binding to a third epitope of the analyte.

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

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

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

According to the present invention, the particle size of the magnet microspheres is 100 nm-1 μm, preferably 200 nm-800 nm, more preferably 300 nm-600 nm.

A second aspect of the present invention provides a homogeneous detection method for detecting whether an analyte exists in a sample to be tested using the kit according to the first aspect of the present invention, comprising the following steps:

S1, separate 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 and the second composition with the first composite to form a second composite;

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 chemiluminescence signal;

S4, analyze the chemiluminescence signal to determine whether the analyte and the content or concentration of the analyte exist in the sample to be tested.

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

According to the present invention, when the analyte is an antigen to be detected, the fourth composition comprises a first antibody or a first antibody fragment capable of specifically binding to the second epitope of the analyte, the first The magnetic microspheres and the analyte in the complex are bound by the first antibody or first antibody fragment.

According to the present invention, when the analyte is an antibody to be detected, the fourth composition comprises a known antigen capable of specifically binding to the third epitope of the analyte, the magnet in the first complex The microspheres and analytes are bound by known antigens.

In some preferred embodiments of the present invention, the method comprises the steps of:

S0, mixing the sample to be tested, the third composition and the fourth composition to obtain a first mixture comprising the first complex formed by the magnetic microspheres-analyte;

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

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.

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

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

In some preferred embodiments of the present invention, in step S2, after removing the magnetic field, the first composition and the second composition are added to the first composite to form the second composite.

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.

The inventors of the present application have surprisingly found that the method of the present invention has changed the current situation that the photo-excited chemiluminescence analysis in the homogeneous immunoassay cannot use an indirect method to measure antibodies. Due to the small proportion of the antibody to be detected in the non-specific antibody, there is no separation and washing process in the existing photo-induced chemiluminescence analysis procedure, and the non-specific antibody cannot be removed. The non-specific antibody can also bind to the anti-antibody-coated receptor microarray. ball, thereby interfering with the detection system. Therefore, conventional light-excited chemiluminescence analysis cannot use the indirect analysis mode of "known antigen-antibody to be detected-labeled anti-antibody", but can only use the double-antigen sandwich or competition analysis mode to detect the antibody to be tested, while the double-antigen sandwich or The competition analysis mode has higher requirements for known antigens or competing antibodies, and cannot distinguish the types of immunoglobulins of the antibodies to be detected. The photo-excited chemiluminescence analysis in the above-mentioned novel 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 interfering substances. Eliminates the effects of non-specific antibodies and other interfering substances.

In addition, the above-described separation and removal of serum/plasma matrix using a magnetic field in the homogeneous immunoassay method does not affect the precision (reproducibility) unlike the separation and washing process of the heterogeneous immunoassay. The "separation and washing" process of heterogeneous immunoassay is carried out after the occurrence of labeled antibody and corresponding antigen to be detected or labeled antigen and corresponding antibody to be detected. Its purpose is to remove the influence of free label on signal detection, and at the same time Interfering substances in serum/plasma are also removed. However, this kind of separation and washing needs to be repeated 3-5 times to completely remove the free label, which not only increases the detection time, but more importantly, because the washing process is difficult to standardize, which will bring large errors (washing errors), thereby affecting The precision of the analytical method. The process of removing serum/plasma matrix in the present invention is performed 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. Part of the interfering substances is sufficient, and there is no excessive requirement for washing accuracy.

The third aspect of the present invention provides a second compound involved in the method according to the second aspect of the present invention, comprising:

a centrosome moiety selected from any of the immune molecules;

A receptor moiety capable of reacting with singlet oxygen to generate a detectable luminescent signal, which is bound to the centrosome by a second antibody or second antibody fragment capable of interacting with the fourth surface of the centrosome site-specific binding;

a donor moiety capable of generating singlet oxygen in the excited state, which binds to the centrosome via a pontosome that specifically binds to a fifth epitope on the centrosome that does not overlap with the fourth epitope;

The magnet portion, which binds to the centrosome via a pontosome that specifically binds to a sixth epitope on the centrosome that does not overlap the fourth epitope.

In some embodiments of the invention, the pontosome is selected from a known antigen, a first antibody or a first antibody fragment capable of specifically binding to a centrosome.

In other embodiments of the present invention, the magnet is indirectly coated with streptavidin through biotin-labeled bovine serum albumin and/or bio-labeled globulin, and the pons is coated with biotin .

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

The fourth aspect of the present invention provides a kit according to the first aspect of the present invention or the method according to the second aspect of the present invention or the second complex according to the third aspect of the present invention in clinical detection of serum or plasma. Antigen or antibody application.

Description of drawings

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

FIG. 1 is a schematic diagram of the principle diagram of the method of the present invention for analyzing antibodies in a sample to be tested. The meanings of the reference numbers in the figure are as follows: 1 Streptavidin-labeled magnet microspheres; 2 Biotin-labeled known antigens; 3 Antibodies to be analyzed; 4 Secondary antibodies bound to acceptor microspheres; 5 Streptavidin Avidin-labeled donor microspheres.

FIG. 2 is a schematic diagram of the principle diagram of the method of the present invention for analyzing antigens in a sample to be tested. The meanings of the reference numbers in the figure are as follows: 1 Streptavidin-labeled magnet microspheres; 2 Biotin-labeled primary antibodies; 3 Antigen to be analyzed; 4 Secondary antibodies bound to receptor microspheres; 5 Chains Mycoavidin-labeled donor microspheres.

FIG. 3 is a schematic diagram of the principle of analyzing antigens in a sample to be tested by using magnetic microspheres indirectly bound to streptavidin. The meanings of the reference numbers in the figure are as follows: 1 Streptavidin-labeled magnet microspheres; 1-1 Magnet microspheres; 1-2 Biotin-labeled bovine serum albumin or globulin; 1-3 Streptavidin 2 primary antibody conjugated to biotin; 3 analyzed antigen; 4 secondary antibody conjugated to acceptor microspheres; 5 streptavidin-labeled donor microspheres.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in detail below. Before the present invention is described in detail, however, it is to be understood that this invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of the stated range and any other stated or intervening value in the stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the preferred methods and materials are now described.

I. Terminology

The term "sample to be tested" in the present invention refers to a mixture that may contain analytes, including but not limited to proteins, hormones, antibodies or antigens. Typical test samples that can be used in the methods disclosed herein include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.

The terms "antibody" and "immunoglobulin" as used herein are used in the broadest sense and include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv , scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Where desired, the antibody may be further conjugated with other moieties, such as specific binding pair members, such as biotin or streptavidin (one of the biotin-streptavidin specific binding pair members), etc. conjugated.

The term "monoclonal antibody" in the present invention refers to immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" in the present invention refers to a collection of immunoglobulins produced by one or more clones of B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "antigen" 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 product antibody and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

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

The term "specific binding" in the present invention refers to the mutual discrimination and selective binding reaction between two substances, which is the conformational correspondence between the corresponding reactants from the perspective of steric structure.

The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, eg, enzyme-substrate, antigen-antibody, ligand-receptor. A specific example of a specific binding pair member pair is the biotin-streptavidin system, in which "biotin" is widely present in animal and plant tissues, and its molecule has two ring structures, namely the imidazolone ring. and thiophene rings, of which the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents; while "streptavidin" is produced by Streptomyces sp. A secreted protein with a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which can bind a biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules simultaneously, resulting in a "tentacle effect" to improve analytical sensitivity.

In any required situation, any reagents used in the present invention, including antigens, antibodies, acceptors or donors, can be conjugated to any member of the biotin-streptavidin specific binding pair according to actual needs.

The term "donor" as used herein refers to a sensitizer capable of generating reactive intermediates such as singlet oxygen that react with acceptors upon activation by energy or reactive compounds. Donors can be photoactivated (eg, dyes and aromatic compounds) or chemically activated (eg, enzymes, metal salts, etc.). In some specific embodiments of the present invention, the donor is a photosensitizer, and the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively photostable and does not react effectively with singlet oxygen, which is non-limiting Examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Patent No. 5,709,994, which is incorporated herein by reference in its entirety, 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 linking groups to members of specific binding partners. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US Pat. No. 6,406,913, which is incorporated herein by reference. In other embodiments of the present invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Some other examples of donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heat these compounds or Direct absorption of light by these compounds releases singlet oxygen.

The term "receptor" as used herein refers to a compound capable of reacting with singlet oxygen to generate a detectable signal. The donor is induced to activate by the energy or active compound and releases high energy singlet oxygen which is captured by the acceptor in close proximity, thereby delivering energy to activate the acceptor. In some embodiments of the invention, the acceptor is a species that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose while simultaneously or glow afterwards. Typical examples of these materials include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridan, vinyl arylene ether, diepoxyethylene, dimethyl Thiophene, aromatic imidazole or lucigenin. In other embodiments of the present invention, the acceptor is an alkene capable of reacting with singlet oxygen to form hydroperoxides or dioxetanes that can be decomposed into ketones or carboxylic acid derivatives; Stable dioxetanes that decompose by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyl compounds, such as luminol ; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the present disclosure and claimed invention are described in US Pat. No. 5,340,716, which is incorporated herein by reference in its entirety. In other specific embodiments of the present invention, the receptors comprise olefin compounds and metal chelates, which are non-particulate and soluble in aqueous media, the case of such receptors can be found in patent PCT/US2010/025433 (This patent document is hereby incorporated by reference in its entirety)

In the present invention, the "donor" and/or "acceptor" can be coated on the substrate by functional groups to form "donor microspheres" and/or "acceptor microspheres". The "matrix" of the present invention is a microsphere or particle known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be swellable or non-swellable, and which may be Porous or non-porous, of any density, but preferably of a density close to that of water, preferably capable of floating in water, and composed of transparent, partially transparent or opaque materials. The matrix may or may not be charged, and when charged, it is preferably negatively charged. The matrix can be solids (eg polymers, metals, glasses, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (eg hydrocarbons, fluorocarbons, siliceous fluids), vesicles (eg Synthetic such as phospholipids, or natural such as cells, and organelles). The matrix can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is usually multifunctional or capable of binding to a donor or acceptor through specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like. A non-limiting example of a matrix suitable for use in the present invention is a carboxyl-modified latex particle. Details of such matrices can be found in US Pat. Nos. 5,709,994 and 5,780,646 (both of which are hereby incorporated by reference in their entirety).

The term "epitope" in the present invention refers to any protein determinant that can specifically bind to immunoglobulins or T cell receptors. In some embodiments of the invention, an epitope is a region of the antigenic surface that can be specifically assembled by an antibody. Epitopic determinants can generally include chemically active surface groups of molecules such as, but not limited to, amino acids, sugar side chains, phosphoryl and/or sulfonyl groups. In some other specific embodiments of the present invention, epitopes may have specific three-dimensional structural characteristics as well as specific charge characteristics.

There are many kinds of the term "immune molecule" in the present invention, some of which have structural and evolutionary homology, mainly including the following categories: membrane surface antigen receptor, major histocompatibility complex antigen, leukocyte differentiation antigen , adhesion molecules, antibodies, complements, cytokines, antigens, etc.; in particular, the "immune molecules" in the present invention mainly refer to antigens and antibodies.

II. Examples

As mentioned above, in the process of antibody detection by the classical indirect method, there must be a separation and washing process to remove the influence of non-specific antibodies and free antibodies, while the light-induced chemiluminescence analysis (LICA) in homogeneous detection is characterized by no separation and washing in the whole process. Therefore, substances in serum or plasma that interfere with the detection of labeled signals or the chemiluminescence reaction cannot be removed, and the indirect method cannot be used to detect antibodies, and it cannot distinguish the types of antibody immunoglobulins to be detected.

The inventors of the present application removed most of the interfering substances from serum/plasma by magnetic separation by introducing magnetic microspheres during the detection process, before adding the first composition containing the donor and the second composition containing the acceptor, The influence of interfering substances on the test results is effectively avoided.

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

A first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or second antibody fragment bound thereto, the second antibody or second antibody fragment capable of interacting with the sample to be tested. specifically binds to the first epitope of the analyte;

a second composition comprising a donor capable of producing singlet oxygen in an excited state;

A third composition comprising magnetic microspheres for separating 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 .

Specifically, the homogeneous detection kit without matrix effect according to the present invention includes the following reagents:

(a) a receptor solution bound to a second antibody or second antibody fragment; the receptor can react with singlet oxygen to generate a detectable signal; the second antibody or second antibody fragment can be combined with the sample to be tested The first epitope of the analyte specifically binds; 0.25mg/ml-0.75mg/ml of the receptor solution;

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

(c) a solution of magnetic microspheres, 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 The concentration of magnet microsphere solution is 15mg/ml-20mg/ml;

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

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.

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

The above reagents can be linked with streptavidin or biotin according to the needs of the reagents, the magnetic microspheres and the donor, the first antibody and the known antigen, so as to pass the specific interaction between the "specific binding pair members" And realize the connection of two molecules. Additionally, the donor and acceptor in the reagent can be coated on matrix particles to form donor microspheres and acceptor microspheres.

The streptavidin can be directly or indirectly bound to the magnetic microspheres; preferably, the streptavidin is bound to the magnetic microspheres indirectly. The way that the streptavidin is indirectly bound to the magnetic microspheres is as follows: firstly connect biotin-labeled bovine serum albumin and/or biologically-labeled globulin on the surface of the magnetic microspheres, and then streptavidin. Copatin binds to the magnetic microspheres indirectly through biotin. This method will further expand the space on the surface of the magnet microspheres to reduce the steric hindrance effect of biomolecular binding. Figure 3 shows a schematic diagram of the principle of analyzing antigens in samples to be tested by using magnetic microspheres indirectly bound to streptavidin.

More specifically, the homogeneous detection kit without matrix effect according to the present invention includes the following reagents:

(a) a solution of acceptor microspheres bound to a second antibody or second antibody fragment; the acceptor microspheres can react with singlet oxygen to generate a detectable signal; the second antibody or second antibody fragment can bind to the specifically binding to the first epitope of the analyte in the test sample;

(b) a solution of biotin-labeled donor microspheres capable of generating singlet oxygen in an excited state;

(c) Streptavidin-labeled magnet microsphere solution, the magnet microspheres are used to separate the matrix in the sample to be tested and the non-specific binding of the first antibody or the first antibody fragment in the sample to be tested other non-specific substances;

(d) streptavidin-labeled first antibody or first antibody fragment solution that specifically binds to the second epitope of the analyte (the analyte is an antigen); or streptavidin-labeled and A solution of a known antigen to which the third epitope of the analyte specifically binds (the analyte is an antibody).

The second aspect of the present invention relates to a homogeneous detection method for detecting the presence of an analyte in a sample to be tested by using the kit according to the first aspect of the present invention, which includes using the kit according to the first aspect of the present invention to detect the presence of an analyte. A method for detecting the presence of antibodies in a sample and a method for detecting the presence or absence of antigens in a sample to be tested using the kit according to the first aspect of the present invention.

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, the schematic diagram of which is shown in Figure 1, specifically includes the following steps:

S0, mix the sample to be tested, the reagent (c) and the reagent (d) to obtain a first mixture comprising the first complex formed by the magnetic microspheres and the antibody to be analyzed; the reagent (d) is streptavidin A solution of a known antigen labeled with and specifically bound to a third epitope of the analyte.

S1, the first complex is adsorbed by a magnetic field, the matrix and other non-specific substances contained in the first mixture are removed, so that the first complex is combined with the matrix in the sample to be tested and not with the first antibody. or other non-specific substances specifically bound by the first antibody fragment are separated.

S2, the magnetic field is removed, and reagent (a) and reagent (b) are contacted with the first complex to form a second complex.

S3, using energy or an active compound to contact the second complex to excite the donor microspheres to generate singlet oxygen, and the acceptor microspheres to react with the received singlet oxygen to generate a detectable chemiluminescence signal.

S4, analyze the chemiluminescence signal to determine whether the antibody to be analyzed exists in the sample to be tested; and determine the content or concentration of the antibody to be analyzed according to the standard curve of the antibody to be analyzed.

In the present invention, "the standard curve of the antibody to be analyzed" refers to the mathematical function curve of "antibody concentration-signal value" obtained by using the standard substance of the antibody to be analyzed.

The schematic diagram of the method for detecting whether there is an antigen in a sample to be tested using the kit according to the first aspect of the present invention is shown in Figure 2, which specifically includes the following steps:

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

S1, the first complex is adsorbed by a magnetic field, the matrix and other non-specific substances contained in the first mixture are removed, so that the first complex is combined with the matrix in the sample to be tested and not with the first antibody. or other non-specific substances specifically bound by the first antibody fragment are separated.

S2, the magnetic field is removed, and reagent (a) and reagent (b) are contacted with the first complex to form a second complex.

S3, using energy or an active compound to contact the second complex to excite the donor microspheres to generate singlet oxygen, and the acceptor microspheres to react with the received singlet oxygen to generate a detectable chemiluminescence signal.

S4, analyze the chemiluminescence signal to determine whether the antigen to be analyzed exists in the sample to be tested; and determine the content or concentration of the antibody to be analyzed according to the standard curve of the antigen to be analyzed.

In the present invention, "the standard curve of the antigen to be analyzed" refers to a mathematical function curve of "antigen concentration-signal value" obtained by using the standard substance of the antigen to be analyzed.

The above-mentioned homogeneous immunodetection analysis method for antigen determination using double antibody sandwich removes irrelevant components in serum or plasma by introducing a magnetic separation process, prevents these components from affecting the subsequent chemiluminescence process, and effectively improves the resistance of analytical reagents. "Hook effect" capability.

Example

In order to make the present invention easier to understand, the present invention will be further described in detail below with reference to the embodiments, which are only for illustrative purposes and do not limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods unless otherwise specified.

Example 1 Indirect detection of sIgE antibody by the method of the present invention

The detection system consists of streptavidin-labeled magnet microspheres (M-SA) solution, streptavidin-labeled donor microspheres (GG-SA) solution, biotin-labeled known antigen (egg albumin (Bio -Ag)) solution and anti-human IgE antibody (FG-anti-hIgE) solution combined with acceptor microspheres.

Main reagents:

(1) Streptavidin-labeled magnet microsphere solution: commercially available, with a concentration of 17.5 mg/ml. The dilution was 0.1M Tris-HCl solution containing 3% (3g/100ml) bovine serum albumin (BSA).

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

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

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

(5) Photo-excited chemiluminescence microplate (96-T): provided by Boyang Biotechnology (Shanghai) Co., Ltd.

(6) The preparation method, composition structure and content of the donor microspheres and acceptor microspheres used in the present invention can refer to Example 1 in Chinese Patent CN100429197C (this patent document is hereby incorporated by reference in its entirety).

Detection steps:

(1) Add 25ml of biotin-labeled egg white protein allergen solution, 25ml of serum/plasma sample to be tested (diluted 40 times with normal saline), and 25ml of streptavidin-labeled magnet microsphere solution into the microplate, and mix well After incubation at 37°C for 20 minutes, a first mixture containing the first complex was obtained. Egg albumin allergen captures the sIgE antibody to be detected to form an immune complex, which is captured by the magnetic microspheres via the biotin-streptavidin system.

(2) Put the microplate close to the magnet, suck the magnet microspheres to one side with the help of the magnetic field, and carefully suck up the liquid with the negative pressure pump from the other side, being careful not to suck the magnet microspheres away. Retain the magnetic microspheres and remove interfering components in the system, especially non-specific IgE antibodies.

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

The anti-human IgE antibody is combined with the sIgE antibody to be detected, and the streptavidin linked to the donor microsphere and the biotin linked to the egg albumin allergen are combined, so that the donor microsphere and the acceptor microsphere are close to each other, as shown in the attached figure 1 shown.

(4) Read the chemiluminescence signal value of the second mixture with a photo-excited chemiluminescence analyzer (excitation wavelength 680 nm, measuring wavelength 615 nm), obtain a standard curve according to the standard solution of sIgE antibody, and calculate the content of sIgE antibody in the sample to be tested, The test results are shown in Table 1.

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

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 the indirect method cannot be used in the light-excited chemiluminescence analysis.

Example 2 Detection of serum total IgE (tIgE) antibody by the method of the present invention

The detection system consists of streptavidin-labeled magnet microsphere solution, streptavidin-labeled donor solution, biotin-labeled anti-human IgE polyclonal antibody solution, and receptor-bound anti-human IgE monoclonal antibody solution composition.

Main reagents:

(1) Streptavidin-labeled magnet microsphere solution: commercially available, with a concentration of 17.5 mg/ml. The dilution was 0.1M Tris-HCl solution containing 3% (3g/100ml) bovine serum albumin (BSA).

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

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

(4) Streptavidin-labeled donor solution: 200 g of chlorophyll A was put into 200 nm carboxyl-modified latex particles according to the method described in the examples of patent US5780646, and streptavidin was coated on the surface to form the streptavidin-labeled donor according to the present invention, and the concentration of the solution is 0.50 mg/ml.

Detection steps:

(1) Add 25ml of biotin-labeled anti-human IgE polyclonal antibody solution, 25ml of serum/plasma sample to be tested, and 25ml of streptavidin-labeled magnet microsphere solution into the microwell plate, mix well and set to 37℃ Incubate for 20 minutes to obtain a first mixture containing the first complex. The IgE in the sample forms an immune complex with the anti-human IgE polyclonal antibody, which is captured by the magnetic microspheres via the biotin-streptavidin system.

(2) Put the microplate close to the magnet, suck the magnet microspheres to one side with the help of the magnetic field, and carefully suck up the liquid with the negative pressure pump from the other side, being careful not to suck the magnet microspheres away. Retain the magnetic microspheres and remove the serum or plasma matrix from the system.

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

(4) Read the chemiluminescence signal value of the second mixture with a photo-excitation chemiluminescence analyzer (excitation wavelength 680nm, measurement wavelength 615nm), obtain a standard curve according to the tIgE antibody standard solution, and calculate the content of the tIgE antibody in the sample to be tested. The test results are shown in Table 2.

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

As can be seen from Table 2, the present invention can accurately detect the tIgE antibody in serum/plasma by using the double antigen sandwich method, and effectively avoid the hook effect when the antigen is detected.

It should be noted that the above-mentioned embodiments 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 is to be understood that the words used therein are words of description and explanation, rather than words of limitation. The present invention may be modified within the scope of the claims of the present invention as specified, and may be modified without departing from the scope and spirit of the present invention. Although the invention described herein refers to the specific methods, materials and embodiments, it is not intended to be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

Claims (24)

1. A homogeneous immunoassay kit without matrix effect comprising:

a first composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second antibody or antibody fragment bound thereto, the second antibody or antibody fragment being capable of specifically binding to a first epitope of an analyte in a test sample;

a third composition comprising magnetic microspheres.

2. The kit of claim 1, further comprising a second composition comprising donor microspheres capable of generating singlet oxygen in an excited state.

3. A kit according to claim 1 or 2, further comprising a fourth composition comprising a first antibody or first antibody fragment capable of specifically binding to a second epitope of an analyte.

4. The kit of any one of claims 1 to 3, wherein the magnet microspheres and the donor microspheres are each coated with one member of a specific binding pair member and the first antibodies are coated with the other member of a specific binding pair member.

5. A kit according to any one of claims 1 to 3, further comprising a fourth composition comprising a known antigen capable of specifically binding to a third epitope of an analyte.

6. The kit of any one of claims 1 to 5, wherein the magnet microspheres and the donor microspheres are each coated with one member of a specific binding pair member and the known antigens are surface coated with the other member of a specific binding pair member.

7. The kit of claim 4 or 6, wherein the specific binding pair member is a biotin-streptavidin system.

8. The kit of claim 7, wherein the magnet microspheres are indirectly coated with streptavidin via biotin-labeled bovine serum albumin and/or biosurface-labeled globulin.

9. The kit according to any one of claims 1 to 8, wherein the magnetic microspheres have a particle size of 100nm to 1 μm, preferably 200nm to 800nm, and more preferably 300nm to 600 nm.

10. A homogeneous immunoassay method for detecting the presence of an analyte in a sample to be tested using the kit of any one of claims 1 to 9, comprising the steps of:

s1, separating a first compound formed by the magnetic microsphere-analyte from the matrix in the sample to be detected and other non-specific substances which are not specifically bound with the first antibody or the first antibody fragment through a magnetic field;

s2, contacting the first composition, the second composition 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 reacting the acceptor with the singlet oxygen to generate a detectable chemiluminescent signal;

and S4, analyzing the chemiluminescence signal, and judging whether the analyte exists in the sample to be detected and the content or concentration of the analyte.

11. The method of claim 10, wherein step S1 is preceded by step S0 of contacting the test sample, the third composition and the fourth composition to generate the first complex.

12. The method according to claim 10 or 11, wherein, when the analyte is the antigen to be detected, the fourth composition comprises a first antibody or a first antibody fragment capable of specifically binding to a second epitope of the analyte, and the magnetic microsphere and the analyte in the first composition are bound by the first antibody or the first antibody fragment.

13. The method according to claim 10 or 11, wherein when the analyte is an antibody to be detected, the fourth composition comprises a known antigen capable of specifically binding to a third epitope of the analyte, and the magnetic microsphere and the analyte in the first composition are bound by the known antigen.

14. A method according to any one of claims 10 to 13, comprising the steps of:

s0, mixing the sample to be tested, the third composition and the fourth composition to obtain a first mixture containing a first complex formed by the magnetic microsphere-analyte;

s1, adsorbing the first complex by using a magnetic field, and removing the matrix and the liquid of other nonspecific substances contained in the first mixture.

15. The method of claim 14, wherein the volume of liquid removed in step S1 is 80% to 95% of the volume of liquid in the first mixture.

16. The method of any one of claims 10 to 15, wherein there is no washing step between the step S1 and the step S2.

17. The method of any one of claims 10 to 16, wherein there is no washing step between the step S2 and the step S3.

18. The method according to any one of claims 10 to 17, wherein in step S2, after removing the magnetic field, the first composition and the second composition are added to the first composite to form the second composite.

19. The method according to any one of claims 1 to 18, wherein the analyte in the sample to be tested is selected from one or more of autoantibodies, allergen specific antibodies and allergen total antibodies in serum or plasma.

20. A second composite comprised in the method of any one of claims 10 to 19, comprising:

a central body portion selected from any one of immune molecules;

a receptor moiety capable of reacting with singlet oxygen to generate a detectable luminescent signal, bound to the centrosome by a second antibody or a second antibody fragment, capable of specifically binding to a fourth epitope of the centrosome;

a donor moiety capable of producing singlet oxygen in an excited state, bound to the centrosome via a bridge that specifically binds to a fifth epitope on the centrosome that does not overlap with the fourth epitope;

a magnet portion bound to the central body by a bridge that specifically binds to a sixth epitope on the central body that does not overlap with the fourth epitope.

21. The second complex of claim 20, wherein the bridge is selected from a known antigen, a first antibody or a first antibody fragment capable of specifically binding to the centrosome.

22. A second complex according to claim 20 or 21, wherein the magnet is indirectly coated with streptavidin via biotin-labelled bovine serum albumin and/or a biosurface-labelled globulin, and the pontic is coated with biotin.

23. A second complex according to any one of claims 20 to 22, wherein the centrosome is selected from one of a pathogenic, autoantibody or allergen specific antibody in human serum or plasma.

24. Use of a kit according to any one of claims 1 to 9 or a method according to any one of claims 10 to 19 or a second complex according to any one of claims 20 to 23 for the clinical detection of an antigen or antibody in serum or plasma.

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