Light-activated chemiluminescence detection reagent composition, kit, detection method and device

Abstract

The invention provides a light-activated chemiluminescence detection reagent composition, a kit, a detection method and a device. The light-excited chemiluminescence detection reagent composition comprises a first reagent and a second reagent, wherein the first reagent comprises a polyantibody A combined with a first marker; the second reagent comprises a light-emitting microsphere, the light-emitting microsphere can react with singlet oxygen to generate a detectable signal, and the surface of the light-emitting microsphere is coated with a monomer antibody B; and the polyantibody A and the monomer antibody B recognize different epitopes of a target molecule to be detected. When the detection reagent composition is used for target molecule detection, the background can be reduced while the positive signal value is not influenced, and the detection accuracy and sensitivity are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a photoinduced chemiluminescence detection reagent composition, a kit, a detection method and a device. Background Art

[0002] Light Initiated Chemiluminesence Assay technology uses homogeneous system chemiluminescence detection technology. One antibody involved in the immune reaction is coated with a photosensitive bead (sensibead) containing phthalocyanine (luminol-type chemiluminescent substance); the other antibody is coated with a luminescent bead (chemibead) containing dimethylthiophene derivatives and Eu chelate. In the presence of the target antigen, a sandwich immune complex can be formed. The target antigen can tightly connect the photosensitive beads and luminescent beads marked on the two antibodies together, and the luminol oxygen pathway chemiluminescence process can be completed under 680nm excitation light.

[0003] In photochemiluminescence analysis, the common methods to reduce the background are: 1) reducing the concentration of the luminescent ball. Although reducing the concentration of the luminescent ball alone will reduce the background, the reaction efficiency will also decrease, and the positive signal value will also decrease; 2) reducing the concentration of the photosensitive ball, but the reduction of the photosensitive ball will cause HOOK caused by excessive biotin.

[0004] Therefore, there is an urgent need in the art to develop a photoinduced chemiluminescence analysis method that does not affect the positive signal value while reducing the background. Summary of the invention

[0005] Based on the deficiencies in the prior art, the present invention provides a photochemiluminescence detection reagent composition that does not affect the positive signal value while reducing the background and a kit containing the composition, as well as a method and device for target molecule detection using the composition or the kit.

[0006] In order to achieve the purpose of the present invention, the following technical scheme is adopted:

[0007] In a first aspect, the present invention provides a photochemiluminescence detection reagent composition, comprising:

[0008] A first reagent, comprising a polyantibody A bound to a first marker; and

[0009] A second reagent, comprising a luminescent microsphere, wherein the luminescent microsphere can react with singlet oxygen to generate a detectable signal, and the surface of the luminescent microsphere is coated with a monomer antibody B;

[0010] The multimeric antibody A and the monomeric antibody B recognize different epitopes of the target molecule to be detected.

[0011] In some embodiments, the multimeric antibody A comprises N monomer antibodies a connected by a cross-linking agent, where N≥2.

[0012] In some specific embodiments, the monomer antibody a is a monoclonal antibody that recognizes a specific epitope of the target molecule to be detected.

[0013] In some embodiments, 2≤N≤7. In some embodiments, 4≤N≤6.

[0014] In some embodiments, the cross-linking agent has a bifunctional group that can react with the carboxyl group or amino group of the monomer antibody a. In some specific embodiments, the cross-linking agent is a dialdehyde compound. In some embodiments, the cross-linking agent is a dihydrazide compound.

[0015] In some embodiments, the first marker has a spacer arm. In some specific embodiments, the length of the spacer arm of the first marker is In some embodiments, the length of the spacer arm of the first marker is In some embodiments, the spacer arm of the first label is polymerized from "-LC-". In some embodiments, the spacer arm of the first label is polymerized from "-PEG-".

[0016] In some embodiments, the first label is biotin or a biotin analog.

[0017] The present invention uses biotin or a biotin analog to label the multimeric antibody, and in a system for reducing the dosage of the photosensitive sphere, it is possible to avoid a decrease in the positive signal value caused by a relative excess of biotin, thereby ensuring the reaction efficiency.

[0018] In some embodiments, the arm length of the biotin is In some embodiments, the arm length of the biotin is

[0019] In some embodiments, the biotin or biotin label is selected from Biotin-LC-LC-NHS, Biotin-PEG3-NHS, Biotin-PEG4-NHS, Biotin-PEG5-NHS, Biotin-PEG6-NHS, Biotin-PEG7-NHS, Biotin-PEG8-NHS, Biotin-PEG9-NHS, Biotin-PEG 10 -NHS, Biotin-PEG 11 - At least one of the NHS.

[0020] In some embodiments, the first label is selected from Biotin-LC-LC-NHS. In some embodiments, the first label is selected from Biotin-PEG4-NHS.

[0021] In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:15-1:60. In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:20-1:50. In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:50. In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:35.

[0022] In the present invention, the multimeric antibody A and the monomeric antibody B bind to different epitopes of the target molecule to be detected, respectively. In the present invention, the specific type of the target molecule to be detected is not particularly limited, for example, the target molecule to be detected may be an influenza A virus antigen.

[0023] In some embodiments, the specific binding partner of the first marker is avidin. In some embodiments, the avidin is selected from at least one of egg white avidin, egg yolk avidin, streptavidin, neutravidin and avidin-like molecules.

[0024] In a second aspect, the present invention provides a photochemiluminescence detection kit, wherein the kit comprises the photochemiluminescence detection reagent composition described in the first aspect of the present invention.

[0025] In some embodiments, in the first reagent, the concentration of the polyantibody A that binds to the first marker is 1 μg / mL-4 μg / mL, preferably 2 μg / mL-3 μg / mL.

[0026] In some embodiments, the kit further comprises a third reagent, wherein the third reagent comprises photosensitive microspheres, wherein the photosensitive microspheres are capable of generating singlet oxygen in an excited state, and wherein the surface of the photosensitive microspheres is coated with a specific binding substance of the first marker.

[0027] In some embodiments, the kit comprises:

[0028] A first reagent, comprising a polyantibody A bound to a first marker;

[0029] A second reagent, comprising a luminescent microsphere, the luminescent microsphere being capable of reacting with singlet oxygen to generate a detectable signal, the surface of the luminescent microsphere being coated with monomeric antibody B; and

[0030] A third reagent includes a photosensitive microsphere, which can generate singlet oxygen in an excited state, and the surface of the photosensitive microsphere is coated with a specific binding substance of the first marker;

[0031] The multimeric antibody A and the monomeric antibody B recognize different epitopes of the target molecule to be detected.

[0032] In some embodiments, in the third reagent, the concentration of the photosensitive microspheres is no higher than 30 μg / mL, preferably 18 μg / mL-30 μg / mL, and more preferably 20 μg / mL-30 μg / mL.

[0033] In the kit of the present invention, the concentration of the photosensitive microspheres is preferably higher than the concentration of the luminescent microspheres. In some embodiments, the ratio of the concentration of the photosensitive microspheres in the third reagent to the concentration of the luminescent microspheres in the second reagent is (1.2-2):1.

[0034] In a third aspect, the present invention provides a photochemiluminescence detection method, which comprises using the photochemiluminescence detection reagent composition described in the first aspect of the present invention or the kit described in the second aspect of the present invention to detect whether an analyte is present in a sample to be tested.

[0035] In some embodiments, the detection method comprises the following steps:

[0036] (1) mixing a sample to be tested with the first reagent and the second reagent to form a first mixture, and subjecting the first mixture to a first stage of incubation to form a double antibody sandwich complex in the presence of an analyte;

[0037] (2) adding the third reagent to the first mixture after the first stage incubation to form a second mixture, and subjecting the second mixture to a second stage incubation to allow the first marker to specifically bind to a specific binding substance of the first marker;

[0038] (3) Perform photoexcitation, detect and analyze the optical signal.

[0039] In some embodiments, in step (1), the volume V0 of the sample to be tested satisfies: 50 μL≤V0≤200 μL, preferably 75 μL≤V0≤125 μL.

[0040] In some embodiments, in step (1), the volume V1 of the first reagent satisfies: 5 μL≤V1≤50 μL, preferably 20 μL≤V1≤40 μL.

[0041] In some embodiments, in step (1), the volume V2 of the second reagent satisfies: 5 μL≤V2≤50 μL, preferably 20 μL≤V1≤40 μL.

[0042] In some embodiments, in step (2), the volume V3 of the third reagent satisfies: 50 μL≤V3≤200 μL, preferably 75 μL≤V2≤125 μL.

[0043] In some embodiments, the volume ratio of the first reagent to the third reagent is V1:V3=(0.1-0.5):1, preferably (0.2-0.4):1.

[0044] In some embodiments, the volume ratio of the second reagent to the third reagent is V2:V3=(0.1-0.5):1, preferably (0.2-0.4):1.

[0045] In some embodiments, the volume ratio of the sample to be tested to the third reagent is V0:V3=(0.5-2):1, preferably (0.8-1.2):1.

[0046] In some embodiments, the volume ratio of the sample to be tested, the first reagent, the second reagent and the third reagent is (0.5-2):(0.1-0.5):(0.1-0.5):1.

[0047] In some embodiments, the volume ratio of the test sample, the first reagent, the second reagent and the third reagent is (0.8-1.2):(0.2-0.4):(0.2-0.4):1.

[0048] In some embodiments, in the second mixture of step (2), the concentration of the photosensitive microspheres is no higher than 11.5 μg / mL, preferably 6.9 μg / mL-11.5 μg / mL, and more preferably 7.7 μg / mL-11.5 μg / mL.

[0049] In some embodiments, in the second mixture of step (2), the concentration of the polyantibody A bound to the first marker is 0.11 μg / mL-0.46 μg / mL, preferably 0.23 μg / mL-0.35 μg / mL.

[0050] In some embodiments, in the second mixture of step (2), the concentration ratio of the photosensitive microspheres to the luminescent microspheres is (40-67):1.

[0051] In a fourth aspect, the present invention provides a photochemiluminescence detection device, which uses the composition of the first aspect or the kit of the second aspect or the method of the third aspect to detect whether an analyte is present in a sample to be tested.

[0052] In some embodiments, the device is a POCT point-of-care testing device.

[0053] In some embodiments, the detection device comprises:

[0054] A reagent card, wherein a plurality of wells are provided, and reagents are pre-installed in some of the wells;

[0055] A pipetting mechanism, used to move the reagents contained in the wells between the wells;

[0056] The detection mechanism is provided with a plurality of placement positions for placing the reagent cards.

[0057] In the fifth aspect, the present invention further provides the use of the composition described in the first aspect, the kit described in the second aspect, the detection method described in the third aspect, or the detection device described in the fourth aspect in photoinduced chemiluminescence detection.

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention uses biotin or biotin analogs to label multimeric antibodies, and by reducing the concentration of photosensitive balls, while keeping the number of antibodies unchanged, it can not only ensure the reaction efficiency and not reduce the positive signal value, but also effectively reduce the background and improve the detection accuracy and sensitivity. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] I. Terminology

[0062] The "photoluminescence detection" described in the present invention is a homogeneous system chemiluminescence detection technology. Taking the double antibody sandwich detection of antigen as an example, one antibody involved in the immune reaction is coated on the surface of the photosensitive microsphere (GG), containing phthalocyanine substances; the other antibody is coated on the surface of the luminescent microsphere (FG), containing dimethylthiophene derivatives and Eu chelates. With the help of antigen-antibody binding, the distance between the photosensitive microsphere and the luminescent microsphere is shortened to within 200nm. When irradiated with 680nm excitation light, the photosensitive microsphere is activated and releases high-energy singlet oxygen to the luminescent microsphere. After 4μs, the luminescent microsphere will emit 610nm light, and the concentration of the object to be tested is estimated by the intensity of the light signal.

[0063] As used herein, "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.

[0064] The "specific binding" mentioned in the present invention refers to the mutual recognition and selective binding reaction between two substances, which is the correspondence of the conformations between the corresponding reactants from the perspective of the three-dimensional structure. Under the technical concept disclosed in the present invention, the detection method of the specific binding reaction includes but is not limited to: double antibody sandwich method, competition method, neutralization competition method, indirect method or capture method.

[0065] The "marker" and "specific binding substance of the marker" described in the present invention refer 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 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, imidazolone ring and thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, etc., 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 is composed of 4 identical peptide chains, each of which can bind to one biotin. Therefore, each antigen or antibody can be coupled with multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the sensitivity of analysis.

[0066] The "antibody" described in the present invention is a type of immunoglobulin that can specifically bind to an antigen. Generally, antibodies exist as one or more Y-shaped monomers, each of which is composed of four polypeptide chains, including two identical heavy chains and two identical light chains. The light chain and the heavy chain are named according to their molecular weight. The top of the Y-shaped structure is the variable region, which is the antigen binding site. Each heavy chain has two regions, namely the constant region and the variable region. All antibodies of the same type have the same constant region, but there are differences between antibodies of different types. Each light chain also has two domains connected front and back, namely a constant region and a variable region.

[0067] The "antigen" of the present invention refers to a substance that can stimulate the body to produce an immune response, and can bind to the immune response product antibodies and sensitized lymphocytes in vivo and in vitro to produce an immune effect. The antigen can be a fusion antigen, and in any case, the antigen can be further conjugated with other parts, such as specific binding pair members, such as biotin or avidin (a member of the biotin-5 avidin specific binding pair member). When there is influenza A virus antigen in the sample to be tested, the influenza A virus antigen is specifically bound to the luminescent microspheres coated with anti-influenza A virus antibodies and the anti-influenza A virus antibodies labeled with biotin at the same time, and forms a double antibody sandwich complex on the surface of the luminescent microspheres; at this time, if avidin-modified photosensitive microspheres are added, biotin and avidin are combined to make the two microspheres close to each other, and under the excitation of the excitation light source, the photosensitive microspheres release active oxygen, and produce chemiluminescence after encountering the luminescent microspheres in the solution, thereby further stimulating the fluorescent group on the same microsphere to produce a cascade amplification reaction to produce fluorescence. At this time, the more influenza A virus antigen markers there are, the stronger the fluorescence intensity.

[0068] The "donor microspheres" or "photosensitive microspheres" or "photosensitive balls" of the present invention can be polymer particles filled with photosensitizers formed by coating functional groups on a substrate, which can generate active oxygen (such as singlet oxygen) under light excitation. The surface of the donor microspheres has hydrophilic aldehyde dextran, and the interior is filled with photosensitizers. The photosensitizer 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, and phthalocyanine, and derivatives of these compounds with 1-50 atomic substituents, and 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. The surface of the donor microspheres can also be filled with other sensitizers, and its non-limiting examples 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 and 9,10-diphenylanthracene-9,10-endoperoxide, which release singlet oxygen when heated or directly absorb light.

[0069] The "acceptor microspheres" or "luminescent microspheres" or "luminescent balls" described in the present invention can be polymer particles filled with luminescent compounds formed by coating functional groups on a substrate. The surface of the luminescent microsphere receptor microspheres has hydrophilic carboxydextran, and the interior is filled with a chemiluminescent agent that can react with active oxygen (such as singlet oxygen). In some specific embodiments of the present invention, the chemiluminescent agent undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate, and the metastable intermediate can decompose and emit light simultaneously or subsequently. In some preferred embodiments of the present invention, the luminescent compound is a europium complex; further preferably, the europium complex is MTTA-EU 3+ .

[0070] The "biotin" mentioned in the present invention is also called vitamin H, coenzyme R, a water-soluble vitamin, and also belongs to the vitamin B family, B7. It is a necessary substance for the synthesis of vitamin C and an indispensable substance for the normal metabolism of fat and protein. It is a nutrient necessary to maintain the natural growth, development and normal health of human body functions. The term "activated biotin" refers to the use of biotin carboxyl group to chemically modify the derivatives of various active groups. For example, Bio-NSH (NHS-Biotin, C 14 H 18 Bio-NSH (NHS) is an N-hydroxysuccinimide biotin that allows simple and efficient biotinylation of antibodies, proteins and any other biomolecules containing primary amines in solution. Bio-NSH offers labeling and detection possibilities where steric hindrance of biotin binding is an important factor. Being uncharged and containing a simple alkyl chain spacer arm, Bio-NSH is membrane permeable and can be used for intracellular labeling. Bio-NSH is the most commonly used biotinylation reagent. NHS-activated biotin can efficiently react with primary amine groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins or antibodies typically contain several primary amines that can serve as targets for labeling, including the side chains of lysine residues and the N-terminus of each polypeptide.

[0071] The "monoclonal antibody" mentioned in the present invention refers to an immunoglobulin secreted by a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

[0072] The "sample to be tested" or "sample to be tested" mentioned in the present invention refers to a mixture that may contain an analyte, and the analyte includes but is not limited to proteins, hormones, antibodies or antigens. Typical samples to be tested that can be used in the method disclosed in the present invention include body fluids, such as whole blood, serum, plasma, saliva and urine. The sample to be tested can be diluted with a diluent as needed before use. For example, in order to avoid the HOOK effect, the sample to be tested can be diluted with a diluent before being tested on the detection instrument.

[0073] II. Specific implementation plan

[0074] In photoluminescence analysis, there are usually two ways to reduce the background: reducing the concentration of the luminescent ball or reducing the concentration of the photosensitive ball. However, only reducing the concentration of the luminescent ball will reduce the background, but the reaction efficiency will also decrease, and the positive signal value will decrease; the reduction of photosensitive balls will cause HOOK caused by excessive biotin, resulting in a decrease in the positive signal value.

[0075] The present invention reduces the concentration of photosensitive balls and uses biotin or biotin analogs to label the polyantibody, optimizes the labeling process and the use concentration of the biotinylated polyantibody, thereby ensuring the positive signal and reducing the background while keeping the number of antibodies unchanged.

[0076] The photoinduced chemiluminescence detection reagent composition described in the first aspect of the present invention comprises:

[0077] A first reagent, comprising a polyantibody A bound to a first marker; and

[0078] A second reagent, comprising a luminescent microsphere, wherein the luminescent microsphere can react with singlet oxygen to generate a detectable signal, and the surface of the luminescent microsphere is coated with a monomer antibody B;

[0079] The multimeric antibody A and the monomeric antibody B recognize different epitopes of the target molecule to be detected.

[0080] In some embodiments, the multimeric antibody A comprises N monomer antibodies a connected by a cross-linking agent, N ≥ 2. In some preferred embodiments, 2 ≤ N ≤ 7, for example, N is 2, 3, 4, 5, 6 or 7. In some more preferred embodiments, 4 ≤ N ≤ 6, for example, N is 4, 5 or 6.

[0081] In some embodiments, the monomer antibody a is a monoclonal antibody that recognizes a specific epitope of the target molecule to be detected.

[0082] In some embodiments, the cross-linking agent has a bifunctional group that can react with the carboxyl or amino group of the monomer antibody a. In some specific embodiments, the cross-linking agent is a dialdehyde compound, such as at least one of glutaraldehyde, glyoxal, malondialdehyde, succinaldehyde, etc. In some specific embodiments, the cross-linking agent is a dihydrazide compound, such as at least one of maleic acid dihydrazide, oxalic acid dihydrazide, adipic acid dihydrazide, etc.

[0083] In some embodiments, the first marker has a spacer arm. In some specific embodiments, the length of the spacer arm of the first marker is For example In some embodiments, the length of the spacer arm of the first marker is In some embodiments, the spacer arm of the first label is polymerized from "-LC-". In some embodiments, the spacer arm of the first label is polymerized from "-PEG-".

[0084] In some embodiments, the first label is biotin. In some embodiments, the first label is a biotin analog. The biotin or biotin analogs of the present invention include but are not limited to Biotin-PEG1-COOH, Biotin-PEG2-COOH, Biotin-PEG3-COOH, Biotin-PEG4-COOH, Biotin-PEG5-COOH, Biotin-PEG6-COOH, Biotin-PEG7-COOH, Biotin-PEG8-COOH, Biotin-PEG9-COOH, Biotin-PEG10-COOH, Biotin-PEG11-COOH, Biotin-PEG12-COOH, Biotin-PEG1-NHS ester, Biotin-PEG2-NHS ester, Biotin-PEG3-NHS ester, Biotin-PEG4-NHS ester, Biotin-PEG5-NHSester, Biotin-PEG6-NHS ester, Biotin-PEG7-NHS ester, Biotin-PEG8-NHS ester, Biotin-PEG9-NHS ester, Biotin-PEG10-NHS ester, Biotin-PEG11-NHS ester, Biotin-PEG12-NHS ester, Biotin-PEG32-NHS ester, Biotin-PEG1-PFP, Biotin-PEG2-PFP, Biotin-PEG3-PFP, Biotin-PEG4-PFP, Biotin-PEG5-PFP, Biotin-PEG6-PFP, Biotin-PEG7-PFP, Biotin-PEG8-PFP, Biotin -PEG9-PFP, Biotin-PEG10-PFP, Biotin-PEG11-PFP, Biotin-PEG12-PFP, Biotin-LC, Biotin-LC-NHS, Biotin-LC-LC-NHS, Biotin-LC-Sulfo-NHS, Biotin-LC-PEG1-NHS ester, Biotin-LC-PEG2-NHS ester, Biotin-LC-PEG3-NHS ester, Biotin-LC-PEG4-NHS ester, Biotin-LC-PEG5-NHS ester, Biotin-LC-PEG6-NHS ester, Biotin-LC-PEG7-NHSester, Biotin-LC-PEG8-NHS ester, Biotin-LC-PEG9-NHS ester, Biotin-LC-PEG10-NHS ester, Biotin-LC-PEG11-NHS ester, Biotin-LC-PEG12-NHSester, Biotin-NHS ester, Biotin-Sulfo-NHS ester, Biotin-PFP, Biotin-SS-COOH or more.

[0085] In some embodiments, the first marker is selected from Biotin-LC-LC-NHS, Biotin-PEG3-NHS, Biotin-PEG4-NHS, Biotin-PEG5-NHS, Biotin-PEG6-NHS, Biotin-PEG7-NHS, Biotin-PEG8-NHS, Biotin-PEG9-NHS, Biotin-PEG 10 -NHS, Biotin-PEG 11 - At least one of the NHS.

[0086] In some preferred embodiments, the first marker is Biotin-LC-LC-NHS.

[0087] In some preferred embodiments, the first marker is Biotin-PEG4-NHS.

[0088] In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:15-1:60, such as 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60 or any value therebetween. In some preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:20-1:50. In some preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:50. In some more preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:35.

[0089] The present invention does not impose any particular limitation on the specific type of the target molecule to be detected. For example, the target molecule to be detected may be an influenza A virus antigen.

[0090] In some embodiments, the specific binding partner of the first marker is avidin, such as at least one of ovalbumin, vitellogenin, streptavidin, neutravidin and avidin-like molecules.

[0091] In some embodiments, the material used for the luminescent microspheres or photosensitive microspheres is a polymer matrix, for example, it can be one or a combination of natural polymers, modified natural polymers or synthetic polymers, these natural polymers, modified natural polymers and synthetic polymers include but are not limited to agarose, cellulose, polypropylene, polystyrene, polymethyl methacrylate. In some embodiments, the polymer matrix has common functional groups, such as carboxyl, amino, thiol, aldehyde, so that biologically active substances can be connected. In some embodiments, the polymer matrix is ​​a polystyrene microsphere with a carboxyl or aldehyde group or a polystyrene microsphere with a carboxyl dextran coated on the outer layer.

[0092] In some embodiments, the photosensitive microspheres are filled with a photosensitizer, and the photosensitizer is selected from one of methylene blue, rose bengal, porphyrin and phthalocyanine. There is no particular limitation on the amount of photosensitizer in the donor microspheres, and it can be an amount commonly used in the art.

[0093] In some embodiments, the luminescent microspheres are filled with luminescent compounds. In some preferred embodiments, the luminescent compound is a europium complex. The europium complex filled in the polystyrene microspheres interacts with the polystyrene microspheres to further improve their luminescence efficiency. In a further preferred embodiment, the europium complex is MTTA-EU 3+ This complex can directly capture the singlet oxygen produced by the phthalocyanine dye in the photosensitive microspheres and then emit red light with a characteristic wavelength of 614-615nm for europium ions.

[0094] The photochemiluminescence detection kit provided in the second aspect of the present invention comprises the photochemiluminescence detection reagent composition described in the first aspect of the present invention.

[0095] In some embodiments, in the first reagent of the kit, the concentration of the multimeric antibody A that binds to the first marker is 1 μg / mL-4 μg / mL, such as 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL or any value therebetween. In some preferred embodiments, in the first reagent of the kit, the concentration of the multimeric antibody that binds to the first marker is 2 μg / mL-3 μg / mL.

[0096] In some embodiments, the kit further comprises a third reagent, wherein the third reagent comprises photosensitive microspheres, wherein the photosensitive microspheres are capable of generating singlet oxygen in an excited state, and wherein the surface of the photosensitive microspheres is coated with a specific binding substance of the first marker.

[0097] In some embodiments, the kit comprises:

[0098] A first reagent, comprising a polyantibody A bound to a first marker;

[0099] A second reagent, comprising a luminescent microsphere, wherein the luminescent microsphere is capable of generating singlet oxygen in an excited state, and the surface of the luminescent microsphere is coated with a monomeric antibody B; and

[0100] A third reagent, comprising a photosensitive microsphere, wherein the photosensitive microsphere can react with singlet oxygen to generate a detectable signal, and the surface of the photosensitive microsphere is coated with a specific binding substance of the first marker;

[0101] The multimeric antibody A and the monomeric antibody B recognize different epitopes of the target molecule to be detected.

[0102] In some embodiments, the concentration of the photosensitive microspheres in the third reagent is not higher than 30 μg / mL. In some embodiments, the concentration of the photosensitive microspheres in the third reagent is 18 μg / mL-30 μg / mL, such as 18 μg / mL, 20 μg / mL, 22 μg / mL, 24 μg / mL, 26 μg / mL, 28 μg / mL, 30 μg / mL or any value therebetween. In some preferred embodiments, the concentration of the photosensitive microspheres in the third reagent is 20 μg / mL-30 μg / mL.

[0103] In the present invention, the concentration of the photosensitive microspheres is preferably higher than the concentration of the luminescent microspheres. In some embodiments, the ratio of the concentration of the photosensitive microspheres in the third reagent to the concentration of the luminescent microspheres in the second reagent is (1.2-2):1, such as 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1 or any value therebetween.

[0104] In some embodiments, the multimeric antibody A comprises N monomer antibodies a connected by a cross-linking agent, N ≥ 2. In some preferred embodiments, 2 ≤ N ≤ 7, for example, N is 2, 3, 4, 5, 6 or 7. In some more preferred embodiments, 4 ≤ N ≤ 6, for example, N is 4, 5 or 6.

[0105] In some embodiments, the monomer antibody a is a monoclonal antibody that recognizes a specific epitope of the target molecule to be detected.

[0106] In some embodiments, the cross-linking agent has a bifunctional group that can react with the carboxyl or amino group of the monomer antibody a. In some specific embodiments, the cross-linking agent is a dialdehyde compound, such as at least one of glutaraldehyde, glyoxal, malondialdehyde, succinaldehyde, etc. In some specific embodiments, the cross-linking agent is a dihydrazide compound, such as at least one of maleic acid dihydrazide, oxalic acid dihydrazide, adipic acid dihydrazide, etc.

[0107] In some embodiments, the first marker has a spacer arm. In some specific embodiments, the length of the spacer arm of the first marker is For example In some embodiments, the length of the spacer arm of the first marker is In some embodiments, the spacer arm of the first label is polymerized from "-LC-". In some embodiments, the spacer arm of the first label is polymerized from "-PEG-".

[0108] In some embodiments, the first label is biotin. In some embodiments, the first label is a biotin analog. The biotin or biotin analogs of the present invention include but are not limited to Biotin-PEG1-COOH, Biotin-PEG2-COOH, Biotin-PEG3-COOH, Biotin-PEG4-COOH, Biotin-PEG5-COOH, Biotin-PEG6-COOH, Biotin-PEG7-COOH, Biotin-PEG8-COOH, Biotin-PEG9-COOH, Biotin-PEG10-COOH, Biotin-PEG11-COOH, Biotin-PEG12-COOH, Biotin-PEG1-NHS ester, Biotin-PEG2-NHS ester, Biotin-PEG3-NHS ester, Biotin-PEG4-NHS ester, Biotin-PEG5-NHSester, Biotin-PEG6-NHS ester, Biotin-PEG7-NHS ester, Biotin-PEG8-NHS ester, Biotin-PEG9-NHS ester, Biotin-PEG10-NHS ester, Biotin-PEG11-NHS ester, Biotin-PEG12-NHS ester, Biotin-PEG32-NHS ester, Biotin-PEG1-PFP, Biotin-PEG2-PFP, Biotin-PEG3-PFP, Biotin-PEG4-PFP, Biotin-PEG5-PFP, Biotin-PEG6-PFP, Biotin-PEG7-PFP, Biotin-PEG8-PFP, Biotin -PEG9-PFP, Biotin-PEG10-PFP, Biotin-PEG11-PFP, Biotin-PEG12-PFP, Biotin-LC, Biotin-LC-NHS, Biotin-LC-LC-NHS, Biotin-LC-Sulfo-NHS, Biotin-LC-PEG1-NHS ester, Biotin-LC-PEG2-NHS ester, Biotin-LC-PEG3-NHS ester, Biotin-LC-PEG4-NHS ester, Biotin-LC-PEG5-NHS ester, Biotin-LC-PEG6-NHS ester, Biotin-LC-PEG7-NHSester, Biotin-LC-PEG8-NHS ester, Biotin-LC-PEG9-NHS ester, Biotin-LC-PEG10-NHS ester, Biotin-LC-PEG11-NHS ester, Biotin-LC-PEG12-NHSester, Biotin-NHS ester, Biotin-Sulfo-NHS ester, Biotin-PFP, Biotin-SS-COOH or more.

[0109] In some embodiments, the first marker is selected from Biotin-LC-LC-NHS, Biotin-PEG3-NHS, Biotin-PEG4-NHS, Biotin-PEG5-NHS, Biotin-PEG6-NHS, Biotin-PEG7-NHS, Biotin-PEG8-NHS, Biotin-PEG9-NHS, Biotin-PEG 10 -NHS, Biotin-PEG 11 - At least one of the NHS.

[0110] In some preferred embodiments, the first marker is Biotin-LC-LC-NHS.

[0111] In some preferred embodiments, the first marker is Biotin-PEG4-NHS.

[0112] In some embodiments, the molar ratio of the polyantibody A to the first marker is 1:15-1:60, such as 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60 or any value therebetween. In some preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:20-1:50. In some preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:50. In some more preferred embodiments, the molar ratio of the polyantibody A to the first marker is 1:25-1:35.

[0113] The present invention does not impose any particular limitation on the specific type of the target molecule to be detected. For example, the target molecule to be detected may be an influenza A virus antigen.

[0114] In some embodiments, the specific binding partner of the first marker is avidin, such as at least one of ovalbumin, vitellogenin, streptavidin, neutravidin and avidin-like molecules.

[0115] In some embodiments, the multimeric antibody A that binds to the first marker is prepared by the following method:

[0116] The monomer antibody a is mixed with a cross-linking agent for reaction, and an amino compound is added to obtain a mixture of polymers containing different coupling numbers;

[0117] Purifying and separating the target polymer in the mixture;

[0118] The target polymer is labeled with a first marker to obtain a polymer antibody A bound to the first marker.

[0119] The multimer A of the present invention is formed by cross-linking at least two monomer antibodies a via the cross-linking agent.

[0120] The amino compound mentioned above functions to cross-link and seal the residual aldehyde groups. Examples of the amino compound include, but are not limited to, amino acids such as lysine, glycine, glutamic acid, arginine, taurine, and cysteine.

[0121] In some embodiments, the material used for the luminescent microspheres or photosensitive microspheres is a polymer matrix, for example, it can be one or a combination of natural polymers, modified natural polymers or synthetic polymers, these natural polymers, modified natural polymers and synthetic polymers include but are not limited to agarose, cellulose, polypropylene, polystyrene, polymethyl methacrylate. In some embodiments, the polymer matrix has common functional groups, such as carboxyl, amino, thiol, aldehyde, so that biologically active substances can be connected. In some embodiments, the polymer matrix is ​​a polystyrene microsphere with a carboxyl or aldehyde group or a polystyrene microsphere with a carboxyl dextran coated on the outer layer.

[0122] In some embodiments, the photosensitive microspheres are filled with a photosensitizer, and the photosensitizer is selected from one of methylene blue, rose bengal, porphyrin and phthalocyanine. There is no particular limitation on the amount of photosensitizer in the donor microspheres, and it can be an amount commonly used in the art.

[0123] In some embodiments, the luminescent microspheres are filled with luminescent compounds. In some preferred embodiments, the luminescent compound is a europium complex. The europium complex filled in the polystyrene microspheres interacts with the polystyrene microspheres to further improve their luminescence efficiency. In a further preferred embodiment, the europium complex is MTTA-EU 3+ This complex can directly capture the singlet oxygen produced by the phthalocyanine dye in the photosensitive microspheres and then emit red light with a characteristic wavelength of 614-615nm for europium ions.

[0124] In some embodiments, the kit further includes a diluent, which is used to dilute the sample to be tested. The diluent includes a buffer, a protein, a stabilizer, a preservative, etc. The diluent has the functions of dilution and buffering, and can improve the accuracy of the final test result and the stability of the sample to be tested.

[0125] In some embodiments, the photoluminescence detection method of the third aspect of the present invention comprises the following steps:

[0126] (1) mixing a sample to be tested having a volume of V0, the first reagent having a volume of V1, and the second reagent having a volume of V2 to form a first mixture, and subjecting the first mixture to a first stage of incubation to form a double antibody sandwich complex in the presence of an analyte;

[0127] (2) adding V3 volume of the third reagent to the first mixture after the first stage incubation to form a second mixture, and subjecting the second mixture to a second stage incubation to allow the first marker to specifically bind to a specific binding substance of the first marker;

[0128] (3) irradiating the second mixture after incubation in step (2) with a laser to excite the photosensitive microspheres to produce singlet oxygen;

[0129] (4) Detecting the intensity of the chemiluminescent signal generated by the reaction of the luminescent microspheres in the second mixture with singlet oxygen in step (3), and analyzing and determining whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested based on the intensity of the chemiluminescent signal.

[0130] In some embodiments, 50 μL≤V0≤200 μL, for example, V0 can be 50 μL, 75 μL, 100 μL, 125 μL, 150 μL, 175 μL, 200 μL, or any value therebetween. In some embodiments, 75 μL≤V0≤125 μL. In some embodiments, 90 μL≤V0≤110 μL.

[0131] In some embodiments, 5 μL≤V1≤50 μL, for example, V1 can be 5 μL, 8 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, or any value therebetween. In some embodiments, 20 μL≤V1≤40 μL. In some embodiments, 25 μL≤V1≤35 μL.

[0132] In some embodiments, 5 μL≤V2≤50 μL, for example, V2 can be 5 μL, 8 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, or any value therebetween. In some embodiments, 20 μL≤V2≤40 μL. In some embodiments, 25 μL≤V2≤35 μL.

[0133] In some embodiments, 50 μL≤V3≤200 μL, for example, V3 can be 50 μL, 75 μL, 100 μL, 125 μL, 150 μL, 175 μL, 200 μL, or any value therebetween. In some embodiments, 75 μL≤V3≤125 μL. In some embodiments, 90 μL≤V3≤110 μL.

[0134] In some embodiments, the volume ratio of the first reagent to the third reagent is V1:V3=(0.1-0.5):1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1 or any value therebetween. In some preferred embodiments, V1:V3=(0.2-0.4):1. In some more preferred embodiments, V1:V3=0.3:1.

[0135] In some embodiments, the volume ratio of the second reagent to the third reagent is V2:V3=(0.1-0.5):1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1 or any value therebetween. In some preferred embodiments, V2:V3=(0.2-0.4):1. In some more preferred embodiments, V2:V3=0.3:1.

[0136] In some embodiments, the volume ratio of the sample to be tested to the third reagent is V0:V3=(0.5-2):1, for example, 0.5:1, 0.8:1, 1.2:1, 1.5:1, 1.8:1, 2:1 or any value therebetween. In some embodiments, V0:V3=(0.8-1.2):1.

[0137] In some embodiments, the volume ratio of the sample to be tested, the first reagent, the second reagent and the third reagent is (0.5-2):(0.1-0.5):(0.1-0.5):1.

[0138] In some preferred embodiments, the volume ratio of the sample to be tested, the first reagent, the second reagent and the third reagent is (0.8-1.2):(0.2-0.4):(0.2-0.4):1.

[0139] In some more preferred embodiments, the volume ratio of the sample to be tested, the first reagent, the second reagent and the third reagent is 1:(0.2-0.4):(0.2-0.4):1.

[0140] In some embodiments, in the second mixture of step (2), the concentration of the photosensitive microspheres is not higher than 11.5 μg / mL. In some preferred embodiments, in the second mixture of step (2), the concentration of the photosensitive microspheres is 6.9 μg / mL-11.5 μg / mL, for example, it can be 6.9 μg / mL, 7.7 μg / mL, 8 μg / mL, 8.5 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 10.5 μg / mL, 11 μg / mL, 11.5 μg / mL or any value therebetween. In some more preferred embodiments, in the second mixture of step (2), the concentration of the photosensitive microspheres is 7.7 μg / mL-11.5 μg / mL.

[0141] In some embodiments, in step (2) the second mixture, the concentration of the polyantibody A bound to the first marker is 0.11 μg / mL-0.46 μg / mL, such as 0.11 μg / mL, 0.15 μg / mL, 0.2 μg / mL, 0.23 μg / mL, 0.28 μg / mL, 0.3 μg / mL, 0.35 μg / mL, 0.4 μg / mL, 0.45 μg / mL or any value therebetween. In some preferred embodiments, in step (2) the second mixture, the concentration of the polyantibody A bound to the first marker is 0.23 μg / mL-0.35 μg / mL.

[0142] In some embodiments, in the second mixture of step (2), the concentration ratio of the photosensitive microspheres to the luminescent microspheres is (40-67):1, for example, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 67:1 or any value therebetween.

[0143] In some preferred embodiments, in step (1), the sample to be tested is first diluted with a diluent and then mixed with the second reagent and the first reagent. The diluent includes a buffer, a protein, a stabilizer, a preservative, etc. The diluent has the functions of dilution and buffering, which can improve the accuracy of the final test result and the stability of the sample to be tested.

[0144] In other embodiments of the present invention, in step (3), laser irradiation is performed using red excitation at 600-700 nm.

[0145] The photochemiluminescence detection device described in the fourth aspect of the present invention detects whether the analyte is present in the sample to be tested using the composition described in the first aspect of the present invention, the kit described in the second aspect, or the detection method described in the third aspect.

[0146] In some embodiments, the device comprises:

[0147] a. A reagent card, wherein a plurality of holes are provided, wherein the holes include at least:

[0148] The sample well to be tested is used to hold the sample to be tested containing the target molecule to be tested;

[0149] Reagent wells for containing a first reagent comprising a multimeric antibody bound to a first marker; and / or a third reagent comprising photosensitive microspheres capable of generating reactive oxygen species under an excited state; and / or a second reagent comprising luminescent microspheres capable of reacting with reactive oxygen species to generate chemiluminescent signals;

[0150] The detection wells are used for reaction and signal detection; wherein each reagent may be in the same reagent well or in different reagent wells, or may be in the detection well.

[0151] b. A pipetting mechanism, which is used to move the reagents contained in the wells between the wells.

[0152] c. A detection mechanism, which is provided with a plurality of placement positions for placing the reagent card, and is used to detect the chemiluminescent signal generated by the reaction of the luminescent microspheres with active oxygen.

[0153] In other embodiments of the present invention, the reagent card covering seals the opening to ensure that the substance therein is not contaminated and remains in a constant state. The covering can be a disposable sealing film or a reusable sealing film.

[0154] In order to facilitate identification and reading of the information of the sample to be tested, a preferred technical solution is that a barcode area is provided on the side of the reagent card, and the barcode area contains the information of the reagent card. The barcode can be a one-dimensional or two-dimensional code.

[0155] Correspondingly, the device further includes a barcode scanning module, and the barcode scanning module is used to identify and read the information in the barcode.

[0156] The barcode scanning module supports IC card scanning and printed barcode medium (paper or reagent card) scanning. Information is read in by contact scanning or non-contact scanning, which can be infrared or radio frequency. The information includes but is not limited to the name of the detection and analysis project, standard curve, reagent composition, batch number, expiration date, and manufacturer information.

[0157] In order to improve the accuracy of the final test result and the stability of the sample to be tested, in some embodiments, the reagent card is further provided with a diluent hole, and the diluent hole is used to hold the diluent.

[0158] In some preferred embodiments of the present invention, the detection device is further provided with a sample adding mechanism. In some preferred embodiments, the sample adding mechanism comprises:

[0159] Pipetting components, used to aspirate or discharge liquids;

[0160] A vertical moving component, the pipetting component is arranged on the vertical moving component, and the vertical moving component is used to drive the pipetting component to move vertically;

[0161] A horizontal moving component, the vertical moving component is arranged on the horizontal moving component, and the horizontal moving component is used to drive the pipetting component to move horizontally.

[0162] In some embodiments, the detection mechanism is electrically connected to the sample adding mechanism.

[0163] In some preferred embodiments, the detection mechanism comprises:

[0164] A base, used for carrying the reagent card;

[0165] A driving assembly, used for driving the base to rotate around its center and driving the reagent card to rotate;

[0166] The detection component is used to detect the chemiluminescent signal generated by the reaction between the luminescent microspheres in the reagent card and the active oxygen.

[0167] In some specific embodiments, the detection component includes an exciter, and the exciter can emit excitation light of 600-700 nm.

[0168] In some embodiments, the detection wavelength of the chemiluminescent signal generated by the reaction of the luminescent microspheres with active oxygen is 450-650 nm.

[0169] In some embodiments, the device further comprises an incubation module, which is used to provide a suitable ambient temperature for the chemiluminescent reaction. During detection, the incubation module uses a metal bath, an air bath, a water bath or an oil bath to keep the temperature of the reagent card and the substance in the reagent card at 20-50°C.

[0170] II. Examples

[0171] The main reagents used in the following examples are:

[0172] Recombinant FluANP protein (purchased from Bio-Bio), FluA monoclonal antibodies (antibody 1, antibody 2, two different antibodies) (purchased from Feipeng), luminescent balls and photosensitive balls (purchased from PerkinElmer), activated biotin (Biotin-LC-NHS, Biotin-LC-LC-NHS, Biotin-PEG4-NHS, Biotin-PEG 12 -NHS, Biotin-PEG 24-NHS) (purchased from Thermo Fisher)

[0173] The antigen used in the following examples is influenza A antigen, and the reaction system used for detection is: 100 μL sample, 30 μL luminescent microsphere labeled antibody, 30 μL biotin labeled polymer antibody, 100 μL photosensitive microsphere. For 1 pg / mL influenza A antigen, excess unbound microspheres will increase the background and affect the detection of low value samples.

[0174] Preparation of reagents

[0175] A. Preparation of Polymeric Antibody A

[0176] 1) Take 4 mg of antibody 2, add pH 7.0 PBS buffer to 1.8 mL, then add 0.1 mL 1 M pH 9.6 CB buffer and 0.1 mL 25% glutaraldehyde, incubate at room temperature overnight, and rotate to mix;

[0177] 2) Add 0.1 mL of 0.2 M lysine (prepared with distilled water) and let stand at 4 °C for 2 h to block the residual aldehyde groups and terminate the reaction;

[0178] 3) Cut a 14KD dialysis bag of a certain length, put it in a boiling water bath for 10 minutes, remove impurities, wash it with ultrapure water, and set it aside;

[0179] 4) Place the blocked coupling complex in a dialysis bag and dialyze twice with 0.01M pH 7.2 PBS at 4°C, each time for 2 h;

[0180] 5) The dialyzed products were collected, purified and tested to obtain polymers with different coupling numbers, and a 3-mer antibody (antibody 3), a 5-mer antibody (antibody 4), and a 7-mer antibody (antibody 5) were selected for use.

[0181] B. Preparation of Biotinylated Polyantibody A

[0182] 1) Take 1 mg of the multi-antibody and add it to a dialysis bag, add NaHCO3 buffer (0.1 M) to an antibody concentration of 1 mg / mL, put it into NaHCO3 buffer (0.1 M) and dialyze it twice at room temperature, each time for 2 hours, take out the dialysis bag, take out the raw material, and measure the antibody concentration by BCA method;

[0183] 2) Add 20 mM biotin to the antibody at a molar ratio of polyantibody: biotin = 1:30 (add biotin at different ratios when screening different labeling ratios), mix well, seal with sealing film, and place at 4°C for 2 hours with rotation mixing;

[0184] 3) Place the biotin-coupled polyantibody in a dialysis bag, clamp it, and dialyze it in PBS buffer (0.02M) at 4°C overnight. Take it out the next day and dialyze it twice at room temperature, changing the solution every 2 hours.

[0185] 4) After dialysis is completed, the labeled antibody is aspirated and the concentration of the biotinylated polyantibody is measured by the BCA method;

[0186] 5) Using the prepared biotin-labeled polyantibody concentrated solution, the concentration is prepared according to the required concentration using Tris-HCl buffer containing BSA at pH 8.0 to obtain a biotin-polyantibody working solution (Bio-polyantibody).

[0187] C. Preparation of photosensitive microsphere reagent: According to the experimental requirements, HEPES buffer (0.05M pH 8.0) containing BSA is prepared into the corresponding concentration to obtain photosensitive microsphere-avidin solution (GG-avidin) for use.

[0188] D. Preparation of luminescent ball labeled antibody reagent:

[0189] 1) Take 1 mg of antibody 1 and add it to a dialysis bag, then add a certain amount of CB buffer (0.05 M) to make the antibody concentration 2 mg / mL, put it into CB buffer (0.05 M) and dialyze it twice at room temperature, each time for 2 hours, take out the dialysis bag, take out the raw materials, and measure the antibody concentration by BCA method;

[0190] 2) Take 10 mg of luminescent spheres, add 1 mL of CB buffer (0.05 M) to the luminescent spheres, mix well, centrifuge at 10,000 rpm for 25 min, discard the supernatant after centrifugation, add 0.2 mL of CB buffer (0.05 M), mix well by ultrasonication and set aside;

[0191] 3) Mix the luminescent ball and the dialyzed antibody 1 at a mass ratio of luminescent ball: antibody = 10:0.5, to a final concentration of luminescent ball of 20-25 mg / mL, and rotate to mix for 14-16 hours;

[0192] 4) Add 20 μL of freshly prepared 8 mg / mL sodium borohydride (CB preparation) to the mixture and rotate and mix at room temperature for 2 hours;

[0193] 5) Add 160 μL of 75 mg / mL glycine (prepared in pure water) to the mixture and rotate and mix at room temperature for 1 hour;

[0194] 6) The mixture was washed by centrifugation with HEPES buffer (0.05 M pH 8.0) containing BSA and prepared into a 10 mg / mL luminescent ball-antibody (FG-antibody 1) solution for later use.

[0195] Example 1: Photoluminescence Detection Kit

[0196] The following first reagent, second reagent and third reagent were prepared by the method for preparing the above reagents, and assembled to obtain the photochemiluminescence detection kit of Example 1, wherein:

[0197] The first reagent includes a biotinylated trimeric FluA monoclonal antibody (Bio-Antibody 3);

[0198] The second reagent includes luminescent microspheres coated with FluA monoclonal antibody (FG-antibody 1);

[0199] The third reagent comprises avidin-coated photosensitive microspheres (GG-avidin).

[0200] Example 2: Photoluminescence Detection Kit

[0201] The following first reagent, second reagent and third reagent were prepared by the method for preparing the above reagents, and assembled to obtain the photochemiluminescence detection kit of Example 2, wherein:

[0202] The first reagent includes a biotinylated pentameric FluA monoclonal antibody (Bio-Antibody 4);

[0203] The second reagent includes luminescent microspheres coated with FluA monoclonal antibody (FG-antibody 1);

[0204] The third reagent comprises avidin-coated photosensitive microspheres (GG-avidin).

[0205] Example 3: Photoluminescence Detection Kit

[0206] The following first reagent, second reagent and third reagent were prepared by the method for preparing the above reagents, and assembled to obtain the photochemiluminescence detection kit of Example 3, wherein:

[0207] The first reagent includes a biotinylated heptameric FluA monoclonal antibody (Bio-Antibody 5);

[0208] The second reagent includes luminescent microspheres coated with FluA monoclonal antibody (FG-antibody 1);

[0209] The third reagent comprises avidin-coated photosensitive microspheres (GG-avidin).

[0210] Example 4 Photoluminescence Detection Method

[0211] The kit in Example 1-3 was used for detection. For the HT instrument test, 100 μL of sample (recombinant Flu ANP protein) was taken, 30 μL of FG-antibody and 30 μL of Bio-polyantibody were added, and the first mixture was obtained after mixing. The first mixture was incubated at 37°C for 15 min, and 100 μL of GG-avidin was added to obtain the second mixture. The second mixture was incubated for 10 min and the reading was taken.

[0212] Experimental data 1:

[0213] In the kits of Examples 1-3, the type of biotin was Biotin-LC-LC-NHS, the labeling ratio (molar ratio) was polyantibody:biotin=1:30, the concentration of FG-antibody 1 was 15 μg / mL, the concentration of GG-avidin was 25 μg / mL, and the concentration of Bio-antibody was 2 μg / mL. For other specific conditions and results, see Table 1.

[0214] Table 1

[0215]

[0216] Experimental results analysis:

[0217] (1) When the concentration of photosensitive balls in the kit is 25 μg / mL, the overall signal value decreases. As the concentration of photosensitive balls decreases, the background will decrease (reduce random collisions) and the positive rate will also decrease. The reaction efficiency of photosensitive balls and luminescent balls will be reduced in the early stage. When the reaction efficiency decreases more later, the number of effective complexes formed is small and the signal value decreases. The preferred range should be slightly more than the number of luminescent balls (the part more than the luminescent balls can bind to excess biotin).

[0218] (2) When the concentration of the photosensitive ball in the kit was reduced to 25μg / mL, the background of the biotin-labeled 5-mer antibody system did not change much, while the positive rate increased slightly. The possible reasons are: ① When the biotin-labeled polyantibody is used at the same concentration, the amount of biotin coupled to it is reduced, avoiding HOOK caused by excessive biotin, while also ensuring the reaction efficiency (polyantibody, the amount of antibody is not reduced). ② If the proportion of polyantibody is too small, the effect is not obvious. If the proportion of polyantibody is too high, many antigen binding sites may be hidden inside and become ineffective, and the activity is poor. Therefore, 4-6 polyantibody is the best choice.

[0219] Experimental data 2:

[0220] The kit in Example 2 was used, and biotin with different arms and different arm lengths was selected, wherein the labeling ratio (molar ratio) was polyantibody:biotin=1:30, the concentration of FG-antibody 1 was 15 μg / mL, the concentration of GG-avidin was 25 μg / mL, and the concentration of Bio-antibody 4 was 2 μg / mL. For other specific conditions and results, see Table 2.

[0221] Table 2

[0222]

[0223] Experimental results analysis:

[0224] Biotin-LC-NHS(Arm Length )、Biotin-LC-LC-NHS(arm length ) and Biotin-PEG4-NHS (arm length ) labeled antibody has a low background signal value, but the positive signal value of the antibody labeled with Biotin-LC-NHS is low, which may be due to the short arm length and the presence of steric hindrance. 12 -NHS(Arm Length ) and Biotin-PEG 24 -NHS(Arm Length ) The background signal value of the labeled antibody is high and the positive signal value is low. Analysis shows that the arm length may be too long, which increases the distance between the luminescent ball and the photosensitive ball, reducing the reaction activity. Therefore, the arm length is selected The biotin detection effect is better.

[0225] Experimental data 3:

[0226] The kit in Example 2 was used, and different labeling ratios were selected. The type of biotin was Biotin-LC-LC-NHS, the concentration of FG-antibody 1 was 15 μg / mL, the concentration of GG-avidin was 25 μg / mL, and the concentration of Bio-antibody was 2 μg / mL. For other specific conditions and results, see Table 3.

[0227] Table 3

[0228]

[0229]

[0230] Experimental results analysis:

[0231] Reducing the amount of biotin labeling, the background is reduced, but the positive signal value is also reduced. Analysis shows that the reaction rate of biotin and avidin is reduced. Increasing the biotin labeling ratio, when the labeling ratio is 1:60, the positive signal value is reduced. Analysis shows that excessive biotin may cause HOOK. Therefore, 1:25-1:50 is selected as the optimal labeling ratio.

[0232] Experimental data 4:

[0233] The kit in Example 2 was used, and Bio-antibody 4 was set to different concentrations (3 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL), biotin type: Biotin-LC-LC-NHS, the labeling ratio (molar ratio) was polyantibody: biotin = 1:30, the concentration of FG-antibody 1 was 15 μg / mL, the concentration of GG-avidin was 25 μg / mL, and other specific conditions and results were shown in Table 4.

[0234] Table 4

[0235]

[0236] Experimental results analysis:

[0237] The titration experiment results show that as the concentration of biotin-labeled multimeric antibody increases, the signal value increases, and the low-end sensitivity also increases. When it reaches 3 μg / mL (equivalent to 0.35 μg / mL of biotin-labeled multimeric antibody in the second mixture), the background decreases and the positive signal value also decreases slightly. The reason for this may be that the biotin content is increased, resulting in the consumption of the photosensitive ball by biotin during detection, which reduces the collision between the luminous ball and the photosensitive ball. When the concentration of biotin-labeled multimeric antibody 4 is 2 μg / mL (equivalent to 0.23 μg / mL of biotin-labeled multimeric antibody in the second mixture), the low-end sensitivity is the highest and the signal value is higher. Therefore, the optimal concentration of biotin-labeled multimeric antibody is 2-3 μg / mL (equivalent to 0.23-0.35 μg / mL of biotin-labeled multimeric antibody in the second mixture).

[0238] Experimental data 5

[0239] The kit in Example 2 was used, and after the parameters of the biotin reagent were determined, the concentration of the photosensitive ball was reduced (the concentrations of GG-avidin were set to 50 μg / mL, 35 μg / mL, 25 μg / mL, and 15 μg / mL, respectively), the type of biotin was Biotin-LC-LC-NHS, the labeling ratio (molar ratio) was polyantibody: biotin = 1:30, the concentration of FG-antibody 1 was 15 μg / mL, and the concentration of Bio-antibody 4 was 2 μg / mL. For other specific conditions and results, see Table 5.

[0240] Table 5

[0241]

[0242] Experimental results analysis:

[0243] As the concentration of photosensitive spheres decreases, both the background and positive signal values ​​decrease. When the concentration of photosensitive spheres is 15μg / mL (equivalent to 5.8μg / mL of photosensitive spheres in the second mixture), the signal value decreases significantly, which is analyzed to be caused by insufficient photosensitive spheres. In summary, when the concentration of photosensitive spheres is in the range of 20-30μg / mL (equivalent to 7.7-11.5μg / mL of photosensitive spheres in the second mixture), labeling multimeric antibodies can reduce the background and does not affect the positive measurement value.

[0244] It can be seen that in the present invention, by reducing the concentration of photosensitive spheres and using biotin-labeled polyantibody reagents, the background can be reduced without affecting the positive signal value.

[0245] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A photochemiluminescence detection reagent composition, comprising: A first reagent, which includes a polyantibody A bound to a first marker; as well as A second reagent, comprising a luminescent microsphere, wherein the luminescent microsphere can react with singlet oxygen to generate a detectable signal, and the surface of the luminescent microsphere is coated with a monomer antibody B; The multimeric antibody A and the monomeric antibody B recognize different epitopes of the target molecule to be detected.

2. The composition according to claim 1, characterized in that The multimeric antibody A comprises N monomer antibodies a connected by a cross-linking agent, N ≥ 2, preferably 2 ≤ N ≤ 7, more preferably 4 ≤ N ≤ 6; The monomer antibody a is a monoclonal antibody that recognizes a specific epitope of the target molecule to be detected; Preferably, the cross-linking agent has a bifunctional group capable of reacting with the carboxyl group or the amino group of the monomer antibody a, such as a dialdehyde compound or a dihydrazide compound.

3. The composition according to claim 1 or 2, characterized in that The first marker has a spacer arm, preferably, the length of the spacer arm is More preferred And / or the spacer arm of the first marker is polymerized by "-LC-" or "-PEG-"; preferably, the first marker is biotin or a biotin analogue.

4. The composition according to claim 1, characterized in that The molar ratio of the polyantibody A to the first marker is 1:15-1:60, preferably 1:20-1:50, and more preferably 1:25-1:50 or 1:25-1:

35.

5. A photochemiluminescence detection kit, comprising the composition according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that In the first reagent, the concentration of the polyantibody A that binds to the first marker is 1 μg / mL-4 μg / mL, preferably 2 μg / mL-3 μg / mL.

7. The kit according to claim 5 or 6, characterized in that It also includes a third reagent, which includes a photosensitive microsphere, which can generate singlet oxygen in an excited state, and the surface of the photosensitive microsphere is coated with a specific binding substance of the first marker; Preferably, the concentration of the photosensitive microspheres in the third reagent is not higher than 30 μg / mL, preferably 18 μg / mL-30 μg / mL, more preferably 20 μg / mL-30 μg / mL; and / or, The ratio of the concentration of the photosensitive microspheres in the third reagent to the concentration of the luminescent microspheres in the second reagent is (1.2-2):

1.

8. A photochemiluminescence detection method, comprising using the composition according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7 to detect whether an analyte is present in a sample to be tested; Preferably, the detection method comprises the following steps: (1) mixing a sample to be tested with the first reagent and the second reagent to form a first mixture, and subjecting the first mixture to a first stage of incubation to form a double antibody sandwich complex in the presence of an analyte; (2) adding the third reagent to the first mixture after the first stage incubation to form a second mixture, and subjecting the second mixture to a second stage incubation to allow the first marker to specifically bind to a specific binding substance of the first marker; (3) Perform photoexcitation, detect and analyze the optical signal.

9. The detection method according to claim 8, characterized in that: In the second mixture, the concentration of the photosensitive microspheres is not higher than 11.5 μg / mL; preferably 6.9 μg / mL-11.5 μg / mL; more preferably 7.7 μg / mL-11.5 μg / mL; and / or, In the second mixture, the concentration of the polyantibody A bound to the first marker is 0.11 μg / mL-0.46 μg / mL; preferably 0.23 μg / mL-0.35 μg / mL; and / or, The concentration ratio of the photosensitive microspheres to the luminescent microspheres is (40-67):

1.

10. A photochemiluminescence detection device, which detects whether an analyte is present in a sample using the composition according to any one of claims 1 to 4, the kit according to any one of claims 5 to 7, or the detection method according to any one of claims 8 to 9; Preferably, the detection device comprises: A reagent card, in which a number of wells are provided, some of which are pre-installed with reagents; A pipetting mechanism, used to move the reagents contained in the wells between the wells; The detection mechanism is provided with a plurality of placement positions for placing the reagent cards.

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