WO2020088697A1 - 一种均相化学发光检测试剂盒 - Google Patents

一种均相化学发光检测试剂盒 Download PDF

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Publication number
WO2020088697A1
WO2020088697A1 PCT/CN2019/128668 CN2019128668W WO2020088697A1 WO 2020088697 A1 WO2020088697 A1 WO 2020088697A1 CN 2019128668 W CN2019128668 W CN 2019128668W WO 2020088697 A1 WO2020088697 A1 WO 2020088697A1
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Prior art keywords
kit according
carrier
molecules
present
biotin
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PCT/CN2019/128668
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English (en)
French (fr)
Inventor
章春奇
金鑫
李临
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博阳生物科技（上海）有限公司
科美诊断技术股份有限公司
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Publication of WO2020088697A1 publication Critical patent/WO2020088697A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the invention belongs to the technical field of chemiluminescence, and particularly relates to a homogeneous chemiluminescence detection kit.
  • Chemiluminescence analysis method is divided into homogeneous chemiluminescence analysis method and heterogeneous chemiluminescence analysis method according to separation or not.
  • Heterogeneous chemiluminescence analysis requires multiple steps such as embedding, elution, and separation. The analysis process is cumbersome and the analysis time is long, which cannot meet the requirements of rapid detection and diagnosis.
  • the homogeneous chemiluminescence analysis method effectively avoids the elaborate steps such as elution and separation, which greatly improves the analysis efficiency and cost performance, and is increasingly used.
  • Biotin-Avidin-System is a new type of biological reaction amplification system developed in the late 1970s.
  • the BAS system has the advantages of high-affinity, high-sensitivity, and high-stability signal amplification and labeling technology. By combining the two, large-molecule biologically active substances such as antigens and antibodies can be coupled. Their combination is rapid, specific, stable, and has a multi-stage amplification effect.
  • the BAS system is mainly used in immunology, molecular biology and other fields. It has great advantages in the practical application of in vitro diagnosis. The biggest disadvantage of this method is the interference of biotin, which causes the detection error.
  • Biotin interference may produce false positives or false negatives.
  • the sandwich method produces false negatives and the competition method produces false positives.
  • the current common solutions 1. Replace the platform that does not use the avidin-biotin system; 2. Retest every other day or one week after discontinuing the relevant drugs / food; 3. Sample pretreatment: Streptavidin coating The particles remove the biotin in the sample.
  • the present invention provides a homogeneous chemiluminescence detection kit for the deficiencies of the prior art, which can solve the problem of biotin interference.
  • the first aspect of the present invention provides a homogeneous chemiluminescence detection kit, which includes:
  • An anti-interference agent which includes a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can specifically bind to biotin molecules;
  • the receptor which is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.
  • the anti-interference agent is capable of recognizing free biotin molecules and biotin markers.
  • the anti-interference agent can selectively adsorb free biotin molecules.
  • the free biotin molecule can diffuse into the carrier and specifically bind to the active molecule therein.
  • the anti-interference agent can restrict the entry of biological macromolecules larger than the size of the active molecule into its carrier.
  • the anti-interference agent can be uniformly distributed in the liquid phase reaction system.
  • the internal surface area of the carrier is greater than its external surface area; preferably, the internal surface area of the carrier is more than 5 times its external surface area, preferably more than 10 times, more preferably 20 More than times.
  • the particle size of the carrier is 15-300 nm, preferably 30-250 nm, and more preferably 50-200 nm.
  • the specific surface area of the carrier is 200 m 2 / g or more, preferably 400 m 2 / g or more, more preferably 600 m 2 / g or more, and most preferably 1000 m 2 / g or more.
  • the minimum porosity of the support is greater than 40%, preferably greater than 50%, and more preferably greater than 60%.
  • the porous medium is selected from one or more of porous metal materials, porous non-metallic materials, and porous polymer materials.
  • the carrier is mesoporous microspheres, preferably ordered mesoporous microspheres.
  • the pore size of the mesoporous microspheres is 2-50 nm, preferably 4-30 nm, and more preferably 5-15 nm.
  • the mesoporous microspheres are cage-like hollow mesoporous microspheres.
  • the mesoporous microspheres are selected from Al 2 O 3 mesoporous materials, WO 3 mesoporous materials, TiO 2 mesoporous materials, ZrO 2 mesoporous materials, silicon-based mesoporous materials and At least one of the mesoporous carbon materials is preferably selected from silicon-based mesoporous materials.
  • the active molecule is selected from avidin and / or streptavidin.
  • the active molecules are filled in the carrier by physical adsorption.
  • the active molecule is filled in the carrier by contacting the carrier in a system containing a buffer.
  • the pH value of the buffer-containing system is 7 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, further preferably 7.3 to 7.6.
  • the active molecule is filled in the carrier by direct or indirect chemical crosslinking.
  • the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein,
  • the chemical group is selected from one or more of carboxyl group, aldehyde group, amino group, mercapto group and hydroxyl group.
  • a biotin molecule is connected to the inner surface of the carrier, and the active molecule is filled in the carrier by specific binding action with the biotin molecule.
  • the anti-interference agent further includes a buffer solution, preferably a PBS buffer solution.
  • the total concentration of the carrier and the active molecules filled in the carrier in the anti-interference agent is 5-50ug / mL, preferably 8-30ug / mL, more preferably 10-20ug / mL.
  • the preparation method of the anti-interference agent includes: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in the first buffer system.
  • the preparation method of the anti-interference agent further includes step S0: the carrier is washed with a second buffer system, and step S0 is performed before step S1.
  • the preparation method of the anti-interference agent further includes step S2: removing active molecules that are not filled into the carrier, step S2 is performed after step S1; preferably, by step S1 A third buffer system is added to the treated carrier, and then solid-liquid separation is performed to remove active molecules not filled in the carrier.
  • the surface of the receptor is directly or indirectly connected to a biologically active substance that can specifically bind to the target molecule to be tested.
  • the biologically active substance is an antigen and / or an antibody;
  • the antigen refers to an immunogenic substance;
  • the antibody refers to an immunoglobulin produced by the body that can recognize a specific foreign object protein.
  • the receptor comprises a chemiluminescent compound and a metal chelate, which is in a non-particulate form and soluble in an aqueous medium.
  • the receptor comprises a luminescent composition and a matrix
  • the luminescent composition is filled in the matrix and / or coated on the surface of the matrix.
  • the luminescent composition is capable of reacting with active oxygen to generate a detectable chemiluminescent signal, which comprises a chemiluminescent compound and a metal chelate.
  • the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, bisbutanedione compounds, dioxane, enol ether, enamine, 9- Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl diethyl etherene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives derivative.
  • olefin compounds preferably selected from dimethylthiophene, bisbutanedione compounds, dioxane, enol ether, enamine, 9- Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl diethyl etherene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives derivative.
  • the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, and more preferably selected from europium.
  • the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl 4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridyl carboxylic acid, aza crown ether, aza ligand and trioctyl oxidation Phosphines and their derivatives.
  • a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl 4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridyl carboxylic acid, aza crown ether, aza ligand and trioctyl oxidation Phosphine
  • the luminescent compound is a derivative of dimethylthiophene, and the metal chelate is a europium chelate.
  • the matrix is selected from polymer microspheres, preferably latex microspheres, and more preferably polystyrene latex microspheres.
  • the kit further includes a donor, the donor is capable of generating active oxygen in an excited state
  • the donor is a light-activated or chemically-activated sensitizer, which is in a non-particulate form and soluble in an aqueous medium.
  • the donor is a polymer particle filled with a photosensitizer, which can generate active oxygen under light excitation.
  • the photosensitizer is selected from one of methylene blue, rose red, porphyrin and phthalocyanine.
  • the donor is linked to streptavidin.
  • the kit further includes a biotin marker, which includes a biomacromolecule that binds to biotin and can directly or indirectly bind to the target molecule to be tested, wherein the biomacromolecule is selected from Protein molecules, nucleic acid molecules, polysaccharide molecules, and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from antigens and / or antibodies; wherein, the antigen refers to a substance with immunogenicity; Antibody refers to the immunoglobulin produced by the body that can recognize specific foreign objects.
  • a biotin marker which includes a biomacromolecule that binds to biotin and can directly or indirectly bind to the target molecule to be tested, wherein the biomacromolecule is selected from Protein molecules, nucleic acid molecules, polysaccharide molecules, and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from antigens and / or antibodies; wherein, the antigen refers to a substance with immunogenicity; Antibody
  • the active oxygen is singlet oxygen.
  • the kit of the present invention includes an anti-interference agent, which uses active molecules such as SA or avidin protein molecules as "guest molecules" to be filled in a porous medium in an appropriate manner Inside the pores, a "mesoporous assembly host-guest" system is formed, which can effectively distinguish free biotin molecules and biotin markers, so that the kit of the present invention can eliminate the interference of free biotin and avoid the chemiluminescence immunoassay. False positive and / or false negative results.
  • the kit of the present invention is also practical and versatile, can be applied on different technical platforms, and has little effect on the performance of the reagent.
  • carrier in the present invention refers to a substance that can carry active molecules to participate in chemical or physical processes together.
  • the chemical composition of the carrier is not particularly limited in the present invention, and may be organic materials or inorganic materials, such as high molecular polymers, metals, glass, mineral salts, diatoms, phospholipid vesicles, silicon particles, microcrystalline dyes and many more.
  • porous medium used in the present invention refers to a substance composed of a skeleton composed of a solid substance and a minute gap formed by the skeleton divided into a large number of densely packed groups.
  • active molecule refers to a molecule having specific binding ability to biotin molecule.
  • active molecules are avidin and streptavidin.
  • target molecule to be detected refers to the substance in the sample to be detected during detection.
  • One or more substances with specific binding affinity to the target molecule to be tested will be used to detect the target molecule.
  • the target molecule to be tested may be a protein, a peptide, an antibody, or a hapten that can bind the antibody.
  • antibody in the present invention is used in the broadest sense and includes any isotype of antibody, antibody fragments that retain specific binding to antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies , Humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins containing the antigen-binding portion of the antibody and non-antibody proteins.
  • antigen refers to a substance that can stimulate the body to produce an immune response, and can combine with antibodies and sensitized lymphocytes of immune response products in vivo and in vivo to produce immune effects.
  • binding in the present invention refers to the direct union between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonds, including but not limited to, such as salt bridges and water bridges. .
  • active oxygen in the present invention refers to a general term for substances in the body or in the natural environment that are composed of oxygen and contain oxygen and are active in nature. It is mainly an excited state of oxygen molecules, including superoxide, an electron reduction product of oxygen Anions (O 2 ⁇ -), two-electron reduction product hydrogen peroxide (H 2 O 2 ), three-electron reduction product hydroxyl radical ( ⁇ OH), nitric oxide and singlet oxygen (1O 2 ), etc.
  • the term "receptor" in the present invention refers to a substance that can react with reactive oxygen to generate a detectable signal.
  • the donor is activated by energy or an active compound and releases active oxygen in a high-energy state, which is captured by a close-range acceptor, thereby transferring energy to activate the acceptor.
  • the receptor is a substance that undergoes a chemical reaction with active oxygen (eg, singlet oxygen) to form an unstable metastable intermediate, the metastable intermediate The body can decompose and emit light simultaneously or subsequently.
  • Typical examples of these substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridine, aromatic vinyl ethers, diethylene oxide, dimethyl Thiophene, aromatic imidazole or gloss.
  • the receptor is capable of reacting with active oxygen (eg, singlet oxygen) to form a hydroperoxide or dioxetane which can be decomposed into ketones or carboxylic acid derivatives Olefins; stable dioxetane that can be decomposed by the action of light; acetylenes that can react with active oxygen (such as singlet oxygen) to form diketones; hydrazones that can form azo compounds or azocarbonyl compounds Or hydrazides, such as luminol; and aromatic compounds that can form internal peroxides.
  • active oxygen eg, singlet oxygen
  • the receptor includes an olefin compound and a metal chelate compound, which is non-particulate and soluble in an aqueous medium.
  • a metal chelate compound which is non-particulate and soluble in an aqueous medium.
  • the "chemiluminescent compound” refers to a compound called a marker that can undergo a chemical reaction to cause luminescence, for example, by being converted to another compound formed under an electronically excited state.
  • the excited state can be a singlet state or a triplet excited state.
  • the excited state can relax to the ground state to directly emit light, or it can restore itself to the ground state by transferring the excitation energy to the emission energy acceptor. In this process, the energy acceptor microspheres will be transitioned to an excited state and emit light.
  • can directly or indirectly bind means that the specified physical object can specifically bind to the physical object (directly), or the specified physical object can specifically bind to the specific binding pair member, or have two or More complexes (indirectly) with specific binding partners capable of binding other entities.
  • the “specific binding pair members” of the present invention are selected from (1) small molecules and binding partners for the small molecules, and (2) large molecules and binding partners for the large molecules.
  • the active oxygen may be provided by a "donor".
  • the term “donor” in the present invention refers to a sensitizer capable of generating an active intermediate such as singlet oxygen that reacts with an acceptor after activation by energy or an active compound.
  • the donor can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.).
  • the donor is a photosensitizer
  • the photosensitizer may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not effectively react with singlet oxygen, which is not limited Examples include compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in US Pat. No. 5,999,994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituent The substituents are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specific binding partners.
  • the donor is a chemically activated other sensitizer.
  • Non-limiting examples are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water.
  • examples of other donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or These compounds directly absorb light and release active oxygen (eg singlet oxygen).
  • the photosensitizer generally activates the chemiluminescent compound by irradiating a medium containing the above reactants.
  • the medium must be irradiated with light having a certain wavelength and sufficient energy to convert the photosensitizer to an excited state, thereby enabling it to activate molecular oxygen into singlet oxygen.
  • the excited state of a photosensitizer capable of exciting molecular oxygen is generally in the triplet state, which has an energy higher than the photosensitizer in the ground state by about 20 Kcal / mol, usually at least 23 Kcal / mol.
  • shorter wavelengths can be used, such as 230-950 nm, preferably, light with a wavelength of approximately 450-950 nm is used to illuminate the medium.
  • the light generated can be measured in any conventional manner, such as photography, visual inspection, photometer, etc., in order to determine the amount related to the analyte content in the medium.
  • the photosensitizer is preferably relatively non-polar to ensure solubility in lipophilic members.
  • the photosensitizer and / or chemiluminescent compound can be selected to dissolve, or non-covalently bind to the surface of the particles. In this case, these compounds are preferably hydrophobic to reduce their ability to dissociate from the particles so that both compounds can bind to the same particles.
  • the kit of the present invention includes an anti-interference agent.
  • the anti-interference agent is filled with a carrier in an appropriate manner by using an active molecule that can specifically bind to a biotin molecule as a "guest molecule” to form a "mesoporous assembly" Host-guest system, so that the kit of the present invention avoids false positive or false negative results due to free biotin.
  • the homogeneous chemiluminescence detection kit according to the first aspect of the present invention includes:
  • An anti-interference agent which includes a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can specifically bind to biotin molecules;
  • the receptor which is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.
  • the active molecules are filled in the carrier means that the active molecules are located in the gaps in the carrier, and may or may not be in contact with the framework.
  • the anti-interference agent is capable of recognizing free biotin molecules and biotin markers.
  • recognition may mean that the active molecules in the anti-interference agent and the free biotin molecules and / or biotin markers achieve mutual binding through the synergistic effect of intermolecular forces.
  • the anti-interference agent can selectively adsorb free biotin molecules.
  • the free biotin molecule can diffuse into the carrier and specifically bind to the active molecule therein.
  • the "diffusion" may mean that free biotin molecules are dispersed into the carrier due to the random movement of the molecules.
  • the anti-interference agent can restrict the entry of biological macromolecules larger than the size of the active molecule into its carrier.
  • the anti-interference agent can be uniformly distributed in the liquid phase reaction system.
  • the carrier satisfies at least one of the following conditions: a) The inner pores of the carrier have a sufficiently large surface area (far beyond the carrier surface area), and the voids only allow active molecules to enter, but limit activity Proteins with larger molecules, such as antibodies or large antigens; b) Active molecules such as SA or Avdin can be filled into the carrier by chemical or physical adsorption methods, such as inside the void; c) The carrier can be stably and uniformly distributed in the solution ( Such as aqueous solution), without precipitation.
  • the internal surface area of the carrier is greater than its external surface area; preferably, the internal surface area of the carrier is more than 5 times its external surface area, preferably more than 10 times, more preferably 20 More than times. In some preferred embodiments of the present invention, the internal surface area of the carrier is a multiple of its external surface area including but not limited to: 5 times, 6 times, 8 times, 10 times, 12 times, 16 times, 18 times, 20 times , 22 times, 24 times, 26 times, 28 times or 30 times.
  • the particle size of the carrier is 15-300nm, such as 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 100nm, 250nm, 300nm, etc., preferably 30-250nm, more It is preferably 50-200 nm. If the particle size of the carrier is too large, it will cause the carrier to settle too quickly, which is not conducive to the formation of a stable uniform solution.
  • the specific surface area of the carrier is 200m 2 / g or more, for example, 200m 2 / g, 400m 2 / g, 600m 2 / g, 800m 2 / g, 1000m 2 / g, 1200 m 2 / g, 1500 m 2 / g and the like are preferably 400 m 2 / g or more, more preferably 600 m 2 / g or more, and most preferably 1000 m 2 / g or more.
  • the minimum porosity of the support is greater than 40%, preferably greater than 50%, and more preferably greater than 60%.
  • the porous medium is selected from one or more of porous metal materials, porous non-metallic materials, and porous polymer materials.
  • the carrier is mesoporous microspheres, preferably ordered mesoporous microspheres.
  • the pore diameter of the mesoporous microspheres is 2-50 nm, such as 2 nm, 5 nm, 10, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 50 nm, etc., preferably 4- 30 nm, more preferably 5-15 nm.
  • the mesoporous microspheres are cage-like hollow mesoporous microspheres.
  • the mesoporous microspheres are selected from Al 2 O 3 mesoporous materials, WO 3 mesoporous materials, TiO 2 mesoporous materials, ZrO 2 mesoporous materials, silicon-based mesoporous materials and At least one of the mesoporous carbon materials is preferably selected from silicon-based mesoporous materials.
  • Silicon-based mesoporous materials are a type of periodic mesoporous materials composed of SiO 2 (CH 2 ) 2 tetrahedral structural units.
  • Mesoporous silica materials can be divided into two categories from the microscopic view: one is disordered mesoporous solids represented by silica xerogels and aerogels.
  • the disordered mesoporous silica can be powder, bulk, flake or thin film.
  • the other type is ordered mesoporous silica represented by MCM41.
  • the structural characteristic of ordered mesoporous silica is that the pore size is uniform, arranged in an orderly manner, and the mesoporous pore size can be adjusted between 2-10 nm.
  • the exchange degree of the silicon-based unit is not high, and the hydrothermal stability is not good. Its specific surface area can reach 1000m 2 / g.
  • SBA series HMM series, TUD series, FSM series, KIT series, CMK series, FDU series, starbon and so on. Among them, there are also more SBA-15 studies, and the hydrothermal stability of the material is better than that of the MCM series.
  • the aperture is adjustable between 5-30nm.
  • HMM is a spherical mesoporous material with an aperture of 4-15nm and an adjustable outer diameter of 20-80nm.
  • the active molecule is selected from avidin and / or streptavidin.
  • Avidin is a glycoprotein that can be extracted from egg white. Its molecular weight is about 60kD. Each molecule is composed of 4 subunits and can be intimately combined with 4 biotin molecules.
  • the avidin includes but is not limited to: avidin, streptavidin, yolk avidin and avidin.
  • Streptavidin (SA) is a protein with similar biological characteristics as avidin (A). It is a protein product secreted by Streptomyces avidin bacteria during the cultivation process. SA can also be genetically engineered produce. The molecular weight of SA is 65000, which is composed of four peptide chains with the same sequence. Each SA peptide chain can be combined with one biotin molecule. Therefore, like avidin, each SA molecule also has four binding sites for biotin molecules, and the binding constant is the same as avidin, which is 1015mol / L.
  • the active molecules are filled in the carrier by physical adsorption.
  • Physical adsorption is also called van der Waals adsorption. It is caused by the force between the adsorbate and the adsorbent molecules. This force is also called van der Waals force.
  • the active molecule is filled in the carrier by contacting the carrier in a system containing a buffer.
  • the pH value of the buffer-containing system is 7 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, further preferably 7.3 to 7.6.
  • the active molecule is filled in the carrier by direct or indirect chemical crosslinking.
  • the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein,
  • the chemical group is selected from one or more of carboxyl group, aldehyde group, amino group, mercapto group and hydroxyl group.
  • a biotin molecule is connected to the inner surface of the carrier, and the active molecule is filled in the carrier by specific binding action with the biotin molecule.
  • the anti-interference agent further includes a buffer solution, preferably a PBS buffer solution.
  • the total concentration of the carrier and the active molecules filled in the carrier in the anti-interference agent is 5-50ug / mL, such as 5ug / mL, 10ug / mL, 15ug / mL, 20ug / mL, 25ug / mL, 30ug / mL, 35ug / mL, 40ug / mL, 45ug / mL, 50ug / mL, etc., preferably 8-30ug / mL, more preferably 10-20ug / mL.
  • the preparation method of the anti-interference agent includes: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in the first buffer system.
  • the pH of the first buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, and most preferably 7.40 .
  • examples of the pH value of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.
  • the pH of the first buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, and most preferably 7.40 .
  • examples of the pH value of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.
  • the pH value of the first buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0.
  • examples of the pH value of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.
  • the method further includes step S0: the carrier is washed with a second buffer system, and step S0 is performed before step S1.
  • the pH value of the second buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, most preferably 7.40 .
  • examples of the pH value of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.
  • the pH value of the second buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0.
  • examples of the pH value of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.
  • the method further includes step S2: removing active molecules that are not filled into the carrier, and step S2 is performed after step S1.
  • step S2 is performed after step S1.
  • step S1 by adding a third buffer system to the carrier treated in step S1, and then performing solid-liquid separation, the active molecules not filled in the carrier are removed.
  • the pH value of the third buffer system is 7.0 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, still more preferably 7.3 to 7.7, further preferably 7.35 to 7.50, and most preferably 7.40 .
  • examples of the pH value of the third buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.
  • the pH value of the third buffer system is 3.0 to 7.0, preferably 3.5 to 6.8, more preferably 4.0 to 6.5, still more preferably 5.0 to 6.4, further preferably 5.5 to 6.3, and most preferably 6.0.
  • examples of the pH value of the third buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, etc.
  • the first buffer system comprises a phosphate buffer, piperazine-1,4-diethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
  • the second buffer system comprises a phosphate buffer, piperazine-1,4-diethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
  • the third buffer system comprises a phosphate buffer, piperazine-1,4-diethylsulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4- One or more of hydroxyethylpiperazine ethanesulfonic acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
  • the third buffer system further includes a surfactant.
  • the surfactant comprises one or more selected from Tween-20, Tween-80, Triton X-405, Triton X-100, BRIJ 35, and Pluronic L64.
  • the surfactant comprises Tween-20.
  • the temperature of the contact is 0-50 ° C, preferably 20-40 ° C, such as 25-30 ° C (ie room temperature); and / or, the contact time is 6- 24 hours, preferably 8-12 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.
  • the temperature of the contact is 0-50 ° C, preferably 20-40 ° C, for example 25-30 ° C (ie room temperature); and / or, the contact time is 1-10 hours It is preferably 2-6 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.
  • step S3 is further included, and a fourth buffer system is added.
  • the fourth buffer system comprises a phosphate buffer, piperazine-1,4-diethylsulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonate One or more of acid buffer and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
  • the surface of the receptor is directly or indirectly connected to a biologically active substance that can specifically bind to the target molecule to be tested.
  • the biologically active substance is an antigen and / or an antibody;
  • the antigen refers to an immunogenic substance;
  • the antibody refers to an immunoglobulin produced by the body that can recognize a specific foreign object protein.
  • the receptor comprises a chemiluminescent compound and a metal chelate, which is in a non-particulate form and soluble in an aqueous medium.
  • the receptor comprises a luminescent composition and a matrix
  • the luminescent composition is filled in the matrix and / or coated on the surface of the matrix.
  • the luminescent composition is capable of reacting with active oxygen to generate a detectable chemiluminescent signal, which comprises a chemiluminescent compound and a metal chelate.
  • the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, bisbutanedione compounds, dioxane, enol ether, enamine, 9- Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl diethyl etherene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives derivative.
  • olefin compounds preferably selected from dimethylthiophene, bisbutanedione compounds, dioxane, enol ether, enamine, 9- Alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl diethyl etherene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives derivative.
  • the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, and more preferably selected from europium.
  • the metal chelate comprises a chelating agent selected from the group consisting of: 4 '-(10-methyl-9-anthryl) -2,2': 6'2 "-coupling Tripyridine-6,6 "-dimethylamine] tetraacetic acid (MTTA), 2- (1 ', 1', 2 ', 2', 3 ', 3'-heptafluoro-4', 6'-hexanedi Ketone-6'-yl) -naphthalene (NHA), 4,4'-bis (2 “, 3", 3 "-heptafluoro-4", 6 "-hexanedione-6” -yl) -o- Terphenyl (BHHT), 4,4'-bis (1 “, 1", 1 ", 2", 2 ", 3", 3 "-heptafluoro-4", 6 "-hexanedione-6”- ) -Chlorosulfo-o-terphenyl (BHHCT)
  • the luminescent compound is a derivative of dimethylthiophene, and the metal chelate is a europium chelate.
  • the matrix is selected from polymer microspheres, preferably latex microspheres, and more preferably polystyrene latex microspheres.
  • the kit further includes a donor, which can generate active oxygen in an excited state.
  • the donor is a light-activated or chemically-activated sensitizer, which is in a non-particulate form and soluble in an aqueous medium.
  • the donor is a polymer particle filled with a photosensitizer, which can generate active oxygen under light excitation.
  • the photosensitizer is selected from one of methylene blue, rose red, porphyrin and phthalocyanine.
  • the donor is linked to streptavidin.
  • the kit further includes a biotin marker, which includes a biomacromolecule that binds to biotin and can directly or indirectly bind to the target molecule to be tested, wherein the biomacromolecule is selected from Protein molecules, nucleic acid molecules, polysaccharide molecules, and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from antigens and / or antibodies; wherein, the antigen refers to a substance with immunogenicity; Antibody refers to the immunoglobulin produced by the body that can recognize specific foreign objects.
  • a biotin marker which includes a biomacromolecule that binds to biotin and can directly or indirectly bind to the target molecule to be tested, wherein the biomacromolecule is selected from Protein molecules, nucleic acid molecules, polysaccharide molecules, and lipid molecules; preferably protein molecules; further preferably, the protein molecules are selected from antigens and / or antibodies; wherein, the antigen refers to a substance with immunogenicity; Antibody
  • the active oxygen is singlet oxygen.
  • the first step in a 2mL centrifuge tube, take 10mg of carboxyl functionalized silicon-based microspheres (particle size 15nm, pore size 2nm), add 0.02M PBS (pH7.4) buffer solution, centrifuge at 10000rpm at 4 ° C, and wash once at 15min.
  • carboxyl functionalized silicon-based microspheres particle size 15nm, pore size 2nm
  • 0.02M PBS (pH7.4) buffer solution centrifuge at 10000rpm at 4 ° C, and wash once at 15min.
  • the SA microspheres were centrifuged with 0.02M PBS (pH7.4) buffer solution containing 0.5% Tween-20, centrifuged at 10000rpm at 4 ° C for 15min, washed three times to remove unadsorbed SA, and finally used 0.02M PBS (pH 7.4) Make the buffer volume to 10 mg / mL.
  • the preparation method is the same as that in Example 1, except that each example uses carboxyl functionalized silicon-based microspheres with different particle sizes and / or pore sizes (see Table 1).
  • the first step take 10mg of carboxyl functionalized silicon-based microspheres in a 2mL centrifuge tube, centrifuge at 10000rpm at 4 ° C with 0.1M MES (pH 6.0) buffer solution, and wash once at 15min.
  • MES pH 6.0
  • the SA microspheres were centrifuged and washed three times with 0.02M PBS (pH7.4) buffer solution containing 0.5% Tween-20 to remove unadsorbed SA. Finally, the volume was adjusted to 10 mg / mL with PBS buffer.
  • the kit includes the following components:
  • Component b donor reagent / photosensitive bead solution / general solution: donor solution labeled with streptavidin coupling;
  • Component c receptor reagent / reagent one: receptor solution coated with diiodothyronine;
  • biotin marker / reagent 2 biotin-labeled anti-triiodothyronine antibody solution.
  • preparation methods of components b to d please refer to Examples 1 to 3 of Chinese Patent CN 101865917 B.
  • Example 10 An experimental procedure for evaluating the effect of the anti-biotin interference homogeneous chemiluminescence detection kit of the present invention:
  • T3 concentrated solution to hormone-free serum to prepare T3 solution with concentration of 1 nmol / L and 2 nmol / L.
  • the signal of the chemiluminescent immune response dropped by 89%, and biotin interference was more serious.
  • the luminescence signal showed almost no significant change.
  • the luminescence signal showed a certain Increase, the decline range is 50% -70%.
  • the concentration of the anti-interference agent is 10ug / mL (comparative comparison of the serial numbers 5 and 6 in Table 1), the luminous signal is further improved, and the decline range is about 25%.
  • the concentration is 10ug / ml, and the particle diameter of the microspheres is increased to 100nm (Sequence Nos. 6 and 7 in Table 1), the deviation of the drop amplitude is within 10%, and the phenomenon of biotin interference disappears.
  • the concentration is increased to 20ug / ml (sequence numbers 7 and 8 in Table 1), the signal drop deviation is within 10%.
  • the concentration of 20ug / mL is unchanged, and the particle size and pore size are increased, the signal drops to a certain extent, with a drop of 20% -40%.
  • the anti-interference agent included in the kit of the present invention is an SA (streptavidin) -filled microsphere with a particle size of 100 nm, a pore size of 10 nm, and a concentration of 10-20 ug / mL
  • SA streptavidin
  • the anti-interference agent of the kit The most capable.
  • the pore size of the added anti-interference agent is small, 2 nm, the kit has no anti-biotin interference capability.
  • the particle size of the added anti-interference agent is greater than 100 nm and the pore size is 10 nm, and the particle size and pore size continue to be increased, the anti-biotin interference ability of the kit will decrease.
  • Example 11 Two experimental steps for evaluating the effect of the anti-biotin interference homogeneous chemiluminescence detection kit of the present invention:
  • T3 concentrated solution to hormone-free serum to prepare T3 solution with concentration of 1 nmol / L and 2 nmol / L.
  • the anti-interference agent prepared by physical adsorption method has strong anti-biotin interference ability, the luminescence signal fell by about 10%, and the luminescence signal of the anti-interference agent prepared by covalent coupling method fell by 50% about.
  • the anti-interference ability of the anti-interference agent prepared by physical adsorption method is also superior to that prepared by covalent coupling.
  • the anti-interference ability of the light-excited chemiluminescence kit including the anti-interference agent prepared by physical adsorption is better than that of the anti-interference agent prepared by covalent coupling Excitation chemiluminescence kit.

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Abstract

一种均相化学发光检测试剂盒，该试剂盒包括：抗干扰剂，其包括载体和活性分子；载体为多孔介质；活性分子填充于载体之中，并能够与生物素分子特异性结合；受体，其能够与活性氧反应产生可检测的化学发光信号。该试剂盒包括一种抗干扰剂，使得该试剂盒避免了因游离生物素造成的假阳性或假阴性结果。

Description

一种均相化学发光检测试剂盒
相关申请的交叉引用
本申请要求享有于2018年10月31日提交的名称为“一种抗生物素干扰的均相化学发光检测试剂盒”的中国专利申请CN201811286611.1的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本发明属于化学发光技术领域，具体涉及一种均相化学发光检测试剂盒。
背景技术
化学发光分析法根据分离与否分为均相化学发光分析法和非均相化学发光分析法。非均相化学发光分析法需要包埋、洗脱、分离等多步操作，分析过程繁琐，分析时间长，不能满足快速检测和诊断的要求。而均相化学发光分析法则有效避免了洗脱、分离等繁琐步骤，极大提高了分析效率和性价比，得到日益广泛的应用。
生物素－亲和素系统(Biotin-Avidin—System，BAS)是70年代末发展起来的一种新型生物反应放大系统。BAS系统具有高亲和力，灵敏度高，稳定性高等优点的信号放大标记技术。结合二者即可偶联抗原抗体等大分子生物活性物质。它们的结合迅速、专一、稳定，并具有多级放大效应。目前BAS系统主要应用于免疫学，分子生物学等领域。在体外诊断的实际应用中更是具有巨大的优越性，该法最大的缺点是有生物素的干扰，造成检测的误差。
生物素干扰可能产生假阳性，也可能产生假阴性。一般来说，夹心法产生假阴性，竞争法产生假阳性。目前常见解决方法：1.更换非使用亲和素-生物素系统的平台；2.停用相关药物/食物后隔日或一周后重测；3.样本预处理：链霉抗生物素蛋白包被的微粒去除样品中的生物素。
但是，目前还没有一种方法在亲和素-生物素系统中能够很好地解决生物素干扰的问题。
发明内容
本发明针对现有技术的不足提供一种均相化学发光检测试剂盒，该试剂盒能够很好地解决生物素干扰的问题。
为此，本发明第一方面提供了一种均相化学发光检测试剂盒，其包括：
抗干扰剂，其包括载体和活性分子；所述载体为多孔介质；所述活性分子填充于所述载体之中，并能够与生物素分子特异性结合；
受体，其能够与活性氧反应产生可检测的化学发光信号。
在本发明的一些实施方式中，所述抗干扰剂能够识别游离生物素分子和生物素标记物。
在本发明的另一些实施方式中，所述抗干扰剂能够选择性吸附游离生物素分子。
在本发明的一些实施方式中，所述游离生物素分子能够扩散到所述载体中，并与其中的所述活性分子特异性结合。
在本发明的另一些实施方式中，所述抗干扰剂能够限制比所述活性分子尺寸更大的生物大分子进入其载体之中。
在本发明的一些实施方式中，所述抗干扰剂能够在液相反应体系中均匀分布。
在本发明的一些优选的实施方式中，所述载体的内表面积大于其外表面积；优选地，所述载体的内表面积为其外表面积的5倍以上，优选为10倍以上，更优选为20倍以上。
在本发明的另一些优选的实施方式中，所述载体的粒径为15-300nm，优选为30-250nm，更优选为50-200nm。
在本发明的一些优选的实施方式中，所述载体的比表面积为200m 2/g以上，优选为400m 2/g以上，更优选为600m 2/g以上，最优选为1000m 2/g以上。
在本发明的另一些优选的实施方式中，所述载体的最小孔隙率大于40％，优选大于50％，更优选大于60％。
在本发明的一些实施方式中，所述多孔介质选自多孔金属材料、多孔非金属材料和多孔高分子材料中的一种或多种。
在本发明的另一些实施方式中，所述载体为介孔微球，优选为有序介孔微球。
在本发明的一些优选的实施方式中，所述介孔微球的孔径为2-50nm，优选为4-30nm，更优选为5-15nm。
在本发明的另一些优选的实施方式中，所述介孔微球为笼状中空介孔微球。
在本发明的一些具体实施方式中，所述介孔微球选自Al 2O 3介孔材料、WO 3介孔材料、TiO 2介孔材料、ZrO 2介孔材料、硅基介孔材料和/或介孔碳材料中的至少一种，优选选自硅基介孔材料。
在本发明的一些实施方式中，所述活性分子选自亲和素和/或链霉亲和素。
在本发明的一些优选的实施方式中，所述活性分子通过物理吸附方式填充于所述载体中。
在本发明的另一些优选的实施方式中，所述活性分子通过在含有缓冲液的体系中与载体接触而填充于所述载体之中。
在本发明的一些优选的实施方式中，所述含有缓冲液的体系的pH值为7至9，优选7.1至8.0，更优选7.2至7.8，进一步优选7.3-7.6。
在本发明的一些实施方式中，所述活性分子通过直接或间接化学交联的方式填充于所述载体之中。
在本发明的一些优选的实施方式中，所述载体内表面修饰有化学基团，所述活性分子通过与所述化学基团的共价偶联而填充于所述载体之中；其中，所述化学基团选自羧基、醛基、氨基、巯基和羟基中的一种或多种。
在本发明的一些实施方式中，所述载体内表面连接有生物素分子，所述活性分子通过与生物素分子的特异结合作用而填充于所述载体之中。
在本发明的另一些实施方式中，所述抗干扰剂还包括缓冲溶液，优选PBS缓冲溶液。
在本发明的一些实施方式中，所述载体以及填充在所述载体中的活性分子在抗干扰剂中的总浓度为5-50ug/mL，优选8-30ug/mL，更优选10-20ug/mL。
在本发明的一些实施方式中，所述抗干扰剂的制备方法包括：步骤S1，使载体与活性分子进行接触；优选地，所述接触在第一缓冲液体系中进行。
在本发明的一些优选的实施方式中，所述抗干扰剂的制备方法还包括步骤S0：用第二缓冲液体系将载体进行清洗，步骤S0在步骤S1之前进行。
在本发明的另一些优选的实施方式中，所述抗干扰剂的制备方法还包括步骤S2：除去未填充到载体中的活性分子，步骤S2在步骤S1之后进行；优选地，通 过向步骤S1处理后的载体中加入第三缓冲液体系，然后进行固液分离，以除去未填充到载体中的活性分子。
在本发明的一些实施方式中，所述受体的表面直接地或间接地与生物活性物质连接，所述生物活性物质能够与待测目标分子特异性结合。
在本发明的另一些实施方式中，所述生物活性物质为抗原和/或抗体；所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
在本发明的一些实施方式中，所述受体包含化学发光化合物和金属螯合物，其为非粒子形式，且在含水介质中可溶。
在本发明的另一些实施方式中，所述受体包含发光组合物和基质，所述发光组合物填充于基质中和/或包被于基质表面。
在本发明的一些实施方式中，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
在本发明的另一些实施方式中，所述化学发光化合物选自烯烃化合物，优选选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
在本发明的一些实施方式中，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
在本发明的另一些实施方式中，所述金属螯合物包含选自下列的螯合剂：NHA、BHHT、BHHCT、DPP、TTA、NPPTA、NTA、TOPO、TPPO、BFTA、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
在本发明的一些实施方式中，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
在本发明的一些实施方式中，所述基质选自高分子微球，优选为乳胶微球，更优选为聚苯乙烯乳胶微球。
在本发明的一些实施方式中，所述试剂盒还包括供体，所述供体能够在激发状态下生成活性氧
在本发明的一些优选的实施方式中，所述供体为光活化的或化学活化的敏化剂，其为非粒子形式，且在含水介质中可溶。
在本发明的一些实施方式中，所述供体为填充有光敏剂的高分子微粒，其在光激发下能够产生活性氧。
在本发明的一些实施方式中，所述光敏剂选自亚甲基蓝、玫瑰红、卟碄和酞菁中的一种。
在本发明的一些实施方式中，供体与链霉亲和素相连。
在本发明的一些实施方式中，所述试剂盒还包括生物素标记物，其包括与生物素结合的能够与待测目标分子直接或间接结合的生物大分子，其中所述生物大分子选自蛋白质分子、核酸分子、多糖分子和脂类分子；优选为蛋白质分子；进一步优选地，所述蛋白质分子选自抗原和/或抗体；其中，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
在本发明的一些实施方式中，所述活性氧为单线态氧。
本发明的有益效果为：本发明所述试剂盒中包括一种抗干扰剂，该抗干扰剂通过将活性分子如SA或avidin蛋白质分子等作为“客体分子”，以恰当方式填充于多孔介质的孔内，形成“介孔组装主客体”体系，能够有效区分游离生物素分子与生物素标记物，从而使得本发明所述试剂盒能够消除游离生物素的干扰，避免了化学发光免疫测定中的假阳性和/或假阴性结果。另外，本发明所述试剂盒还具有实用性和通用性，可以在不同的技术平台上应用，对试剂的性能影响小。
具体实施方式
为使本发明容易理解，下面将详细说明本发明。但在详细描述本发明前，应当理解本发明不限于描述的具体实施方式。还应当理解，本文中使用的术语仅为了描述具体实施方式，而并不表示限制性的。
在提供了数值范围的情况下，应当理解所述范围的上限和下限和所述规定范围中的任何其他规定或居间数值之间的每个居间数值均涵盖在本发明内。这些较小范围的上限和下限可以独立包括在较小的范围中，并且也涵盖在本发明内，服 从规定范围中任何明确排除的限度。在规定的范围包含一个或两个限度的情况下，排除那些包括的限度之任一或两者的范围也包含在本发明中。
除非另有定义，本文中使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可以在本发明的实施或测试中使用，但是现在描述了优选的方法和材料。
I.术语
本发明所述用语“载体”是指能载带活性分子共同参与化学或物理过程的物质。本发明对载体的化学成分没有特别的限制，可以是有机材质的、也可以无机材质的，如高分子聚合物、金属、玻璃、矿物盐、硅藻、磷脂囊泡、硅颗粒、微晶染料等等。
本发明所使用的术语“多孔介质”是指由固体物质组成的骨架和由骨架分隔成大量密集成群的微小空隙所构成的物质。
本发明所使用的术语“活性分子”是指具有与生物素分子特异性结合能力的分子。示例性的活性分子为亲和素和链霉亲和素。
本发明所述用语“待测目标分子”是指检测时待检测样本中的物质。与待测目标分子具有特异性结合亲合力的一种或多种物质会被用于检测该目标分子。待测目标分子可以是蛋白、肽、抗体或可以使其与抗体结合的半抗原。
本发明所述用语“抗体”以最广含义使用，包括任何同种型的抗体，保留对抗原的特异性结合的抗体片段，包括但不限于Fab、Fv、scFv、和Fd片段、嵌合抗体、人源化抗体、单链抗体、双特异性抗体、和包含抗体的抗原结合部分和非抗体蛋白的融合蛋白。
本发明所述用语“抗原”是指能够刺激机体产生免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体内外结合，发生免疫效应的物质。
本发明所述用语“结合”指由于例如共价、静电、疏水、离子和/或氢键等相互作用，包括但不限于如盐桥和水桥等相互作用引起的两个分子间的直接联合。
本发明所述用语“特异性结合”，是指两种物质之间的相互辨别和选择性结合反应，从立体结构角度上说就是相应的反应物之间构象的对应性。
本发明所述用语“活性氧”是指机体内或者自然环境中由氧组成，含氧并且性质活泼的物质的总称，主要为一种激发态的氧分子，包括氧的一电子还原产物超氧阴离子(O 2·-)、二电子还原产物过氧化氢(H 2O 2)、三电子还原产物羟基自由基(·OH)以及一氧化氮和单线态氧(1O 2)等。
本发明中，本发明所述用语“受体”是指能够与活性氧反应可以产生可检测信号的物质。供体被能量或者活性化合物诱导激活并释放高能态的活性氧，该高能态的活性氧被近距离的受体俘获，从而传递能量以激活所述受体。在本发明的一些具体实施例中，所述受体是这样的物质，其经历与活性氧(例如单线态氧)的化学反应以形成不稳定的亚稳态中间体，所述亚稳态中间体可以分解，同时或随后发光。这些物质的典型例子包括但不限于：烯醇醚、烯胺、9-烷叉黄原胶、9-烷叉-N-烷基吖啶满、芳乙烯醚、双环氧乙烯、二甲基噻吩、芳香性咪唑或光泽精。在本发明的另一些具体实施例中，所述受体是能够与活性氧(例如单线态氧)反应以形成可以分解成酮类或羧酸衍生物的氢过氧化物或二氧环丁烷的烯烃类；可以通过光的作用分解的稳定二氧环丁烷；可以与活性氧(例如单线态氧)反应以形成二酮类的乙炔类；可以形成偶氮化合物或偶氮羰基化合物的腙类或酰肼类，诸如鲁米诺；和可以形成内过氧化物类的芳族化合物。可以根据本公开和要求保护的发明利用的受体的具体的、非限制性实例记载于美国专利号US5340716(该专利文献在此全文引为参考)。在本发明另一些具体实施例中，所述受体包含烯烃化合物和金属螯合物，其是非粒子化的并且在含水介质中可溶，这种受体的情况可参见专利PCT/US2010/025433(该专利文献在此全文引为参考)。
本发明中，所述“化学发光化合物”即一种被称作为标记物的化合物，可进行化学反应以便引起发光，比如通过被转化为在电子激发态下形成的另一种化合物。激发态可以是单线态或是三重激发态。激发态可弛豫到基态直接发光，或者是通过将激发能量传递到发射能量受体，从而自身恢复到基态。在此过程中，能量受体微球将被跃迁为激发态而发光。
用语“能够直接或间接地结合”是指指定的实体物能够特异地结合到实体物上(直接地)，或者指定的实体物能够特异性地结合至特异性结合配对成员、或具有两个或更多能够结合其他实体物的特异性结合伴侣的复合物上(间接地)。
本发明所述“特异性结合配对成员”选自(1)小分子和对于所述小分子的结合配偶体，以及(2)大分子和对于所述大分子的结合配偶体。
在本发明中，所述活性氧可以由“供体”提供。本发明所述用语“供体”是指通过能量或者活性化合物的激活后能够产生与受体反应的诸如单线态氧的活 性中间体的敏化剂。供体可以是光活化的(如染料和芳香化合物)或者化学活化的(如酶、金属盐等)。在本发明一些具体实施例中，所述供体是光敏剂，所述光敏剂可以是本领域已知的光敏剂，优选相对光稳定且不与单线态氧有效反应的化合物，其非限定性的例子包括例如美国专利US5709994(该专利文献在此全文引为参考)公开的亚甲基蓝、玫瑰红、卟啉、酞菁和叶绿素等化合物，以及这些化合物的具有1-50个原子取代基的衍生物，所述取代基用于使得这些化合物更具有亲脂性或更具有亲水性、和/或作为连接至特异性结合配对物的连接基团。本领域技术人员已知的其他光敏剂的例子也可以在本发明中使用，例如美国专利US6406913中记载的内容，该专利文献并入本文以供参考。在本发明另一些具体实施例中，所述供体是化学活化的其他敏化剂，其非限定性的例子是某些化合物，它们催化过氧化氢转化为单线态氧和水。其他一些供体的例子包括：1,4-二羧基乙基-1,4-萘内过氧化物、9,10-二苯基蒽-9,10-内过氧化物等，加热这些化合物或者这些化合物直接吸收光会释放活性氧(例如单线态氧)。
光敏剂一般通过照射含有上述反应物的介质来活化化学发光化合物。介质必须用具有一定的波长、且能量足以将光敏剂转化至激发态、进而使得它能够将分子氧活化为单线态氧的光照射。能够激发分子氧的光敏剂的激发态一般位于三重态，其比处于基态的光敏剂的能量高大约20Kcal/mol、通常至少23Kcal/mol。虽然可以使用较短的波长，如230-950nm，但是优选地，使用波长大约是450-950nm的光来照射介质。可以以任何传统方式，诸如照相、肉眼直观、光度计等，来测定产生的光，以便确定其与介质中分析物含量相关的量。光敏剂优选是相对非极性的，以确保可溶解到亲脂性成员中。可以选择光敏剂和/或化学发光化合物溶解在、或非共价地结合到颗粒的表面。在这种情况下，这些化合物优选是疏水性的，以降低它们从颗粒解离下来的能力，从而使两种化合物都能和相同的颗粒结合。
II.具体实施方案
下面将更详细地说明本发明。
本发明所述试剂盒包括一种抗干扰剂，该抗干扰剂通过将能够与生物素分子特异性结合的活性分子作为“客体分子”，以恰当方式填充于载体之中，形成“介孔组装主客体”体系，从而使得本发明所述试剂盒避免了因游离生物素造成的假阳性或假阴性结果。
本发明第一方面所涉及的均相化学发光检测试剂盒，其包括：
抗干扰剂，其包括载体和活性分子；所述载体为多孔介质；所述活性分子填充于所述载体之中，并能够与生物素分子特异性结合；
受体，其能够与活性氧反应产生可检测的化学发光信号。
其中，“活性分子填充于载体之中”是指活性分子位于载体中的空隙中，可以与骨架接触，也可以与骨架不接触。
在本发明的一些实施方式中，所述抗干扰剂能够识别游离生物素分子和生物素标记物。在本发明中，“识别”可以是指抗干扰剂中的活性分子与游离生物素分子和/或生物素标记物通过分子间作用力的协同作用达到相互结合。
在本发明的另一些实施方式中，所述抗干扰剂能够选择性吸附游离生物素分子。
在本发明的一些实施方式中，所述游离生物素分子能够扩散到所述载体中，并与其中的所述活性分子特异性结合。在本发明中，所述“扩散”可以是指由于分子的无规则运动而导致的游离生物素分子分散到载体中。
在本发明的另一些实施方式中，所述抗干扰剂能够限制比所述活性分子尺寸更大的生物大分子进入其载体之中。
在本发明的一些实施方式中，所述抗干扰剂能够在液相反应体系中均匀分布。
在本发明的一些实施方式中，所述载体满足以下条件中的至少一个：a)载体的内孔有足够大的表面积(远超载体表面积)，并且空隙仅能让活性分子进入，但限制活性分子更大的蛋白质，比如抗体或大的抗原等；b)能将活性分子如SA或Avdin通过化学或物理吸附方法填充于载体中，例如空隙内部；c)载体能稳定地均匀分布在溶液(如水溶液)中，而不发生沉淀。
在本发明的一些优选的实施方式中，所述载体的内表面积大于其外表面积；优选地，所述载体的内表面积为其外表面积的5倍以上，优选为10倍以上，更优选为20倍以上。在本发明的一些优选实施方式中，所述载体的内表面积为其外表面积的倍数包括但不限于：5倍、6倍、8倍、10倍、12倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍或30倍。
在本发明的另一些优选的实施方式中，所述载体的粒径为15-300nm，例如15nm、20nm、25nm、30nm、40nm、50nm、100nm、250nm、300nm等，优选为 30-250nm，更优选为50-200nm。载体粒径过大，会造成载体沉降过快，不利于形成稳定的均匀溶液。
在本发明的一些优选的实施方式中，所述载体的比表面积为200m 2/g以上，例如200m 2/g、400m 2/g、600m 2/g、800m 2/g、1000m 2/g、1200m 2/g、1500m 2/g等，优选为400m 2/g以上，更优选为600m 2/g以上，最优选为1000m 2/g以上。
在本发明的另一些优选的实施方式中，所述载体的最小孔隙率大于40％，优选大于50％，更优选大于60％。
在本发明的一些实施方式中，所述多孔介质选自多孔金属材料、多孔非金属材料和多孔高分子材料中的一种或多种。
在本发明的另一些实施方式中，所述载体为介孔微球，优选为有序介孔微球。
在本发明的一些优选的实施方式中，所述介孔微球的孔径为2-50nm，例如2nm、5nm、10、15nm、20nm、25nm、30nm、35nm、40nm、50nm等，优选为4-30nm，更优选为5-15nm。
在本发明的另一些优选的实施方式中，所述介孔微球为笼状中空介孔微球。
在本发明的一些具体实施方式中，所述介孔微球选自Al 2O 3介孔材料、WO 3介孔材料、TiO 2介孔材料、ZrO 2介孔材料、硅基介孔材料和/或介孔碳材料中的至少一种，优选选自硅基介孔材料。
硅基介孔材料是一类由SiO 2(CH 2) 2四面体结构单元所构成的周期性介孔物质。介孔二氧化硅材料从微观上可以分为两类：一类是以二氧化硅干凝胶和气凝胶类为代表的无序介孔固体。无序介孔二氧化硅宏观上可以是粉体，块体，片状或者薄膜。另一类是以MCM41为代表的有序介孔二氧化硅。有序介孔二氧化硅的结构特点是孔径大小均匀，按六方有序排列，介孔孔径可以在2-10nm之间可调。由于其孔壁较薄，硅基单元交流度不高，水热稳定性不好。其比表面积可达1000m 2/g。还有SBA系列，HMM系列，TUD系列，FSM系列，KIT系列，CMK系列，FDU系列，starbon等。其中SBA-15研究也较多，材料的水热稳定性比MCM系列要好。孔径在5-30nm可调。HMM是球形的介孔材料，其孔径是4-15nm，外径20-80nm可调。
在本发明的一些实施方式中，所述活性分子选自亲和素和/或链霉亲和素。亲和素是一种糖蛋白，可由蛋清中提取，其分子量约60kD，每个分子由4个亚基组成，可以和4个生物素分子亲密结合。所述亲和素包括但不限于：卵白亲和素、 链亲和素、卵黄亲和素及类亲和素。链霉亲和素(SA)是与亲和素(A)有类似生物学特性的一种蛋白质，其是由Streptomyces avidin菌在培养过程中分泌的一种蛋白质产物，SA也可以通过基因工程手段生产。SA的分子量为65000，由4条序列相同的肽链构成，每条SA肽链可以结合1个生物素分子。因此与亲和素一样，每个SA分子也具有4个可与生物素分子结合位点，其结合常数与亲和素相同为1015mol/L。
在本发明的一些优选的实施方式中，所述活性分子通过物理吸附方式填充于所述载体中。物理吸附也称范德华吸附，它是由吸附质和吸附剂分子间作用力所引起，此力也称作范德华力。
在本发明的另一些优选的实施方式中，所述活性分子通过在含有缓冲液的体系中与载体接触而填充于所述载体之中。
在本发明的一些优选的实施方式中，所述含有缓冲液的体系的pH值为7至9，优选7.1至8.0，更优选7.2至7.8，进一步优选7.3-7.6。
在本发明的一些实施方式中，所述活性分子通过直接或间接化学交联的方式填充于所述载体之中。
在本发明的一些优选的实施方式中，所述载体内表面修饰有化学基团，所述活性分子通过与所述化学基团的共价偶联而填充于所述载体之中；其中，所述化学基团选自羧基、醛基、氨基、巯基和羟基中的一种或多种。
在本发明的一些实施方式中，所述载体内表面连接有生物素分子，所述活性分子通过与生物素分子的特异结合作用而填充于所述载体之中。
在本发明的另一些实施方式中，所述抗干扰剂还包括缓冲溶液，优选PBS缓冲溶液。
在本发明的一些实施方式中，所述载体以及填充在所述载体中的活性分子在抗干扰剂中的总浓度为5-50ug/mL，例如5ug/mL、10ug/mL、15ug/mL、20ug/mL、25ug/mL、30ug/mL、35ug/mL、40ug/mL、45ug/mL、50ug/mL等，优选8-30ug/mL，更优选10-20ug/mL。
在本发明的一些实施方式中，所述抗干扰剂的制备方法包括：步骤S1，使载体与活性分子进行接触；优选地，所述接触在第一缓冲液体系中进行。在上述方法的一些实施方式中，所述第一缓冲液体系的pH值为7.0至9，优选7.1至8.0，更优选7.2至7.8，再优选7.3至7.7，进一步优选7.35至7.50，最优选7.40。在 该实施方式中，所述第一缓冲液体系的pH值的示例包括7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、9.0等。
在上述方法的一些实施方式中，所述第一缓冲液体系的pH值为7.0至9，优选7.1至8.0，更优选7.2至7.8，再优选7.3至7.7，进一步优选7.35至7.50，最优选7.40。在该实施方式中，所述第一缓冲液体系的pH值的示例包括7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、9.0等。
在上述方法的另外一些实施方式中，所述第一缓冲液体系的pH值为3.0至7.0，优选3.5至6.8，更优选4.0至6.5，再优选5.0至6.4，进一步优选5.5至6.3，最优选6.0。在该实施方式中，所述第一缓冲液体系的pH值的示例包括3.0、3.5、4.0、4.5、5.0、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.1、6.2、6.3、6.4、6.5、6.6等。
根据一些具体实施方式，所述方法还包括步骤S0：用第二缓冲液体系将载体进行清洗，步骤S0在步骤S1之前进行。在上述方法的一些实施方式中，所述第二缓冲液体系的pH值为7.0至9，优选7.1至8.0，更优选7.2至7.8，再优选7.3至7.7，进一步优选7.35至7.50，最优选7.40。在该实施方式中，所述第一缓冲液体系的pH值的示例包括7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、9.0等。在上述方法的另外一些实施方式中，所述第二缓冲液体系的pH值为3.0至7.0，优选3.5至6.8，更优选4.0至6.5，再优选5.0至6.4，进一步优选5.5至6.3，最优选6.0。在该实施方式中，所述第一缓冲液体系的pH值的示例包括3.0、3.5、4.0、4.5、5.0、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.1、6.2、6.3、6.4、6.5、6.6等。
根据一些具体实施方式，所述方法还包括步骤S2：除去未填充到载体中的活性分子，步骤S2在步骤S1之后进行。优选地，通过向步骤S1处理后的载体中加入第三缓冲液体系，然后进行固液分离，以除去未填充到载体中的活性分子。
在上述方法的一些实施方式中，所述第三缓冲液体系的pH值为7.0至9，优选7.1至8.0，更优选7.2至7.8，再优选7.3至7.7，进一步优选7.35至7.50，最优选7.40。在该实施方式中，所述第三缓冲液体系的pH值的示例包括7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、9.0等。在上述方法的另外一些实施方式中，所述第三缓冲液体系的pH值为3.0至7.0，优选3.5至6.8，更优选4.0至6.5，再优选5.0至6.4，进一步优选5.5至6.3，最优选6.0。在该实施方式 中，所述第三缓冲液体系的pH值的示例包括3.0、3.5、4.0、4.5、5.0、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.1、6.2、6.3、6.4、6.5、6.6等。
在上述方法的一些实施方式中，所述第一缓冲液体系包含选自磷酸盐缓冲剂、哌嗪-1,4-二乙磺酸缓冲剂、3-吗啉丙磺酸缓冲剂、4-羟乙基哌嗪乙磺酸缓冲剂、3-(羟乙基哌嗪)-2-羟基丙磺酸缓冲剂中的一种或多种。在上述方法的一些实施方式中，所述第二缓冲液体系包含选自磷酸盐缓冲剂、哌嗪-1,4-二乙磺酸缓冲剂、3-吗啉丙磺酸缓冲剂、4-羟乙基哌嗪乙磺酸缓冲剂和3-(羟乙基哌嗪)-2-羟基丙磺酸缓冲剂中的一种或多种。在上述方法的一些实施方式中，所述第三缓冲液体系包含选自磷酸盐缓冲剂、哌嗪-1,4-二乙磺酸缓冲剂、3-吗啉丙磺酸缓冲剂、4-羟乙基哌嗪乙磺酸缓冲剂和3-(羟乙基哌嗪)-2-羟基丙磺酸缓冲剂中的一种或多种。
在上述方法的一些实施方式中，所述第三缓冲液体系还包含表面活性剂。根据一些实施例，所述表面活性剂包含选自吐温-20、Tween-80、Triton X-405、Triton X-100、BRIJ 35和Pluronic L64中的一种或多种。根据一些实施例，所述表面活性剂包含吐温-20。
在上述方法的一些实施方式中，步骤S1中，所述接触的温度为0-50℃，优选20-40℃，例如25-30℃(即室温)；和/或，接触的时间为6-24小时，优选为8-12小时，例如8小时、9小时、10小时、11小时、12小时等。在上述方法的一些另一些实施方式中，所述接触的温度为0-50℃，优选20-40℃，例如25-30℃(即室温)；和/或，接触的时间为1-10小时，优选为2-6小时、例如2小时、3小时、4小时、5小时、6小时等。
在上述方法的一些实施方式中，还包括步骤S3，加入第四缓冲体系。优选地，所述第四缓冲体系包含选自磷酸盐缓冲剂、哌嗪-1,4-二乙磺酸缓冲剂、3-吗啉丙磺酸缓冲剂、4-羟乙基哌嗪乙磺酸缓冲剂和3-(羟乙基哌嗪)-2-羟基丙磺酸缓冲剂中的一种或多种。
在本发明的一些实施方式中，所述受体的表面直接地或间接地与生物活性物质连接，所述生物活性物质能够与待测目标分子特异性结合。
在本发明的另一些实施方式中，所述生物活性物质为抗原和/或抗体；所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
在本发明的一些实施方式中，所述受体包含化学发光化合物和金属螯合物，其为非粒子形式，且在含水介质中可溶。
在本发明的另一些实施方式中，所述受体包含发光组合物和基质，所述发光组合物填充于基质中和/或包被于基质表面。
在本发明的一些实施方式中，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
在本发明的另一些实施方式中，所述化学发光化合物选自烯烃化合物，优选选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
在本发明的一些实施方式中，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
在本发明的另一些实施方式中，所述金属螯合物包含选自下列的螯合剂：4’-(10-甲基-9-蒽基)-2,2’:6’2”-联三吡啶-6,6”-二甲胺]四乙酸(MTTA)、2-(1’,1’,2’,2’,3’,3’-七氟-4’,6’-己二酮-6’-基)-萘(NHA)、4,4’-二(2”,3”,3”-七氟-4”,6”-己二酮-6”-基)-邻-三联苯(BHHT)、4,4’-二(1”,1”,1”,2”,2”,3”,3”-七氟-4”,6”-己二酮-6”-基)-氯代磺基-邻-三联苯(BHHCT)、4,7-联苯-1,10-菲咯啉(DPP)、1,1,1-三氟丙酮(TTA)、3-萘酰-1,1,1-三氟丙酮(NPPTA)、萘基三氟丁二酮(NTA)、三辛基氧化膦(TOPO)、三苯基氧化膦(TPPO)、3-苯甲酰-1,1,1-三氟丙酮(BFTA)、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
在本发明的一些实施方式中，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
在本发明的一些实施方式中，所述基质选自高分子微球，优选为乳胶微球，更优选为聚苯乙烯乳胶微球。
在本发明的一些实施方式中，所述试剂盒还包括供体，所述供体能够在激发状态下生成活性氧。
在本发明的一些优选的实施方式中，所述供体为光活化的或化学活化的敏化剂，其为非粒子形式，且在含水介质中可溶。
在本发明的一些实施方式中，所述供体为填充有光敏剂的高分子微粒，其在光激发下能够产生活性氧。
在本发明的一些实施方式中，所述光敏剂选自亚甲基蓝、玫瑰红、卟碄和酞菁中的一种。
在本发明的一些实施方式中，供体与链霉亲和素相连。
在本发明的一些实施方式中，所述试剂盒还包括生物素标记物，其包括与生物素结合的能够与待测目标分子直接或间接结合的生物大分子，其中所述生物大分子选自蛋白质分子、核酸分子、多糖分子和脂类分子；优选为蛋白质分子；进一步优选地，所述蛋白质分子选自抗原和/或抗体；其中，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
在本发明的一些实施方式中，所述活性氧为单线态氧。
Ⅲ.实施例
为使本发明更加容易理解，下面将结合实施例来进一步详细说明本发明，这些实施例仅起说明性作用，并不局限于本发明的应用范围。本发明中所使用的原料或组分若无特殊说明均可以通过商业途径或常规方法制得。
试剂和仪器：
SA(Sigma Aldrich公司)，羧基官能团化的硅基微球(粒径15-200nm,孔径2-15nm,Sigma Aldrich公司)，磷酸盐缓冲液(0.02M PBS，pH 7.4)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDAC(Thermo fisher)，Tween-20，0.1M MES缓冲液(pH 6.0)，生物素(D-biotin)，去激素血清，三碘甲状腺原氨酸(T3)检测试剂盒(博阳生物科技(上海)有限公司)，光激化学发光分析系统通用液(感光珠溶液/链霉亲和素标记的供体溶液)。LiCA HT(博阳生物科技(上海)有限公司)，日立高速冷冻离心机。
物理吸附方式制备本发明的抗干扰剂
实施例1
第一步，2mL离心管中，取10mg羧基官能团化的硅基微球(粒径15nm， 孔径2nm)，加入0.02M PBS(pH 7.4)的缓冲液，4℃离心10000rpm，15min清洗一次。
第二步，加入200uL PBS缓冲液超声分散均匀，再加入150uL 10mg/mL SA的水溶液，补充PBS缓冲液至微球反应浓度20mg/mL,室温搅拌过夜。
第三步，用含有0.5％Tween-20的0.02M PBS(pH 7.4)缓冲溶液将SA微球离心，4℃离心10000rpm，15min，清洗三次，去除未吸附的SA，最后用0.02M PBS(pH 7.4)缓冲液定容至10mg/mL。
实施例2-7
制备方法同实施例1，区别在于各实施例采用了不同粒径和/或孔径的羧基官能团化的硅基微球(参见表1)。
共价偶联方式制备本发明的抗干扰剂
实施例8(共价偶联方式)
第一步，2mL离心管中，取10mg羧基官能团化的硅基微球，用0.1M MES(pH 6.0)的缓冲溶液，4℃离心10000rpm，15min清洗一次。
第二步，加入200uL 0.1M MES(pH 6.0)缓冲液超声分散均匀，再加入150uL 10mg/mL SA的水溶液，接着加入100uL 10mg/mLEDAC(0.1M MES)溶液，室温搅拌4h。
第三步，用含有0.5％Tween-20的0.02M PBS(pH 7.4)缓冲溶液将SA微球离心清洗三次，去除未吸附的SA，最后用PBS缓冲液定容至10mg/mL。
实施例9：三碘甲状腺原氨酸均相化学发光检测试剂盒
试剂盒中包括以下组分：
组分a，实施例1-8所制备的抗干扰剂；
组分b，供体试剂/感光珠溶液/通用液：链霉亲和素偶联标记的供体溶液；
组分c，受体试剂/试剂一：二碘甲状腺氨酸包被的受体溶液；
组分d，生物素标记物/试剂二：生物素标记的抗三碘甲状腺原氨酸抗体溶液。其中，组分b～d的制备方法请参见中国专利CN 101865917 B实施例1～3。
实施例10：本发明抗生物素干扰的均相化学发光检测试剂盒的效果评价一实验步骤：
1.添加T3浓溶液于去激素血清中，配制成浓度为1nmol/L、2nmol/L的T3溶液。
2.配制感光珠溶液40ug/mL，采用实施例1-7中制备的抗干扰剂配制不同浓度的溶液(用PBS稀释)，见表1。
3.添加生物素于上述T3溶液中，配制成生物素浓度分别为0，128ng/ml的样本溶液。
4.加入25ul样本溶液，再依次加入T3试剂盒中的试剂一(含有二碘甲状腺氨酸包被的受体溶液)和试剂二(含有生物素标记的抗三碘甲状腺原氨酸抗体溶液)各25uL(按反应模式手工加入25ul样本溶液及25uL试剂一和25uL试剂二)，再按照如下表1加入25uL步骤2制备的抗干扰剂溶液，其中条件1、2不加入抗干扰剂。
5.放入LICA HT中，进行第一阶段温育：37℃温育17min。
6.手工加入175ul通用液(含有链霉亲和素标记的供体)。
7.进行第二阶段温育：37℃温育15min，温育后得到相应的待测样品。
8.利用能量激发上述待测样品并对产生的发光信号读数。读数结果参见表2和3。
表1
Figure PCTCN2019128668-appb-000001
Figure PCTCN2019128668-appb-000002
Figure PCTCN2019128668-appb-000003
数据分析：
当T3浓度为1nM、生物素浓度为128ng/mL时，化学发光免疫反应的信号下跌89％，生物素干扰较严重。加入孔径2nm的微球(表1中的序号3)时，发光信号几乎无明显变化，当采用粒径50nm，孔径5nm，10nm(表1中的序号4和5)时，发光信号出现一定的提升，下跌幅度50％-70％。
当粒径和孔径不变时，提高抗干扰剂的浓度至10ug/mL(表1中的序号5和6对比对比)，发光信号进一步提升，下跌幅度25％左右。当孔径10nm，浓度10ug/ml，增大微球粒径至100nm(表1中的序号6和7)时，下跌幅度偏差10％以内，生物素干扰现象消失。当浓度提高至20ug/ml(表1中的序号7和8)时，信号下跌偏差10％以内。当浓度20ug/mL不变，增大粒径和孔径时，信号出现一定程度下跌，下跌幅度20％-40％。
实验结论：
当本发明所述试剂盒包括的抗干扰剂为，填充SA(链霉亲和素)的微球粒 径100nm，孔径10nm，浓度10-20ug/mL时，所述试剂盒的抗生物素干扰能力最强。当所添加的抗干扰剂的孔径较小，2nm时，所述试剂盒无抗生物素干扰能力。当所添加的抗干扰剂的粒径大于100nm，孔径10nm，继续增大粒径和孔径时，所述试剂盒的抗生物素干扰能力会下降。
实施例11：本发明抗生物素干扰的均相化学发光检测试剂盒的效果评价二实验步骤：
1.添加T3浓溶液于去激素血清中，配制成浓度为1nmol/L、2nmol/L的T3溶液。
2.配制感光珠溶液40ug/mL，采用实施例5和8中制备的抗干扰剂配制不同浓度的溶液(用PBS稀释)，见表4(用PBS稀释)10ug/mL、20ug/mL。
3.添加生物素于上述T3溶液中，配制成生物素浓度分别为0、128ng/ml的样本溶液。
4.加入25ul样本溶液，再依次加入T3试剂盒中的试剂一和试剂二各25uL(按反应模式手工加入25ul样本溶液及25uL试剂一和25uL试剂二)，再按照如下表格加入25uL步骤2中制备的抗干扰剂溶液，条件1和2不加入抗干扰剂。
5.放入LICA HT中，进行第一阶段温育：37℃温育17min。
6.手工加入175ul感光珠溶液。
7.进行第二阶段温育：37℃温育15min，温育后得到相应的待测样品。
8.利用能量激发上述待测样品和并对产生的发光信号读数。读数结果参见表5和6。
表4
Figure PCTCN2019128668-appb-000004
Figure PCTCN2019128668-appb-000005
数据分析：
从表4中的序号3和5对比，物理吸附方式制备的抗干扰剂的抗生物素干扰能力强，发光信号下跌10％左右，共价偶联方式制备的抗干扰剂的发光信号下跌50％左右。表4中的序号4和6对比，物理吸附方式制备的抗干扰剂的抗生物素干扰能力也优于共价偶联的方式制备的抗干扰剂。
实验结论：
对于相同粒径，孔径，浓度，比表面积，包括物理吸附方式制备的抗干扰剂的光激化学发光试剂盒的抗生物素干扰能力优于包括共价偶联的方式制备的抗干扰剂的光激化学发光试剂盒。
应当注意的是，以上所述的实施例仅用于解释本发明，并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述，但应当理解为其中所用的词语为描述性和解释性词汇，而不是限定性词汇。可以按规定在本发明权利要求的范围内对本发明作出修改，以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的方法、材料和实施例，但是并不意味着本发明限于其中公开的特定例，相反，本发明可扩展至其他所有具有相同功能的方法和应用。

Claims (38)

  1. 一种均相化学发光检测试剂盒，其包括：
    抗干扰剂，其包括载体和活性分子；所述载体为多孔介质；所述活性分子填充于所述载体之中，并能够与生物素分子特异性结合；
    受体，其能够与活性氧反应产生可检测的化学发光信号。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述抗干扰剂能够识别游离生物素分子和生物素标记物。
  3. 根据权利要求1或2所述的试剂盒，其特征在于，所述抗干扰剂能够选择性吸附游离生物素分子。
  4. 根据权利要求3所述的试剂盒，其特征在于，所述游离生物素分子能够扩散到所述载体中，并与其中的所述活性分子特异性结合。
  5. 根据权利要求1-4中任意一项所述的试剂盒，其特征在于，所述抗干扰剂能够限制比所述活性分子尺寸更大的生物大分子进入其载体之中。
  6. 根据权利要求1-5中任意一项所述的试剂盒，其特征在于，所述抗干扰剂能够在液相反应体系中均匀分布。
  7. 根据权利要求1-6中任意一项所述的抗试剂盒，其特征在于，所述载体的内表面积大于其外表面积；优选地，所述载体的内表面积为其外表面积的5倍以上，优选为10倍以上，更优选为20倍以上；和/或，
    所述载体的粒径为15-300nm，优选为30-250nm，更优选为50-200nm；和/或，
    所述载体的比表面积为200m 2/g以上，优选为400m 2/g以上，更优选为600m 2/g以上，最优选为1000m 2/g以上；和/或，
    所述载体的最小孔隙率大于40％，优选大于50％，更优选大于60％。
  8. 根据权利要求1-7中任意一项所述的试剂盒，其特征在于，所述多孔介质选自多孔金属材料、多孔非金属材料和多孔高分子材料中的一种或多种。
  9. 根据权利要求1-8中任意一项所述的抗试剂盒，其特征在于，所述载体为介孔微球，优选为有序介孔微球。
  10. 根据权利要求9所述的试剂盒，其特征在于，所述介孔微球的孔径为2-50nm，优选为4-30nm，更优选为5-15nm。
  11. 根据权利要求9或10所述的试剂盒，其特征在于，所述介孔微球为笼状中空介孔微球。
  12. 根据权利要求9-11中任意一项所述的试剂盒，其特征在于，所述介孔微球选自Al 2O 3介孔材料、WO 3介孔材料、TiO 2介孔材料、ZrO 2介孔材料、硅基介孔材料和/或介孔碳材料中的至少一种，优选选自硅基介孔材料。
  13. 根据权利要求1-12中任意一项所述的试剂盒，其特征在于，所述活性分子选自亲和素和/或链霉亲和素。
  14. 根据权利要求1-13中任意一项所述的试剂盒，其特征在于，所述活性分子通过物理吸附方式填充于所述载体中。
  15. 根据权利要求1-14中任意一项所述的试剂盒，其特征在于，所述活性分子通过在含有缓冲液的体系中与载体接触而填充于所述载体之中。
  16. 根据权利要求1-13中任意一项所述的试剂盒，其特征在于，所述活性分子通过直接或间接化学交联的方式填充于所述载体之中。
  17. 根据权利要求1-16中任意一项所述的试剂盒，其特征在于，所述载体内表面修饰有化学基团，所述活性分子通过与所述化学基团的共价偶联而填充于所述载体之中；其中，所述化学基团选自羧基、醛基、氨基、巯基和羟基中的一种或多种。
  18. 根据权利要求1-17中任意一项所述的试剂盒，其特征在于，所述载体内表面连接有生物素分子，所述活性分子通过与生物素分子的特异结合作用而填充于所述载体之中。
  19. 根据权利要求1-18中任意一项所述的试剂盒，其特征在于，所述抗干扰剂还包括缓冲溶液，优选PBS缓冲溶液。
  20. 根据权利要求1-19中任意一项所述的试剂盒，其特征在于，所述抗干扰剂的制备方法包括：步骤S1，使载体与活性分子进行接触；优选地，所述接触在第一缓冲液体系中进行。
  21. 根据权利要求20所述的试剂盒，其特征在于，所述抗干扰剂的制备方法还包括步骤S0：用第二缓冲液体系将载体进行清洗，步骤S0在步骤S1之前进行。
  22. 根据权利要求20或21所述的试剂盒，其特征在于，所述抗干扰剂的制备方法还包括步骤S2：除去未填充到载体中的活性分子，步骤S2在步骤S1之后 进行；优选地，通过向步骤S1处理后的载体中加入第三缓冲液体系，然后进行固液分离，以除去未填充到载体中的活性分子。
  23. 根据权利要求1-22中任意一项所述的试剂盒，其特征在于，所述受体的表面直接地或间接地与生物活性物质连接，所述生物活性物质能够与待测目标分子特异性结合。
  24. 根据权利要求1-23中任意一项所述的试剂盒，其特征在于，所述生物活性物质为抗原和/或抗体；所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
  25. 根据权利要求1-24中任意一项所述的试剂盒，其特征在于，所述受体包含化学发光化合物和金属螯合物，其为非粒子形式，且在含水介质中可溶。
  26. 根据权利要求1-24中任意一项所述的试剂盒，其特征在于，所述受体包含发光组合物和基质，所述发光组合物填充于基质中和/或包被于基质表面。
  27. 根据权利要求26所述的试剂盒，其特征在于，所述发光组合物能够与活性氧反应产生可检测的化学发光信号，其包含化学发光化合物和金属螯合物。
  28. 根据权利要求25-27中任意一项所述的试剂盒，其特征在于，所述化学发光化合物选自烯烃化合物，优选选自二甲基噻吩、双丁二酮化合物、二氧杂环己烯、烯醇醚、烯胺、9-亚烷基苍耳烷、9-亚烷基-N-9,10二氢化吖啶、芳基乙醚烯、芳基咪唑和光泽精以及它们的衍生物，更优选选自二甲基噻吩及其衍生物。
  29. 根据权利要求25-28中任意一项所述的试剂盒，其特征在于，所述金属螯合物的金属是稀土金属或VIII族金属，优选选自铕、铽、镝、钐、锇和钌，更优选选自铕。
  30. 根据权利要求25-29中任意一项所述的试剂盒，其特征在于，所述金属螯合物包含选自下列的螯合剂：NHA、BHHT、BHHCT、DPP、TTA、NPPTA、NTA、TOPO、TPPO、BFTA、2,2-二甲基-4-全氟丁酰-3-丁酮(fod)、2,2’-联吡啶(bpy)、联吡啶基羧酸、氮杂冠醚、氮杂穴状配体和三辛基氧化膦以及它们的衍生物。
  31. 根据权利要求25-30中任意一项所述的试剂盒，其特征在于，所述发光化合物是二甲基噻吩的衍生物，所述金属螯合物是铕螯合物。
  32. 根据权利要求26-31中任意一项所述的试剂盒，其特征在于，所述基质选自高分子微球，优选为乳胶微球，更优选为聚苯乙烯乳胶微球。
  33. 根据权利要求1-32中任意一项所述的试剂盒，其特征在于，所述试剂盒 中还包括供体，所述供体能够在激发状态下生成活性氧；优选地，所述供体为光活化的或化学活化的敏化剂，其为非粒子形式，且在含水介质中可溶。
  34. 根据权利要求33所述的试剂盒，其特征在于，所述供体为填充有光敏剂的高分子微粒，其在光激发下能够产生活性氧。
  35. 根据权利要求34所述的试剂盒，其特征在于，所述光敏剂选自亚甲基蓝、玫瑰红、卟碄和酞菁中的一种。
  36. 根据权利要求33-35中任意一项所述的试剂盒，其特征在于，供体与链霉亲和素相连。
  37. 根据权利要求1-36中任意一项所述的试剂盒，其特征在于，所述试剂盒还包括生物素标记物，其包括与生物素结合的能够与待测目标分子直接或间接结合的生物大分子，其中所述生物大分子选自蛋白质分子、核酸分子、多糖分子和脂类分子；优选为蛋白质分子；进一步优选地，所述蛋白质分子选自抗原和/或抗体；其中，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
  38. 根据权利要求1-37中任意一项所述的试剂盒，其特征在于，所述活性氧为单线态氧。
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