CN111122852A - Homogeneous phase chemiluminescence analysis method and application thereof - Google Patents

Homogeneous phase chemiluminescence analysis method and application thereof Download PDF

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CN111122852A
CN111122852A CN201811452234.4A CN201811452234A CN111122852A CN 111122852 A CN111122852 A CN 111122852A CN 201811452234 A CN201811452234 A CN 201811452234A CN 111122852 A CN111122852 A CN 111122852A
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carrier
molecule
biotin
sample
active
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章春奇
金鑫
赵卫国
刘宇卉
李临
其他发明人请求不公开姓名
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Beyond Diagnostics Shanghai Co ltd
Chemclin Diagnostics Corp
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Chemclin Diagnostics Corp
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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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

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Abstract

The invention relates to a homogeneous phase chemiluminescence analysis method and application thereof in the technical field of homogeneous phase chemiluminescence. The method comprises the following steps: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a chemiluminescent signal generated by the reaction of a receptor in the sample to be detected and active oxygen; wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecules are filled in the carrier and can be specifically combined with biotin molecules. The method can avoid false positive or false negative result caused by free biotin.

Description

Homogeneous phase chemiluminescence analysis method and application thereof
Technical Field
The invention belongs to the technical field of chemiluminescence, and particularly relates to a homogeneous phase chemiluminescence analysis method and application thereof.
Background
The chemiluminescence analysis methods are classified into homogeneous chemiluminescence analysis methods and heterogeneous chemiluminescence analysis methods according to whether separation is performed or not. Heterogeneous chemiluminescence analysis methods require multiple operations such as embedding, elution, separation and the like, are complicated in analysis process and long in analysis time, and cannot meet the requirements of rapid detection and diagnosis. The homogeneous chemiluminescence analysis method effectively avoids complicated steps such as elution and separation, greatly improves the analysis efficiency and the cost performance, and is increasingly widely applied. The Biotin-Avidin System (BAS) is a new type of amplification System for biological reactions developed in the late 70 s. The BAS system has the advantages of high affinity, high sensitivity, high stability and the like. Combining the two to couple macromolecular bioactive substances such as antigen and antibody. Their combination is rapid, specific, stable and has multi-stage amplification effect. At present, the BAS system is mainly applied to the fields of immunology, molecular biology and the like. The method is particularly advantageous in the practical application of in vitro diagnosis, and the greatest defect of the method is that the interference of biotin causes detection errors.
Biotin interference may produce false positives and may also produce false negatives. Generally, the sandwich method produces false negatives and the competition method produces false positives. The current common solutions are: 1. replacing platforms that do not use an avidin-biotin system; 2. retesting every other day or after one week after the withdrawal of the relevant drugs/foods; 3. sample pretreatment: the streptavidin-coated microparticles remove the biotin from the sample.
However, there is no method that can solve the problem of biotin interference well in the avidin-biotin system.
Disclosure of Invention
The invention provides a homogeneous phase chemiluminescence analysis method aiming at the defects of the prior art, and the method can well solve the problem of biotin interference.
To this end, the present invention provides in a first aspect a homogeneous chemiluminescent assay method comprising the steps of: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of a receptor in the sample to be detected and active oxygen;
wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecules are filled in the carrier and can be specifically combined with biotin molecules.
In some embodiments of the invention, the anti-interference agent is capable of recognizing a free biotin molecule and a biotin label.
In some embodiments of the invention, the anti-interference agent is capable of selectively adsorbing free biotin molecules.
In other embodiments of the invention, the free biotin molecule is capable of diffusing into the carrier and specifically binding to the active molecule therein.
In some embodiments of the invention, the anti-interference agent is capable of restricting the entry of a biological macromolecule larger in size than the active molecule into its carrier.
In other embodiments of the present invention, the anti-interference agent can be uniformly distributed in the liquid phase reaction system.
In some embodiments of the invention, the porous medium is selected from one or more of a porous metal material, a porous non-metal material, and a porous polymer material.
In other embodiments of the present invention, the support is a mesoporous microsphere, preferably an ordered mesoporous microsphere.
In some embodiments of the present invention, the pore size of the mesoporous microsphere is 2 to 50nm, preferably 4 to 30nm, and more preferably 5 to 15 nm.
In other embodiments of the present invention, the mesoporous microsphere is a cage-shaped hollow mesoporous microsphere.
In some preferred embodiments of the present invention, the mesoporous microspheres are selected from Al2O3Mesoporous material and WO3Mesoporous material and TiO2Mesoporous material, ZrO2At least one of the mesoporous material, the silicon-based mesoporous material and/or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
In some embodiments of the invention, the active molecule is selected from avidin and/or streptavidin.
In other embodiments of the present invention, the active molecule is packed in the carrier by physical adsorption.
In some preferred embodiments of the invention, the active molecule is loaded into the carrier by contacting the carrier in a system comprising a buffer.
In other preferred embodiments of the invention, the pH of the buffer containing system is from 7 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, even more preferably from 7.3 to 7.6.
In some embodiments of the invention, the active molecule is loaded into the carrier by direct or indirect chemical crosslinking.
In some preferred embodiments of the present invention, 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, aldehyde group, amino, sulfhydryl and hydroxyl.
In other preferred embodiments of the present invention, the carrier has a biotin molecule attached to its inner surface, and the active molecule is filled in the carrier by specific binding to the biotin molecule.
In some embodiments of the invention, the anti-interference agent further comprises a buffer solution, preferably a PBS buffer solution.
In some embodiments of the present invention, the preparation method of the anti-interference agent comprises: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in a first buffer system.
In some preferred embodiments of the present invention, the preparation method of the anti-interference agent further includes step S0: the carrier is washed with the second buffer system, and step S0 is performed before step S1.
In other preferred embodiments of the present invention, the method for preparing the anti-interference agent further includes step S2: removing the active molecules not filled into the carrier, step S2 being performed after step S1; preferably, the active molecules not filled in the carrier are removed by adding a third buffer solution system to the carrier treated in step S1 and then performing solid-liquid separation.
In some embodiments of the invention, the receptor surface is directly or indirectly linked to a biomacromolecule capable of specifically binding to a target molecule to be detected.
In other embodiments of the present invention, the substance in the sample to be tested comprises a biotin label comprising a biomacromolecule that binds to biotin and is capable of binding directly or indirectly to a target molecule to be tested.
In some preferred embodiments of the invention, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; preferably a protein molecule; further preferably, the protein molecule is selected from the group consisting of an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.
In some embodiments of the invention, the test sample further comprises a donor; the donor surface is directly or indirectly bound to a biotin-specific binding substance and is capable of generating reactive oxygen species in an excited state.
In some embodiments of the invention, the method comprises the steps of:
s1, mixing the sample to be detected with a reagent a containing a receptor, a reagent b containing a biotin marker, a reagent c containing an anti-interference agent and a reagent d containing a donor to obtain a sample to be detected;
s2, contacting the sample to be tested obtained in the step S1 with energy or active compounds to excite the donor to generate active oxygen;
and S3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of the receptor and the active oxygen in the sample to be detected.
In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with a reagent a containing a receptor, a reagent b containing a biotin label, and a reagent c containing an anti-interference agent, and then mixed with a reagent d containing a donor, thereby obtaining a sample to be tested.
In some embodiments of the invention, in step S2, the sample to be tested obtained in step S1 is irradiated with 600-700 nm red excitation light to excite the sample to be tested to generate chemiluminescence.
In some embodiments of the present invention, in step S3, the detection wavelength of the luminescence signal value is recorded to be 520-620 nm.
In a second aspect, the present invention provides a homogeneous chemiluminescent assay device for performing homogeneous chemiluminescent assay using the method of the first aspect of the present invention.
In a third aspect, the present invention provides a method of controlling a homogeneous chemiluminescent detection apparatus according to the second aspect of the present invention.
In a fourth aspect, the present invention provides the use of a method according to the first aspect of the present invention or a detection apparatus according to the second aspect of the present invention or a control method according to the third aspect of the present invention in a detection of a biotin-streptavidin system; preferably in thyroid function detection; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.
The invention has the beneficial effects that: the method is carried out in the presence of an anti-interference agent, and the anti-interference agent can effectively distinguish free biotin molecules from biotin markers by taking active molecules such as SA or avidin protein molecules and the like as 'guest molecules' and filling the 'guest molecules' in pores of a porous medium in a proper manner to form a 'mesoporous assembled host-guest' system, so that the method can eliminate the interference of the free biotin and avoid false positive and/or false negative results in chemiluminescence immunoassay. In addition, the method disclosed by the invention also has practicability and universality, can be applied to different technical platforms, and has small influence on the performance of the reagent.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Term(s) for
The term "homogeneous phase chemiluminescence reaction" refers to a process in which under homogeneous phase conditions, a receptor, a donor and a target molecule to be detected are combined to form a compound, and the formed compound generates chemiluminescence under the irradiation of excitation light.
The term "carrier" as used herein refers to a substance capable of carrying active molecules together to participate in a chemical or physical process. The chemical composition of the carrier in the present invention is not particularly limited, and may be organic or inorganic, such as high molecular polymer, metal, glass, mineral salt, diatom, phospholipid vesicle, silicon particle, microcrystalline dye, etc.
The term "porous medium" as used herein refers to a substance composed of a skeleton composed of a solid substance and a plurality of fine voids densely grouped and partitioned by the skeleton.
The term "active molecule" as used herein refers to a molecule having the ability to specifically bind to a biotin molecule. Exemplary reactive molecules are avidin and streptavidin.
The term "test sample" as used herein refers to a mixture containing or suspected of containing a target molecule to be tested. Samples to be tested that can be used in the present disclosure include bodily fluids such as blood (which can be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected will be used for the detection of the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody or a hapten which allows it to bind to an antibody.
The term "sample to be measured" as used herein refers to a multi-component mixed liquid to be measured, which contains a sample to be measured, a reagent containing a donor, a reagent containing an acceptor, and a reagent containing an anti-interference agent, before being subjected to on-machine detection analysis.
The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.
The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.
The term "active oxygen" as used herein refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited state of oxygen molecules, including the reduction of one electron of oxygenProduct superoxide anion (O)2(-) and the two-electron reduction product hydrogen peroxide (H)2O2) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and singlet oxygen (1O)2) And the like.
In the present invention, the term "acceptor" refers to a substance capable of reacting with reactive oxygen species to generate a detectable signal. The donor is induced by energy or an active compound to activate and release a high energy state of reactive oxygen species, which is captured by a close proximity acceptor, thereby transferring energy to activate the acceptor. In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with reactive oxygen species (e.g., singlet oxygen) to form an unstable metastable intermediate that can decompose with or following luminescence. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin. In other embodiments of the invention, the acceptor is an olefin capable of reacting with a reactive oxygen species (e.g., singlet oxygen) to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes that can react with reactive oxygen species (e.g., singlet oxygen) to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety. In other embodiments of the invention, the receptor comprises an olefinic compound and a metal chelate, which is non-particulated and soluble in an aqueous medium, as in the case of the receptor described in patent PCT/US2010/025433 (which is incorporated herein by reference in its entirety). .
In the present invention, the "chemiluminescent compound", i.e., a compound referred to as a label, may undergo a chemical reaction to cause luminescence, such as by being converted to another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly, or may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy-acceptor microsphere will be transitioned to an excited state to emit light.
The phrase "capable of binding, directly or indirectly," means that the specified entity is capable of specifically binding to the entity (directly), or that the specified entity is capable of specifically binding to a specific binding pair member, or a complex having two or more specific binding partners capable of binding to other entities (indirectly).
The "specific binding pair member" of the present invention is selected from the group consisting of (1) a small molecule and a binding partner for the small molecule, and (2) a macromolecule and a binding partner for the macromolecule
In the present invention, the active oxygen may be provided by a "donor". The term "donor" as used herein refers to a sensitizer capable of generating a reactive intermediate such as singlet oxygen that reacts with an acceptor upon activation by energy or an active compound. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor is a photosensitizer which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No. 5,5709994, which is incorporated herein by reference in its entirety, as well as derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group for attachment to a specific binding partner. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference. In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or which absorb light directly to release active oxygen (e.g., singlet oxygen).
Photosensitizers generally activate chemiluminescent compounds by irradiating the medium containing the reactant. The medium must be irradiated with light having a wavelength and an energy sufficient to convert the photosensitizer to an excited state, thereby enabling it to activate molecular oxygen to singlet oxygen. The excited state of a photosensitizer capable of exciting molecular oxygen is generally in the triplet state, which is about 20Kcal/mol, usually at least 23Kcal/mol higher than the energy of the photosensitizer in the ground state. Preferably, the medium is irradiated with light having a wavelength of about 450 and 950nm, although shorter wavelengths, such as 230 and 950nm, may be used. The light generated can be measured in any conventional manner, such as by photography, visual inspection, photometer, etc., to determine its amount relative to the amount of analyte in the medium. The photosensitizer is preferably relatively non-polar to ensure solubility into the lipophilic member.
The photosensitizer and/or chemiluminescent compound may be selected to be dissolved in, or non-covalently bound to, the surface of the particle. In this case, the compounds are preferably hydrophobic to reduce their ability to dissociate from the particles, thereby allowing both compounds to bind to the same particle.
Detailed description of the preferred embodiments
The present invention will be described in more detail below.
According to the method, an anti-interference agent is added, and the anti-interference agent is filled in a carrier in a proper mode by taking an active molecule capable of being specifically combined with a biotin molecule as an object molecule, so that a mesoporous assembly host-object system is formed, and therefore the homogeneous chemiluminescence analysis method avoids false positive or false negative results caused by free biotin.
The homogeneous chemiluminescence analysis method comprises the following steps: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of a receptor in the sample to be detected and active oxygen;
wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecules are filled in the carrier and can be specifically combined with biotin molecules. The expression "the active molecule is filled in the carrier" means that the active molecule is located in a void in the carrier, and may or may not be in contact with the skeleton.
In some embodiments of the invention, the anti-interference agent is capable of recognizing a free biotin molecule and a biotin label. In the present invention, "recognition" may mean that the active molecule in the anti-interference agent and the free biotin molecule and/or biotin label are combined with each other through the synergistic effect of intermolecular forces.
In some embodiments of the invention, the anti-interference agent is capable of selectively adsorbing free biotin molecules.
In other embodiments of the invention, the free biotin molecule is capable of diffusing into the carrier and specifically binding to the active molecule therein. In the present invention, the "diffusion" may mean that free biotin molecules are dispersed into a carrier due to random movement of the molecules.
In some embodiments of the invention, the anti-interference agent is capable of restricting the entry of a biological macromolecule larger in size than the active molecule into its carrier.
In other embodiments of the present invention, the anti-interference agent can be uniformly distributed in the liquid phase reaction system.
In some embodiments of the invention, the vector satisfies at least one of the following conditions: a) the inner pores of the carrier have a sufficiently large surface area (far exceeding the surface area of the carrier), and the voids can only allow the entry of active molecules, but limit the larger proteins of the active molecules, such as antibodies or large antigens, etc.; b) active molecules such as SA or avidin can be filled in the carrier, for example, inside the voids, by a chemical or physical adsorption method; c) the carrier can be stably and uniformly distributed in a solution (e.g., an aqueous solution) without precipitation.
In some embodiments of the invention, the inner surface area of the support is greater than the outer surface area thereof; preferably, the internal surface area of the support is more than 5 times, preferably more than 10 times, more preferably more than 20 times the external surface area thereof. In some preferred embodiments of the invention, the internal surface area of the support 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.
In other embodiments of the present invention, the particle size of the support is 15-300nm, such as 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 100nm, 250nm, 300nm, etc., preferably 30-250nm, more preferably 50-200 nm. Too large a carrier particle size can cause the carrier to settle too quickly, which is not conducive to forming a stable, uniform solution.
In some embodiments of the invention, the support has a specific surface area of 200m2Per g or more, e.g. 200m2/g、400m2/g、600m2/g、800m2/g、1000m2/g、1200m2/g、1500m2G, etc., preferably 400m2More preferably 600 m/g or more2More than g, most preferably 1000m2More than g.
In other embodiments of the present invention, the support has a minimum porosity of greater than 40%, preferably greater than 50%, more preferably greater than 60%.
In some embodiments of the invention, the porous medium is selected from one or more of a porous metal material, a porous non-metal material, and a porous polymer material.
In other embodiments of the present invention, the support is a mesoporous microsphere, preferably an ordered mesoporous microsphere.
In some embodiments of the present invention, the pore size of the mesoporous microsphere is 2 to 50nm, such as 2nm, 5nm, 10, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 50nm, etc., preferably 4 to 30nm, and more preferably 5 to 15 nm.
In other embodiments of the present invention, the mesoporous microsphere is a cage-shaped hollow mesoporous microsphere.
In some preferred embodiments of the present invention, the mesoporous microspheres are selected from Al2O3Mesoporous material and WO3Mesoporous material and TiO2Mesoporous material, ZrO2At least one of the mesoporous material, the silicon-based mesoporous material and/or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
The silicon-based mesoporous material is made of SiO2(CH2)2Periodic mesoporous material composed of tetrahedral structural units. The mesoporous silica materials can be microscopically classified into two types: one is disordered mesoporous solids represented by silica xerogels and aerogels. The disordered mesoporous silica can be powder, block, sheet or film in macroscopic view. The other is an ordered mesoporous silica represented by MCM 41. The ordered mesoporous silica has the structural characteristics that the pore size is uniform, the ordered mesoporous silica is arranged in a hexagonal order, and the pore size of the mesoporous silica can be adjusted between 2nm and 10 nm. Because the hole wall is thin, the silicon-based unit has low alternating current degree and poor hydrothermal stability. The specific surface area can reach 1000m2(ii) in terms of/g. Also SBA series, HMM series, TUD series, FSM series, KIT series, CMK series, FDU series, starbon series, etc. Among them, SBA-15 has more research, and the hydrothermal stability of the material is better than that of MCM series. The aperture is adjustable between 5nm and 30 nm. HMM is a spherical mesoporous material, the aperture of which is 4-15nm, and the outer diameter of which is 20-80nm and is adjustable.
In some embodiments of the invention, the active molecule is selected from avidin and/or streptavidin. Avidin is a glycoprotein extracted from egg white, has a molecular weight of about 60kD, is composed of 4 subunits per molecule, and can be closely bound to 4 biotin molecules. Such avidin includes, but is not limited to: avidin, streptavidin, vitellin, and avidin-like. Streptavidin (SA), a protein with biological properties similar to those of avidin (A), is a protein product secreted by Streptomyces avidins during culture, and SA can also be produced by genetic engineering means. The molecular weight of SA is 65000, and consists of 4 peptide chains with the same sequence, and each SA peptide chain can combine with 1 biotin molecule. Thus, like avidin, each SA molecule also has 4 binding sites for biotin molecules with a binding constant of 1015mol/L as avidin.
In other embodiments of the present invention, the active molecule is packed in the carrier by physical adsorption. Physical adsorption, also known as van der waals adsorption, is caused by intermolecular forces between the adsorbate and the adsorbent, also known as van der waals forces.
In some preferred embodiments of the invention, the active molecule is loaded into the carrier by contacting the carrier in a system comprising a buffer.
In other preferred embodiments of the invention, the pH of the buffer containing system is from 7 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, even more preferably from 7.3 to 7.6.
In some embodiments of the invention, the active molecule is loaded into the carrier by direct or indirect chemical crosslinking.
In some preferred embodiments of the present invention, 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, aldehyde group, amino, sulfhydryl and hydroxyl.
In other preferred embodiments of the present invention, the carrier has a biotin molecule attached to its inner surface, and the active molecule is filled in the carrier by specific binding to the biotin molecule.
In some embodiments of the invention, the anti-interference agent further comprises a buffer solution, preferably a PBS buffer solution.
In other embodiments of the invention, the total concentration of the carrier and the active molecule 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 10-20 ug/mL.
In some embodiments of the present invention, the preparation method of the anti-interference agent comprises: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in a first buffer system. In some embodiments of the above method, the pH of the first buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH 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.
In still other embodiments of the above method, the pH of the first buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH 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, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
According to some embodiments, the method further comprises step S0: the carrier is washed with the second buffer system, and step S0 is performed before step S1. In some embodiments of the above method, the pH of the second buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH 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. In still other embodiments of the above method, the pH of the second buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH 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, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
According to some embodiments, the method further comprises step S2: the active molecules not filled in the carrier are removed, and step S2 is performed after step S1. Preferably, the active molecules not filled in the carrier are removed by adding a third buffer solution system to the carrier treated in step S1 and then performing solid-liquid separation.
In some embodiments of the above methods, the pH of the third buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, even more preferably from 7.3 to 7.7, even more preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH 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. In still other embodiments of the above method, the pH of the third buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH 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, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
In some embodiments of the above methods, the first buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above methods, the second buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above methods, the third buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
In some embodiments of the above methods, the third buffer system further comprises a surfactant. According to some embodiments, the surfactant comprises one or more selected from the group consisting of Tween-20, Tween-80, Triton X-405, Triton X-100, BRIJ 35 and Pluronic L64. According to some embodiments, the surfactant comprises tween-20.
In some embodiments of the above method, the contacting temperature is 0-50 ℃, preferably 20-40 ℃, such as 25-30 ℃ (i.e., room temperature) in step S1; 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. In some other embodiments of the above method, the contacting temperature is from 0 to 50 ℃, preferably from 20 to 40 ℃, e.g., from 25 to 30 ℃ (i.e., room temperature); and/or the contact time is 1 to 10 hours, preferably 2 to 6 hours, such as 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.
In some embodiments of the above method, further comprising step S3, adding a fourth buffer system. Preferably, the fourth buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulphonic acid buffer, a 3-morpholinopropanesulphonic acid buffer, a 4-hydroxyethylpiperazine ethanesulphonic acid buffer and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulphonic acid buffer.
In some embodiments of the invention, the receptor surface is directly or indirectly linked to a biomacromolecule capable of specifically binding to a target molecule to be detected.
In other embodiments of the present invention, the substance in the sample to be tested comprises a biotin label comprising a biomacromolecule that binds to biotin and is capable of binding directly or indirectly to a target molecule to be tested.
In some preferred embodiments of the invention, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; preferably a protein molecule; further preferably, the protein molecule is selected from the group consisting of an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.
In some embodiments of the invention, the test sample further comprises a donor; the donor surface is directly or indirectly bound to a biotin-specific binding substance and is capable of generating reactive oxygen species in an excited state. Preferably, the biotin-specific binding agent is streptavidin.
In some embodiments of the invention, the method comprises the steps of:
s1, mixing the sample to be detected with a reagent a containing a receptor, a reagent b containing a biotin marker, a reagent c containing an anti-interference agent and a reagent d containing a donor to obtain a sample to be detected;
s2, contacting the sample to be tested obtained in the step S1 with energy or active compounds to excite the donor to generate active oxygen;
and S3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of the receptor and the active oxygen in the sample to be detected.
In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with a reagent a containing a receptor, a reagent b containing a biotin label, and a reagent c containing an anti-interference agent, and then mixed with a reagent d containing a donor, thereby obtaining a sample to be tested.
In some embodiments of the invention, in step S2, the sample to be tested obtained in step S1 is irradiated with 600-700 nm red excitation light to excite the sample to be tested to generate chemiluminescence.
In other embodiments of the present invention, in step S3, the detection wavelength of the luminescence signal value is recorded to be 520 to 620 nm.
It is to be noted that the method of the present invention is not limited to the sandwich method detection, and can be used for detection by a capture method, a competition method, or the like.
The second aspect of the present invention relates to a homogeneous chemiluminescent assay device utilizing the method of the first aspect of the present invention for homogeneous chemiluminescent assay; homogeneous chemiluminescent detection of the anti-biotin interference is preferably performed.
A third aspect of the present invention relates to a method for controlling the homogeneous chemiluminescence detection apparatus according to the second aspect of the present invention.
The fourth aspect of the present invention relates to the use of a method according to the first aspect of the present invention or a detection apparatus according to the second aspect of the present invention or a control method according to the third aspect of the present invention in a detection of the biotin-streptavidin system; preferably in thyroid function detection; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.
Example III
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Reagents and instrumentation:
SA (Sigma Aldrich), carboxyl-functionalized silica-based microspheres (particle size 15-200nm, pore size 2-15nm, Sigma Aldrich), phosphate buffer (0.02M PBS, pH 7.4), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDAC (thermo fisher), Tween-20, 0.1M MES buffer (pH 6.0), biotin (D-biotin), serenoid, triiodothyronine (T3) detection kit (Boyang Biotech (Shanghai) Co., Ltd.), photospherd-solution (photosphere solution/streptavidin-labeled donor solution). LiCA HT (boyang biotechnology (shanghai) ltd), hitachi high speed refrigerated centrifuge.
The anti-interference agent is prepared by a physical adsorption mode
Example 1
In the first step, 10mg of carboxyl-functionalized silica-based microspheres (particle size 15nm, pore size 2nm) were taken in a 2mL centrifuge tube, 0.02M PBS (pH 7.4) buffer was added, centrifuged at 10000rpm at 4 ℃ and washed once for 15 min.
And secondly, adding 200uL PBS buffer solution, performing ultrasonic dispersion uniformly, adding 150uL 10mg/mL SA water solution, supplementing the PBS buffer solution until the microsphere reaction concentration is 20mg/mL, and stirring at room temperature overnight.
Thirdly, centrifuging the SA microspheres by using 0.02M PBS (pH 7.4) buffer solution containing 0.5% Tween-20, centrifuging at 10000rpm at 4 ℃, washing for three times, removing unadsorbed SA, and finally diluting to 10mg/mL by using 0.02M PBS (pH 7.4) buffer solution.
Examples 2 to 7
The preparation method is the same as example 1, except that carboxyl-functionalized silica-based microspheres with different particle sizes and/or pore sizes are used in each example (see table 1).
Covalent coupling method for preparing anti-interference agent
Example 8 (covalent coupling mode)
In the first step, 10mg of carboxyl functionalized silica-based microspheres were taken in a 2mL centrifuge tube and washed once with 0.1M MES (pH 6.0) buffer at 4 ℃ at 10000rpm for 15 min.
In the second step, 200uL of 0.1M MES (pH 6.0) buffer was added and dispersed by sonication, followed by 150uL of 10mg/mL SA in water, followed by 100uL of 10mg/mLEDAC (0.1M MES) solution and stirring at room temperature for 4 h.
Thirdly, the SA microspheres are centrifugally washed three times by using 0.02M PBS (pH 7.4) buffer solution containing 0.5% Tween-20 to remove unadsorbed SA, and finally, the volume is adjusted to 10mg/mL by using PBS buffer solution.
Example 9: effect evaluation of homogeneous chemiluminescence analysis method of anti-biotin interference
The experimental steps are as follows:
1. a concentrated solution of T3 was added to the serum of degerming serum to prepare a solution of T3 at concentrations of 1nmol/L and 2 nmol/L.
2. A40 ug/mL solution of beads was prepared and solutions of different concentrations (diluted in PBS) were prepared using the anti-interference agents prepared in examples 1-7, as shown in Table 1.
3. Biotin was added to the above T3 solution to prepare sample solutions having biotin concentrations of 0 and 128ng/ml, respectively.
4. Adding 25uL of sample solution, sequentially adding 25uL of reagent I (containing diiodothyronine-coated receptor) and reagent II (containing biotin-labeled anti-triiodothyronine antibody) in the T3 kit (manually adding 25uL of sample solution, 25uL of reagent I and 25uL of reagent II according to the reaction mode), and adding 25uL of the anti-interference agent solution prepared in the step 2 according to the following table 1, wherein the anti-interference agent is not added under the conditions 1 and 2.
5. Put into LICA HT, a first stage incubation is performed: incubate at 37 ℃ for 17 min.
6. Add 175ul of universal solution (containing streptavidin labeled donor) manually.
7. Performing a second stage incubation: incubation is carried out for 15min at 37 ℃ and the corresponding sample to be tested is obtained after incubation.
8. And exciting the sample to be detected by using energy and reading the generated luminescent signal. See tables 2 and 3 for reading results.
TABLE 1
Figure BDA0001886946640000161
Figure BDA0001886946640000171
Figure BDA0001886946640000172
And (3) data analysis:
when the concentration of T3 is 1nM and the concentration of biotin is 128ng/mL, the signal of chemiluminescence immune response drops by 89%, and biotin interference is severe. When the microspheres with the pore diameter of 2nm (the number 3 in the table 1) are added, the luminescent signals are almost not obviously changed, and when the microspheres with the particle diameters of 50nm, the pore diameters of 5nm and 10nm (the numbers 4 and 5 in the table 1) are adopted, the luminescent signals are improved to a certain extent, and the falling amplitude is 50-70%.
When the particle size and the aperture are not changed, the concentration of the anti-interference agent is increased to 10ug/mL (serial numbers 5 and 6 in Table 1 are compared), the luminous signal is further increased, and the falling amplitude is about 25%. When the aperture is 10nm and the concentration is 10ug/ml, and the particle size of the microsphere is increased to 100nm (serial numbers 6 and 7 in table 1), the drop amplitude deviation is within 10%, and the biotin interference phenomenon disappears. When the concentration was increased to 20ug/ml (numbers 7 and 8 in Table 1), the signal dropped within 10% of the deviation. When the concentration is not changed by 20ug/mL and the particle size and the aperture are increased, the signal falls to a certain extent, and the falling amplitude is 20-40%.
And (4) experimental conclusion:
when the anti-interference agent added by the method is the microspheres filled with SA (streptavidin) and has the particle size of 100nm, the pore diameter of 10nm and the concentration of 10-20ug/mL, the anti-biotin interference capability of the method is strongest. When the aperture of the added anti-interference agent is smaller and is 2nm, the method has no anti-biotin interference capability. When the added anti-interference agent has a particle size of more than 100nm and a pore size of 10nm, the anti-biotin interference ability of the method is reduced when the particle size and the pore size are continuously increased.
Example 10: effect evaluation of homogeneous chemiluminescence analysis method of anti-biotin interference of the invention
The experimental steps are as follows:
1. a concentrated solution of T3 was added to the serum of degerming serum to prepare a solution of T3 at concentrations of 1nmol/L and 2 nmol/L.
2. A40 ug/mL solution of beads was prepared, and different concentrations of the anti-interference agent prepared in examples 5 and 8 (diluted in PBS) were prepared, as shown in Table 4 (diluted in PBS) at 10ug/mL and 20 ug/mL.
3. Biotin was added to the above T3 solution to prepare sample solutions having biotin concentrations of 0 and 128ng/ml, respectively.
4. Adding 25uL of sample solution, sequentially adding 25uL of reagent I and reagent II in the T3 kit (adding 25uL of sample solution, 25uL of reagent I and 25uL of reagent II manually according to the reaction mode), and adding 25uL of the anti-interference agent solution prepared in the step 2 according to the following table, wherein the anti-interference agent is not added under the conditions 1 and 2.
5. Put into LICA HT, a first stage incubation is performed: incubate at 37 ℃ for 17 min.
6. Add 175ul of universal solution by hand.
7. Performing a second stage incubation: incubation is carried out for 15min at 37 ℃ and the corresponding sample to be tested is obtained after incubation.
8. Exciting the sample to be tested by using energy and reading the generated luminescent signal. See tables 5 and 6 for reading results.
TABLE 4
Figure BDA0001886946640000191
Figure BDA0001886946640000192
And (3) data analysis:
from serial numbers 3 and 5 in table 4, the anti-biotin interference ability of the anti-interference agent prepared by the physical adsorption method was strong, the luminescent signal dropped by about 10%, and the luminescent signal of the anti-interference agent prepared by the covalent coupling method dropped by about 50%. In numbers 4 and 6 in table 4, the anti-biotin interference ability of the anti-interference agent prepared by physical adsorption is better than that of the anti-interference agent prepared by covalent coupling.
And (4) experimental conclusion:
the anti-biotin interference capability of the light-activated chemiluminescence analysis method of the anti-interference agent prepared by adding the physical adsorption mode with the same particle size, pore diameter, concentration and specific surface area is superior to that of the light-activated chemiluminescence analysis method of the anti-interference agent prepared by adding the covalent coupling mode.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims (33)

1. A homogeneous chemiluminescent assay method comprising the steps of: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of a receptor in the sample to be detected and active oxygen;
wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecules are filled in the carrier and can be specifically combined with biotin molecules.
2. The method of claim 1, wherein the anti-interference agent is capable of recognizing a free biotin molecule and a biotin label.
3. The method of claim 1 or 2, wherein the anti-interference agent is capable of selectively adsorbing free biotin molecules.
4. The method of claim 3, wherein said free biotin molecule is capable of diffusing into said carrier and specifically binding to said active molecule therein.
5. The method of any one of claims 1 to 4, wherein the anti-interference agent is capable of restricting the passage of a biological macromolecule larger than the size of the active molecule into its carrier.
6. The method according to any one of claims 1 to 5, wherein the anti-interference agent is capable of being uniformly distributed in a liquid phase reaction system.
7. The method of any one of claims 1 to 6, wherein the porous medium is selected from one or more of a porous metallic material, a porous non-metallic material and a porous polymeric material.
8. The method according to any one of claims 1 to 7, wherein the support is a mesoporous microsphere, preferably an ordered mesoporous microsphere.
9. The method according to claim 8, wherein the pore size of the mesoporous microsphere is 2-50nm, preferably 4-30nm, and more preferably 5-15 nm.
10. The method of claim 8 or 9, wherein the mesoporous microspheres are cage-shaped hollow mesoporous microspheres.
11. The method according to any one of claims 8 to 10, wherein the mesoporous microspheres are selected from Al2O3Mesoporous material and WO3Mesoporous material and TiO2Mesoporous material, ZrO2At least one of the mesoporous material, the silicon-based mesoporous material and/or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
12. The method according to any one of claims 1 to 11, wherein the active molecule is selected from avidin and/or streptavidin.
13. The method according to any one of claims 1 to 12, wherein the active molecules are packed in the carrier by physical adsorption.
14. The method according to any one of claims 1 to 13, wherein the active molecule is loaded into the carrier by contacting the carrier in a system comprising a buffer.
15. The method according to claim 14, wherein the pH of the buffer containing system is 7 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, further preferably 7.3-7.6.
16. The method according to any one of claims 1 to 12, wherein the active molecules are loaded into the carrier by direct or indirect chemical cross-linking.
17. The method of claim 16, wherein the carrier is modified on its inner surface with chemical groups, and the active molecule is filled in the carrier by covalent coupling with the chemical groups; wherein the chemical group is selected from one or more of carboxyl, aldehyde group, amino, sulfhydryl and hydroxyl.
18. The method according to any one of claims 1 to 17, wherein the carrier has a biotin molecule attached to its inner surface, and the active molecule is packed in the carrier by specific binding to the biotin molecule.
19. The method of any one of claims 1 to 18, wherein the anti-interference agent further comprises a buffer solution, preferably a PBS buffer solution.
20. The method of any one of claims 1-19, wherein the anti-interference agent is prepared by a method comprising: step S1, contacting the carrier with the active molecule; preferably, the contacting is performed in a first buffer system.
21. The method according to claim 20, wherein the preparation method of the anti-interference agent further comprises step S0: the carrier is washed with the second buffer system, and step S0 is performed before step S1.
22. The method according to claim 20 or 21, wherein the preparation method of the anti-interference agent further comprises step S2: removing the active molecules not filled into the carrier, step S2 being performed after step S1; preferably, the active molecules not filled in the carrier are removed by adding a third buffer solution system to the carrier treated in step S1 and then performing solid-liquid separation.
23. The method of any one of claims 1 to 22, wherein the receptor surface is directly or indirectly linked to a biomacromolecule capable of specifically binding to a target molecule to be detected.
24. The method according to any one of claims 1 to 23, wherein the substance in the sample to be tested comprises a biotin label comprising a biomacromolecule capable of binding directly or indirectly to the target molecule to be tested, which binds to biotin.
25. The method of claim 23 or 24, wherein the biological macromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; preferably a protein molecule; further preferably, the protein molecule is selected from the group consisting of an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.
26. The method of any one of claims 1-25, wherein the test sample further comprises a donor; the donor surface is directly or indirectly bound to a biotin-specific binding substance and is capable of generating reactive oxygen species in an excited state.
27. The method according to any one of claims 1-26, characterized in that the method comprises the steps of:
s1, mixing the sample to be detected with a reagent a containing a receptor, a reagent b containing a biotin marker, a reagent c containing an anti-interference agent and a reagent d containing a donor to obtain a sample to be detected;
s2, contacting the sample to be tested obtained in the step S1 with energy or active compounds to excite the donor to generate active oxygen;
and S3, analyzing and judging whether the sample to be detected contains the target molecules to be detected and/or the concentration of the target molecules to be detected by detecting the intensity of a luminescent signal generated by the reaction of the receptor and the active oxygen in the sample to be detected.
28. The method according to claim 27, wherein in step S1, the sample to be tested is mixed with a reagent a containing an acceptor, a reagent b containing a biotin label, and a reagent c containing an anti-interference agent, and then mixed with a reagent d containing a donor, thereby obtaining a sample to be tested.
29. The method according to claim 27 or 28, wherein in step S2, the sample to be tested obtained in step S1 is irradiated with 600-700 nm red excitation light to excite chemiluminescence.
30. The method according to any one of claims 27 to 29, wherein in step S3, the detection wavelength of the luminescence signal value is recorded to be 520 to 620 nm.
31. A homogeneous chemiluminescent assay device utilizing the method of any one of claims 1-30 for homogeneous chemiluminescent assay.
32. A method of controlling the homogeneous chemiluminescent assay device of claim 31.
33. Use of a method according to any one of claims 1 to 30 or a detection device according to claim 31 or a control method according to claim 32 in the detection of a biotin-streptavidin system; preferably in thyroid function detection; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102448995A (en) * 2009-04-29 2012-05-09 茵维特罗根戴纳股份公司 Monodisperse submicron polymer particles
CN102735833A (en) * 2012-07-09 2012-10-17 沃克(天津)生物科技有限公司 Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof
CN103837675A (en) * 2014-03-07 2014-06-04 常艳敏 Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method
EP3176580A1 (en) * 2015-12-01 2017-06-07 Roche Diagnostics GmbH Method for reduction of interferences in immunoassays
CN107976535A (en) * 2017-11-03 2018-05-01 北京科美生物技术有限公司 The homogeneous immunological detection reagent box of target IgM antibody and its application method and application in a kind of detection sample
CN108051585A (en) * 2017-11-27 2018-05-18 北京科美生物技术有限公司 A kind of homogeneous immunological detection reagent box, detection method and its application
CN108333344A (en) * 2017-12-26 2018-07-27 南京迪格诺斯生物技术有限公司 Highly sensitive chemical luminescence immune analysis reagent box and its preparation method and application

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160025736A1 (en) * 2013-03-15 2016-01-28 Siemens Healthcare Diagnostics Inc. Heterogeneous luminescent oxygen channeling immunoassays and methods of production and use thereof
CN108152490A (en) * 2017-12-22 2018-06-12 太原瑞盛生物科技有限公司 A kind of chemiluminescence detection kit of Big ET-1 and preparation method thereof
CN108445223A (en) * 2018-02-11 2018-08-24 北京科美生物技术有限公司 Detect homogeneous immunological detection reagent box and its application of the anti-Carp antibody of target

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102448995A (en) * 2009-04-29 2012-05-09 茵维特罗根戴纳股份公司 Monodisperse submicron polymer particles
CN102735833A (en) * 2012-07-09 2012-10-17 沃克(天津)生物科技有限公司 Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof
CN103837675A (en) * 2014-03-07 2014-06-04 常艳敏 Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method
EP3176580A1 (en) * 2015-12-01 2017-06-07 Roche Diagnostics GmbH Method for reduction of interferences in immunoassays
CN107976535A (en) * 2017-11-03 2018-05-01 北京科美生物技术有限公司 The homogeneous immunological detection reagent box of target IgM antibody and its application method and application in a kind of detection sample
CN108051585A (en) * 2017-11-27 2018-05-18 北京科美生物技术有限公司 A kind of homogeneous immunological detection reagent box, detection method and its application
CN108333344A (en) * 2017-12-26 2018-07-27 南京迪格诺斯生物技术有限公司 Highly sensitive chemical luminescence immune analysis reagent box and its preparation method and application

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHRISTINA TRAMBAS ET AL: "Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin–biotin immunoassays", ANNALS OF CLINICAL BIOCHEMISTRY, vol. 55, no. 2, pages 216 - 226, XP055698199, DOI: 10.1177/0004563217707783 *
刘佳等: "介孔材料固定化酶的研究进展及其应用前景", 《石油化工》 *
刘佳等: "介孔材料固定化酶的研究进展及其应用前景", 《石油化工》, vol. 43, no. 4, 31 December 2014 (2014-12-31), pages 358 *
田修营等: "介孔材料的研究进展及应用前景", 《山东轻工业学院学报(自然科学版)》 *
田修营等: "介孔材料的研究进展及应用前景", 《山东轻工业学院学报(自然科学版)》, no. 02, 15 June 2008 (2008-06-15), pages 20 - 28 *

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