CN118010979A

CN118010979A - Homogeneous chemiluminescence analysis method and application thereof

Info

Publication number: CN118010979A
Application number: CN202410282111.XA
Authority: CN
Inventors: 金鑫; 王程荣; 刘宇卉; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2018-10-31
Filing date: 2018-11-30
Publication date: 2024-05-10
Also published as: CN111122852A; WO2020088698A1

Abstract

The invention relates to a homogeneous chemiluminescence analysis method and application thereof in the technical field of homogeneous 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 a target molecule to be detected and/or the concentration of the target molecule 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 molecule is filled in the carrier and can be specifically combined with biotin molecules. The method can avoid false positive or false negative results caused by free biotin.

Description

Homogeneous chemiluminescence analysis method and application thereof

Technical Field

The invention belongs to the technical field of chemiluminescence, and particularly relates to a homogeneous chemiluminescence analysis method and application thereof.

Background

Chemiluminescent assays are classified into homogeneous chemiluminescent assays and heterogeneous chemiluminescent assays depending on whether they are separated or not. The heterogeneous chemiluminescent analysis method requires multi-step operations such as embedding, eluting, separating and the like, has complicated analysis process and long analysis time, and cannot meet the requirements of rapid detection and diagnosis. The homogeneous phase chemiluminescence analysis method effectively avoids complex steps such as elution and separation, greatly improves analysis efficiency and cost performance, and is increasingly widely applied. The Biotin-Avidin System (BAS) was a novel biological reaction amplification System developed at the end of the 70 s. The BAS system has the advantages of high affinity, high sensitivity, high stability and the like. The combination of the two can couple macromolecular bioactive substances such as antigen and antibody. Their combination is rapid, specific and stable, and has multistage amplification effect. The BAS system is mainly applied to the fields of immunology, molecular biology and the like at present. The method has great superiority in the practical application of in vitro diagnosis, and the biggest disadvantage of the method is that the method has biotin interference, which causes detection errors.

Biotin interference may produce false positives as well as false negatives. In general, sandwich methods produce false negatives and competition methods produce false positives. The current common solution is: 1. replacing the platform without using the avidin-biotin system; 2. retesting every other day or one week after the withdrawal of the relevant drug/food; 3. sample pretreatment: streptavidin coated microparticles remove biotin from the sample.

However, there is no method to solve the problem of biotin interference in avidin-biotin systems.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a homogeneous chemiluminescence analysis method which can well solve the problem of biotin interference.

To this end, the first aspect of the present invention provides a homogeneous chemiluminescent assay comprising the steps of: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the luminous signal intensity generated by the reaction of the receptor in the sample to be detected and the active oxygen;

Wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecule is 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 free biotin molecules and biotin labels.

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 biological macromolecules larger than the size of 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 metallic material, a porous nonmetallic material, and a porous polymeric material.

In other embodiments of the invention, the carrier is a mesoporous microsphere, preferably an ordered mesoporous microsphere.

In some embodiments of the invention, the mesoporous microspheres have a pore size of from 2 to 50nm, preferably from 4 to 30nm, more preferably from 5 to 15nm.

In other embodiments of the invention, the mesoporous microspheres are cage-like hollow mesoporous microspheres.

In some preferred embodiments of the invention, the mesoporous microspheres are selected from at least one of Al ₂O₃ mesoporous material, WO ₃ mesoporous material, tiO ₂ mesoporous material, zrO ₂ mesoporous material, silicon-based mesoporous material and/or mesoporous carbon material, preferably from silicon-based mesoporous material.

In some embodiments of the invention, the active molecule is selected from avidin and/or streptavidin.

In other embodiments of the invention, the active molecule is packed in the carrier by physical adsorption.

In some preferred embodiments of the invention, the active molecule is filled in the carrier by contacting it in a system comprising a buffer.

In other preferred embodiments of the invention, the pH of the buffer-containing system is 7 to 9, preferably 7.1 to 8.0, more preferably 7.2 to 7.8, even more preferably 7.3-7.6.

In some embodiments of the invention, the active molecule is filled in the carrier by means of direct or indirect chemical crosslinking.

In some preferred embodiments of the invention, the inner surface of the carrier is modified with chemical groups, and the active molecules are filled in the carrier by covalent coupling with the chemical groups; wherein the chemical group is selected from one or more of carboxyl, aldehyde, amino, mercapto and hydroxyl.

In other preferred embodiments of the present invention, biotin molecules are attached to the inner surface of the carrier, and the active molecules are filled in the carrier by specific binding to the biotin molecules.

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 invention, the method for preparing the anti-interference agent comprises: step S1, contacting a carrier with an active molecule; preferably, the contacting is performed in a first buffer system.

In some preferred embodiments of the present invention, the method for preparing the anti-interference agent further comprises step S0: the carrier is washed with a second buffer system, step S0 being performed before step S1.

In other preferred embodiments of the present invention, the method for preparing the anti-interference agent further comprises step S2: removing active molecules not filled in the carrier, and performing step S2 after step S1; preferably, the active molecules that are not filled in the carrier are removed by adding a third buffer 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 biological macromolecule capable of specifically binding to the target molecule to be detected.

In other embodiments of the invention, the substance in the test sample comprises a biotin label comprising a biological macromolecule capable of binding directly or indirectly to the test target molecule, bound to biotin.

In some preferred embodiments of the invention, 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 an antigen and/or an antibody; wherein the antigen refers to a substance having immunogenicity; the antibody refers to an immunoglobulin produced by an organism and capable of recognizing a specific foreign object.

In some embodiments of the invention, the sample to be tested further comprises a donor; the donor surface is directly or indirectly bound to a biotin-specific binding agent and is capable of generating active oxygen in an excited state.

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

s1, mixing a sample to be tested with a reagent a containing an acceptor, a reagent b containing a biotin label, a reagent c containing an anti-interference agent and a reagent d containing a donor to obtain a sample to be tested;

S2, contacting the sample to be detected obtained in the step S1 by using energy or an active compound, and exciting a donor to generate active oxygen;

S3, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the luminous signal intensity generated by the reaction of the receptor in the sample to be detected and the active oxygen.

In some preferred embodiments of the present invention, in step S1, a sample to be tested is mixed with a reagent a comprising a receptor, a reagent b comprising a biotin label, a reagent c comprising an anti-interference agent, and then mixed with a reagent d comprising a donor, thereby obtaining a sample to be tested.

In some embodiments of the present invention, in step S2, the sample to be tested obtained in step S1 is irradiated with 600-700 nm of red excitation light to excite the sample to generate chemiluminescence.

In some embodiments of the present invention, in step S3, the detection wavelength for recording the luminescence signal value is 520 to 620nm.

In a second aspect the invention provides a homogeneous chemiluminescent detection device which utilises the method of the first aspect of the invention for homogeneous chemiluminescent detection.

In a third aspect the present invention provides a method of controlling a homogeneous chemiluminescent detection assembly 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 invention or a detection device according to the second aspect of the invention or a control method according to the third aspect of the invention in the detection of a biotin-streptavidin system; preferably in the detection of thyroid function; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.

The beneficial effects of the invention are as follows: the method is carried out in the presence of an anti-interference agent, and active molecules such as SA or avidin protein molecules and the like are used as 'guest molecules', so that 'mesoporous assembled host-guest' systems are formed by filling the active molecules in the pores of a porous medium in a proper mode, and free biotin molecules and biotin markers can be effectively distinguished, so that the method can eliminate the interference of the free biotin, and false positive and/or false negative results in chemiluminescent immunoassay are avoided. In addition, the method provided by the invention has practicability and universality, can be applied to different technical platforms, and has little influence on the performance of the reagent.

Detailed Description

In order that the invention may be readily understood, the invention will be described in detail. Before the present 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, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

I. terminology

The term "homogeneous chemiluminescent reaction" refers to a process in which under homogeneous conditions, an acceptor, a donor and a target molecule to be detected are combined to form a complex, and the complex formed under irradiation of excitation light produces chemiluminescence.

The term "carrier" as used herein refers to a substance that is capable of carrying an active molecule together in a chemical or physical process. The chemical composition of the carrier 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 minute voids separated by the skeleton into a large number of densely packed groups.

The term "active molecule" as used herein refers to a molecule having the ability to specifically bind to a biotin molecule. Exemplary active molecules are avidin and streptavidin.

The term "test sample" as used herein refers to a mixture that is tested for the presence or suspected presence of a test target molecule. Samples to be tested that may be used in the present disclosure include body fluids such as blood (which may be anticoagulated blood as is commonly found 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 measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing 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 may be used to detect the target molecule. The target molecule to be tested may be a protein, peptide, antibody or hapten which can be conjugated to an antibody.

The term "sample to be measured" 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, a reagent containing an anti-interference agent, and the like, before on-machine detection and analysis.

The term "antibody" as used herein is used in its 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 binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.

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

The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants.

The term "active oxygen" refers to a general term of substances which contain oxygen and are active in nature and consist of oxygen in a body or in a natural environment, and mainly comprises oxygen molecules in an excited state, including a one-electron reduction product superoxide anion (O ₂ -), a two-electron reduction product hydrogen peroxide (H ₂O₂), a three-electron reduction product hydroxyl radical (OH), nitric oxide, singlet oxygen (1O ₂) and the like.

In the present invention, the term "receptor" refers to a substance that can react with active oxygen to produce a detectable signal. The donor is induced to activate by energy or an active compound and releases active oxygen in a high energy state which is captured by the acceptor in close proximity, thereby transferring energy to activate the acceptor. In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with active oxygen (e.g., singlet oxygen) to form an unstable metastable intermediate that may decompose while or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan, 9-alkylidene-N-alkyl acridines, arylvinyl ethers, bisoxyethylene, dimethylthiophene, aromatic imidazoles or gloss concentrates. In other embodiments of the invention, the acceptor is an olefin capable of reacting with active oxygen (e.g., singlet oxygen) to form a hydroperoxide or dioxetane that can decompose to a ketone or carboxylic acid derivative; stable dioxetanes that 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 of azo compounds or azocarbonyl compounds, such as luminol, may be formed; and aromatic compounds which can form endoperoxides. A specific, non-limiting example of a receptor that can be utilized in accordance with the present disclosure and claimed invention is described in U.S. patent No. US5340716 (which is incorporated herein by reference in its entirety). In other embodiments of the present invention, the acceptor comprises an olefinic compound and a metal chelate that is non-particulated and soluble in an aqueous medium, as described in PCT/US2010/025433 (which is incorporated herein by reference in its entirety). .

In the present invention, the "chemiluminescent compound", a compound known 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 by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor microsphere will be transitioned to an excited state to emit light.

The term "capable of binding directly or indirectly" means that a given entity is capable of specifically binding to the entity (directly), or that the given 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 invention is selected from (1) a small molecule and a binding partner for the small molecule, and (2) a large molecule and a binding partner for the large molecule

In the present invention, the active oxygen may be provided by a "donor". The term "donor" as used herein refers to a sensitizer that upon activation of energy or an active compound is capable of generating an active intermediate such as singlet oxygen that reacts with the acceptor. 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, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Pat. No. 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 atom substituents for making these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding partners. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in U.S. Pat. No. 3, 6406913, 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. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and active oxygen (e.g., singlet oxygen) may be released by heating these compounds or by direct absorption of light by these compounds.

Photosensitizers generally activate chemiluminescent compounds by irradiating a medium containing the reactants described above. The medium must be irradiated with light having a wavelength and energy sufficient to convert the photosensitizer into 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 typically in the triplet state, which is about 20Kcal/moL, typically at least 23Kcal/moL, higher than the energy of the photosensitizer in the ground state. Preferably, light having a wavelength of about 450-950nm is used to illuminate the medium, although shorter wavelengths, such as 230-950nm, may be used. The light produced may be measured in any conventional manner, such as photographic, visual, photometer, etc., to determine its amount related to the analyte content of the medium. The photosensitizer is preferably relatively non-polar to ensure dissolution 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 particles.

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 active molecules capable of being specifically combined with biotin molecules are used as 'guest molecules', so that a 'mesoporous assembled host-guest' system is formed by filling the active molecules into a carrier in a proper mode, and therefore false positive or false negative results caused by free biotin are avoided in the homogeneous chemiluminescent analysis method.

The homogeneous chemiluminescent analysis method according to the first aspect of the present invention comprises the steps of: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the luminous signal intensity generated by the reaction of the receptor in the sample to be detected and the active oxygen;

wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the active molecule is filled in the carrier and can be specifically combined with biotin molecules. The active molecule is filled in the carrier, and the active molecule is positioned in the gaps in the carrier and can be contacted with the framework or not contacted with the framework.

In some embodiments of the invention, the anti-interference agent is capable of recognizing free biotin molecules and biotin labels. In the present invention, "recognition" may mean that the active molecules in the anti-interference agent and the free biotin molecules and/or biotin labels are bound to each other by the synergistic action of intermolecular forces.

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 refer to the dispersion of free biotin molecules into a carrier due to the random movement of the molecules.

In some embodiments of the invention, the carrier meets at least one of the following conditions: a) The inner pores of the carrier have a sufficiently large surface area (far overload body surface area) and the interstices only allow access of the active molecules, but limit larger proteins of the active molecules, such as antibodies or large antigens; b) Active molecules such as SA or Avdin can be filled in the carrier by chemical or physical adsorption method, such as the inside of the gaps; c) The carrier is 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 carrier is greater than the outer surface area thereof; preferably, the inner surface area of the support is 5 times or more, preferably 10 times or more, more preferably 20 times or more the outer surface area thereof. In some preferred embodiments of the present invention, the inner surface area of the support is a multiple of its outer surface area including, but not limited to: 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 16-fold, 18-fold, 20-fold, 22-fold, 24-fold, 26-fold, 28-fold or 30-fold.

In other embodiments of the invention, the particle size of the support is 15-300nm, e.g., 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 100nm, 250nm, 300nm, etc., preferably 30-250nm, more preferably 50-200nm. The excessive particle size of the carrier can cause excessive rapid sedimentation of the carrier, which is unfavorable for forming stable and uniform solution.

In some embodiments of the invention, the support has a specific surface area of 200m ²/g or more, such as 200m²/g、400m²/g、600m²/g、800m²/g、1000m²/g、1200m²/g、1500m²/g, etc., preferably 400m ²/g or more, more preferably 600m ²/g or more, most preferably 1000m ²/g or more.

In other embodiments of the invention, the minimum porosity of the support is greater than 40%, preferably greater than 50%, more preferably greater than 60%.

In some embodiments of the invention, the mesoporous microspheres have a pore size of from 2 to 50nm, for example from 2nm, 5nm, 10, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 50nm, etc., preferably from 4 to 30nm, more preferably from 5 to 15nm.

The silicon-based mesoporous material is a periodic mesoporous substance formed by SiO ₂(CH₂)₂ tetrahedral structural units. Mesoporous silica materials can be microscopically divided into two categories: one type is a disordered mesoporous solid represented by silica xerogels and aerogels. The disordered mesoporous silica can be macroscopically powder, block, sheet or film. The other is 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 according to a hexagonal order, and the mesoporous pore size can be adjustable between 2 nm and 10 nm. Because the pore wall is thinner, the silicon-based unit has low alternating current degree and poor hydrothermal stability. The specific surface area can reach 1000m ²/g. Also SBA series, HMM series, TUD series, FSM series, KIT series, CMK series, FDU series, starbon, etc. The SBA-15 has more researches, 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 spherical mesoporous material, its aperture is 4-15nm, and its external diameter is 20-80 nm.

In some embodiments of the invention, the active molecule is selected from avidin and/or streptavidin. Avidin is a glycoprotein which is extracted from egg white and has a molecular weight of about 60kD and consists of 4 subunits per molecule which can be intimately associated with 4 biotin molecules. The avidin includes, but is not limited to: ovalbumin, streptavidin, vitelline avidin and avidin-like elements. Streptavidin (SA) is a protein with similar biological properties to avidin (A), which is a protein product secreted by Streptomyces avidin bacteria during culture, and SA can also be produced by genetic engineering means. The molecular weight of SA is 65000, and the SA consists of 4 peptide chains with the same sequence, and each SA peptide chain can be combined with 1 biotin molecule. Thus, as with avidin, each SA molecule also has 4 biotin-binding sites, with a binding constant of 1015moL/L as with avidin.

In other embodiments of the invention, the active molecule is packed in the carrier by physical adsorption. Physical adsorption, also known as van der waals adsorption, is caused by the forces exerted between the adsorbate and the adsorbent molecules, also known as van der waals forces.

In other embodiments of the invention, the total concentration of the carrier and active molecules loaded in the carrier in the anti-interference agent is 5-50ug/mL, e.g., 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.

In some embodiments of the invention, the method for preparing the anti-interference agent comprises: step S1, contacting a carrier with an active molecule; preferably, the contacting is performed in a first buffer system. In some embodiments of the above methods, 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, still more preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of pH values 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, etc.

In further 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, still more preferably from 5.0 to 6.4, still more preferably from 5.5 to 6.3, most preferably 6.0. In this embodiment, examples of pH values 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, etc.

According to some embodiments, the method further comprises step S0: the carrier is washed with a second buffer system, step S0 being performed before step S1. In some embodiments of the above methods, 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, still more preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of pH values 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, etc. In further embodiments of the above method, the pH 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, still more preferably 5.5 to 6.3, most preferably 6.0. In this embodiment, examples of pH values 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, etc.

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 that are not filled in the carrier are removed by adding a third buffer system to the carrier treated in step S1 and then performing solid-liquid separation.

In some embodiments of the above method, 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, still more preferably from 7.3 to 7.7, still more preferably from 7.35 to 7.50, most preferably 7.40. In this embodiment, examples of pH values 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, etc. In further embodiments of the above method, the pH 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, still more preferably 5.5 to 6.3, most preferably 6.0. In this embodiment, examples of pH values 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, etc.

In some embodiments of the above methods, the first buffer liquid system comprises one or more selected from the group consisting of phosphate buffer, piperazine-1, 4-diethylsulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 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 phosphate buffer, piperazine-1, 4-diethylsulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 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 phosphate buffer, piperazine-1, 4-diethylsulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 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, in step S1, the temperature of the contacting is from 0 to 50 ℃, preferably from 20 to 40 ℃, such as from 25 to 30 ℃ (i.e., room temperature); and/or the time of contact is 6 to 24 hours, preferably 8 to 12 hours, for example 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc. In some other embodiments of the above process, the temperature of the contacting is from 0 to 50 ℃, preferably from 20 to 40 ℃, such as from 25 to 30 ℃ (i.e., room temperature); and/or the time of contact is 1 to 10 hours, preferably 2 to 6 hours, for example 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 phosphate buffer, piperazine-1, 4-diethylsulfonic acid buffer, 3-morpholinopropansulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.

In some embodiments of the invention, the sample to be tested further comprises a donor; the donor surface is directly or indirectly bound to a biotin-specific binding agent and is capable of generating active oxygen in an excited state. Preferably, the biotin-specific binding agent is streptavidin.

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

In other embodiments of the present invention, in step S3, the detection wavelength for recording the luminescence signal value is 520 to 620nm.

It is noted that the method of the present invention is not limited to sandwich detection, but can be used for detection by capture, competition, etc.

A second aspect of the invention relates to a homogeneous chemiluminescent detection device for homogeneous chemiluminescent detection by the method according to the first aspect of the invention; preferably, a homogeneous chemiluminescent detection of the avidin interference is performed.

A third aspect of the invention relates to a method of controlling a homogeneous chemiluminescent detection device according to the second aspect of the invention.

A fourth aspect of the invention relates to the use of a method according to the first aspect of the invention or a detection device according to the second aspect of the invention or a control method according to the third aspect of the invention in the detection of a biotin-streptavidin system; preferably in the detection of thyroid function; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.

III. Examples

In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.

Reagents and instrumentation:

SA (SIGMA ALDRICH Co.) and carboxyl functionalized silica-based microspheres (particle size 15-200nm, pore size 2-15nm,Sigma Aldrich Co.) 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), norhormonal serum, triiodothyronine (T3) detection kit (Boyang biosciences, shanghai), photo-activated chemiluminescent assay System Universal (photosensitive bead solution/streptavidin labeled donor solution). LiCA HT (Boyang Biotechnology (Shanghai) Inc.), hitachi high-speed refrigerated centrifuge.

Physical adsorption mode for preparing the anti-interference agent

Example 1

In the first step, 10mg of carboxyl functionalized silica-based microspheres (particle size 15nm, pore size 2 nm) were taken in a 2mL centrifuge tube, 0.02M PBS (pH 7.4) buffer was added, and the mixture was centrifuged at 10000rpm at 4℃for 15min to wash once.

Secondly, 200uL of PBS buffer solution is added for ultrasonic dispersion uniformly, 150uL of 10mg/mL SA aqueous solution is added, PBS buffer solution is supplemented to the microsphere reaction concentration of 20mg/mL, and stirring is carried out at room temperature overnight.

Third, SA microspheres were centrifuged with 0.02M PBS (pH 7.4) containing 0.5% Tween-20, centrifuged at 10000rpm at 4℃for 15min, washed three times to remove unadsorbed SA, and finally fixed to a volume of 10mg/mL with 0.02M PBS (pH 7.4) buffer.

Examples 2 to 7

The preparation was identical to example 1, except that carboxyl-functionalized silicon-based microspheres of different particle sizes and/or pore sizes were used in each example (see Table 1).

Covalent coupling mode for preparing the 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, centrifuging 10000rpm at 4℃for 15 min.

Secondly, 200uL of 0.1M MES (pH 6.0) buffer solution is added for ultrasonic dispersion uniformly, then 150uL of 10mg/mL SA aqueous solution is added, and then 100uL of 10mg/mLEDAC (0.1M MES) solution is added and stirred for 4 hours at room temperature.

Third, SA microspheres were washed three times with 0.02M PBS (pH 7.4) buffer containing 0.5% Tween-20, unadsorbed SA was removed, and finally the volume was fixed to 10mg/mL with PBS buffer.

Example 9: evaluation of the effects of the homogeneous chemiluminescent assay of the present invention on the interference of avidin

The experimental steps are as follows:

1. Adding the concentrated solution of T3 into the hormone-removed serum to prepare the T3 solution with the concentration of 1nmoL/L and 2 nmoL/L.

2. A40 ug/mL solution of the photoreceptor beads was prepared, and solutions of different concentrations (diluted with 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 each of reagent one (containing diiodothyronine coated receptor) and reagent two (containing biotin-labeled anti-triiodothyronine antibody) in a T3 kit (manually adding 25uL of sample solution and 25uL of reagent one and 25uL of reagent two according to a reaction mode), and adding 25uL of anti-interference agent solution prepared in step 2 according to the following table 1, wherein the conditions are as follows

1.2 Without adding an anti-interference agent.

5. Placed in LICA HT, first stage incubation: incubation was carried out at 37℃for 17min.

6. 175UL of universal solution (streptavidin labeled donor) was added manually.

7. Incubation in the second stage was performed: incubation is carried out for 15min at 37 ℃, and the corresponding sample to be detected is obtained after the incubation.

8. The sample to be tested is excited by energy and the generated luminous signal is read. The results of the readings are shown in tables 2 and 3.

TABLE 1

Data analysis:

The signal of chemiluminescent immune response drops by 89% with a T3 concentration of 1nM and a biotin concentration of 128ng/mL, with severe biotin interference. When the microsphere with the aperture of 2nm (the sequence number 3 in the table 1) is added, the luminescence signal almost has no obvious change, and when the microsphere with the particle diameter of 50nm, the aperture of 5nm and the aperture of 10nm (the sequence numbers 4 and 5 in the table 1) are adopted, the luminescence signal is improved to a certain extent, and the dropping amplitude is 50% -70%.

When the particle size and the pore diameter are unchanged, the concentration of the anti-interference agent is increased to 10ug/mL (the numbers 5 and 6 in the table 1 are compared), the luminous signal is further improved, and the falling range is about 25%. When the particle diameter of the microspheres was increased to 100nm (SEQ ID NO: 6 and 7 in Table 1) at a concentration of 10ug/ml at a pore diameter of 10nm, the biotin interference disappeared within 10% of the drop width deviation. When the concentration was increased to 20ug/ml (numbers 7 and 8 in Table 1), the signal drop was within 10%. When the concentration is 20ug/mL, and the particle size and the pore diameter are increased, the signal is dropped to a certain extent, and the drop width is 20% -40%.

Conclusion of experiment:

When the anti-interference agent added by the method is 100nm of microsphere particle size, 10nm of pore size and 10-20ug/mL of concentration filled with SA (streptavidin), the method has the strongest anti-biotin interference capability. When the pore diameter of the added anti-interference agent is smaller and is 2nm, the method has no anti-biotin interference capability. When the particle size of the added anti-interference agent is larger than 100nm and the pore size is 10nm, the anti-biotin interference capability of the method is reduced when the particle size and the pore size are continuously increased.

Example 10: the effect evaluation II of the homogeneous chemiluminescence analysis method of the invention for resisting the interference of the biotin

The experimental steps are as follows:

2. A40 ug/mL solution of the beads was prepared, and solutions of different concentrations (diluted with PBS) were prepared using the anti-interference agents prepared in examples 5 and 8, see Table 4 (diluted with PBS) for 10ug/mL and 20ug/mL.

4. 25UL of sample solution was added, then 25uL of each of the first reagent and the second reagent in the T3 kit was sequentially added (25 uL of sample solution and 25uL of each of the first reagent and the second reagent were manually added according to the reaction mode), and then 25uL of the anti-interference agent solution prepared in step 2 was added according to the following table, without adding the anti-interference agent in conditions 1 and 2.

6. 175UL of universal solution was added manually.

8. The sample to be tested is excited by energy and the generated luminous signal is read. The results of the readings are shown in tables 5 and 6.

TABLE 4 Table 4

TABLE 5 TABLE 6

Data analysis:

From the serial numbers 3 and 5 in table 4, the anti-interference agent prepared by physical adsorption method has strong anti-biotin interference ability, the luminous signal drops by about 10%, and the luminous signal drops by about 50% of the anti-interference agent prepared by covalent coupling method. The anti-biotin interference ability of the anti-interference agent prepared by physical adsorption is also better than that of the anti-interference agent prepared by covalent coupling in the numbers 4 and 6 in the table 4.

Conclusion of experiment:

For the same particle size, pore diameter, concentration and specific surface area, the anti-biological interference capability of the photo-activated chemiluminescence analysis method of the anti-interference agent prepared by adding a physical adsorption mode is better than that of the photo-activated chemiluminescence analysis method of the anti-interference agent prepared by adding a covalent coupling mode.

It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims

1. A homogeneous chemiluminescent assay comprising the steps of: in the presence of an anti-interference agent, analyzing and judging whether the sample to be detected contains the target molecule to be detected and/or the concentration of the target molecule to be detected by detecting the luminous signal intensity generated by the reaction of the receptor in the sample to be detected and the active oxygen;

Wherein the anti-interference agent comprises a carrier and an active molecule; the carrier is a porous medium; the porous medium is selected from carboxyl-functionalized silicon-based microspheres; the active molecules are filled in the carrier and can be mutually combined with free biotin molecules and/or biotin markers through the synergistic effect of intermolecular forces; the active molecule is selected from avidin and/or streptavidin; the active molecules are filled in the interstices of the carrier by contact with the carrier in a system comprising a buffer; the anti-interference agent can be uniformly distributed in the liquid phase reaction system; the free biotin molecule is capable of diffusing into the carrier and specifically binding to the active molecule therein; the anti-interference agent is capable of limiting the entry of biological macromolecules of larger size than the active molecules into their carriers;

Wherein the method comprises the steps of:

s1, mixing a sample to be tested with a buffer solution, a reagent a containing an acceptor, a reagent b containing a biotin marker, a reagent c containing an anti-interference agent and a reagent d containing a donor to obtain the sample to be tested;

2. The method according to claim 1, wherein the carrier is a mesoporous microsphere, preferably an ordered mesoporous microsphere; and/or the mesoporous microsphere is a cage-shaped hollow mesoporous microsphere.

3. The method according to claim 2, wherein the mesoporous microspheres have a pore size of 2-50nm, preferably 4-30nm, more preferably 5-15nm.

4. The method according to claim 1, characterized in that 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; and/or, the anti-interference agent further comprises a buffer solution, preferably a PBS buffer solution.

5. The method according to any one of claims 1 to 4, wherein the method for preparing the anti-interference agent comprises: step S1, contacting a carrier with an active molecule; preferably, the contacting is performed in a first buffer system.

6. The method of claim 5, wherein the method of preparing the anti-tamper agent further comprises step S0: washing the carrier by using a second buffer system, wherein the step S0 is performed before the step S1; and/or, the preparation method of the anti-interference agent further comprises the step S2: removing active molecules not filled in the carrier, and performing step S2 after step S1; preferably, the active molecules that are not filled in the carrier are removed by adding a third buffer system to the carrier treated in step S1 and then performing solid-liquid separation.

7. The method according to claim 1, wherein in step S1, the sample to be measured is mixed with a reagent a containing a receptor, a reagent b containing a biotin label, a reagent c containing an anti-interference agent, and then with a reagent d containing a donor, thereby obtaining the sample to be measured.

8. A homogeneous chemiluminescent detection device utilizing the method of any one of claims 1-7 for homogeneous chemiluminescent detection.

9. A control method of the homogeneous chemiluminescent detection apparatus of claim 8.

10. Use of a method according to any one of claims 1-7 or a detection device according to claim 8 or a control method according to claim 9 in the detection of a biotin-streptavidin system; preferably in the detection of thyroid function; further preferred is the use in the detection of triiodothyronine and/or tetraiodothyronine.