CN112114131A

CN112114131A - Homogeneous phase chemiluminescence detection method and application thereof

Info

Publication number: CN112114131A
Application number: CN201910544962.6A
Authority: CN
Inventors: 康蔡俊; 吴晨; 杨阳; 刘宇卉; 李临
Original assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Beyond Diagnostics Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2019-06-21
Filing date: 2019-06-21
Publication date: 2020-12-22
Also published as: WO2020252870A1

Abstract

The invention relates to a homogeneous phase chemiluminescence detection method and application thereof in the technical field of chemiluminescence detection. The method comprises the following steps: step S1, contacting the sample to be tested with an acceptor reagent containing acceptor particles and a donor reagent containing donor particles, and generating a mixture to be tested after reaction; controlling the variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent to be more than or equal to 5%. And step S2, exciting the mixture to be detected to perform chemiluminescence by using energy or active compounds, analyzing the signal intensity of the chemiluminescence, 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 in the sample to be detected. Compared with the prior art, the detection performance of the method is greatly improved, and the method has ultrahigh sensitivity and wide detection range.

Description

Homogeneous phase chemiluminescence detection method and application thereof

Technical Field

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

Background

Immunoassays have evolved in many varieties over half a century. The separation of the substance to be measured from the reaction system during the assay can be classified into heterogeneous immunoassay (Heterogenous immunoassay) and Homogeneous immunoassay (Homogeneous immunoassay). Heterogeneous immunoassay refers to the operation process of introducing a probe for labeling, wherein various related reagents are required to be separated after mixed reaction, and an object to be detected is separated from a reaction system and then detected, and is the mainstream method in the existing immunoassay. Such as enzyme-linked immunosorbent assay (ELISA method) and magnetic particle chemiluminescence method. Homogeneous immunoassay refers to direct measurement after mixing and reacting an analyte with a relevant reagent in a reaction system in the measurement process, and no redundant separation or cleaning step is needed. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, and the like.

For example, Light-activated chemiluminescence Assay (LiCA) is a typical homogeneous immunoassay. It is based on two kinds of antigen or antibody coated on the surface of microsphere, and immune complex is formed in liquid phase to draw two kinds of microsphere. Under the excitation of laser, the transfer of singlet oxygen between the microspheres occurs, so that high-level red light is generated, and the number of photons is converted into the concentration of target molecules through a single photon counter and mathematical fitting. When the sample does not contain the target molecules, immune complexes cannot be formed between the two microspheres, the distance between the two microspheres exceeds the propagation range of singlet oxygen, the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy level red light signal is generated during detection. It has the characteristics of high speed, homogeneous phase (no flushing), high sensitivity and simple operation. Light-activated chemiluminescence has been used in a number of detection projects.

The light-activated chemiluminescence detection is basically characterized by 'double spheres', wherein the 'double spheres' means that a system consists of luminescent microspheres and photosensitive microspheres, and the two microspheres have good suspension characteristics in a liquid phase. The liquid dynamic characteristics of the microspheres are completely met when the microspheres meet antigens or antibodies in a liquid phase. The luminescent microsphere is formed by connecting or embedding a luminescent substance in the surface of a monodisperse polymer microsphere, and an active functional group on the surface of the microsphere can be directly or indirectly coupled with an antigen or an antibody for immunoassay. Monodisperse polymeric microspheres broadly refer to polymeric microspheres that have a uniform particle appearance, e.g., morphology and size. Thus, it is also referred to as uniformly sized polymeric microspheres.

According to the conventional optical detection theory knowledge, the more uniform the particle size of the microsphere used in homogeneous chemiluminescence detection, the better the performance of chemiluminescence detection using the microsphere, and therefore, those skilled in the art tend to make efforts to obtain monodisperse system microspheres with more uniform particle size. However, with the progress of the detection industry, the demand for a hypersensitivity reagent is increasing, the requirement for sensitivity is extremely high, and the requirement for detection range is very wide, so that the existing homogeneous chemiluminescence detection method is difficult to meet the detection conditions.

Therefore, there is a need to develop a homogeneous chemiluminescence detection method that can meet both the sensitivity requirement and the linear range requirement.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a homogeneous phase chemiluminescence detection method aiming at the defects of the prior art, and the method has ultrahigh sensitivity and wide detection range when used for detection.

To this end, the present invention provides in a first aspect a homogeneous chemiluminescent detection method comprising the steps of:

step S1, contacting the sample to be tested with an acceptor reagent containing acceptor particles and a donor reagent containing donor particles, and generating a mixture to be tested after reaction; controlling the variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent to be more than or equal to 5 percent;

and step S2, exciting the mixture to be detected to perform chemiluminescence by using energy or active compounds, analyzing the signal intensity of the chemiluminescence, 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 in the sample to be detected. In some embodiments of the invention, the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 10%.

In some preferred embodiments of the present invention, the acceptor particles are controlled to have a coefficient of variation C.V value in the size distribution of the acceptor reagent of 40% or less; still more preferably, the acceptor particles are controlled to have a variation coefficient C.V value of 20% or less in their particle size distribution in the acceptor reagent.

In some embodiments of the invention, the acceptor particles exhibit a particle size distribution in the acceptor agent that is polydisperse.

In some embodiments of the invention, the value of the variation coefficient C.V of the particle size distribution is calculated by Gaussian distribution.

In other embodiments of the invention, the acceptor particle exhibits two or more peaks in the acceptor agent's Gaussian distribution curve using a Gaussian distribution analysis.

In some preferred embodiments of the invention, the receptive agent comprises at least two distributions of average particle size receptive particles.

In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, the luminescent composition being filled in and/or attached to the carrier.

In some embodiments of the invention, the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal comprising a chemiluminescent compound and a metal chelate.

In other embodiments of the present invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxins, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, and more preferably from the group consisting of dimethylthiophene and derivatives thereof.

In some embodiments of the invention, the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from the group consisting of europium, terbium, dysprosium, samarium osmium and ruthenium, more preferably europium.

In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides and derivatives thereof.

In some embodiments of the invention, the carrier is selected from the group consisting of a tape, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; beads and microspheres are preferred.

In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

In some embodiments of the invention, the support material is selected from natural, synthetic or modified naturally occurring polymers including, but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate.

In other embodiments of the present invention, the support is an aldehydized latex particle.

In some preferred embodiments of the invention, the support has an average particle size in the range of 50nm to 1 μm; preferably 100nm to 500 nm; more preferably 150nm to 400 nm; most preferably 190nm to 300 nm.

In some embodiments of the invention, the surface of the support is coated with at least two successive polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.

In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

In some embodiments of the invention, the polysaccharide has pendant functional groups, and the functional groups of the continuous polysaccharide layer are oppositely charged from the functional groups of the preceding polysaccharide layer.

In other embodiments of the present invention, the polysaccharide has pendant functional groups and the continuous layer of polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer.

In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

In other embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the support.

In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

In other embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly linked to a reporter molecule capable of specifically binding to the target molecule to be detected.

In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to one of the members of the specific binding pair.

In other embodiments of the invention, the specific binding pair member is selected from the group consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin; preferably, the specific binding pair member is biotin-avidin.

In some embodiments of the invention, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

Note that receptor particles attached directly or indirectly to a reporter molecule or to one of the members of the specific binding pair still have a variation coefficient of size distribution C.V in the receptor reagent of greater than or equal to 5%. In some embodiments of the invention, the receptor particles attached directly or indirectly to one of the reporter molecule or the specific binding pair member have a particle size distribution variation coefficient C.V value in the receptor agent of greater than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution is more than or equal to 10%; more preferably, the variation coefficient C.V value of the particle size distribution is less than or equal to 40%; more preferably, the coefficient of variation C.V for the particle size distribution is less than or equal to 20%. In some preferred embodiments of the invention, the particle size distribution of the receptor particles as described above, attached directly or indirectly to a reporter molecule or to one of the members of the specific binding pair, exhibits polydispersity.

In some embodiments of the invention, the donor particle is linked directly or indirectly to a reporter molecule or is bound to one of the members of the specific binding pair.

In other embodiments of the present invention, the sample to be tested is diluted with a diluent and then contacted with the receptor reagent including the receptor particles and the donor reagent including the donor particles.

In some embodiments of the invention, the chemiluminescence has a detection wavelength of 520 to 620 nm.

In other embodiments of the present invention, the laser irradiation is performed using red excitation light of 600 to 700 nm.

In some embodiments of the invention, the concentration of the receptor particle in the receptor agent is from 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500 ug/mL; more preferably 20ug/mL to 200 ug/mL.

In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

A second aspect of the invention provides a clinical use of a method according to the first aspect of the invention for in vitro diagnosis of a disease in a patient.

In a third aspect, the present invention provides a homogeneous chemiluminescent analyzer for detecting the presence and/or concentration of a target molecule to be detected in a sample to be detected by the method according to the first aspect of the present invention.

In some embodiments of the invention, the homogeneous chemiluminescent detector comprises the following components:

the sample adding mechanism is used for adding a sample to be detected into the reaction container;

a reagent addition mechanism for adding an acceptor reagent and/or a donor reagent to a reaction vessel;

an incubation module for providing a suitable temperature for a homogeneous chemiluminescent reaction in a reaction vessel;

and the detection module is used for detecting a chemiluminescent signal generated by the reaction of the receptor particles and the active oxygen.

In some preferred embodiments of the invention, the homogeneous chemiluminescent detector is a POCT analyzer.

The fourth aspect of the present invention provides a method for controlling the POCT analyzer to perform homogeneous chemiluminescence analysis, comprising the following steps:

step Q1, contacting the sample to be tested with an acceptor reagent containing acceptor particles and a donor reagent containing donor particles, and generating a mixture to be tested after reaction; the donor particles are capable of generating reactive oxygen species in an excited state; the receptor particles can react with active oxygen to generate a detectable chemiluminescent signal, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent is more than or equal to 5%;

step Q2, exciting the mixture to be detected to carry out chemiluminescence by using exciting light with the wavelength of 600-700 nm, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520-620 nm;

and step Q3, analyzing the signal intensity of the chemiluminescence, 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 in the sample to be detected.

The invention has the beneficial effects that: according to the homogeneous phase chemiluminescence detection method, the receptor reagent containing the receptor particles is added into a sample to be detected, wherein the variation coefficient C.V value of the particle size distribution of the receptor particles is more than or equal to 5%, so that the detection performance of the method is greatly improved compared with the prior art, and the method has ultrahigh sensitivity and wide detection range. The receptor reagent provided by the invention is low in production cost, and the detection precision of homogeneous phase chemiluminescence detection is high.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is a graph showing a Gaussian distribution of aldehyde-based polystyrene latex microspheres prepared in example 1.

Fig. 2 is a graph showing a Gaussian distribution of aldehyde-based polystyrene latex microspheres embedded with a light-emitting composition prepared in example 1.

FIG. 3 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres with embedded luminescent composition coated with dextran prepared in example 1

FIG. 4 is a Gaussian distribution plot of acceptor particles prepared in example 1 with an average particle size around 250 nm.

FIG. 5 is a Gaussian distribution plot of acceptor particles prepared in example 1 with a particle size around 110 nm.

FIG. 6 is a Nicomp distribution plot of acceptor particles with a particle size around 110nm prepared in example 1.

FIG. 7 is a Gaussian distribution plot of acceptor particles prepared in example 1 with a particle size around 350 nm.

FIG. 8 is a Nicomp distribution plot of acceptor particles with a particle size of around 350nm prepared in example 1.

FIG. 9 is a Gaussian distribution diagram of the particle size distribution of the mixed acceptor particles of example 2.

FIG. 10 is a Nicomp distribution plot of the mixed acceptor particle size distribution in example 2.

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. The practice of the invention is not limited to the following examples, and any variations and/or modifications made thereto are intended to fall within the scope of the invention.

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.

Term (I)

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 oxygen molecule, including superoxide anion (O) which is an electron reduction product of oxygen₂(-) 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.

The term "donor particles" as used herein refers to particles containing a sensitizer capable of generating a reactive intermediate, such as a reactive oxygen species, upon activation by energy or a reactive compound, to react with the acceptor particles. The donor particles may be light activated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor particles are polymeric microspheres filled with photosensitizers, which may be known in the art, preferably relatively light stable and not reactive with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls, as disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as linkers to specific binding partner members. 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.

The term "acceptor particle" as used herein refers to a particle that contains a compound that reacts with reactive oxygen species to produce a detectable signal. The donor particles are induced by energy or an active compound to activate and release reactive oxygen species in a high energy state that are captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and/or coated on the surface of the carrier. The "carrier" according to the present invention is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which may have any density, but preferably has a density close to that of water, preferably is capable of floating in water, and which are composed of a transparent, partially transparent or opaque material. The carrier may or may not have a charge, and when charged, is preferably a negative charge. The carrier may be a latex particle or other particle containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.

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 particle will be transitioned to an excited state to emit light.

In the present invention, 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 member of a specific binding pair (indirectly).

The term "specific binding pair member" as used herein refers to a pair of substances capable of specifically binding to each other.

The "C.V value of variation coefficient of particle size distribution" in the present invention refers to the variation coefficient of particle size in Gaussian distribution in the detection result of the nanometer particle size analyzer. The coefficient of variation is calculated as: C.V value (standard deviation SD/Mean) x 100%.

The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the US PSS nanometer particle sizer, NICOMP. Compared with a Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages in the analysis of multi-component liquid dispersion systems with nonuniform particle size distribution and the stability analysis of colloidal systems.

The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. The test sample that can be used in the present invention includes body fluids such as blood (which may 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 target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a member of a specific binding pair. Other examples of typical target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteroazoids, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a receptor. Analytes also include cells, viruses, bacteria, and fungi.

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. In any case desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.

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. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

Description of the preferred embodiments

The present invention will be described in more detail with reference to examples.

The prior common sense is as follows: the more uniform the particle size of the microspheres, the better the performance of homogeneous chemiluminescent detection using the microspheres. Current research on microspheres employed in homogeneous chemiluminescence therefore tends to result in microspheres of more uniform particle size. After research, the inventor of the application finds that when the microspheres with uniform particle size are used for homogeneous chemiluminescence detection, the sensitivity and the detection range of the detection result cannot be guaranteed at the same time. However, by adopting the microspheres with proper particle size uniformity (for example, the variation coefficient of the particle size distribution of the microspheres is more than 5%), the sensitivity of the light-activated chemiluminescence detection can be ensured, and the detection range can be widened.

The inventor of the present invention controls the particle size distribution of the receptor particles in the receptor reagent, and further controls the amount of the reporter molecules (e.g., antibody/antigen) on the surface of each receptor particle (small-particle size microspheres have a large specific surface area, large reporter molecules on the surface of unit-mass microspheres, large-particle size microspheres have a small specific surface area, and small reporter molecules on the surface of unit-mass microspheres). The larger the variation coefficient of the particle size distribution of the receptor particles in the receptor reagent is, the higher the nonuniformity is, which is equivalent to the fact that the receptor particles with different sizes exist in a system, so that the method disclosed by the invention has higher sensitivity and a wide detection range.

Accordingly, the invention in a first aspect relates to a homogeneous chemiluminescent detection method comprising the steps of:

and step S2, exciting the mixture to be detected to perform chemiluminescence by using energy or active compounds, analyzing the signal intensity of the chemiluminescence, 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 in the sample to be detected.

In some embodiments of the invention, the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 10%.

It should be noted that the value of C.V for the variation coefficient of the particle size distribution of the acceptor particles in the present invention refers to the value of C.V for the variation coefficient of the particle size distribution of the acceptor particles after the acceptor particles are coated with the desired substance.

In some embodiments of the invention, the recipient particle has a coefficient of variation of particle size distribution C.V value of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, etc. in the recipient agent controlled.

In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4 ' - (10-methyl-9-anthracenyl) -2,2 ': 6 ' 2 "-bipyridine-6, 6" -dimethylamine ] tetraacetic acid (MTTA), 2- (1 ', 1 ', 2 ', 2 ', 3 ', 3 ' -heptafluoro-4 ', 6 ' -hexanedion-6 ' -yl) -Naphthalene (NHA), 4 ' -bis (2 ', 3 ', 3 "-heptafluoro-4 ', 6" -hexanedion-6 "-yl) -o-terphenyl (BHHT), 4 ' -bis (1 ', 2 ', 3 ', 3" -heptafluoro-4 ', 6 "-hexanedion-6" -yl) -chlorosulphonyl-o-terphenyl (BHHCT), 4, 7-biphenyl-1, 10-phenanthroline (DPP), 1,1, 1-trifluoroacetone (TTA), 3-naphthoyl-1, 1, 1-trifluoroacetone (NPPTA), Naphthyltrifluorobutanedione (NTA), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO), 3-benzoyl-1, 1, 1-trifluoroacetone (BFTA), 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acid, azacrown ether, azacryptand trioctylphosphine oxide and derivatives thereof.

In other embodiments of the invention, the specific binding pair member is selected from the group consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin; preferably, the specific binding pair member is biotin-avidin. Wherein the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin, preferably neutravidin and/or streptavidin.

Specifically, receptor particles that have been linked, directly or indirectly, to a reporter molecule or a member of a specific binding pair have a coefficient of variation in particle size distribution C.V in the receptor reagent that is greater than or equal to 5%. In some embodiments of the invention, the receptor particles attached directly or indirectly to one of the reporter molecule or the specific binding pair member have a particle size distribution variation coefficient C.V value in the receptor agent of greater than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution is more than or equal to 10%; more preferably, the variation coefficient C.V value of the particle size distribution is less than or equal to 40%; more preferably, the coefficient of variation C.V for the particle size distribution is less than or equal to 20%. In some preferred embodiments of the invention, the particle size distribution of the receptor particles as described above, attached directly or indirectly to a reporter molecule or to one of the members of the specific binding pair, exhibits polydispersity.

In some preferred embodiments of the present invention, in step S1, the sample to be tested is mixed with the receptor reagent and then mixed with the donor reagent.

To further improve the accuracy of the final test result and the stability of the sample to be tested, in other embodiments of the present invention, the sample to be tested is diluted with a diluent and then contacted with the receptor reagent including the receptor particles and the donor reagent including the donor particles.

In some embodiments of the invention, the chemiluminescence has a detection wavelength of 520 to 620 nm; preferably 610-620nm, more preferably 615 nm.

In other embodiments of the present invention, the laser irradiation is performed with 600 to 700nm red excitation light; preferably, red exciting light with 640-680nm is adopted for laser irradiation; more preferably, the laser irradiation is performed with 660nm red excitation light.

It should be noted that the method of the present invention can be used for a double-antigen sandwich antibody measurement or a double-antibody sandwich antigen measurement. The method can also be used for detection of an antigen or antibody by a competition method, detection of an antigen or antibody by an indirect method, detection of an antigen or antibody by a capture method, and the like.

A second aspect of the invention relates to the clinical use of a method according to the first aspect of the invention for in vitro diagnosis of a disease in a patient.

A third aspect of the present invention relates to a homogeneous chemiluminescent analyzer for detecting the presence and/or concentration of a target molecule to be detected in a sample to be detected using the method according to the first aspect of the present invention.

a detection module for detecting a chemiluminescent signal produced by the homogeneous chemiluminescent reaction.

In some preferred embodiments of the invention, the homogeneous chemiluminescent detector is a POCT analyzer. The POCT analyzer is a point-of-care testing (point-of-care testing) instrument for clinical testing (bedside testing) performed beside a patient. The principle of the homogeneous immunoassay POCT analyzer is as follows: the biomolecule to be detected in the sample to be detected reacts with the donor particles and the acceptor particles to form immune complexes, the interaction draws the donor particles and the acceptor particles closer, and under the irradiation of laser (the wavelength is 680nm), the sensitizer in the donor particles converts oxygen in the surrounding environment into more active monomer oxygen. The monomer oxygen diffuses to the acceptor particle and reacts with the chemiluminescence agent in the acceptor particle to further activate the luminescent group on the acceptor particle to enable the luminescent group to emit light with the wavelength of 520-620 nm. The half-life of the monomeric oxygen is 4 [ mu ] Sec, and the diffusion distance in the solution is about 200 nm. If there is no interaction between the biomolecules and singlet oxygen cannot diffuse to the receptor particle, no light signal is generated. Therefore, the concentration of the target molecule to be detected in the sample to be detected can be calculated by measuring the intensity of light emitted by the mixture.

The POCT analyzer comprises a sample adding mechanism, an incubation module, a detection module and a circuit control module; the sample adding mechanism, the incubation module and the detection module are all electrically connected with the circuit control module. Under the control of the circuit control module, the incubation module is used for adjusting the temperature of the reagent card and substances in the reagent card, the reagent adding mechanism is used for transferring the substances in the reagent card, and the detection module is used for emitting laser and measuring the light intensity emitted by a sample to be detected.

In other embodiments of the present invention, the sample to be tested is selected from materials suspected of containing the target molecule to be tested, which include but are not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, or spinal fluid.

The fourth aspect of the present invention relates to a method for controlling the POCT analyzer to perform homogeneous chemiluminescence analysis, which comprises the following steps:

Example III

Example 1: preparation of a solution of antibody I (PCT antibody) conjugated receptor particles

(I) preparation of antibody-conjugated receptor particles having an average particle diameter of about 250nm

1.1 preparation and characterization Process of aldehyde polystyrene latex microspheres

1) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10min, N was introduced thereinto₂ 30min；

2) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system in the step 1, and continuously introducing N₂ 30min；

3) Heating the reaction system to 70 ℃ and reacting for 15 hours;

4) the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion with deionized water by secondary centrifugal sedimentation until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting with water, and storing in an emulsion form;

5) the mean particle size of the latex microspheres in a Gaussian distribution measured by a nanometer particle sizer was 202.2nm, the coefficient of variation (C.V.) -4.60%, and the Gaussian distribution curve is shown in fig. 1. The aldehyde group content of the latex microsphere is 280nmol/mg measured by an electric conductivity titration method.

1.2 landfill procedures and characterization of luminescent compositions

1) A25 ml round-bottom flask was prepared, and 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) were added³⁺) 10ml of 95% ethanol, magnetically stirring, heating in a water bath to 70 ℃ to obtain a complex solution;

2) preparing a 100ml three-neck flask, adding 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde polystyrene latex microspheres with the concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;

3) slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling;

4) and centrifuging the emulsion for 1 hour at 30000G, and removing supernatant after centrifugation to obtain the aldehyde polystyrene microspheres embedded with the luminescent composition.

5) The average particle size of the resulting microspheres in a Gaussian distribution was 204.9nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) (see fig. 2) was 5.00%.

1.3 surface coating of receptor particles with dextran

1) Taking 50mg of aminodextran solid, putting the aminodextran solid in a 20mL round-bottom flask, adding 5mL of 50mM/pH 10 carbonate buffer solution, and stirring and dissolving the aminodextran solid at 30 ℃ in the dark;

2) adding 100mg of prepared aldehyde polystyrene microspheres in which the luminescent composition is embedded into an aminodextran solution, and stirring for 2 hours;

3) dissolving 10mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

4) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeated centrifugal washing for three times, the solution is fixed by 50mM/pH 10 carbonate buffer solution to a final concentration of 20 mg/ml;

5) adding 100mg aldehyde dextran solid into a 20mL round-bottom flask, adding 5mL 50mM/pH 10 carbonate buffer, and stirring and dissolving at 30 ℃ in the dark;

6) adding the microspheres into an aldehyde dextran solution and stirring for 2 hours;

7) dissolving 15mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;

8) after the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH 10 carbonate buffer was added thereto for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH 10 carbonate buffer.

9) The average particle size of Gaussian distribution of the particle size of the microspheres at this time was 241.6nm as measured by a nanometer particle sizer, and the coefficient of variation (C.V.) (see fig. 3) was 12.90%.

1.4 conjugation procedure for antibodies

1) The conjugated antibody I was dialyzed into 50mM CB buffer at pH 10 to give a concentration of 1 mg/ml.

2) Adding 0.5ml of the receptor particles obtained in step (3) and 0.5ml of the conjugated antibody I obtained in step 1) into a 2ml centrifuge tube, mixing uniformly, adding 100 mu l of 10mg/ml NaBH₄The solution (50mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

3) After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50mM CB buffer) was added thereto, and the reaction was carried out at 2 to 8 ℃ for 2 hours.

4) After completion of the reaction, the reaction mixture was centrifuged at 30000G for 45min, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted to a final concentration of 100. mu.g/ml to obtain a receptor particle solution of conjugated antibody I.

5) The average particle size of the resulting microspheres was 253.5nm in a Gaussian distribution as measured by a nanometer particle sizer, and the coefficient of variation (C.V value) was 9.60% (see FIG. 4).

(II) preparation of antibody-conjugated receptor particles having an average particle diameter of about 110nm

The preparation method is the same as the preparation process of the acceptor particles with the average particle size of about 250nm in the step (I), the average particle size value of the acceptor particles is 107.1nm by Gaussian distribution (shown in figure 5) of the particle size of the acceptor particles measured by a nanometer particle sizer, and the coefficient of variation (C.V.) is 7.6%. Nicomp distribution is unimodal (as shown in fig. 6).

(III) preparation of antibody-coupled receptor particles having an average particle diameter of about 350nm

The preparation method is the same as the preparation process of the acceptor particles with the average particle size of about 250nm in the step (I), the average particle size value of the acceptor particles measured by a nanometer particle sizer (shown in figure 7) is 347.5nm, the variation coefficient (C.V.) -3.9%, and the Nicomp distribution is unimodal (shown in figure 8).

Example 2: determination of sensitivity and detection Upper Limit for the methods of the invention

The sensitivity point is defined as when the signal at concentration Cx is higher than the signal at twice the concentration of C0, i.e., RLU (Cx) >2RLU (C0), then the corresponding detection reagent sensitivity is Cx. The upper detection limit point is defined as the upper range limit determined using the method in the american clinical laboratory standards committee (NCCLS) Evaluation Protocol (EP) series 6 documents.

(1) The receptor agent (concentration 100ug/ml) comprising separately different mean particle size (110nm and 350nm) particles of receptor coupled with PCT antibody I prepared in example 1 was used by diluting the PCT antigen to a series of concentrations of 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 60pg/ml, 80pg/ml, 160pg/ml, 500pg/ml, 1000pg/ml, 5000pg/ml, 20000pg/ml, 50000pg/ml, 100000pg/ml and 200000pg/ml, the concentration series of PCT antigens were then assayed with the same biotin-labeled PCT antibody 2 (diluted to 2ug/ml) and universal solution (reagent containing donor particles), and the detection sensitivity and upper limit of detection using a light-activated chemiluminescence assay system developed by Boyang Biotechnology (Shanghai) Inc. are shown in Table 1.

TABLE 1

As can be seen from Table 1, the upper limit of detection is high for the acceptor particles with an average particle size of 110nm but the sensitivity is poor, while the upper limit of detection is low for the acceptor particles with an average particle size of 350nm, which is the best sensitivity.

(2) And (3) mixing the receptor particle solution of the coupled PCT antibody I with the average particle size of 110nm with the receptor particle solution of the coupled PCT antibody I with the average particle size of 350nm to obtain the new receptor reagent. In the novel receptor reagent, the result of the measurement of the particle size of the receptor particles is as follows:

gaussian distribution mean particle size 317.7nm, and coefficient of variation of particle size distribution (C.V value) 37.2% (as shown in fig. 9);

nicomp distribution is bimodal: # 1: the coefficient of variation (C.V value) of the average particle diameter of 103.1nm was 11.8%; # 2: the average particle diameter was 328.8nm, and the coefficient of variation in particle size distribution (C.V value) was 13.0% (see fig. 10).

The sensitivity and upper limit of detection of the above concentration series of PCT antigens using a light-activated chemiluminescence analysis system developed by bosyang biotechnology (shanghai) ltd are shown in table 2, in which the above novel receptor reagent, biotin-labeled PCT mab 2 (diluted to 2ug/ml) and a universal solution (reagent containing donor particles) were used to detect the above concentration series of PCT antigens.

TABLE 2

As can be seen from table 2, the detection performance of the method is significantly improved by increasing the variation coefficient of the particle size distribution of the receptor particles in the receptor reagent, i.e., by properly increasing the heterogeneity of the particle sizes of the receptor particles.

Example 3: preparation of receptor particle solutions of a series of conjugated antibodies I (PCT antibodies) with average particle size of about 250nm and different coefficient of variation of particle size distribution

Receptor particle solutions of conjugated antibody I (PCT antibody) having different coefficients of variation in particle size distribution were obtained according to the method described in (A) of example 1.

The method specifically comprises the following steps:

receptor particle 1: the average grain diameter of Gaussian distribution is 251.2nm, and the variation coefficient of grain diameter distribution C.V is 3.7%; nicomp distribution is unimodal.

Receptor particle 2: the average grain diameter of Gaussian distribution is 254.9nm, and the variation coefficient of grain diameter distribution C.V is 5.0%; nicomp distribution is unimodal.

Receptor particle 3: the average grain diameter of Gaussian distribution is 251.3nm, and the variation coefficient of grain diameter distribution C.V is 8.0%; nicomp distribution is unimodal.

Receptor particle 4: the average grain diameter of Gaussian distribution is 251.9nm, and the variation coefficient of grain diameter distribution C.V is 10.5%; nicomp distribution is unimodal.

Receptor particle 5: the average grain diameter of Gaussian distribution is 252.3nm, and the variation coefficient of grain diameter distribution C.V is 16.8%; nicomp distribution is unimodal.

Receptor particle 6: the average grain diameter of Gaussian distribution is 240.8nm, and the variation coefficient of grain diameter distribution C.V is 34.5%; the Nicomp distribution is bimodal.

Example 4: determination of sensitivity and detection Upper Limit for the methods of the invention

The sensitivity point is defined as when the signal at concentration Cx is higher than the signal at twice the concentration of C0, i.e., RLU (Cx) >2RLU (C0), then the corresponding detection reagent sensitivity is Cx. The detection upper limit point is defined as the upper limit of the range determined using the method in the NCCLS EP-6 document.

(1) PCT antigens were diluted to a series of concentrations of 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 60pg/ml, 80pg/ml, 160pg/ml, 500pg/ml, 1000pg/ml, 5000pg/ml, 20000pg/ml, 50000pg/ml, 100000pg/ml and 200000pg/ml, and the above concentration series of PCT antigens were detected using the acceptor reagent containing the acceptor particle conjugated to PCT antibody I prepared in example 3 (concentration of 100ug/ml), respectively, and then with the same biotin-labeled PCT mab 2 (diluted to 2ug/ml) and a universal solution (reagent containing the donor particle), and the detection sensitivity and detection upper limit were determined using a photostimulation chemiluminescence analysis system developed by Boyang Biotech (Shanghai) Ltd, as shown in Table 3.

TABLE 3

As can be seen from table 3, when the variation coefficient of the size distribution of the receptor particles is 5% or more, the method of adding the receptor reagent containing the receptor particles has a wide detection range as well as a suitable sensitivity.

Example 5: detection of cTnI and PCT marker standards

5.1 detection of cTnI antigen standards

cTnI antigen is diluted to 1pg/ml, 2pg/ml, 5pg/ml, 10pg/ml, 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 100pg/ml, 1000pg/ml, 5000pg/ml, 10000pg/ml, 50000pg/ml and 1000ng/ml, receptor particles with different particle sizes (50nm, 80nm, 110nm, 140nm, 170nm, 200nm, 250nm, 300nm, 350nm and 400nm) are adopted to coat the receptor agent of cTnI monoclonal antibody 1 (the concentration is 100ug/ml, the C.V value is about 10 percent), the detection sensitivity and the upper limit of detection of the above concentration series of cTnI antigen with the same biotin-labeled cTnI monoclonal antibody 2 (diluted to 2ug/ml) and universal solution (solution containing donor particles) using a light-activated chemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Ltd are shown in Table 4.

TABLE 4

As can be seen from table 4, the detection results of the cTnI item: the upper limit of detection is high for the acceptor particles with a particle size of 50nm, 80nm, but the sensitivity is poor, while the upper limit of detection is low for the acceptor particles with a particle size of 300nm, which has the best sensitivity. Acceptor particles having a particle size of 50nm and 80nm were mixed with acceptor particles having a particle size of 300nm, respectively, to form acceptor reagents, and the sensitivity and upper limit of detection of the method of adding the corresponding acceptor reagent were measured, and the results are shown in Table 5.

TABLE 5

As can be seen from table 5, the addition of the receptor reagent formed by combining the receptor particles having a smaller average particle size and the receptor particles having a larger average particle size has both high sensitivity and a high upper limit of detection (wide detection range), exhibits the advantages of the receptor particles having a larger particle size and the receptor particles having a smaller particle size, and greatly improves the performance of the receptor reagent containing the receptor particles having two or more average particle sizes as compared with the receptor particles having a single average particle size distribution.

5.2 detection of PCT antigen standards

PCT antigen is diluted to 20pg/ml, 30pg/ml, 40pg/ml, 50pg/ml, 60pg/ml, 80pg/ml, 160pg/ml, 500pg/ml, 1000pg/ml, 5000pg/ml, 20000pg/ml, 100000pg/ml and 2000ng/ml, acceptor particles with different particle sizes (50nm, 80nm, 110nm, 140nm, 170nm, 200nm, 250nm, 300nm, 350nm and 400nm) are used for coating acceptor reagent (the concentration is 100ug/ml, and the C.V value is about 10%), the detection sensitivity and upper limit of detection of the above concentration series of PCT antigens using the photo-activated chemiluminescence assay system developed by Boyang Biotechnology (Shanghai) Ltd, are shown in Table 6, together with the same biotin-labeled PCT antibody 2 (diluted to 2ug/ml) and a universal solution (reagent containing donor particles).

TABLE 6

As can be seen from the results of PCT test in Table 6, the upper limit of detection of the receptor particles having a particle size of 110nm is high but the sensitivity is poor, while the upper limit of detection of the receptor particles having a particle size of 300nm or 350nm is low although the sensitivity is optimal. Acceptor particles having a particle size of 110nm were mixed with acceptor particles having a particle size of 300nm and 350nm, respectively, to form an acceptor reagent, and the sensitivity and the upper limit of detection of the method of adding the corresponding acceptor reagent were detected using a light-activated chemiluminescence analysis system developed by bosyang biotechnology (shanghai) ltd, and the results are shown in table 7.

TABLE 7

As can be seen from table 7, the addition of the receptor reagent formed by combining the small-sized receptor particles and the large-sized receptor particles distributed at approximately C.V values has both high sensitivity and high upper limit of detection (wide detection range), exhibits the advantages of the large-sized receptor particles and the small-sized receptor particles, and greatly improves the performance of the receptor reagent containing two or more kinds of receptor particles compared to the receptor particles of a single particle size.

Example 6: detection of cTnI marker levels in samples from normal and suspected patients with myocardial injury

In this example, 40 clinical samples (13 negative samples and 27 positive samples) were tested, and a cTnI quantitative determination test kit (light-activated chemiluminescence method) was used which consisted of reagent 1(R1) containing acceptor particles coated with a first anti-cTnI monoclonal antibody, reagent 2(R2) containing a biotin-labeled second anti-cTnI monoclonal antibody, and further included a universal solution (R3) containing donor particles. Wherein R1 is receptor agent (concentration of 100ug/ml) prepared from receptor particles with average particle diameter of about 250nm and particle size distribution variation coefficient of C.V of 11%.

The detection process is completed on a full-automatic light-activated chemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Inc. and a detection result is output, and the specific detection steps comprise:

a. adding a clinical sample into the reaction hole;

b. sequentially adding R1 and R2 into the reaction hole;

c. incubation;

d. adding R3 into the reaction hole;

e. incubation;

f. irradiating the reaction holes by laser and calculating the quantity of light-emitting photons of each hole;

g. and calculating the concentration of cTnI in the sample to be detected.

When the cTnI marker exists in a clinical sample, the cTnI is specifically combined with the receptor particles coated with the first anti-cTnI monoclonal antibody and a biotin-labeled second anti-cTnI monoclonal antibody at the same time, and a double-antibody sandwich complex is formed on the surface of the receptor particles; at this time, if a donor particle modified by streptavidin is added, biotin and streptavidin are combined to enable the two particles to approach each other, under the excitation of an excitation light source, the donor particle releases singlet oxygen, and chemiluminescence is generated after the donor particle touches an acceptor particle in a solution, so that a fluorophore on the same particle is further excited to generate a cascade amplification reaction to generate fluorescence. At this time, the more the content of the cTnI marker is, the stronger the fluorescence intensity is, and the amount of cTnI in the serum of the patient is quantitatively detected according to the intensity of luminescence, and the specific detection results are shown in table 8 below:

TABLE 8

By comparison, the Abbott measurement correlated with the above measurement of example 6 by 0.9973 with a slope of 1.0495. The sample No. 1-13 is a patient for normal physical examination, the distribution range is 1.77 pg/ml-25.3 pg/ml, and the median value is 6.77 pg/ml; samples No. 14-40 identified patients with myocardial injury, with a distribution range of 30.94pg/ml to 29896.88pg/ml and a median of 450.54 pg/ml.

Example 7: detection of PCT marker levels in samples from normal and suspected patients with inflammation

In this example, 40 clinical specimens were tested, and a PCT quantitative assay kit (photo-activated chemiluminescence method) was used which consisted of a reagent 1(R1 ') containing acceptor microparticles coated with a primary anti-PCT antibody, a reagent 2(R2 ') containing a biotin-labeled secondary anti-PCT antibody, and additionally contained a general-purpose liquid (R3 ') containing donor particles. Wherein R1 is the receptor reagent (concentration 200ug/ml) prepared using the receptor particle 4 (particle size distribution variation coefficient C.V value: 10.5%) in example 3.

The specific experimental steps are as follows:

1. selecting 40 parts of clinical samples, balancing to room temperature, and uniformly mixing;

2. respectively adding the uniformly mixed sample, the prepared R1 'and the prepared R2' into an 8X 12 white board;

3. putting the white board added with the sample into a LiCA HT instrument for reaction in the following reaction mode;

(1) mixing 40ul sample, 15ul R1 'and 15ul R2' well;

(2) incubating at 37 ℃ for 8 min;

(3) 160ul of the universal solution (R3') was added;

(4) incubating at 37 ℃ for 2 min;

(5) the excitation readings and the specific detection results are shown in table 9 below.

TABLE 9

By comparison, the correlation between the Roche measurement and the above measurement of example 7 was 0.9977, with a slope of 0.9984. The sample No. 1-11 is a patient for normal physical examination, the distribution range is 30 pg/ml-70 pg/ml, and the median value is 50 pg/ml; sample No. 12-40 identified patients with inflammation, the distribution range was 120pg/ml to 63.23ng/ml, and the median was 580 pg/ml.

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

1. A homogeneous chemiluminescent assay method comprising the steps of:

2. The method as claimed in claim 1, wherein the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution of the acceptor particles in the acceptor reagent is controlled to be more than or equal to 10%.

3. The method of claim 1 or 2, wherein the acceptor particle has a particle size distribution coefficient of variation C.V value of 40% or less; still more preferably, the acceptor particles are controlled to have a variation coefficient C.V value of 20% or less in their particle size distribution in the acceptor reagent.

4. The method of any one of claims 1 to 3, wherein the acceptor particles exhibit a polydispersity in their particle size distribution in the acceptor agent.

5. The receptor reagent of any one of claims 1-4, wherein the value of the coefficient of variation C.V in particle size distribution is calculated using a Gaussian distribution.

6. The method according to any one of claims 1 to 5, wherein the acceptor particles exhibit two or more peaks in a Gaussian distribution curve in the acceptor reagent by a Gaussian distribution analysis method.

7. The method of any one of claims 1 to 6, wherein the receptor agent comprises at least two distributions of average particle size of receptor particles.

8. The method according to any one of claims 1 to 7, wherein the receptor particles comprise a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and/or attached to the carrier.

9. The method of claim 8, wherein the luminescent composition is capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal and comprises a chemiluminescent compound and a metal chelate.

10. The method according to claim 9, wherein the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably from the group consisting of dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins and derivatives thereof, more preferably from the group consisting of dimethylthiophene and its derivatives.

11. The process according to claim 9 or 10, characterized in that the metal of the metal chelate is a rare earth metal or a group VIII metal, preferably selected from europium, terbium, dysprosium, samarium osmium and ruthenium, more preferably europium.

12. The method of any one of claims 9 to 11, wherein the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2-dimethyl-4-perfluorobutanoyl-3-butanone (fod), 2' -bipyridine (bpy), bipyridylcarboxylic acids, azacrown ethers, azacryptands and trioctylphosphine oxides and derivatives thereof.

13. The method of any one of claims 8 to 12, wherein the carrier is selected from the group consisting of a tape, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle and a microsphere; beads and microspheres are preferred.

14. The method according to any one of claims 8 to 13, wherein the carrier is a magnetic or non-magnetic particle.

15. The method of any one of claims 8 to 14, wherein the support material is selected from natural, synthetic or modified naturally occurring polymers including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate.

16. The method of claim 15, wherein the support is an aldehydized latex particle.

17. The method according to any one of claims 8 to 16, wherein the support has an average particle size in the range of 50nm to 1 μm; preferably 100nm to 500 nm; more preferably 150nm to 400 nm; most preferably 190nm to 300 nm.

18. The method according to any one of claims 8 to 17, wherein the surface of the support is coated with at least two successive polysaccharide layers, wherein a first polysaccharide layer is associated spontaneously with a second polysaccharide layer.

19. The method of claim 18, wherein each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

20. The method of claim 18 or 19, wherein said polysaccharide has pendant functional groups, said functional groups of said successive polysaccharide layers being oppositely charged from said functional groups of said previous polysaccharide layer.

21. The method of any one of claims 18 to 20, wherein the polysaccharide has pendant functional groups and the continuous layer of polysaccharide is covalently linked to the previous polysaccharide layer by a reaction between the functional groups of the continuous layer and the functional groups of the previous layer.

22. The method of claim 21, wherein the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

23. The method of claim 22, wherein the amine-reactive functional group is an aldehyde group or a carboxyl group.

24. The method of any one of claims 18 to 23, wherein the first polysaccharide layer spontaneously associates with the support.

25. The method of any one of claims 18 to 24, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group.

26. The method of any one of claims 18 to 25, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, thiol groups, amino groups, hydroxyl groups, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.

27. The method of claim 25 or 26, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly linked to a reporter molecule capable of specifically binding to the target molecule to be detected.

28. The method of claim 25 or 26, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to one of the members of the specific binding pair.

29. The method of claim 28, wherein the specific binding pair member is selected from the group consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, or biotin; preferably, the specific binding pair member is biotin-avidin.

30. The method according to any one of claims 18 to 29, wherein the polysaccharide is selected from the group consisting of carbohydrates comprising three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.

31. The method of any one of claims 1 to 30, wherein the donor particles are linked directly or indirectly to a reporter molecule or to one of the members of a specific binding pair.

32. The method of any one of claims 1 to 31, wherein the sample to be tested is diluted with a diluent and then contacted with the receptor reagent comprising receptor particles and the donor reagent comprising donor particles.

33. The method according to any one of claims 1 to 32, wherein the chemiluminescence is detected at a wavelength of 520 to 620 nm.

34. The method according to any one of claims 1 to 33, wherein the laser irradiation is performed with red excitation light of 600 to 700 nm.

35. The method of any one of claims 1 to 34, wherein the concentration of the receptor particles in the receptor agent is from 1ug/mL to 1000 ug/mL; preferably 10ug/mL to 500 ug/mL; more preferably 20ug/mL to 200 ug/mL.

36. The method according to any one of claims 1 to 35, wherein the active oxygen is singlet oxygen.

37. A clinical use of the method of any one of claims 1 to 36 for the in vitro diagnosis of a disease in a patient.

38. A homogeneous chemiluminescent analyzer which utilizes the method of any one of claims 1 to 36 to detect the presence and/or concentration of target molecules to be detected in a sample to be detected.

39. The homogeneous chemiluminescent analyzer of claim 38 wherein the homogeneous chemiluminescent detector comprises the following components:

a detection module for detecting a chemiluminescent signal generated by the homogeneous chemiluminescent reaction.

40. The homogeneous chemiluminescent detector of claim 38 or 39 wherein the homogeneous chemiluminescent detector is a POCT analyzer.

41. A method of controlling the POCT analyzer of claim 40 for homogeneous chemiluminescent analysis comprising the steps of:

and step Q3, 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 in the sample to be detected according to the analysis of the chemiluminescence signal intensity.