CN112240937B

CN112240937B - Application of donor reagent in diagnosis of bacterial inflammatory diseases of subject infection

Info

Publication number: CN112240937B
Application number: CN201910656169.5A
Authority: CN
Inventors: 刘苗; 杨阳; 康蔡俊; 赵卫国; 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: 2019-07-19
Filing date: 2019-07-19
Publication date: 2023-06-13
Anticipated expiration: 2039-07-19
Also published as: CN112240937A

Abstract

The present invention relates to the use of a donor agent for the preparation of a kit for use in a method for in vitro diagnosis of whether a subject is infected with a bacterial inflammatory disease, wherein the method comprises: contacting body fluid from a subject with an acceptor reagent and a donor reagent, and reacting to produce a mixture to be tested; exciting the test mixture at least once with excitation light, detecting the signal intensity of the chemiluminescence produced thereby; quantitatively calculating the concentration of procalcitonin in the body fluid according to the signal intensity of the chemiluminescence, so as to judge whether the main body is infected with bacterial inflammatory diseases; wherein the donor agent comprises donor particles capable of generating active oxygen in an excited state, the donor particles comprising a first support, the interior of which is filled with a sensitizer, the surface of which is chemically bound to one of the members of the specific binding pair. The method has ultrahigh sensitivity and wide detection range.

Description

Application of donor reagent in diagnosis of bacterial inflammatory diseases of subject infection

Technical Field

The invention belongs to the field of chemiluminescence analysis, and particularly relates to application of a donor reagent in diagnosing bacterial inflammatory diseases of a subject.

Background

Procalcitonin (PCT) is a protein consisting of 116 amino acids with a molecular weight of about 13kDa, is a propeptide of Calcitonin (CT), has no calcitonin-like hormonal activity, and its molecule consists of calcitonin, calcitonin and an N-terminal fragment of 57 amino acids. PCT reflects the activity of systemic inflammatory response, and PCT has very low PCT content (< 0.05 mg/L) in normal human serum, which does not increase or slightly increases PCT concentration in the case of local infection, viral infection, chronic nonspecific inflammation, cancer fever, graft-host rejection or autoimmune diseases, but significantly increases its level in plasma in the case of severe bacterial, fungal, parasitic infection, and sepsis and multiple organ failure. Thus, bacterial and non-bacterial infections and inflammations can be differentially diagnosed as an acute parameter based on the results of PCT assays.

Procalcitonin is detected by a number of methods, such as chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography (gic), but has a certain disadvantage. The CLIA method is an immunodetection technology developed on the basis of an enzyme-linked immunoassay method, has the advantages of high sensitivity, wide detection linear range, simplicity and convenience in operation, high automation degree and the like, and is widely applied in recent years. Detecting procalcitonin antigen in the sample, and reacting procalcitonin antibody coated on superparamagnetic particles with enzyme-labeled procalcitonin antibody conjugate to form sandwich (antibody-antigen-antibody) complex. Under the action of the magnetic field, the magnetic particles are adsorbed to the wall of the reaction tube, and unbound substances are washed away by the washing liquid. Chemiluminescent substrate solution was added to the reaction mixture and the test results were expressed as relative luminescence intensity (RLU). The amount of procalcitonin antigen in the test sample is proportional to the relative luminescence intensity (RLU) detected by the analyzer optics.

At present, the domestic chemiluminescent immunoassay market mainly uses an imported closed type full-automatic chemiluminescent detection system (reagent and instrument), however, the domestic PCT detection reagent starts later, and the performance of the reagent is always different from that of the imported reagent optimized for years or even decades, particularly in repeatability, stability and sensitivity.

Disclosure of Invention

The invention aims to solve the technical problems of the prior art, and provides application of a donor reagent in preparing a kit used for in-vitro diagnosis of whether a main body is infected with bacterial inflammatory diseases, and the donor reagent has ultrahigh sensitivity and wide detection range when being used for detection.

To this end, a first aspect of the invention provides the use of a donor agent in the preparation of a kit for use in a method for in vitro diagnosis of whether a subject is infected with a bacterial inflammatory disease, wherein the method comprises: contacting body fluid from a subject with an acceptor reagent and a donor reagent, and reacting to produce a mixture to be tested; exciting the test mixture at least once with excitation light, detecting the signal intensity of the chemiluminescence produced thereby; quantitatively calculating the concentration of procalcitonin in the body fluid according to the signal intensity of the chemiluminescence, so as to judge whether the main body is infected with bacterial inflammatory diseases;

Wherein the donor agent comprises donor particles capable of generating active oxygen in an excited state, the donor particles comprising a first support, the interior of which is filled with a sensitizer, the surface of which is chemically bound to one of the members of the specific binding pair.

In some embodiments of the invention, the surface of the first carrier is not coated or attached with a polysaccharide substance that directly chemically binds to one of the members of the specific binding pair.

In other embodiments of the invention, the surface of the first support carries a binding functionality for chemically binding one member of a specific binding pair member to the surface of the first support.

In some embodiments of the invention, the bonding functional group is selected from the group consisting of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group; preferably selected from aldehyde groups and/or carboxyl groups.

In some embodiments of the invention, the content of binding functionalities on the surface of the first support is 100 to 500nmol/mg, preferably 200 to 400nmol/mg.

In some embodiments of the invention, the surface of the first carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein the first polysaccharide layer is spontaneously associated with the second polysaccharide layer.

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

In some embodiments of the invention, the polysaccharide has pendant functional groups, the functional groups of the continuous polysaccharide layer being oppositely charged to the functional groups of the preceding polysaccharide layer.

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

In other embodiments of the 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 groups, carboxyl groups, mercapto groups, amino groups, hydroxyl groups, and maleamine groups; preferably selected from aldehyde groups and/or carboxyl groups.

In other embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are bound directly or indirectly by a member of a specific binding pair.

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

In some embodiments of the invention, the particle size of the first support is selected from 100 to 400nm, preferably 150 to 350nm, more preferably 180 to 220nm.

In other embodiments of the invention, the first carrier is magnetic or non-magnetic, preferably non-magnetic.

In some embodiments of the invention, the first carrier is in a shape 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; preferably microspheres.

In other embodiments of the invention, the first carrier is selected from natural, synthetic or modified naturally occurring polymers; artificially synthesized polymers are preferred.

In some embodiments of the present invention, the first carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyethylene butyrate, or polyacrylate; preferably selected from polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

In some embodiments of the invention, the first carrier is a polystyrene latex microsphere.

In other embodiments of the invention, the sensitizer is a photoactivated photosensitizer and/or a chemically activated initiator, preferably a photoactivated photosensitizer.

In some embodiments of the invention, the sensitizer is selected from methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll.

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, which are capable of specifically binding to each other.

In some embodiments of the invention, the specific binding pair member is avidin-biotin.

In other embodiments of the invention, the avidin is selected from the group consisting of avidin, streptavidin, vitelline avidin, neutravidin and avidin-like, preferably neutravidin and/or streptavidin.

In some embodiments of the invention, the avidin is chemically bound to the surface of the first support by reacting an amino group with an aldehyde group on the surface of the first support to form a schiff base.

In some embodiments of the invention, the coefficient of variation C.V of the particle size distribution of the donor particles in the donor agent is controlled to be greater than or equal to 5%.

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

In some embodiments of the invention, the coefficient of variation C.V value of the particle size distribution of the donor particles in the donor agent is controlled to be less than or equal to 40%; still more preferably, the value of the coefficient of variation C.V of the particle size distribution of the donor particles in the donor agent is controlled to be 20% or less.

In other embodiments of the invention, the particle size distribution of the donor particles in the donor agent exhibits polydispersity.

In some embodiments of the invention, the concentration of the donor particles in the donor agent is between 10 μg/ml and 1mg/ml, preferably between 20 μg/ml and 500 μg/ml, more preferably between 50 μg/ml and 200 μg/ml.

In other embodiments of the present invention, the donor agent further comprises a buffer solution having a pH of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

In some embodiments of the invention, the buffer solution contains a polysaccharide 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, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.

In other embodiments of the invention, the dextran has a molecular weight distribution Mw selected from 10000 ～ 1000000kDa, preferably from 100000 ～ 800000kDa, more preferably from 300000 ～ 700000kDa.

In some embodiments of the invention, the dextran is present in the buffer solution in an amount of 0.01 to 1wt%, preferably 0.05 to 0.5wt%.

In other embodiments of the present invention, the acceptor particles in the acceptor reagent include a second carrier, the second carrier is internally filled with the light-emitting composition, the surface of the second carrier is coated with at least one polysaccharide layer, and a reporter molecule is connected to the surface of the polysaccharide layer, and the reporter molecule can be specifically bound to the target molecule to be detected.

In some embodiments of the invention, the luminescent composition comprises a chemiluminescent compound and a metal chelate.

In other embodiments of the invention, the chemiluminescent compound is selected from the group consisting of olefinic compounds, preferably selected from the group consisting of dimethylthiophene, bis-butanedione compounds, dioxins, enol ethers, enamines, 9-alkylene xanthenes, 9-alkylene-N-9, 10-acridinium dihydrogenate, aryletherenes, arylimidazoles, and lucigenin and their derivatives, more preferably selected from the group consisting of dimethylthiophene and its derivatives.

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 europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably europium.

In other embodiments of the 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, 2-dimethyl-4-perfluorobutanoyl-3-butanone, 2' -bipyridine, bipyridylcarboxylic acid, azacrown ether, azacryptand and trioctylphosphine oxide, and derivatives thereof.

In some embodiments of the invention, the body fluid from the subject is diluted with a diluent prior to contact with the acceptor reagent and the donor reagent.

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

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

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

In other embodiments of the invention, the active oxygen is singlet oxygen.

In other embodiments of the invention, the bodily fluid from the subject includes, but is not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, stool, urine, or spinal fluid.

The beneficial effects of the invention are as follows: the invention provides a use of a donor reagent in preparing a kit used for a method for in vitro diagnosing whether a main body is infected with bacterial inflammatory diseases, wherein the donor particles in the donor reagent have high active oxygen generating efficiency, active oxygen is more easily transferred to receptor particles in a homogeneous system and is not easily interfered by other substances, and the donor particles have higher stability, can stably exist in the donor reagent and are not easy to inactivate. When the donor reagent is used for detection, the detection has ultrahigh sensitivity and wide detection range. In addition, the donor particles in the donor reagent have low production cost, are convenient to use and can be commonly used in various detection projects.

Drawings

The invention will be further described with reference to the accompanying drawings.

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

FIG. 2 is a Nicomp profile of the aldehyde-based polystyrene latex microspheres prepared in example 1.

FIG. 3 is a Gaussian distribution plot of donor particles prepared in example 1.

FIG. 4 is a Gaussian distribution diagram of dextran-coated microspheres prepared in example 2

FIG. 5 is a Gaussian distribution plot of donor particles prepared in example 2.

FIG. 6 is a graph showing the Gaussian distribution of the aldehyde-based polystyrene latex microspheres prepared in example 3.

FIG. 7 is a graph showing the Gaussian distribution of the aldehyde-based polystyrene latex microspheres impregnated with the luminescent composition prepared in example 3.

FIG. 8 is a Gaussian distribution diagram of dextran-coated aldehyde-based polystyrene latex microspheres embedded with a luminescent composition prepared in example 3.

FIG. 9 is a Gaussian distribution diagram of the receptor particles having an average particle diameter of about 250nm prepared in example 3.

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. The practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.

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 "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O ₂ Hydrogen peroxide (H), a two-electron reduction product ₂ O ₂ ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) ₂ ) Etc.

The term "donor particle" as used herein refers to particles containing a sensitizer which upon activation of energy or an active compound is capable of generating an active intermediate such as active oxygen which reacts with the acceptor particle. The donor particles 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 particles are polymeric microspheres filled with 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 well as 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, as disclosed in U.S. patent No. 5709994 (which is incorporated herein by reference in its entirety). Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as described in U.S. patent No. 6406913, which is incorporated herein by reference.

The term "acceptor particle" as used herein refers to a particle comprising a compound capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced to activate by energy or an active compound and release active oxygen in a high energy state which is captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the invention, the acceptor particle comprises a luminescent composition and a carrier, the luminescent composition being filled in the carrier and/or coated on the surface of the carrier.

The "carrier" according to the 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 well known to the person 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 be magnetic or non-magnetic, which has any density, but preferably has a density close to that of water, preferably is floatable in water, and which is composed of transparent, partially transparent or opaque materials.

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

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

The term "C.V value of the particle size distribution coefficient of variation" as used herein refers to the coefficient of variation of the particle size in the Gaussian distribution in the result of the detection by the nanoparticle analyzer. The calculation formula of the variation coefficient is as follows: C.V values = (standard deviation SD/Mean) x 100%.

The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the united states PSS nanoparticle sizer Nicomp. The Nicomp multimodal algorithm has unique advantages over the Gaussian unimodal algorithm for the analysis of multicomponent, non-uniform particle size distribution liquid dispersions and stability analysis of colloidal systems.

The term "body fluid from a subject" as used herein refers to a mixture to be tested that contains or is suspected of containing a myocardial marker. The body fluid is derived from whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, stool, urine, tears, or spinal fluid of the subject. The body fluid from the body may be diluted with a diluent as needed before use. For example, in order to avoid the HOOK effect, the body fluid from the subject may be diluted with a diluent before the on-machine test and then tested on the test instrument.

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. In any desired case, 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 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. Under the technical ideas disclosed in the present invention, the detection method of the specific binding reaction includes, but is not limited to: a diabody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.

II. Detailed description of the preferred embodiments

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

Those skilled in the art generally recognize that the more uniform the particle size of a microsphere, the better the performance of a homogeneous chemiluminescent assay using the microsphere. Thus, current research on microspheres employed in homogeneous chemiluminescence tends to obtain microspheres of more uniform particle size. The inventor of the application finds that when the microsphere with uniform particle size is adopted for homogeneous chemiluminescence detection after research, the sensitivity and the detection range of the detection result are difficult to ensure simultaneously. However, by adopting the microsphere with proper uniformity of particle size (for example, the variation coefficient of microsphere particle size distribution is more than 5%), the sensitivity of the photo-excitation chemiluminescence detection can be ensured, and the detection range can be widened.

Thus, the present invention relates to the use of a donor agent for the preparation of a kit for use in a method for in vitro diagnosis of whether a subject is infected with a bacterial inflammatory disease, wherein the method comprises: contacting body fluid from a subject with an acceptor reagent and a donor reagent, and reacting to produce a mixture to be tested; exciting the test mixture at least once with excitation light, detecting the signal intensity of the chemiluminescence produced thereby; quantitatively calculating the concentration of procalcitonin in the body fluid according to the signal intensity of the chemiluminescence, so as to judge whether the main body is infected with bacterial inflammatory diseases;

It is noted that the value of C.V of the particle size distribution of the donor particle refers to the value of C.V of the particle size distribution of the donor particle coated with the desired substance.

In some embodiments of the invention, the donor particle may have a particle size distribution coefficient of variation C.V value of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35% or 40% in the acceptor reagent.

In some embodiments of the invention, the acceptor particles have a particle size distribution coefficient of variation C.V value of 5% or more in the acceptor reagent.

In some embodiments of the invention, the receptor particle has a particle size distribution coefficient of variation C.V value of greater than or equal to 8% in the receptor agent; preferably, the receptor particles have a value of C.V of the coefficient of variation of the particle size distribution in the receptor agent of not less than 10%.

In other embodiments of the invention, the receptor particles have a particle size distribution coefficient of variation C.V value of 40% or less in the receptor agent; still more preferably, the acceptor particles have a value of a variation coefficient C.V of the particle size distribution in the acceptor reagent of 20% or less.

It is noted that the C.V value of the particle size distribution coefficient of variation of the receptor particles refers to C.V value of the particle size distribution coefficient of variation of the receptor particles coated with the desired substance.

In some embodiments of the invention, the receptor particle may have a particle size distribution coefficient of variation C.V value of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35% or 40% in the receptor agent.

In some embodiments of the invention, the particle size distribution of the acceptor particles in the acceptor agent exhibits polydispersity.

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

In other embodiments of the invention, the receptor particles exhibit two or more peaks in the Gaussian distribution curve of the receptor reagent using a Gaussian distribution assay.

In some embodiments of the invention, the acceptor reagent comprises at least two acceptor particles having an average particle size distribution.

In some embodiments of the invention, the surface of the carrier is coated with a coating of 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, the polysaccharide has pendant functional groups and the continuous polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between the functional groups and the functional groups of the previous polysaccharide layer.

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

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

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

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

In order to further improve the accuracy of the final test result and the stability of the sample to be tested, in some embodiments of the present invention, the body fluid from the subject is diluted with a diluent and then contacted with the acceptor reagent and the donor reagent.

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

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

In other embodiments of the invention, the active oxygen is singlet oxygen.

III. Examples

Example 1: preparation of donor particles with surface uncoated or bound polysaccharide and donor reagent

Preparation of (one) aldehyde polystyrene latex microsphere

a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced ₂ 30min。

b) 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 of the step a), and continuing to introduce N ₂ 30min。

c) The reaction system was warmed to 70℃and reacted for 15 hours.

d) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. The obtained emulsion is washed by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, and then diluted with water, and is stored in the form of emulsion.

e) The average particle size of the Gaussian distribution of the latex microsphere particle size was 201.3nm as measured by a nanoparticle analyzer, the coefficient of variation (c.v.) was=8.0%, the Gaussian distribution was shown in fig. 1, and the Nicomp distribution was multimodal (shown in fig. 2). The aldehyde group content of the latex microsphere is 260nmol/mg measured by a conductivity titration method.

(II) filling of sensitizer

a) A25 ml round bottom flask was prepared, 0.11g copper phthalocyanine, 10ml N, N-dimethylformamide was added thereto, and magnetically stirred, and the temperature was raised to 75℃in a water bath to obtain a photosensitizer solution.

b) A100 ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water and 10ml of the aldehyde-based polystyrene latex microspheres obtained in (I) at a concentration of 10% were added, and the mixture was magnetically stirred and heated to 70℃in a water bath.

c) Slowly dripping the solution in the step a) into the three-neck flask in the step b), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling to obtain emulsion.

d) The emulsion was centrifuged for 1 hour, 30000G, after which the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugation washing three times, the mixture was resuspended in 50mM CB buffer having a pH value of 10 to a final concentration of 20mg/ml.

(III) microsphere surface modification avidin, preparation of donor reagent

a) Microsphere suspension treatment: sucking a certain amount of microspheres prepared in the step (II), centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, adding a certain amount of MES buffer, performing ultrasonic treatment on an ultrasonic cell disruption instrument until particles are resuspended, and adding the MES buffer to adjust the concentration of the microspheres to 100mg/ml.

b) Avidin solution preparation: a quantity of streptavidin was weighed and dissolved to 8mg/ml in MES buffer.

c) Mixing: mixing the treated microsphere suspension, avidin of 8mg/ml and MES buffer in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.

d) The reaction: 25mg/ml NaBH is prepared by MES buffer solution ₃ The CN solution was added in a volume ratio of 1:25 to the reaction solution, and the mixture was rapidly and uniformly mixed. The reaction was rotated at 37℃for 48 hours.

e) Closing: preparing 75mg/ml Gly solution and 25mg/ml NaBH in MES buffer ₃ CN solution is added into the solution according to the volume ratio of 2:1:10 with the reaction solution, and the mixture is uniformly mixed and rotated at 37 ℃ for 2 hours. Then 200mg/ml BSA solution (MES buffer) was added thereto in a volume ratio of 5:8, and the mixture was swiftly mixed and reacted at 37℃for 16 hours.

f) Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, suspending again by an ultrasonic method, centrifuging again, washing for 3 times, suspending with a small amount of donor particle buffer solution, measuring the solid content, and regulating the concentration to 150 mug/ml with the donor particle buffer solution to obtain the donor reagent containing donor particles.

g) The donor particles had a gaussian distribution average particle diameter of 227.7nm as measured by a nanoparticle analyzer, and a coefficient of variation (c.v.) of=6.5, as shown in fig. 3.

Example 2: preparation of polysaccharide coated donor particles and donor reagents

The procedure for the preparation of the aldehyde-based polystyrene latex microspheres and the filling of the sensitizer is the same as in the preparation steps (one) and (two) of example 1.

Preparation of (one) aminodextran

a) The 500mL four-necked flask was placed in an oil bath, equipped with a condenser, and purged with nitrogen.

b) 10g of dextran having an average molecular weight distribution of 500000kDa, 100ml of deionized water, 2g of NaOH, 10g N- (2, 3-epoxypropyl) phthalimide were added in this order and stirred mechanically.

c) After oil bath at 90 ℃ for 2 hours, heating is closed, stirring is maintained, and natural cooling is carried out.

d) The reaction mixture was separated out of the main mixture in 2L of methanol, and the solid was collected and dried.

e) 200mL four-necked flask was placed in an oil bath, equipped with a condenser, and purged with nitrogen.

f) The dried solid, 100mL of deionized water, 1.8g of sodium acetate, 5mL of 50% hydrazine hydrate were added sequentially, the pH was adjusted to 4, and the mixture was stirred mechanically.

g) After oil bath at 85 ℃ for 1 hour, heating is closed, stirring is maintained, and natural cooling is carried out.

h) The reaction solution was adjusted to neutral pH and then filtered, and the filtrate was collected.

i) The filtrate is placed in a dialysis bag, dialyzed for 2 days at 4 ℃ with deionized water, and changed for 3-4 times per day.

j) After completion of dialysis, the gel was lyophilized to obtain 9.0g of an aminodextran solid.

k) The amino group content was found to be 0.83mmol/g by TNBSA method.

Preparation of (di) aldehyde dextran

a) 10g of dextran having an average molecular weight distribution of 500000kDa was weighed into a 250 beaker, 100mL of 0.1M/pH=6.0 phosphate buffer was added and dissolved by stirring at room temperature.

b) 1.8g of sodium metaperiodate is weighed into a 50mL beaker, 10 mL of 0.1M/pH=6.0 phosphate buffer is added, and the mixture is stirred and dissolved at room temperature.

c) Slowly dripping the sodium metaperiodate solution into the dextran solution, and continuously stirring for 1 hour after the reaction is carried out until no bubbles are generated.

d) The reaction mixture is placed in a dialysis bag, dialyzed for 2 days at 4 ℃ with deionized water, and changed for 3-4 times per day.

e) After completion of dialysis, the resulting mixture was lyophilized to obtain 9.6g of an aldehyde dextran solid.

f) The aldehyde group content was found to be 0.94mmol/g by BCA Kit.

(III) microsphere coating dextran

a) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added and dissolved by stirring away from light at 30 ℃.

b) 100mg of donor particles were taken and added to the aminodextran solution and stirred for 2 hours.

c) 10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the reaction mixture, followed by reaction at 30℃overnight in the absence of light.

d) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/ml by constant volume with 50 mM/pH=10 carbonate buffer.

e) 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added and dissolved by stirring away from light at 30 ℃.

f) The above particles were added to the aldehyde dextran solution and stirred for 2 hours.

g) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and the solution was added dropwise to the reaction mixture, followed by reaction at 30℃overnight in the absence of light.

h) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/ml by constant volume with 50 mM/pH=10 carbonate buffer.

i) The average gaussian distribution particle diameter of the microsphere was 235.6nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=8.1%, as shown in fig. 4.

(IV) microsphere surface modification avidin, preparing donor reagent

h) Microsphere suspension treatment: sucking a certain amount of microspheres prepared in the step (III), centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, adding a certain amount of MES buffer, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the microspheres are resuspended, and adding the MES buffer to adjust the concentration of donor particles to 100mg/ml.

i) Avidin solution preparation: a certain amount of neutravidin was weighed and dissolved in MES buffer to 8mg/ml.

j) Mixing: mixing the treated microsphere suspension, avidin of 8mg/ml and MES buffer in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.

k) The reaction: 25mg/ml NaBH is prepared by MES buffer solution ₃ The CN solution was added in a volume ratio of 1:25 to the reaction solution, and the mixture was rapidly and uniformly mixed. The reaction was rotated at 37℃for 48 hours.

l) closing: preparing 75mg/ml Gly solution and 25mg/ml NaBH in MES buffer ₃ CN solution is added into the solution according to the volume ratio of 2:1:10 with the reaction solution,mixing well, and rotating at 37 ℃ for 2 hours. Then 200mg/ml BSA solution (MES buffer) was added thereto in a volume ratio of 5:8, and the mixture was swiftly mixed and reacted at 37℃for 16 hours.

m) cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, suspending again by an ultrasonic method, centrifuging again, washing for 3 times, suspending with a small amount of donor particle buffer solution, measuring the solid content, and regulating the concentration to 150 mug/ml with the donor particle buffer solution to obtain the donor reagent containing donor particles.

n) the donor particles had a gaussian distribution average particle diameter of 249.9nm as measured by a nanoparticle analyzer, and a coefficient of variation (c.v.) of 11.6%, as shown in fig. 5.

Example 3: preparation of acceptor particles

1. Preparation and characterization process of aldehyde polystyrene latex microsphere

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 10 minutes, N was introduced ₂ 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 of the step 1, and continuing to introduce N ₂ 30min；

3) Heating the reaction system to 70 ℃ for reaction 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 by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;

5) The average particle diameter of the Gaussian distribution of the latex microsphere particle diameter at this time was 202.2nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=4.60%, and the Gaussian distribution curve is shown in fig. 6. The aldehyde group content of the latex microsphere is 280nmol/mg measured by a conductivity titration method.

2. Process and characterization of embedding luminescent compositions inside microspheres

1) A25 ml round bottom flask was prepared and charged with 0.1g of the dimethylthiophene derivative and 0.1gEuropium (III) complexes (MTTA-EU) ³⁺ ) 10ml of 95% ethanol is magnetically stirred, and the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;

2) Preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres with concentration of 10% obtained in the step 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), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;

4) Centrifuging the emulsion for 1 hour, 30000G, and discarding supernatant after centrifuging to obtain aldehyde polystyrene microsphere filled with luminous composition.

5) The average particle diameter of the Gaussian distribution of the microsphere particle diameter at this time was 204.9nm as measured by a nanoparticle sizer, and the coefficient of variation (c.v.) =5.00% (as shown in fig. 7)

3. Process and characterization of coating polysaccharide coating on microsphere surface

1) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and the solution was stirred at 30 ℃ in the absence of light;

2) Taking 100mg of the aldehyde-based polystyrene microspheres prepared in the step 2 and embedded with the luminous composition, adding the microspheres into an aminodextran solution, and stirring for 2 hours;

3) 10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;

4) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/ml;

5) 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and stirred at 30 ℃ in the absence of light for dissolution;

6) Adding the microsphere into an aldehyde dextran solution, and stirring for 2 hours;

7) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;

8) After the reaction mixture was centrifuged at 30000G, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/ml by constant volume with 50 mM/pH=10 carbonate buffer.

9) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 241.6nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=12.90% (as shown in fig. 8).

Conjugation procedure of antibodies to PCT antibodies

1) The paired PCT antibodies were dialyzed to 50mM CB buffer at ph=10, and the concentration was measured to be 1mg/ml.

2) Adding 0.5ml of the microspheres obtained in step 3 and 0.5ml of the paired antibodies I obtained in step 1) into a 2ml centrifuge tube, uniformly mixing, and adding 100. Mu.l of 10mg/ml NaBH ₄ The solution (50 mM CB buffer) was reacted at 2-8℃for 4 hours.

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

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

5) The average particle diameter of the Gaussian distribution of the receptor particle diameter at this time was 253.5nm as measured by a nanoparticle analyzer, and the coefficient of variation (C.V value) =9.60% (as shown in fig. 9).

Example 4: preparation of a Donor reagent comprising the following series of Donor particles by the method of example 1

Donor reagent 1: the average particle diameter of the donor particles in the Gaussian distribution curve is 226.5nm, and the variation coefficient of the particle diameter distribution is C.V value=3.8; nicomp distribution is unimodal.

Donor reagent 2: the average particle diameter of the donor particles in the Gaussian distribution curve is 225.3nm, and the variation coefficient C.V value of the particle diameter distribution=4.6; nicomp distribution is unimodal.

Donor reagent 3: the average particle diameter of the donor particles in the Gaussian distribution curve is 225.2nm, and the variation coefficient of the particle diameter distribution is C.V value=5.0; nicomp distribution is unimodal.

Donor reagent 4: the average particle diameter of the donor particles in the Gaussian distribution curve is 226.7nm, and the variation coefficient of the particle diameter distribution is C.V value=8.1; nicomp distribution is unimodal.

Donor reagent 5: the average particle diameter of the donor particles in the Gaussian distribution curve is 227.8nm, and the variation coefficient C.V value of the particle diameter distribution=15.6; nicomp distribution is unimodal.

Donor reagent 6: the average particle diameter of the donor particles in the Gaussian distribution curve is 225.9nm, and the variation coefficient of the particle diameter distribution is C.V value=26.1; nicomp distribution is unimodal.

Donor reagent 7: the average particle diameter of the donor particles in the Gaussian distribution curve is 225.1nm, and the variation coefficient C.V value of the particle diameter distribution=32.4; nicomp distribution is unimodal.

Example 5: photo-excitation chemiluminescence immunity analyzer

The principle of the photoexcitation chemiluminescence immunoassay analyzer in this embodiment is as follows: the target molecules to be detected in the sample to be detected suspected to contain the PCT marker react with the donor particles and the acceptor particles to form immune complexes, the interaction can pull the donor particles and the acceptor particles closer, and under the irradiation of laser (with the wavelength of 680 nm), the sensitizer in the donor particles converts oxygen in the surrounding environment into more active monomer oxygen. The monomer oxygen diffuses into the acceptor particle and reacts with the chemiluminescent agent in the acceptor particle to further activate the luminescent group on the acceptor particle to emit light with a wavelength of 520-620nm. The half-life of the monomeric oxygen was 4. Mu.Sec and the diffusion distance in the solution was about 200 nm. If there is no interaction of the biomolecules, singlet oxygen cannot diffuse to the acceptor particles, no optical signal will be generated. Therefore, the concentration of the target molecule to be detected in the sample to be detected can be calculated by measuring the light intensity emitted by the mixture. Wherein the donor particle comprises a first carrier, the interior of the first carrier is filled with a sensitizer, and the surface of the first carrier is chemically bonded to one of the specific binding pair members.

One preferred structure of the photoexcitation chemiluminescent immunoassay analyzer according to this embodiment includes the following components:

a reagent sample adding module for adding a sample to be tested and/or an acceptor reagent and a donor reagent to the reaction vessel; wherein the donor reagent comprises donor particles, and the variation coefficient C.V value of the particle size distribution of the donor particles in the donor reagent is more than or equal to 5%;

an incubation module for providing a suitable temperature environment for the homogeneous chemiluminescent reaction in the reaction vessel; the incubation module can adopt a metal bath, a water bath or an oil bath and other modes;

a detection module comprising a laser exciter and a photon detector (PMT) for optical signal detection for detecting chemiluminescent signals generated by the homogeneous chemiluminescent reaction.

And the reagent sample adding module, the incubation module and the detection module are all electrically connected with the circuit control module. The incubation module is used for adjusting the temperature of immunoreactive substances under the control of the circuit control module, the reagent sampling module is used for transferring the substances in the reaction container, and the detection module is used for emitting laser and measuring the light intensity emitted by a sample to be detected.

Example 6: on-machine detection result and analysis (detection substance: PCT antigen)

(1) PCT antigens were detected by the analyzer of example 5 by simultaneously loading the donor reagent of example 1 and the acceptor reagent of example 3 with the donor reagent of example 2, respectively, and the detection results are shown in table 1. The PCT quantitative assay kit (photoexcitation chemiluminescence method) used in this example consisted of reagent 1 (R1 ') containing the acceptor particle coated with the first anti-PCT antibody, reagent 2 (R2 ') containing the second anti-PCT antibody labeled with biotin, and further included a universal solution (R3 ') containing the donor particle. Wherein R1' is a receptor reagent prepared using the receptor particles (the particle size distribution coefficient of variation C.V value=9.6%) in example 3; r3' is a donor agent prepared using the donor particles of examples 1 and 2.

TABLE 1

From the results of table 1, the analytical methods provided herein were excellent in both sensitivity and upper limit of detection. And the sensitivity and upper limit of detection of the analytical method using the donor reagent in example 1 are both superior to those of the analytical method using the donor reagent in example 2. It can be seen that the performance of the donor particles with surface not coated with polysaccharide is more excellent.

(2) The donor reagent of example 4 was tested on-press with the acceptor reagent of example 3

The PCT quantitative assay kit (photoexcitation chemiluminescence method) used in this example consisted of reagent 1 (R1 ') containing the acceptor particle coated with the first anti-PCT antibody, reagent 2 (R2 ') containing the second anti-PCT antibody labeled with biotin, and further included a universal solution (R3 ') containing the donor particle. Wherein R1' is a receptor reagent prepared using the receptor particles (the particle size distribution coefficient of variation C.V value=9.6%) in example 3; r3' is a series of donor reagents prepared using example 4.

The detection process is completed on a fully automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang biotechnology (Shanghai) limited company, and the detection result is output, and the specific experimental steps are as follows:

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

2. placing the white board with the sample into a LiCA HT instrument for reaction in the following reaction mode;

(1) Mixing 40ul of sample to be tested, 15ul of R1 'and 15ul of R2';

(2) Incubation at 37℃for 8min;

(3) 160ul of reagent R3' are added;

(4) Incubation at 37℃for 2min;

(5) Excitation readings, and specific detection results are shown in table 2 below.

TABLE 2

As is clear from Table 2, when the variation coefficient of the particle size distribution of the donor particles used is 5% or more, the on-machine detection using the donor reagent containing the donor particles has a relatively good sensitivity and a wide detection range.

Example 7: clinical detection of PCT in normal and suspected inflammatory patients

In this example, 40 clinical samples were tested, and the PCT quantitative assay kit (photoexcitation chemiluminescence method) used included: reagent 1 (R1 ') comprising a first anti-PCT antibody coated acceptor particle, reagent 2 (R2 ') comprising a biotin-labeled second anti-PCT antibody, and universal liquid (R3 ') comprising donor particles. Wherein the concentration of donor particles in the reagent R3 is 120ug/ml, and the variation coefficient C.V value of the particle size distribution of the donor particles in the reagent R3 is=11%.

The specific experimental steps are as follows:

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

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

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

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

(2) Incubation at 37℃for 8min;

(3) 160ul of R3' was added;

(4) Incubation at 37℃for 2min;

(5) Excitation readings, and specific detection results are shown in table 3 below.

TABLE 3 Table 3

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The correlation between the Roche measurement and the measurement in example 7 was 0.9977 and the slope was 0.9984. Sample numbers 1-11 are normal physical examination patients, the distribution range is 30 pg/ml-70 pg/ml, and the median value is 50pg/ml; samples 12-40 were identified as having inflammatory disease and had a distribution ranging from 120pg/ml to 63.23ng/ml with a median value of 580pg/ml.

Procalcitonin (PCT) is a member of the Calcitonin (CT) superfamily, consisting of 116 amino acids of a polypeptide having a molecular weight of about 14.5 kDa. When severely bacterial, fungal, parasitic infections, sepsis and multiple organ failure, their levels in the plasma of the subject are elevated. PCT reflects the activity level of the systemic inflammatory response. Factors affecting PCT levels include the size and type of the organ being infected, the type of bacteria, the extent of inflammation, and the condition of the immune response. The test results of example 7 of the present invention demonstrate that the use of the donor agent of the present invention in the preparation of a kit for use in a method for in vitro diagnosis of a subject for the infection of a bacterial inflammatory disease, the quantitative determination of PCT markers in body fluids of a subject using the donor agent of the present invention and the corresponding method allows the differential diagnosis of bacterial and non-bacterial infections and inflammations as an acute parameter, monitoring patients at risk of infection (e.g., post-surgical and post-organ-transplant immunosuppression periods, multiple post-trauma) and patients in need of intensive care for detecting the systemic effects of bacterial infections or for detecting septic complications; clinical course and prognosis of severe inflammatory diseases, such as peritonitis, sepsis, SIRS and MODS, are assessed.

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. Use of a donor agent in the preparation of a kit for use in a method for in vitro diagnosis of whether a subject is infected with a bacterial inflammatory disease, wherein the method comprises: contacting body fluid from a subject with an acceptor reagent and a donor reagent, and reacting to produce a mixture to be tested; exciting the test mixture at least once with excitation light, detecting the signal intensity of the chemiluminescence produced thereby; quantitatively calculating the concentration of procalcitonin in the body fluid according to the signal intensity of the chemiluminescence, so as to judge whether the main body is infected with bacterial inflammatory diseases;

Wherein the donor reagent comprises donor particles capable of generating active oxygen in an excited state, the donor particles comprise a first carrier, the particle size of the first carrier is selected from 100-400 nm, the material of the first carrier is selected from polystyrene, the interior of the first carrier is filled with a sensitizer, and the surface of the first carrier is not coated or connected with a polysaccharide substance, and is directly chemically bonded with one member of a specific binding pair member; the donor reagent also comprises a buffer solution with the pH value of 7.0-9.0, and the donor particles are suspended in the buffer solution; the buffer solution contains polysaccharide selected from dextran, starch, glycogen, inulin, levan, mannan, agarose and galactan;

the donor particles have a particle size distribution coefficient of variation C.V value in the donor agent selected from 5% to 32.4%.

2. The use according to claim 1, wherein the surface of the first support carries a binding functionality for chemically binding one member of a specific binding pair member to the surface of the first support.

3. Use according to claim 2, characterized in that the bonding functional groups are selected from amine groups, amide groups, hydroxyl groups, aldehyde groups, carboxyl groups, maleimide groups and mercapto groups.

4. Use according to claim 3, characterized in that the bonding functional groups are selected from aldehyde groups and/or carboxyl groups.

5. Use according to claim 2, characterized in that the content of binding functions of the first support surface is between 100 and 500nmol/mg.

6. The use according to claim 5, characterized in that the content of binding functions of the first support surface is 200 to 400nmol/mg.

7. The use according to any one of claims 1 to 6, wherein the particle size of the first carrier is selected to be 150 to 350nm.

8. The use according to claim 7, characterized in that the particle size of the first carrier is selected to be 180-220 nm.

9. The use according to any one of claims 1 to 6, wherein the first carrier is a polystyrene latex microsphere.

10. Use according to any one of claims 1 to 6, characterized in that the sensitizer is a photoactivated photosensitizer and/or a chemically activated initiator.

11. The use according to claim 10, wherein the sensitizer is a photoactivated photosensitizer.

12. The use according to any one of claims 1 to 6, wherein the sensitizer is selected from methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll.

13. The use according to any one of claims 1 to 6, 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, and a pair of substances capable of specifically binding to each other.

14. The use according to claim 13, wherein the specific binding pair member is avidin-biotin.

15. Use according to claim 14, wherein the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like.

16. Use according to claim 15, characterized in that the avidin is neutravidin and/or streptavidin.

17. The use according to claim 16, wherein the avidin is chemically bound to the surface of the first support by reacting amino groups with aldehyde groups on the surface of the first support to form schiff bases.

18. The use according to any one of claims 1 to 6, wherein the particle size distribution of the donor particles in the donor agent exhibits polydispersity.

19. The use according to any one of claims 1 to 6, characterized in that the concentration of the donor particles in the donor agent is between 10 μg/ml and 1mg/ml.

20. Use according to claim 19, characterized in that the concentration of the donor particles in the donor agent is 20 μg/ml to 500 μg/ml.

21. Use according to claim 20, characterized in that the concentration of the donor particles in the donor agent is 50 μg/ml to 200 μg/ml.

22. The use according to any one of claims 1 to 6, wherein the dextran has a molecular weight distribution Mw selected from 10000 ～ 1000000KDa.

23. The use according to claim 22, characterized in that the molecular weight distribution Mw of the dextran is selected from 100000 ～ 800000KDa.

24. The use according to claim 23, characterized in that the molecular weight distribution Mw of the dextran is selected from 300000 ～ 700000KDa.

25. The use according to any one of claims 1 to 6, characterized in that the dextran content of the buffer solution is 0.01 to 1wt%.

26. Use according to claim 25, characterized in that the dextran content of the buffer solution is 0.05-0.5 wt%.

27. Use according to any one of claims 1 to 6, characterized in that the dextran is selected from carboxydextran and/or aminodextran.

28. The use according to any one of claims 1 to 6, wherein the acceptor particles in the acceptor reagent comprise a second carrier, the second carrier is internally filled with a luminescent composition, the surface of the second carrier is coated with at least one polysaccharide layer, the surface of the polysaccharide layer is connected with a reporter molecule, and the reporter molecule can be specifically combined with a target molecule to be detected.

29. The use according to claim 28, wherein the luminescent composition comprises a chemiluminescent compound and a metal chelate.

30. The use according to claim 29, wherein the chemiluminescent compound is selected from the group consisting of alkene compounds.

31. The use according to claim 30, wherein the chemiluminescent compound is selected from the group consisting of dimethylthiophene, a bis-butanedione compound, dioxins, enol ethers, enamines, 9-alkylene xanthenes, 9-alkylene-N-9, 10 acridinium dihydrogenates, aryl ether alkenes, aryl imidazoles and lucigenin and derivatives thereof.

32. The use according to claim 31, wherein the chemiluminescent compound is selected from the group consisting of dimethylthiophene and derivatives thereof.

33. Use according to claim 29, characterized in that the metal of the metal chelate is a rare earth metal or a group VIII metal.

34. The use according to claim 33, wherein the metal of the metal chelate is selected from europium, terbium, dysprosium, samarium, osmium and ruthenium.

35. The use according to claim 34, wherein the metal of the metal chelate is europium.

36. Use according to claim 29, characterized in that the metal chelate comprises a chelating agent selected from the group consisting of: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA 2, 2-dimethyl-4-perfluorobutanoyl-3-butanone, 2' -bipyridine, bipyridylcarboxylic acid, azacrown ether, azacryptand and trioctylphosphine oxide, and derivatives thereof.

37. The use according to any one of claims 1 to 6, wherein the body fluid from the subject is diluted with a diluent and contacted with the acceptor reagent and the donor reagent.

38. The use according to any one of claims 1 to 6, wherein the chemiluminescent detection wavelength is 520 to 620nm.

39. The use according to any one of claims 1 to 6, characterized in that the laser irradiation is performed with 600 to 700nm of red excitation light.

40. The use according to any one of claims 1 to 6, wherein the concentration of the acceptor particles in the acceptor reagent is from 1ug/mL to 1000ug/mL.

41. The use according to claim 40, wherein the concentration of the acceptor particles in the acceptor reagent is from 10ug/mL to 500ug/mL.

42. The use according to claim 41, wherein the concentration of the acceptor particles in the acceptor reagent is from 20ug/mL to 200ug/mL.

43. The use according to any one of claims 1 to 6, wherein the active oxygen is singlet oxygen.

44. The use according to any one of claims 1 to 6, wherein the body fluid from the subject includes, but is not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, stool, urine, or spinal fluid.