Use of a receptor reagent in diagnosing bacterial inflammatory diseases in a subject

Abstract

The present application relates to a kind of acceptor reagent in the preparation for the use of kit for the method for diagnosing whether subject is infected bacterial inflammatory disease in vitro, wherein the method comprises: the body fluid from subject is contacted with acceptor reagent and donor reagent, and generates the mixture to be measured after reaction;The intensity of the chemiluminescence signal generated thereby is detected by exciting the mixture to be measured at least once with excitation light;The concentration of procalcitonin in the body fluid is quantitatively calculated according to the intensity of the chemiluminescence signal, to determine whether subject is infected bacterial inflammatory disease;Wherein, the acceptor reagent comprises the acceptor particle capable of generating chemiluminescence by reacting with active oxygen, and the particle size distribution variation coefficient C.V value of the acceptor particle in acceptor reagent is controlled to be greater than or equal to 5%;The donor reagent comprises donor particle capable of generating active oxygen under excitation state.The method has both ultra-high sensitivity and very wide detection range.

Description

Technical Field

[0001] The present invention belongs to the field of chemiluminescence detection, and in particular relates to the use of a receptor reagent in diagnosing bacterial inflammatory diseases in a subject. Background Art

[0002] Procalcitonin (PCT) is a 116-amino acid protein with a molecular weight of approximately 13 kDa. It is the propeptide of calcitonin (CT) and lacks calcitonin-like hormonal activity. Its molecule consists of calcitonin, a calcium inhibitory peptide, and a 57-amino acid N-terminal fragment. PCT reflects the activity of systemic inflammatory responses. PCT levels in normal human serum are extremely low (<0.05 mg / L). PCT concentrations are minimally elevated or do not increase in conditions such as localized infections, viral infections, chronic nonspecific inflammation, cancer fever, graft-versus-host rejection, and autoimmune diseases. However, PCT levels are significantly elevated in severe bacterial, fungal, and parasitic infections, as well as in sepsis and multiorgan failure. Therefore, PCT measurement can be used as an acute diagnostic parameter to differentiate between bacterial and nonbacterial infections and inflammation.

[0003] Numerous methods exist for measuring procalcitonin, including chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and colloidal gold immunochromatography (GICA), but all have limitations. CLIA, an immunoassay developed from ELISA, offers advantages such as high sensitivity, a wide linear range, ease of use, and a high degree of automation, and has gained widespread application in recent years. Procalcitonin antigen in the test sample reacts with procalcitonin antibodies coated on superparamagnetic microparticles and an enzyme-labeled procalcitonin antibody conjugate to form a sandwich (antibody-antigen-antibody) complex. Under the influence of a magnetic field, the magnetic microparticles adhere to the walls of the reaction tube, while unbound material is washed away with a wash solution. A chemiluminescent substrate solution is added to the reaction mixture, and the test results are expressed as relative luminescence intensity (RLU). The amount of procalcitonin antigen in the test sample is directly proportional to the relative luminescence intensity (RLU) detected by the analyzer's optical system.

[0004] At present, the domestic chemiluminescence immunoassay market is mainly dominated by imported closed fully automatic chemiluminescence detection systems (reagents and instruments). However, domestic PCT detection reagents started late, and their performance has always been lagging behind imported reagents that have been optimized for several years or even decades, especially in terms of repeatability, stability and sensitivity. Summary of the Invention

[0005] The present application aims to solve the technical problem of the prior art, and provides a use of a receptor reagent in a kit for preparing a method for diagnosing whether a subject is infected with a bacterial inflammatory disease in vitro, which has both ultra-high sensitivity and a wide detection range when the receptor reagent is used for detection.

[0006] To this end, the present application provides, in a first aspect, a use of a receptor reagent in a kit for preparing a method for diagnosing whether a subject is infected with a bacterial inflammatory disease in vitro, wherein the method comprises: contacting a body fluid from a subject with a receptor reagent and a donor reagent to generate a to-be-tested mixture after reaction; exciting the to-be-tested mixture at least once with excitation light to detect the signal intensity of the chemiluminescence generated thereby; and quantitatively calculating the concentration of procalcitonin in the body fluid according to the signal intensity of the chemiluminescence, so as to determine whether the subject is infected with a bacterial inflammatory disease.

[0007] In the present application, the receptor reagent comprises receptor particles capable of generating chemiluminescence by acting on active oxygen, and the coefficient of variation C.V of the particle size distribution of the receptor particles in the receptor reagent is controlled to be greater than or equal to 5%; and the donor reagent comprises donor particles capable of generating active oxygen in an excited state.

[0008] In some embodiments of the present application, the coefficient of variation C.V of the particle size distribution of the receptor particles in the receptor reagent is controlled to be greater than or equal to 8%; preferably, the coefficient of variation C.V of the particle size distribution of the receptor particles in the receptor reagent is controlled to be greater than or equal to 10%.

[0009] In some preferred embodiments of the present application, the coefficient of variation C.V of the particle size distribution of the receptor particles in the receptor reagent is controlled to be less than or equal to 40%; more preferably, the coefficient of variation C.V of the particle size distribution of the receptor particles in the receptor reagent is controlled to be less than or equal to 20%.

[0010] In some embodiments of the present application, the particle size distribution of the receptor particles in the receptor reagent exhibits polydispersity.

[0011] In some specific embodiments of the present application, the coefficient of variation C.V of the particle size distribution is calculated by Gaussian distribution.

[0012] In some specific embodiments of the present application, the Gaussian distribution curve of the receptor particles in the receptor reagent exhibits two or more peaks by Gaussian distribution analysis.

[0013] In some preferred embodiments of the present application, the receptor reagent comprises at least two kinds of receptor particles with different average particle size distributions.

[0014] In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or attached to the carrier.

[0015] In some embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

[0016] In other specific embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0017] In some specific embodiments of the present 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.

[0018] In other specific 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, 2,2-dimethyl-4-perfluorobutyryl-3-butanone (FOD), 2,2'-bipyridine (BPY), bipyridyl carboxylic acid, azacrown ethers, azacryptands and trioctylphosphine oxide and derivatives thereof.

[0019] In some embodiments of the present invention, the carrier is selected from the group consisting of a strip, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably a bead and a microsphere.

[0020] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0021] In some embodiments of the present 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.

[0022] In other embodiments of the present invention, the carrier is formaldehyde-modified latex particles.

[0023] In some preferred embodiments of the present invention, the average particle size of the carrier is in the range of 50 nm to 1 μm; preferably 100 nm to 500 nm; more preferably 150 nm to 400 nm; most preferably 190 nm to 300 nm.

[0024] In some embodiments of the present invention, the surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

[0025] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0026] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers have charges opposite to those of the functional groups of the previous polysaccharide layer.

[0027] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and the successive layers of the polysaccharide are covalently linked to the previous polysaccharide layer by reaction between the functional groups of the successive layers and the functional groups of the previous layer.

[0028] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0029] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0030] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0031] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0032] In some embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0033] In other specific embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

[0034] In some embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to a member of the specific binding pair.

[0035] In other specific embodiments of the present invention, the specific binding pair member is selected from a pair of substances consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, avidin or biotin; preferably, the specific binding pair member is biotin-avidin.

[0036] In some specific embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose.

[0037] It should be noted that the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is still ≥5%. In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution is ≥10%; further preferably, the coefficient of variation (CV) of the particle size distribution is ≤40%; and even more preferably, the coefficient of variation (CV) of the particle size distribution is ≤20%. In some preferred embodiments of the present invention, the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair exhibits polydispersity.

[0038] In some embodiments of the invention, the donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

[0039] In other embodiments of the present invention, the body fluid from the subject is diluted with a diluent before contacting with the receptor reagent and the donor reagent.

[0040] In some embodiments of the present invention, the detection wavelength of the chemiluminescence is 520-620 nm.

[0041] In other embodiments of the present invention, red excitation light of 600 to 700 nm is used for laser irradiation.

[0042] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL; more preferably 20 ug / mL-200 ug / mL.

[0043] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0044] In some embodiments of the present invention, the body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

[0045] The beneficial effects of the present invention are as follows: the present invention provides a use of a receptor reagent in preparing a kit for use in a method for in vitro diagnosis of whether a subject is infected with a bacterial inflammatory disease. The method comprises adding a receptor reagent containing receptor particles to body fluids from the subject, wherein the coefficient of variation CV value of the receptor particle size distribution is ≥5%, so that the detection performance of the method of the present invention for PCT is greatly improved compared with the existing technology, with both ultra-high sensitivity and a wide detection range. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings.

[0047] Figure 1 This is a Gaussian distribution curve of the formaldehyde polystyrene latex microspheres prepared in Example 1.

[0048] Figure 2 This is a Gaussian distribution curve of the formaldehyde polystyrene latex microspheres filled with the luminescent composition prepared in Example 1.

[0049] Figure 3 Gaussian distribution diagram of the dextran-coated aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 1

[0050] Figure 4 This is the Gaussian distribution diagram of the receptor particles prepared in Example 1 with an average particle size of about 250 nm.

[0051] Figure 5 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0052] Figure 6 This is the Nicomp distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0053] Figure 7 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0054] Figure 8 This is the Nicomp distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0055] Figure 9 This is the Gaussian distribution diagram of the particle size distribution of the mixed receptor particles in Example 2.

[0056] Figure 10 This is the Nicomp distribution diagram of the particle size distribution of the mixed receptor particles in Example 2. DETAILED DESCRIPTION

[0057] To facilitate understanding of the present invention, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to be limiting. The practice of the present invention is not limited to the following examples, and any modifications and / or variations made to the present invention fall within the scope of the present invention.

[0058] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. Where a specified range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0059] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present 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, preferred methods and materials are now described.

[0060] I. Terminology

[0061] The term "active oxygen species" as used in the present invention refers to a general term for substances composed of oxygen, containing oxygen and having active properties in the body or the natural environment, mainly an excited state of oxygen molecules, including the one-electron reduction product of oxygen, the superoxide anion (O2·-), the two-electron reduction product of oxygen, the three-electron reduction product of oxygen, the hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2).

[0062] The term "donor particle" as used herein refers to a particle containing a sensitizer that, upon activation by energy or an active compound, is capable of generating an active intermediate, such as a reactive oxygen species, that reacts with the acceptor particle. The donor particle can be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the present application, the donor particle is a polymeric microsphere filled with a photosensitizer, which can be any photosensitizer known in the art, preferably a compound that is relatively photostable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose Bengal, porphyrins, phthalocyanines, and chlorophylls, as disclosed in U.S. Patent No. 5,709,994 (incorporated herein by reference in its entirety), as well as derivatives of these compounds having 1-50 atom substituents that serve to make the compounds more lipophilic or more hydrophilic, and / or as linking groups to specific binding partners. Examples of other photosensitizers known to those skilled in the art can also be used in the present application, such as those described in U.S. Patent No. 6,406,913, which is incorporated herein by reference.

[0063] The term "acceptor particle" as used herein refers to a particle containing a compound that is capable of reacting with a reactive oxygen species to produce a detectable signal. The donor particle is induced to activate by energy or an active compound and releases a reactive oxygen species in a high-energy state that is captured by a proximal acceptor particle, thereby transferring energy to activate the acceptor particle. In some embodiments of the present application, 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" as used herein is selected from the group consisting of strips, sheets, rods, tubes, wells, microtiter plates, beads, particles, and microspheres, which can be microspheres or microparticles known to those skilled in the art, which can be of any size, which can be organic or inorganic, which can be swellable or non-swellable, which can be porous or non-porous, which can have any density, but preferably has a density close to that of water, preferably is capable of floating in water, and is composed of a transparent, partially transparent, or opaque material. The carrier can be charged or uncharged, and when charged, is preferably negatively charged. The carrier can be a latex particle or other particle containing an organic or inorganic polymer, a lipid bilayer such as a liposome, a phospholipid vesicle, a small oil droplet, a silica particle, a metal sol, a cell, and a microcrystalline dye.

[0064] In the present application, the "chemiluminescent compound" is a compound known as a label that undergoes a chemical reaction to cause luminescence, such as by being converted to another compound that forms in an electronically excited state. The excited state can be a singlet or a triplet excited state. The excited state can either luminesce directly by relaxing to the ground state, or by transferring the excitation energy to an energy acceptor, thereby recovering to the ground state. In this process, the energy acceptor particle is transitioned to an excited state and luminesces.

[0065] In the present invention, "capable of binding directly or indirectly" means that the specified entity can specifically bind to the entity (directly), or the specified entity can specifically bind to a specific binding pair member (indirectly).

[0066] The "specific binding pair members" of the present invention refer to a pair of substances that can specifically bind to each other.

[0067] The "coefficient of variation of particle size distribution CV value" mentioned in the present invention refers to the coefficient of variation of particle size in Gaussian distribution in the detection results of nanoparticle size analyzer. The formula for calculating the coefficient of variation is: CV value = (standard deviation SD / mean value) × 100%.

[0068] The term "Nicomp distribution" as used herein refers to an algorithmic distribution used in the American PSS nanoparticle size analyzer NICOMP. Compared to the Gaussian unimodal algorithm, the Nicomp multimodal algorithm offers unique advantages for analyzing multicomponent liquid dispersions with uneven particle size distributions, as well as for analyzing the stability of colloidal systems.

[0069] As used herein, the term "body fluid from a subject" refers to a mixture containing or suspected of containing a myocardial marker to be tested. The body fluid may be whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears, or spinal fluid from a subject. The body fluid from a subject may be diluted with a diluent as needed prior to use. For example, to avoid the hook effect, the body fluid from a subject may be diluted with a diluent prior to testing on a test instrument.

[0070] 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. Where desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair), etc.

[0071] The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0072] As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.

[0073] The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances. From a stereostructural perspective, this refers to the conformational correspondence between the reactants. Within the technical concepts disclosed herein, methods for detecting specific binding reactions include, but are not limited to, double antibody sandwich assays, competitive assays, neutralization competition assays, indirect assays, and capture assays.

[0074] II. Implementation Method

[0075] The present invention will be described in more detail below with reference to the embodiments.

[0076] The existing common sense is that the more uniform the particle size of the microspheres, the better the performance of the homogeneous chemiluminescence detection using the microspheres. Therefore, the current research on microspheres used in homogeneous chemiluminescence tends to obtain microspheres with more uniform particle sizes. After research, the inventors of this application found that when using microspheres with uniform particle size for homogeneous chemiluminescence detection, the sensitivity and detection range of the detection results cannot be guaranteed at the same time. However, by using microspheres with appropriate particle size uniformity (such as the coefficient of variation of the microsphere particle size distribution is greater than 5%), the sensitivity of the photoinduced chemiluminescence detection can be guaranteed and the detection range can be widened.

[0077] The inventors of the present invention control the particle size distribution of the receptor particles in the receptor reagent, thereby controlling the amount of reporter molecules (e.g., antibodies / antigens) on the surface of each receptor particle (small-diameter microspheres have a large specific surface area, resulting in a high amount of reporter molecules on the surface per unit mass of the microspheres, while large-diameter microspheres have a small specific surface area, resulting in a low amount of reporter molecules on the surface per unit mass of the microspheres). A larger coefficient of variation in the particle size distribution of the receptor particles in the receptor reagent indicates a higher degree of heterogeneity, equivalent to the presence of receptor particles of various sizes in the system. This allows the method of the present invention to have both high sensitivity and a wide detection range.

[0078] Therefore, the first aspect of the present invention relates to the use of a receptor reagent in preparing 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 a body fluid from a subject with a receptor reagent and a donor reagent to generate a test mixture after reaction; exciting the test mixture at least once with excitation light, and detecting the intensity of the chemiluminescence signal generated thereby; and quantitatively calculating the concentration of procalcitonin in the body fluid based on the intensity of the chemiluminescence signal, thereby determining whether the subject is infected with a bacterial inflammatory disease.

[0079] The receptor reagent comprises receptor particles capable of reacting with active oxygen to produce chemiluminescence, and the particle size distribution coefficient of variation CV value of the receptor particles in the receptor reagent is controlled to be ≥5%; the donor reagent comprises donor particles capable of generating active oxygen in an excited state.

[0080] In some embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

[0081] In some preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤20%.

[0082] It is worth noting that the coefficient of variation CV value of the particle size distribution of the receptor particles described in the present invention refers to the coefficient of variation CV value of the particle size distribution after the receptor particles are coated with the required substance.

[0083] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% and 40%, etc.

[0084] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0085] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0086] In other specific embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0087] In some preferred embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0088] In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or attached to the carrier.

[0089] In some embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

[0090] In other specific embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0091] In some specific embodiments of the present 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.

[0092] In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4'-(10-methyl-9-anthryl)-2,2':6'2"-terpyridine-6,6"-dimethylamine]tetraacetic acid (MTTA), 2-(1',1',2',2',3',3'-heptafluoro-4',6'-hexanedione-6'-yl)-naphthalene (NHA), 4,4'-bis(2",3",3"-heptafluoro-4",6"-hexanedione-6"-yl)-o-terphenyl (BHHT), 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedione- 6"-yl)-chlorosulfonyl-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), 2,2-dimethyl-4-perfluorobutyryl-3-butanone (FOD), 2,2'-bipyridine (BPY), bipyridylcarboxylic acid, azacrown ethers, azacryptands and trioctylphosphine oxide and their derivatives.

[0093] In some embodiments of the present invention, the carrier is selected from the group consisting of a strip, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably a bead and a microsphere.

[0094] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0095] In some embodiments of the present 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.

[0096] In other embodiments of the present invention, the carrier is formaldehyde-modified latex particles.

[0097] In some preferred embodiments of the present invention, the average particle size of the carrier is in the range of 50 nm to 1 μm; preferably 100 nm to 500 nm; more preferably 150 nm to 400 nm; most preferably 190 nm to 300 nm.

[0098] In some embodiments of the present invention, the surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

[0099] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0100] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers have charges opposite to those of the functional groups of the previous polysaccharide layer.

[0101] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and the successive layers of the polysaccharide are covalently linked to the previous polysaccharide layer by reaction between the functional groups of the successive layers and the functional groups of the previous layer.

[0102] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0103] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0104] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0105] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0106] In some embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0107] In other specific embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

[0108] In some embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to a member of the specific binding pair.

[0109] In other embodiments of the present invention, the specific binding pair member is selected from a pair of substances consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, avidin, or biotin; preferably, the specific binding pair member is biotin-avidin. The avidin is selected from avidin, streptavidin, vitellogenin, neutravidin, and avidin-like substances, preferably neutravidin and / or streptavidin.

[0110] In some specific embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; it is selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; it is preferably selected from dextran, starch, glycogen and polyribose.

[0111] It should be noted that the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥5%. In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution is ≥10%; further preferably, the coefficient of variation (CV) of the particle size distribution is ≤40%; even more preferably, the coefficient of variation (CV) of the particle size distribution is ≤20%. In some preferred embodiments of the present invention, the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair is polydisperse.

[0112] In some preferred embodiments of the present invention, in step S1, the sample to be tested is first mixed with an acceptor reagent and then mixed with a donor reagent.

[0113] In some embodiments of the invention, the donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

[0114] To further improve the accuracy of the final test results and the stability of the test sample, in other embodiments of the present invention, the body fluid from the subject is diluted with a diluent before contacting with the receptor reagent and the donor reagent.

[0115] In some embodiments of the present invention, the chemiluminescence detection wavelength is 520-620 nm, preferably 610-620 nm, and more preferably 615 nm.

[0116] In other embodiments of the present invention, red excitation light of 600-700 nm is used for laser irradiation; preferably, red excitation light of 640-680 nm is used for laser irradiation; and more preferably, red excitation light of 660 nm is used for laser irradiation.

[0117] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL; more preferably 20 ug / mL-200 ug / mL.

[0118] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0119] In some embodiments of the present invention, the body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

[0120] III. Examples

[0121] Example 1: Preparation of Receptor Particle Solution Conjugated with Antibody I (PCT Antibody)

[0122] (1) Preparation of antibody-coupled receptor particles with an average particle size of approximately 250 nm

[0123] 1.1 Preparation and characterization of formaldehyde-based polystyrene latex microspheres

[0124] 1) Prepare a 100ml three-necked flask, add 40mmol of styrene, 5mmol of acrolein, and 10ml of water, stir for 10min, and then pass N2 for 30min;

[0125] 2) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride and dissolve them in 40ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step 1 and continue to flow N2 for 30 minutes.

[0126] 3) The reaction system was heated to 70°C and reacted for 15 hours;

[0127] 4) Cool the emulsion after the reaction to room temperature and filter it through a suitable filter cloth. Wash the resulting emulsion with deionized water by centrifugation until the conductivity of the supernatant is close to that of deionized water. Then dilute it with water and store it as an emulsion.

[0128] 5) The Gaussian distribution average particle size of the latex microspheres at this time was 202.2 nm, and the coefficient of variation (C.V.) was 4.60% as measured by a nanoparticle size analyzer. The Gaussian distribution curve is shown in FIG. 2. Figure 1 The aldehyde group content of the latex microspheres was 280 nmol / mg as measured by conductometric titration.

[0129] 1.2 Embedding process of the luminescent composition and characterization

[0130] 1) A 25 ml round-bottom flask was prepared, 0.1 g of dimethylthiophene derivative and 0.1 g of europium (III) complex (MTTA-EU 3+ ) were added, 10 ml of 95% ethanol was added, and magnetic stirring was performed. The complex solution was obtained by heating the water bath to 70°C;

[0131] 2) A 100 ml three-necked flask was prepared, 10 ml of 95% ethanol, 10 ml of water, and 10 ml of aldehyde group polystyrene latex microspheres with a concentration of 10% obtained in step 1.1 were added, and magnetic stirring was performed. The water bath was heated to 70°C;

[0132] 3) The complex solution in step 1) was slowly added to the three-necked flask in step 2), and the stirring was stopped after 2 hours of reaction at 70°C, and the natural cooling was performed;

[0133] 4) The above emulsion was centrifuged for 1 hour at 30000 G, and the supernatant was discarded after centrifugation to obtain the aldehyde group polystyrene microspheres embedded with the luminescent composition.

[0134] 5) The Gaussian distribution average particle size of the microspheres at this time was 204.9 nm, and the coefficient of variation (C.V.) was 5.00% as measured by a nanoparticle size analyzer (as shown in FIG. 3). Figure 2

[0135] 1.3 Surface coating of the receptor particles with dextran

[0136] 1) 50 mg of solid aminodextran was taken in a 20 mL round-bottom flask, 5 mL of 50 mM / pH=10 carbonate buffer was added, and the stirring was performed at 30°C in the dark until the dissolution was completed;

[0137] 2) 100 mg of the aldehyde group polystyrene microspheres embedded with the luminescent composition prepared above was added to the aminodextran solution and stirred for 2 hours;

[0138] 3) 10 mg of sodium borohydride was dissolved in 0.5 mL of 50 mM / pH=10 carbonate buffer and added dropwise to the above reaction solution, and the reaction was performed overnight at 30°C in the dark;

[0139] ​4) Centrifuge the reaction mixture at 30,000g, discard the supernatant, and add 50mM / pH 10 carbonate buffer for ultrasonic dispersion. Repeat the centrifugation and washing three times, then adjust the volume with 50mM / pH 10 carbonate buffer to a final concentration of 20mg / ml.

[0140] 5) Place 100 mg of aldehyde dextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0141] 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours;

[0142] 7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight.

[0143] 8) The reaction mixture was centrifuged at 30,000 G, the supernatant discarded, and 50 mM / pH 10 carbonate buffer was added for ultrasonic dispersion. Repeat the centrifugation and washing three times, and then the volume was adjusted to 20 mg / ml with 50 mM / pH 10 carbonate buffer.

[0144] 9) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 241.6 nm, and the coefficient of variation (CV) is 12.90% (as shown in FIG. Figure 3 shown).

[0145] 1.4 Antibody Conjugation Process

[0146] 1) The paired antibody I was dialyzed into 50 mM CB buffer at pH 10, and the concentration was measured to be 1 mg / ml.

[0147] 2) Add 0.5 ml of the receptor particles obtained in step (3) and 0.5 ml of the paired antibody I obtained in step 1) to a 2 ml centrifuge tube, mix well, add 100 μl of 10 mg / ml NaBH4 solution (50 mM CB buffer), and react at 2-8°C for 4 hours.

[0148] 3) After the reaction is complete, add 0.5 ml of 100 mg / ml BSA solution (50 mM CB buffer) and react at 2-8°C for 2 hours.

[0149] 4) After the reaction is complete, centrifuge at 30,000g for 45 minutes. Discard the supernatant and resuspend in 50mM MES buffer. Repeat the centrifugation and washing process four times. Dilute to a final concentration of 100 μg / ml to obtain a solution of receptor particles conjugated to Antibody I.

[0150] 5) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 253.5 nm, and the coefficient of variation (CV) is 9.60%. Figure 4 shown).

[0151] (II) Preparation of antibody-coupled receptor particles with an average particle size of approximately 110 nm

[0152] The preparation method is the same as the preparation process of the receptor particles with an average particle size of about 250nm in the above (1), and the Gaussian distribution of the receptor particle size is measured by a nanoparticle size analyzer (such as Figure 5 The average particle size is 107.1 nm, and the coefficient of variation (CV) is 7.6%. The Nicomp distribution is unimodal (as shown in FIG. Figure 6 (III) Preparation of antibody-coupled receptor particles with an average particle size of approximately 350 nm

[0153] The preparation method is the same as the preparation process of the receptor particles with an average particle size of about 250nm in the above (1), and the Gaussian distribution of the receptor particle size is measured by a nanoparticle size analyzer (such as Figure 7 The average particle size is 347.5 nm, the coefficient of variation (CV) is 3.9%, and the Nicomp distribution is unimodal (as shown in FIG. Figure 8 shown).

[0154] Example 2: Sensitivity and upper limit of detection of PCT markers using the receptor reagent of the present invention

[0155] The sensitivity point is defined as the point where the signal at concentration Cx is twice that at concentration C0, i.e., RLU(Cx) > 2RLU(C0). The corresponding sensitivity of the assay is Cx. The upper limit of detection is defined as the upper limit of the range determined using the method in the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocol (EP) Series 6 document.

[0156] (1) PCT antigen was diluted to a series of concentrations of 20 pg / ml, 30 pg / ml, 50 pg / ml, 40 pg / ml, 60 pg / ml, 80 pg / ml, 160 pg / ml, 500 pg / ml, 1000 pg / ml, 5000 pg / ml, 20000 pg / ml, 50000 pg / ml, 100000 pg / ml and 200000 pg / ml. The receptor reagent (concentration of 100 μg / ml) containing receptor particles of different average particle sizes (110 nm and 350 nm) coupled to PCT antibody I prepared in Example 1 was used, and then the PCT antigen of the above concentration series was detected with the same biotin-labeled PCT monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (reagent containing donor particles). The detection sensitivity and detection limit of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 1.

[0157] Table 1

[0158]

[0159] As can be seen from Table 1, the upper detection limit of the receptor particles with an average particle size of 110 nm is higher, but the sensitivity is lower, while the receptor particles with an average particle size of 350 nm have the best sensitivity, but the upper detection limit is lower.

[0160] (2) A solution of receptor particles with an average particle size of 110 nm coupled to PCT antibody I was mixed with a solution of receptor particles with an average particle size of 350 nm coupled to PCT antibody I to obtain a new receptor reagent. The results of the measurement of the receptor particle size in the new receptor reagent are as follows:

[0161] The average particle size of the Gaussian distribution is 317.7 nm, and the coefficient of variation (CV value) of the particle size distribution is 37.2% (e.g. Figure 9 shown);

[0162] The Nicomp distribution is bimodal: #1: average particle size 103.1nm, coefficient of variation (CV value) = 11.8%; #2: average particle size 328.8nm, coefficient of variation (CV value) of particle size distribution = 13.0% (e.g. Figure 10 shown).

[0163] The above-mentioned new receptor reagent was used with biotin-labeled PCT monoclonal antibody 2 (diluted to 2ug / ml) and universal solution (reagent containing donor particles) to detect the above-mentioned concentration series of PCT antigen. The detection sensitivity and detection limit of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 2.

[0164] Table 2

[0165]

[0166] As can be seen from Table 2, by increasing the coefficient of variation of the particle size distribution of the receptor particles in the receptor reagent, that is, appropriately increasing the heterogeneity of the particle size of the receptor particles, the detection performance of the method is significantly improved.

[0167] Example 3: Preparation of a series of receptor particle solutions of coupled antibody I (PCT antibody) with an average particle size of about 250 nm and different coefficients of variation of particle size distribution

[0168] According to the method described in Example 1 (I), receptor particle solutions of coupled antibody I (PCT antibody) with different coefficients of variation of particle size distribution were obtained.

[0169] Specifically:

[0170] Receptor particles 1: Gaussian distribution average particle size is 251.2 nm, particle size distribution coefficient of variation CV value = 3.7%; Nicomp distribution is unimodal.

[0171] Receptor particles 2: Gaussian distribution average particle size is 254.9 nm, particle size distribution coefficient of variation CV value = 5.0%; Nicomp distribution is unimodal.

[0172] Receptor particles 3: Gaussian distribution average particle size is 251.3 nm, particle size distribution coefficient of variation CV value = 8.0%; Nicomp distribution is unimodal.

[0173] Receptor particles 4: Gaussian distribution average particle size is 251.9 nm, particle size distribution coefficient of variation CV value = 10.5%; Nicomp distribution is unimodal.

[0174] Receptor particles 5: Gaussian distribution average particle size is 252.3 nm, particle size distribution coefficient of variation CV value = 16.8%; Nicomp distribution is unimodal.

[0175] Receptor particles 6: Gaussian distribution average particle size is 240.8 nm, particle size distribution coefficient of variation CV value = 34.5%; Nicomp distribution is bimodal.

[0176] Example 4: Sensitivity and upper limit of detection of PCT markers using the receptor reagent of the present invention

[0177] The sensitivity point is defined as when the signal at concentration Cx is higher than twice the signal at concentration C0, that is, RLU(Cx)>2RLU(C0), and the corresponding detection reagent sensitivity is Cx. The upper detection limit is defined as the upper limit of the range determined using the method in the NCCLS EP-6 document.

[0178] The PCT antigen was diluted to a concentration series of 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 80 pg / ml, 160 pg / ml, 500 pg / ml, 1000 pg / ml, 5000 pg / ml, 20000 pg / ml, 50000 pg / ml, 100000 pg / ml and 200000 pg / ml. The receptor reagent (concentration of 100 ug / ml) containing receptor particles coupled to PCT antibody I prepared in Example 3 was used, and then the PCT antigen in the above concentration series was detected with the same biotin-labeled PCT monoclonal antibody 2 (diluted to 2 ug / ml) and universal solution (reagent containing donor particles). The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 3.

[0179] Table 3

[0180]

[0181] As can be seen from Table 3, when the coefficient of variation of the receptor particle size distribution is greater than or equal to 5%, the method of adding a receptor reagent containing the above receptor particles has both relatively suitable sensitivity and a wide detection range.

[0182] Example 5: Detection of PCT marker standards

[0183] PCT antigen was 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, 100000pg / ml and 2000ng / ml, and different particle sizes (50nm, 80nm, 110nm, 140nm, 170nm, 20 The PCT antigen in the above concentration series was detected using a receptor reagent (concentration of 100 μg / ml, CV values ​​of about 10%) coated with acceptor particles (0 nm, 250 nm, 300 nm, 350 nm, and 400 nm) of PCT monoclonal antibody 1, and the same biotinylated PCT monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (reagent containing donor particles). The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 4.

[0184] Table 4

[0185]

[0186]

[0187] From the PCT project detection results of Table 4, it can be seen that the detection upper limit of the 110 nm receptor particles is high, but the sensitivity is poor, while the 300 nm and 350 nm receptor particles have the best sensitivity, but the detection upper limit is lower. The 110 nm receptor particles were mixed with the 300 nm and 350 nm receptor particles respectively to form receptor reagents, and the sensitivity and detection upper limit of the method were detected by adding the corresponding receptor reagents using the light-induced chemiluminescence analysis system developed by Boyang Biotech (Shanghai) Co., Ltd. The results are shown in Table 5.

[0188] Table 5

[0189]

[0190] From Table 5, it can be seen that by adding the receptor reagent formed by combining small particle size receptor particles with approximately C.V value distribution and large particle size receptor particles, the method has high sensitivity and high detection upper limit (wide detection range) at the same time, and exhibits the advantages of large particle size receptor particles and small particle size receptor particles. Compared with a single average particle size distribution of receptor particles, the performance of the receptor reagent containing two or more average particle size distributions of receptor particles is greatly improved.

[0191] Example 6: Clinical detection of PCT of normal people and patients suspected of having inflammation

[0192] This example detects 40 clinical samples, and the PCT quantitative detection reagent kit (light-induced chemiluminescence method) used is composed of reagent 1 (R1') containing first anti-PCT antibody coated receptor microparticles, reagent 2 (R2') containing biotin labeled second anti-PCT antibody, and in addition, a universal liquid (R3') containing donor particles. Among them, R1 is a receptor reagent (concentration of 200 ug / ml) prepared by using the receptor particles 4 (particle size distribution coefficient of variation C.V value = 10.5%) in Example 3.

[0193] The specific experimental steps are as follows:

[0194] 1. Select 40 clinical samples, balance to room temperature, and mix well;

[0195] 2. Add the mixed sample, prepared R1' and R2' to an 8x12 whiteboard respectively;

[0196] 3. Put the sample whiteboard into the LiCA HT instrument for reaction, and the reaction mode used is as follows;

[0197] (1) Mix 40 ul of sample, 15 ul of R1' and 15 ul of R2';

[0198] (2) Incubate at 37°C for 8 min;

[0199] (3) Add 160 μl of universal solution (R3');

[0200] (4) Incubate at 37°C for 2 min;

[0201] (5) Excitation readings. The specific test results are shown in Table 6 below.

[0202] Table 6

[0203]

[0204]

[0205] Data comparison revealed a correlation of 0.9977 between the Roche values ​​and those measured in Example 6, with a slope of 0.9984. Samples 1-11 were obtained from healthy patients, with a distribution range of 30 pg / ml to 70 pg / ml, and a median of 50 pg / ml. Samples 12-40 were obtained from patients with confirmed inflammation, with a distribution range of 120 pg / ml to 63.23 ng / ml, and a median of 580 pg / ml.

[0206] Procalcitonin (PCT) is a member of the calcitonin (CT) superfamily, a 116-amino acid polypeptide with a molecular weight of approximately 14.5 kDa. Its plasma levels are elevated in patients with severe bacterial, fungal, or parasitic infections, as well as sepsis and multiple organ failure. PCT reflects the activity of the systemic inflammatory response. Factors influencing PCT levels include the size and type of the infected organ, the type of bacteria, the degree of inflammation, and the state of the immune response. The experimental results of Example 6 of the present invention demonstrate the feasibility of using the receptor reagent of the present invention in the preparation of a kit for in vitro diagnosis of bacterial inflammatory diseases in a subject. The quantitative results of PCT markers measured in body fluids using the receptor reagent of the present invention and the corresponding method can be used as an acute parameter to differentiate between bacterial and non-bacterial infections and inflammations, monitor patients at risk of infection (e.g., those in the immunosuppressive period after surgery and organ transplantation, and those requiring intensive care), detect the systemic effects of bacterial infection or detect septic complications, and assess the clinical course and prognosis of severe inflammatory diseases such as peritonitis, sepsis, SIRS, and MODS.

[0207] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. Use of a receptor reagent in preparing 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 a body fluid from a subject with a receptor reagent and a donor reagent to react to form a test mixture; Exciting the test mixture at least once with excitation light, and detecting the intensity of the chemiluminescence signal generated thereby; quantitatively calculating the concentration of procalcitonin in the body fluid based on the intensity of the chemiluminescence signal, thereby determining whether the subject is infected with a bacterial inflammatory disease; The receptor reagent comprises receptor particles capable of reacting with active oxygen to produce chemiluminescence, the receptor particles comprising a luminescent composition and a carrier, the luminescent composition being filled in the carrier and / or attached to the carrier, the receptor reagent comprising at least two receptor particles with average particle size distributions, and the coefficient of variation of the particle size distribution of the receptor particles in the receptor reagent is controlled to be 5%≤CV value≤16.8%; the donor reagent comprises donor particles capable of generating active oxygen in an excited state.

2. The use according to claim 1, characterized in that The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥8%.

3. The use according to claim 2, characterized in that The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

4. The use according to any one of claims 1 to 3, characterized in that The particle size distribution of the receptor particles in the receptor reagent is polydisperse.

5. The use according to any one of claims 1 to 3, characterized in that The coefficient of variation CV value of the particle size distribution is calculated through Gaussian distribution.

6. The use according to any one of claims 1 to 3, characterized in that Using the Gaussian distribution analysis method, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

7. The use according to claim 1, characterized in that The luminescent composition can react with active oxygen to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

8. The use according to claim 7, characterized in that The chemiluminescent compound is selected from olefin compounds.

9. The use according to claim 7, characterized in that The chemiluminescent compound is selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives.

10. The use according to claim 9, characterized in that The chemiluminescent compound is selected from dimethylthiophene and its derivatives.

11. The use according to any one of claims 7 to 10, characterized in that The metal of the metal chelate is a rare earth metal or a Group VIII metal.

12. The use according to claim 11, characterized in that The metal of the metal chelate is selected from the group consisting of europium, terbium, dysprosium, samarium, osmium and ruthenium.

13. The use according to claim 12, characterized in that The metal of the metal chelate is europium.

14. The use according to any one of claims 7 to 10, 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-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridyl carboxylic acid, azacrown ethers, azacryptands, trioctylphosphine oxide, and derivatives thereof.

15. The use according to claim 1, characterized in that The carrier is selected from beads.

16. The use according to claim 1, characterized in that The carrier is selected from microspheres.

17. The use according to claim 1, characterized in that The carrier is a magnetic or non-magnetic particle.

18. The use according to claim 1, characterized in that The support material is selected from natural, synthetic or modified naturally occurring polymers.

19. The use according to claim 18, characterized in that The carrier material includes: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

20. The use according to claim 18, characterized in that The carrier is aldehyde-modified latex particles.

21. The use according to claim 1, characterized in that The average particle size of the carrier is in the range of 50 nm to 1 μm.

22. The use according to claim 21, characterized in that The average particle size of the carrier is in the range of 100 nm to 500 nm.

23. The use according to claim 22, characterized in that The average particle size of the carrier is in the range of 150 nm to 400 nm.

24. The use according to claim 23, characterized in that The average particle size of the carrier is in the range of 190 nm to 300 nm.

25. The use according to claim 1, characterized in that The surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

26. The use according to claim 25, characterized in that Each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

27. The use according to claim 25 or 26, characterized in that The polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers are opposite in charge to the functional groups of the previous polysaccharide layer.

28. The use according to claim 25 or 26, characterized in that The polysaccharide has pendant functional groups, and the successive polysaccharide layer is covalently linked to the preceding polysaccharide layer by reaction between the functional groups of the successive polysaccharide layer and the functional groups of the preceding polysaccharide layer.

29. The use according to claim 28, characterized in that The functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

30. The use according to claim 29, characterized in that The amine-reactive functional group is an aldehyde group or a carboxyl group.

31. The use according to claim 25, characterized in that The first polysaccharide layer is spontaneously associated with the carrier.

32. The use according to claim 25, characterized in that The outermost polysaccharide layer of the coating has at least one pendant functional group.

33. The use according to claim 25, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein.

34. The use according to claim 33, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and / or carboxyl groups.

35. The use according to any one of claims 32 to 34, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to the reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

36. The use according to any one of claims 32 to 34, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to one of the specific binding pair members.

37. The use according to claim 36, characterized in that The specific binding pair members are selected from a pair of substances consisting of antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, haptens, antigens, immunoglobulin binding proteins, avidin or biotin.

38. The use according to claim 37, characterized in that The specific binding pair member is biotin-avidin.

39. The use according to claim 25, characterized in that The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.

40. The use according to claim 39, characterized in that The polysaccharide is selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran.

41. The use according to claim 40, characterized in that The polysaccharide is selected from the group consisting of dextran, starch, glycogen and polyribose.

42. The use according to claim 1, characterized in that The donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

43. The use according to claim 1, characterized in that The body fluid from the subject is diluted with a diluent and then contacted with the receptor reagent and the donor reagent.

44. The use according to claim 1, characterized in that The detection wavelength of the chemiluminescence is 520-620 nm.

45. The use according to claim 1, characterized in that Red excitation light of 600-700 nm was used for laser irradiation.

46. ​​The use according to claim 1, characterized in that The concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL.

47. The use according to claim 46, characterized in that The concentration of the receptor particles in the receptor reagent is 10ug / mL-500ug / mL.

48. The use according to claim 1, characterized in that The concentration of the receptor particles in the receptor reagent is 20ug / mL-200ug / mL.

49. The use according to claim 1, characterized in that The active oxygen is singlet oxygen.

50. The use according to claim 1, characterized in that The body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

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