CN116337851A

CN116337851A - Homogeneous chemiluminescence detection kit and application thereof

Info

Publication number: CN116337851A
Application number: CN202310486914.2A
Authority: CN
Inventors: 杨阳; 刘宇卉; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2018-07-18
Filing date: 2019-07-18
Publication date: 2023-06-27
Also published as: CN110736735A

Abstract

The invention relates to a homogeneous phase chemiluminescence detection kit and application thereof. The kit comprises: a donor agent comprising donor microspheres capable of generating active oxygen in an excited state, the donor microspheres having a surface coated with a label; and, a receptor reagent comprising a receptor microsphere capable of reacting with active oxygen to produce a detectable chemiluminescent signal, and the surface of the receptor microsphere is coated with a biomolecule capable of specifically binding to a target molecule to be detected; wherein the donor microspheres have a particle size greater than the acceptor microspheres. The particle size of the donor microsphere in the kit is larger than that of the acceptor microsphere, so that the precision and the sensitivity of the kit are improved.

Description

Homogeneous chemiluminescence detection kit and application thereof

The application is that the application date is 2019, 7, 18, the application number is 201910651533.9, and the invention is named as one A homogeneous chemiluminescent detection kit and application thereof are disclosed in the divisional application of Chinese patent application.

Technical Field

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

Background

Methods for measuring trace amounts of biologically active substances in living bodies have been carried out to date by Radioimmunoassay (RIA), fluoroimmunoassay (FIA), enzyme-linked immunoassay (EIA), and chemiluminescent assay. This evolution is mainly determined on the basis of increasing demands on sensitivity, accuracy and simplicity of the detection method.

Chemiluminescent analysis is a method of detection using light waves emitted by chemiluminescent substances. Chemiluminescent substances are used as labels in nucleic acid detection and immunodetection. For example, a molecule of a specific binding pair may be bound to a luminescent substance to form a luminescent complex by a variety of routes. The complex is capable of reacting with the analyte (the other molecule in the specific binding pair) in the sample, partitioning into a solid phase and a liquid phase, and the partitioning ratio is related to the amount of the analyte. The corresponding concentration of the detection object in the sample can be obtained by measuring the luminescence amount in the solid phase or the liquid phase.

The luminescence efficiency of the donor and acceptor microspheres in a chemiluminescent assay determines the detection sensitivity of the chemiluminescent assay. In order to increase the light-emitting efficiency of the donor and/or acceptor microspheres, methods are generally employed in the art to increase the light-sensing efficiency of the dye in the donor microspheres and/or the light-receiving and light-emitting efficiency of the light-emitting compound in the acceptor microspheres.

Although the detection sensitivity of chemiluminescent detection methods can be improved to some extent by the methods described in the art, the detection range or linear range is relatively narrow. Therefore, it is required to develop a kit capable of further improving the luminous efficiency based on the prior art, which not only has higher sensitivity, but also has wider detection range.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a kit for homogeneous chemiluminescence detection, which has higher sensitivity and wider detection range when being used for homogeneous chemiluminescence detection.

To this end, a first aspect of the invention provides a homogeneous chemiluminescent detection kit comprising:

a donor agent comprising donor microspheres capable of generating active oxygen in an excited state, the donor microspheres having a surface coated with a label; and, a step of, in the first embodiment,

a receptor reagent comprising a receptor microsphere capable of reacting with active oxygen to produce a detectable chemiluminescent signal, and the surface of the receptor microsphere is coated with a biomolecule capable of specifically binding to a target molecule to be detected;

wherein the donor microspheres have a particle size greater than the acceptor microspheres.

In some embodiments of the invention, the donor microspheres have an average particle size of 100nm to 400nm, the acceptor microspheres have an average particle size of 100nm to 350nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.1 to 4.0.

Preferably, the average particle diameter of the donor microspheres is 190nm to 250nm, the average particle diameter of the acceptor microspheres is 180nm to 240nm, and the ratio of the average particle diameter of the donor microspheres to the average particle diameter of the acceptor microspheres is 1.2 to 3.0.

Further preferably; the average particle diameter of the donor microsphere is 100nm-200nm, the average particle diameter of the acceptor microsphere is 50nm-150nm, and the ratio of the average particle diameter of the donor microsphere to the average particle diameter of the acceptor microsphere is 1.3-2.0.

Still more preferably; the average particle diameter of the donor microsphere is 150-200 nm, the average particle diameter of the acceptor microsphere is 100-140 nm, and the ratio of the average particle diameter of the donor microsphere to the average particle diameter of the acceptor microsphere is 1.4-1.6.

In some particularly preferred embodiments of the invention, the donor microspheres have an average particle size of 150nm, the acceptor microspheres have an average particle size of 100nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.5.

In some embodiments of the invention, the donor microsphere comprises a first support, the interior of the first support is filled with a sensitizer, and the surface of the first support is chemically bonded to a label.

In other embodiments of the invention, the surface of the first carrier is not coated or attached with a polysaccharide substance, which is directly chemically bound to the label.

In some embodiments of the invention, the label is avidin.

In other embodiments of the invention, the avidin is selected from the group consisting of avidin, streptavidin, vitelline avidin, neutravidin and avidin-like, preferably selected from the group consisting of neutravidin and 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 other embodiments of the invention, the surface of the first support carries a bonding functionality for chemically bonding a label 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 other embodiments of the invention, the content of binding functionalities on the surface of the first support is between 100 and 500nmol/mg, preferably between 200 and 400nmol/mg.

In some embodiments of the invention, the surface of the first carrier is coated with a hydrophilic aldehyde dextran, the aldehyde groups of which chemically bond to the labels.

In some preferred embodiments of the invention, the photosensitizer is selected from one of methylene blue, rose bengal, porphine, and phthalocyanine.

In other embodiments of the present invention, the receptor microsphere comprises a second carrier, the interior of the second carrier is filled with a light-emitting composition, the surface of the second carrier is coated with at least one polysaccharide layer, and the surface of the polysaccharide layer is connected with biomolecules.

In some embodiments of the invention, the surface of the second carrier is coated with hydrophilic carboxydextran.

In other embodiments of the present invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ 。

In some embodiments of the present invention, the first carrier and/or the second carrier is/are 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 other embodiments of the invention, the donor microspheres have a coefficient of variation C.V in the particle size distribution of the donor agent of 5% or more.

In other embodiments of the invention, the receptor microspheres have a coefficient of variation C.V in the particle size distribution of the receptor agent of greater than or equal to 5%.

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

In some embodiments of the invention, the kit specifically comprises:

a receptor reagent comprising a receptor microsphere that binds to a first antigen, the epitope of the first antigen being capable of specifically binding to a first binding site of an epitope of an antibody of interest to be detected; the receptor microsphere is capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

a first reagent comprising a second antigen that binds biotin, the second antigen being capable of specifically binding to an epitope second binding site of the antibody of interest to be tested, and the epitope first binding site and the epitope second binding site of the antibody of interest to be tested being non-overlapping; and, a step of, in the first embodiment,

a donor agent comprising donor microspheres bound to avidin; the donor microsphere is capable of generating singlet oxygen in an excited state;

In other embodiments of the invention, the kit specifically comprises:

a receptor reagent comprising a receptor microsphere bound to a first antibody capable of specifically binding to an epitope of an antigen of interest to be tested; the receptor microsphere is capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

a first reagent comprising a second antibody that binds biotin, said second antibody being capable of specifically binding to an epitope of an antigen of interest to be tested; and, a step of, in the first embodiment,

In some embodiments of the invention, the kit specifically comprises:

a receptor reagent comprising a receptor microsphere that binds to an antigen capable of specifically binding to an antibody of interest, the receptor microsphere capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

a first reagent comprising a second antibody that binds biotin, the second antibody being capable of specifically binding to the antigen; and

A donor agent comprising donor microspheres bound to avidin, said donor microspheres being capable of generating singlet oxygen in an excited state;

In other embodiments of the invention, the kit specifically comprises:

a first reagent comprising a first antigen that binds biotin, said first antigen being capable of specifically binding to an epitope binding site of an antibody of interest;

a receptor agent comprising a receptor microsphere that binds to an anti-immune complex antibody capable of specifically binding to an antibody of interest in an immune complex formed by a first antigen and the antibody of interest, the receptor microsphere capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal; and, a step of, in the first embodiment,

In some embodiments of the invention, the kit specifically comprises:

a receptor reagent comprising a receptor microsphere that binds to a first antigen, said first antigen capable of specifically binding to an antibody of interest, said receptor microsphere capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal;

A first reagent comprising an anti-immune complex antibody that binds to biotin, the anti-immune complex antibody being capable of specifically binding to an antibody of interest in an immune complex formed by a first antigen and the antibody of interest;

a donor agent comprising donor microspheres bound to avidin, said donor microspheres being capable of generating singlet oxygen in an excited state.

According to the invention, the anti-immune complex antibody does not bind to the target antibody, either free or unbound to the first antigen.

In a second aspect, the present invention provides a homogeneous chemiluminescent detection method for detecting a test target molecule in a test sample using a kit according to the first aspect of the present invention.

In a third aspect, the present invention provides a homogeneous chemiluminescent detection device for detecting a test object molecule in a test sample using a kit according to the first aspect of the present invention or a method according to the second aspect of the present invention.

In some preferred embodiments of the invention, the device is a POCT instant detection device.

The beneficial effects of the invention are as follows: the kit disclosed by the invention has the advantages that the matrix and the particle size of the acceptor microsphere and the donor microsphere are controlled, so that the luminous efficiency of detection is improved when the kit is used for chemiluminescent detection, and the kit has good detection sensitivity. In addition, the surface of the acceptor microsphere is coated with the hydrophilic carboxyl glucan, and the surface of the donor microsphere is coated with the hydrophilic aldehyde glucan, so that the nonspecific adsorption is greatly reduced, the influence of other environmental factors outside the system such as pH value and electrolyte is reduced, and the detection accuracy can be further improved.

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 7.

FIG. 2 is a Nicomp profile of the aldehyde-based polystyrene latex microspheres prepared in example 7.

FIG. 3 is a Gaussian distribution plot of donor microspheres prepared in example 7.

FIG. 4 is a Gaussian distribution diagram of dextran-coated microspheres prepared in example 8

FIG. 5 is a Gaussian distribution plot of donor microspheres prepared in example 8.

Detailed Description

In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

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

I terminology

The term "homogeneous" as used herein is defined as "homogeneous" and refers to the fact that the detection is accomplished without the need to separate the bound antigen-antibody complex from the remaining free antigen or antibody.

In the present invention, the "donor microsphere" may be polymer particles filled with a photosensitizer formed by coating a functional group on a carrier, and can generate active oxygen (e.g., singlet oxygen) under light excitation, and the donor microsphere may also be called a photosensitive microsphere or a photosensitive particle. The donor microsphere is internally filled with a photosensitizer. The photosensitizers may be photosensitizers known in the art, preferably compounds that are relatively light stable and do not effectively react with singlet oxygen, non-limiting examples of which include methylene blue, rose bengal, porphyrin, and phthalocyanine compounds, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. The donor microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.

The "receptor microsphere" may be polymer particles filled with a light-emitting compound formed by coating a carrier with a functional group, and may be referred to as light-emitting microsphere or light-emitting particle, wherein the surface of the light-emitting microsphere receptor microsphere has hydrophilic carboxyl glucan, and the inside of the light-emitting microsphere receptor microsphere is filled with a light-emitting composition capable of reacting with active oxygen (e.g., singlet oxygen). In some embodiments of the invention, the luminescent composition undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate, which may decompose while or subsequently emit light. In some preferred embodiments of the invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ 。

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.

The term "test sample" as used herein refers to a mixture that may contain a test target molecule, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that may be used in the methods disclosed herein include body fluids such as whole blood, serum, plasma, saliva, urine, and the like. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.

The term "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.

The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibodies are capable of specifically binding to the antigen to be tested.

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 "biotin" is widely used in animal and plant tissues, and has two cyclic structures, namely an imidazolone ring and a thiophene ring, on the molecule, wherein the imidazolone ring is the main part combined with streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. Any agent used in the present invention, including an antigen, an antibody, an oxygen-bearing microsphere, or an oxygen-supplying microsphere, may be conjugated to any one of the biotin-streptavidin specific binding pair members, as desired.

The term "particle size" as used herein refers to the average particle size of the microspheres as determined by conventional particle size measurement.

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.

II. Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The chemiluminescent homogeneous assay kit according to the first aspect of the present invention comprises:

Further preferably, the ratio of the average particle diameter of the donor microspheres to the average particle diameter of the acceptor microspheres may be 1.3 to 2.0.

Still more preferably, the ratio of the average particle diameter of the donor microspheres to the average particle diameter of the acceptor microspheres may be 1.4 to 1.6.

For example, in some embodiments of the invention, the donor microspheres may have an average particle size of 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm; and their corresponding receptor microspheres have an average particle size of 20nm, 50nm, 100nm, 150nm, 200nm, 250nm, 300nm and 350nm.

In some preferred embodiments of the invention, the donor microspheres have an average particle size of 150nm, the acceptor microspheres have an average particle size of 100nm, and the ratio of the average particle size of the donor microspheres to the average particle size of the acceptor microspheres is 1.5.

In some embodiments of the invention, the label is avidin.

In some preferred embodiments of the invention, the photosensitizer is selected from one of methylene blue, rose bengal, porphine, and phthalocyanine. The loading amount of the photosensitizer is not particularly limited, and it may be an amount commonly used in the art.

When the microsphere containing the carrier is used for detection, nonspecific adsorption can be greatly reduced, and the influence of other environmental factors outside the system such as pH value, electrolyte and the like is reduced, so that the detection accuracy is improved.

In the present inventionIn other embodiments of the invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ . The europium complex filled in the polystyrene microsphere interacts with the polystyrene microsphere to further improve the luminous efficiency. In a further preferred embodiment of the present invention, the europium complex is MTTA-EU ³⁺ The complex can directly capture singlet oxygen generated by phthalocyanine dye in photosensitive microsphere and then emit red light with europium ion characteristic wavelength of 614-615 nm.

MTTA: [4' - (10-methyl-9-anthryl) -2,2':6'2 ' -bipyridine-6, 6' -dimethylamine ] tetraacetic acid, the structural formula of which is shown in formula I, and the synthesis reference CN200510130851.9.

Europium complex MTTA-EU ³⁺ The synthesis of (europium (III) complexes) is as follows:

(1) A500 mL three-necked flask was taken, 732mg of MTTA (1 mmoL) and 366mg of EuCl ₃ ·6H ₂ O (1 mmoL) was dissolved in 100mL of methanol and refluxed at 70℃for 2 hours under stirring.

(2) The solvent was distilled off under reduced pressure.

(3) 50mL of diethyl ether was added to the resultant, and the cake was collected by filtration and washed three times with acetone.

(4) After drying in vacuo 830mg MTTA-EU were obtained ³⁺ 。

In some embodiments of the invention, the donor and acceptor microspheres are polystyrene microspheres.

In some embodiments of the invention, the biomolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; preferably a protein molecule. Of course, the biomacromolecule is not limited to a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule, and any substance that can be designed to satisfy the above conditions can be used as the biomacromolecule in the present invention as long as it is combined with the prior art under the technical ideas disclosed in the present invention, and the details thereof will not be repeated.

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

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

In addition, it is generally recognized by those skilled in the art 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.

In some embodiments of the invention, the donor microspheres have a coefficient of variation C.V in the particle size distribution of the donor agent of 5% or more.

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

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

In some embodiments of the invention, the donor microspheres 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.

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

In some embodiments of the invention, the receptor microspheres have a coefficient of variation C.V in the particle size distribution of the receptor agent of greater than or equal to 5%.

In some embodiments of the invention, the receptor microsphere has a particle size distribution coefficient of variation C.V of greater than or equal to 8% in the receptor agent; preferably, the variation coefficient C.V value of the particle size distribution of the receptor microspheres in the receptor reagent is more than or equal to 10%.

In other embodiments of the invention, the receptor microsphere has a coefficient of variation C.V value of particle size distribution in the receptor agent of less than or equal to 40%; still more preferably, the receptor microspheres have a coefficient of variation C.V in the particle size distribution of the receptor agent of 20% or less.

In some embodiments of the invention, the receptor microsphere may have a particle size distribution coefficient of variation C.V in the receptor reagent 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% or the like.

It is noted that the C.V value of the particle size distribution variation coefficient of the receptor microsphere according to the present invention refers to C.V value of the particle size distribution variation coefficient of the receptor microsphere after being coated with the desired substance.

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 some embodiments of the invention, the kit further comprises a first reagent. The first reagent according to the present invention is not particularly limited to a certain type of reagent, and is added to ensure that certain detection methods based on specific reactions are performed smoothly or optimally.

In some embodiments of the invention, the kit specifically comprises:

In other embodiments of the invention, the kit specifically comprises:

In some embodiments of the invention, the kit specifically comprises:

In other embodiments of the invention, the kit specifically comprises:

In some embodiments of the invention, the kit specifically comprises:

The second aspect of the present invention relates to a homogeneous chemiluminescent detection method for detecting a target molecule to be detected in a sample to be detected using a kit according to the first aspect of the present invention.

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

s1, mixing a sample to be tested with an acceptor reagent and a first reagent, and then mixing with a donor reagent to obtain a mixture to be tested;

s2, carrying out laser irradiation on the mixture to be detected obtained in the step S1, and exciting a donor to generate singlet oxygen;

s3, analyzing and judging whether the sample to be tested contains the target molecule to be tested and/or the concentration of the target molecule to be tested by detecting the intensity of a chemiluminescent signal generated by the reaction of the receptor microsphere in the mixture to be tested and the singlet oxygen.

In some preferred embodiments of the present invention, in step S1, the sample to be tested is diluted with a diluent and then mixed with the acceptor reagent and the donor reagent to form a mixture to be tested. The diluent comprises buffer solution, protein, stabilizer, preservative and the like. The diluent has the functions of dilution and buffering, and the accuracy of a final detection result and the stability of a sample to be detected are improved.

In other embodiments of the present invention, in step S2, laser irradiation is performed using red excitation of 600 to 700 nm.

A third aspect of the present invention relates to a homogeneous chemiluminescent detection device for detecting a test object molecule in a test sample by means of a kit according to the first aspect of the present invention or a method according to the second aspect of the present invention.

In some embodiments of the invention, the apparatus comprises:

a. the reagent cup strip is provided with a plurality of hole sites for containing reagents, and the hole sites at least comprise:

the sample hole site to be measured is used for accommodating a sample to be measured containing target molecules to be measured;

a first reagent well for holding a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state;

a second reagent well for containing an acceptor reagent comprising acceptor microspheres capable of reacting with active oxygen to produce a chemiluminescent signal, the donor microspheres having a particle size greater than the particle size of the acceptor microspheres;

b. the sampling mechanism is used for mutually moving the reagents contained in the hole sites among the hole sites; the material transferred by the sampling mechanism is 1-500 mu L each time;

c. And the detection mechanism is electrically connected with the sampling mechanism and is used for detecting chemiluminescent signals generated by the reaction of the receptor microspheres and the active oxygen.

In other embodiments of the invention, the sample site to be tested, the donor reagent site and the acceptor reagent site are all covered with a membrane to close the orifice to ensure that the material therein is not contaminated. The coating film can be a disposable sealing film or a repeatedly used sealing film.

In order to facilitate the identification and reading of the information of the sample to be measured, the preferred technical scheme is that the lateral surface of the reagent cup strip along the width direction is provided with a bar code area, and the bar code area contains the information of the reagent cup strip. The bar code may be a one-dimensional or two-dimensional code.

Correspondingly, the POCT device further comprises a bar code scanning module, and the bar code scanning module is used for identifying and reading information in the bar code.

The bar code scanning module supports IC card scanning and printing bar code medium (paper or reagent card) scanning, and the information reading adopts contact scanning or non-contact scanning, and the way can be infrared or radio frequency; such information includes, but is not limited to, assay item names, standard curves, reagent components, lot numbers, expiration dates, manufacturer information.

In order to improve the accuracy of the final detection result and the stability of the sample to be detected, in some embodiments of the present invention, a diluent hole is further formed on the reagent cup strip, and the diluent hole is used for containing diluent.

In some embodiments of the invention, the reagent cup strip is further provided with an additional reagent hole site for holding an additional reagent (first reagent), and the additional reagent hole site is covered with a film to close the hole.

In some preferred embodiments of the present invention, the sampling mechanism comprises:

a pipetting assembly for aspirating or draining a liquid;

the vertical moving assembly is arranged on the liquid transferring assembly and is used for driving the liquid transferring assembly to move vertically;

the horizontal movement assembly is arranged on the horizontal movement assembly and is used for driving the pipetting assembly to move horizontally.

In some preferred embodiments of the invention, the detection mechanism comprises:

the base is used for bearing the reagent cup strips;

the driving assembly is used for driving the base to rotate around the center of the base and driving the reagent cup strips to rotate;

And the detection component is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere in the reagent cup strip and the active oxygen.

In some embodiments of the invention, the detection assembly comprises an exciter capable of emitting red excitation light at 600-700 nm.

In some embodiments of the invention, the detection wavelength of the chemiluminescent signal generated by the reaction of the acceptor microsphere with active oxygen is in the range of 450-650nm.

In some preferred embodiments of the present invention, the pipetting assembly comprises a piston mechanism, a connector and a pipette arranged in sequence from top to bottom, the piston mechanism being connected to the connector, the pipette being arranged at the end edge of the base; when liquid transfer is required, the connecting piece descends and is connected with the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

In some embodiments of the invention, the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction. During detection, the temperature of the reagent cup strips and substances in the reagent cup strips is 20-50 ℃ by adopting a metal bath, a water bath or an oil bath and other modes.

In other embodiments of the invention, the cross-sectional shapes of the sample well to be tested, the donor reagent well, and the acceptor reagent well are different from one another.

The using flow of the device is as follows: after the hole site of the sample to be detected, the hole site of the donor reagent and the hole site of the acceptor reagent are respectively filled with the sample to be detected, the donor reagent and the acceptor reagent, the reagent card is placed in the POCT analyzer, a sampling mechanism is used for taking the sample to be detected with corresponding volume, the first reagent hole site is added, after a certain time reaction, a certain volume of mixed liquid is continuously taken and added into the second reagent hole site, an exciter in the detection assembly is used for emitting laser to irradiate the second reagent hole site, after a certain time reaction, the detection mechanism detects a chemiluminescent signal generated by the reaction of the acceptor microsphere and the active oxygen, and the concentration of procalcitonin in the sample to be detected is calculated.

III. Examples

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

Example 1: preparation of homogeneous chemiluminescent detection kit

1. Preparation of receptor microspheres

1. A25 mL round-bottomed flask was prepared, 0.1g of europium (III) complex was added, 10mL of 95% ethanol was magnetically stirred, and the temperature was raised to 70℃in a water bath to obtain a europium (III) complex solution.

2. A100 mL three-necked flask was prepared, 10mL of 95% ethanol, 10mL of water and 10mL of 10% concentration polystyrene microsphere coated with carboxyl dextran hydrogel having a particle size of 200nm were added, and the mixture was magnetically stirred and heated to 70℃in a water bath.

3. Slowly dripping the europium (III) 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 to obtain emulsion.

4. The emulsion was centrifuged for 1 hour, 30000g, the supernatant discarded after centrifugation, and resuspended in 50% ethanol. After repeating the centrifugal washing 3 times, the resultant solution was resuspended in 50mM CB buffer having a pH of 10 to a final concentration of 20mg/mL to obtain a receptor microsphere solution having a particle diameter of 200 nm.

5. The same procedure was used to prepare receptor microsphere solutions having particle sizes of 100nm, 150nm, 250nm, 300nm and 350nm, respectively.

2. Receptor microsphere conjugated antibodies

1. 10mg of the receptor microsphere coated with the carboxyl dextran hydrogel is measured according to the preparation amount, and the receptor microsphere is placed in a centrifuge tube and centrifuged at 10000rpm for 60min.

2. The supernatant was discarded, and 2mg of Anti-PCT antibody I (which may be any of the antibody examples corresponding to the other analysis items (Anti-cTnI antibody I and Anti-PCT antibody I)), 50. Mu.L of Tween-20 (50 mg/mL) was added to the pellet, and a volume of 0.05M MES pH=6.0 was added thereto to give a final concentration of 10mg/mL of the receptor microspheres.

3. And (5) quickly and uniformly mixing by ultrasonic waves.

4. Add 50. Mu.L of NaBH to centrifuge tube ₃ CN (50 mg/mL,0.05M MES pH=6.0) was mixed and placed in a rotary mixer at 37℃for 36-48h.

5. Closing: 1mL of BSA (50 mg/mL,0.05M MES pH=6.0 formulation) was added and the mixture was reacted at 37℃in a rotary mixer for 12-16h.

6. Cleaning: wash 3 times with 0.05M MES buffer.

7. The washed antibody-coated receptor microspheres were sampled and assayed for concentration, particle size, and signal value.

3. Preparation of donor microspheres

1. A25 mL round bottom flask was prepared, 0.1g copper (II) phthalocyanine, 10mL DMF was added, magnetically stirred, and the temperature was raised to 70℃in a water bath to obtain a copper (II) phthalocyanine solution.

2. A100 mL three-necked flask was prepared, 10mL of 95% ethanol, 10mL of water and 10mL of 10% strength polystyrene microspheres coated with an aldehyde dextran hydrogel having a particle size of 200nm were added, and the mixture was magnetically stirred and heated to 70℃in a water bath.

3. Slowly dripping the copper (II) phthalocyanine solution in the step 1 into the three-neck flask in the step 2, reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling to obtain emulsion.

4. The emulsion was centrifuged for 1 hour, 30000g, and after centrifugation the supernatant was discarded and resuspended in 50% ethanol. After repeated centrifugation washing 3 times, the resultant solution was resuspended in 50mM CB buffer having a pH of 10 to a final concentration of 20mg/mL to obtain a donor microsphere solution having a particle size of 200 nm.

5. Donor microsphere solutions having particle sizes of 80nm, 100nm, 150nm, 250nm, 300nm and 350nm, respectively, were prepared in the same manner.

4. Donor microsphere coupled avidin

1. Donor microsphere suspension treatment: sucking a certain amount of donor microspheres, centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, adding a certain amount of MES buffer, performing ultrasonic treatment on an ultrasonic cytoclasis instrument until the particles are resuspended, and adding the MES buffer to adjust the concentration of the donor microspheres to 100mg/mL.

2. Avidin solution preparation: an amount of avidin (which may also be streptavidin or neutralizing avidin) was weighed and dissolved in MES buffer to 8mg/mL.

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

4. The reaction: 25mg/mL NaBH is prepared from MES buffer 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.

5. Closing: preparing 75mg/mL Gly solution and 25mg/mL NaBH with MES buffer solution 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. 200mg/mL BSA solution (MES buffer) was added thereto, and the mixture was rapidly mixed with the reaction mixture at a volume ratio of 5:8, and the mixture was subjected to rotary reaction at 37℃for 16 hours.

6. Cleaning: and adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, re-suspending by an ultrasonic method, centrifuging again, washing for 3 times, suspending by a small amount of buffer solution, measuring the solid content, and regulating the concentration to 10mg/mL by the buffer solution.

5. Preparation of biotin-labeled antibody (first reagent)

1. Antibody treatment: anti-PCT antibody II (which may be any other assay item corresponding to antibody examples (Anti-cTnI antibody II and Anti-PCT antibody II)) was dialyzed against 0.1M NaHCO ₃ Solution, antibody concentration was determined and adjusted to 1mg/mL.

2. A16.17 mg/mL biotin solution was prepared in DMSO.

3. Marking: mixing 1mg/mL of the treated Anti-PCT antibody II (which can be any antibody example (Anti-cTnI antibody II and Anti-PCT antibody II) corresponding to any other analysis item) with the prepared biotin solution according to the volume ratio of 10000:54, and rapidly and uniformly mixing the two solutions. Standing at 2-8 deg.c for reaction for 12-16 hr.

4. And (3) dialysis: the reacted biotin-labeled antibody was dialyzed against biotin-labeled dialysis buffer (ph=8.00).

5. And sucking out the dialyzed biotinylated antibody, transferring the dialyzed biotinylated antibody into a clean centrifuge tube, and sampling to determine the concentration of the antibody. The concentration of the biotin-labeled antibody which is qualified in quality inspection is regulated to 0.5mg/mL.

6. Assembly

And assembling the prepared reagent to obtain the chemiluminescent homogeneous detection kit.

Example 2: chemiluminescence homogeneous detection method

After respectively adding 25 mu L of sample to be detected, 25 mu L of biotin-labeled antibody, 175 mu L of donor reagent and 25 mu L of acceptor reagent into a sample hole site to be detected, an additional reagent hole site, a first reagent hole site and a second reagent hole site of a reagent cup strip, placing the reagent cup strip into a POCT analyzer developed by Boyang biotechnology (Shanghai) limited company, taking a corresponding volume of sample to be detected by a sampling mechanism, adding the sample to be detected into the additional reagent hole site, vibrating, and incubating for 10 minutes at 37 ℃; adding the liquid after incubation in the additional reagent hole site into the first reagent hole site, vibrating, and incubating at 37 ℃ for 10 minutes to form mixed liquid; the mixed liquid is added into the second reagent hole site, vibrated and incubated for 10 minutes at 37 ℃ to form a mixture to be tested. And irradiating the second reagent hole site by using laser emitted by an exciter in the detection assembly, reacting for a certain time, detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen by using a detection mechanism, and calculating the concentration of the target molecule to be detected in the sample to be detected.

Example 3: detection of luminescence signal quantity

PCT to be tested in a sample is detected by the method described in example 2 and the kit described in example 1 (using a kit comprising a receptor microsphere coated with Anti-PCT antibody I and biotin-labeled Anti-PCT antibody II as an example), and the detection results are shown in Table 1.

TABLE 1

As can be seen from table 1, when the particle size of the acceptor microsphere is larger than that of the donor microsphere under the condition that the particle size of the donor microsphere is fixed, the detected luminescence signal value gradually decreases as the particle size of the donor microsphere increases. Under the same conditions, when the particle size of the donor microsphere is larger than that of the acceptor microsphere, the detected luminescence signal value is excellent. And when the particle size of the donor microsphere is 150nm and the particle size of the acceptor microsphere is 100nm, the detected luminescence signal value is optimal, and the detection sensitivity is the best.

Example 4: preparation of quality control product and calibrator

1. Preparation of quality control product

The new born calf serum is used as a diluent, and antigen pure products are respectively diluted into 2 working solutions with different concentrations, and the working solutions are quality control products Q1 and Q2. And (5) taking quality control products Q1 and Q2 to be detected, and repeatedly calibrating the quality control products on the instrument system of the company for three times. And measuring 10 holes each time, and calculating the overall average value and SD, wherein the average value + -3 SD is the measurement allowable range of the quality control product concentration.

2. Preparation of a calibrator

The antigen pure product is diluted into a series of concentration by calf serum (containing preservative), and is frozen and preserved for standby after being calibrated by the national standard for immunoassay. The effective period is 2 years when the product is preserved at-20 ℃.

Simultaneously analyzing and measuring the calibrator and the national standard with corresponding concentration, and fitting by using 4 parameters or other models, wherein the absolute value of the correlation coefficient (r) of the calibrator dose-response curve is required to be not lower than 0.9900; at the same time, the two dose-response curves do not deviate significantly from parallel (t-test); the ratio of the measured potency to the calibrated potency of the calibrator is between 0.90 and 1.10 by taking the national standard as a standard.

Example 5: detection of precision between batches of chemiluminescent homogeneous detection kit

Sample to be measured: quality control Q1 prepared in example 4;

the process comprises the following steps: repeating the detection for 20 times to obtain a light intensity value (RLU)

Decision criteria for outliers: not less than 3SD

The kit adopted in the detection comprises the following reagents:

(1) A receptor reagent comprising receptor microspheres (the particle size of the receptor microspheres is 200nm and linked to the anti-cTnI antibody i and the anti-PCT antibody i, respectively);

(2) A first reagent (biotin-labeled antibody, anti-cTnI antibody II and anti-PCT antibody II, respectively));

(3) A donor agent comprising donor microspheres (avidin-coated donor microspheres of different particle sizes (80 nm, 200 nm)).

The reagents (1) - (3) were matched to form reagent sets 1 and 2 shown in table 2, cTnI and PCT were detected separately (after dilution to the appropriate concentrations, reagent sets 1 and 2 were used to detect the same samples simultaneously), 20 wells were repeatedly tested, the test procedure was as described in example 2, and the test results were shown in table 3.

TABLE 2

Reagent grouping	Reagent set 1	Reagent set 2
			Receptor microsphere particle size/nm	200	200
Donor microsphere particle size/nm	80	300

TABLE 3 Table 3

As can be seen from table 3, the light intensity of reagent set 2 was increased compared to that of reagent set 1, and when the particle size of the donor microspheres was larger than that of the acceptor microspheres, the light intensity detected by this method was increased. Meanwhile, compared with the reagent set 1, the reagent set 2 has smaller variation Coefficient (CV), namely, the precision is higher when the particle size of the donor microsphere is larger than that of the acceptor microsphere.

Example 6: detection of analytical sensitivity of chemiluminescent homogeneous assay kit

Sample to be measured: zero value calibration material;

Sensitivity: RLU substitution calibration curve

The reagents and the detection procedure used in the detection are the same as in example 5, and the detection results are shown in Table 4.

TABLE 4 Table 4

As can be seen from table 4, the sensitivity of the reagent set 2 was better than that of the reagent set 1, i.e., the sensitivity of the reagent was improved when the particle size of the donor microspheres was larger than that of the acceptor microspheres.

Comparative example 1: preparation of contrast kit

1. Receptor microsphere coated antibodies

1. anti-PCT antibody i was dialyzed to 50mM CB buffer at ph=10 to give a concentration of 1mg/mL.

2. 0.5mL of the receptor microsphere prepared in example 1 and 0.5mL of anti-PCT antibody I are added into a 2mL centrifuge tube, and 100 mu L of 10mg/mL NaBH is added after uniform mixing 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 of a solution of mL of SA (50 mM CB buffer) was added, and the reaction was carried out at 2-8℃for 2 hours.

4. After completion of the reaction, the mixture was centrifuged for 45min,30000g, and the supernatant was discarded after centrifugation, and resuspended in 50mM MES buffer. The centrifugation and washing were repeated 4 times, and diluted to a final concentration of 100. Mu.g/mL, to obtain anti-PCT antibody I coated receptor microspheres having particle diameters of 100nm, 150nm, 200nm, 250nm and 350nm, respectively.

2. Donor microsphere coated antibodies

1. anti-PCT antibody ii was dialyzed to 50mM CB buffer at ph=10 to give a concentration of 1mg/mL.

2. Adding 0.5mL of photosensitive microsphere and 0.5mL of paired antibody II 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 completion of the reaction, the mixture was centrifuged for 45min and 30000g, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugation wash was repeated 4 times and diluted to a final concentration of 100. Mu.g/mL. Obtaining the donor microspheres coated with anti-PCT antibody II with the particle sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

Comparative example 2: detection of luminescence signal quantity of contrast kit

The test procedure was as in example 3, except that the microsphere composition used was replaced with the kit prepared in comparative example 1, and the test results are shown in Table 5.

TABLE 5

As can be seen from table 5, the luminescence signal amount of the comparative kit was significantly reduced, and the detection sensitivity was significantly reduced. And the luminescence signal value detected in the comparative kit is not optimal when the particle size of the donor microspheres is larger than that of the acceptor microspheres.

Example 7: preparation of (one) aldehyde-based polystyrene latex microspheres with surface of 250nm average particle size and no coating or binding of polysaccharide to donor microspheres and donor reagent

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 the microspheres 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 by a small amount of donor microsphere buffer solution, measuring the solid content, and regulating the concentration to 150 mug/ml by the donor microsphere buffer solution to obtain the donor reagent containing the donor microspheres.

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

Example 8: preparation of polysaccharide coated donor microspheres and donor reagents having an average particle size of 250nm

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 microspheres were 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 microspheres 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

g) 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 the donor microspheres to 100mg/ml.

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

i) 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.

j) 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.

k) 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.

l) 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 microsphere buffer solution, measuring the solid content, and regulating the concentration to 150 mug/ml by using the donor microsphere buffer solution to obtain the donor reagent containing the donor microspheres.

The donor microspheres had a gaussian distribution average particle size of 249.9nm as measured by a nanoparticle sizer, and a coefficient of variation (c.v.) of 11.6%, as shown in fig. 5.

Example 9: preparation of acceptor microsphere with average particle size of 200nm

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 187.5nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) =6.30%. 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-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (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 particle diameters of the microspheres at this time was 187.9nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=6.00%.

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 202.7nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=13.80%.

Coupling procedure of 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 a buffer solution to a final concentration of 50. Mu.g/ml to obtain a receptor microsphere solution coupled with PCT antibody.

The average particle diameter of the Gaussian distribution of the particle diameter of the receptor microsphere at this time was 204.3nm as measured by a nanoparticle analyzer, and the coefficient of variation (C.V value) =10.20%.

Example 10: on-machine detection result and analysis (detection substance: PCT antigen)

The PCT homogeneous chemiluminescent detection kit (photoexcitation chemiluminescent method) used in this example consisted of reagent 1 (R1 ') comprising a first anti-PCT antibody-coated acceptor microsphere, reagent 2 (R2 ') comprising a biotin-labeled second anti-PCT antibody, and additionally included a universal solution (R3 ') comprising donor microspheres. Wherein R1' is a receptor reagent prepared using the receptor microsphere (particle size distribution coefficient of variation C.V value=10.20%) in example 9; r3' is a donor agent prepared using the donor microspheres of examples 7 and 8.

The detection process was completed on a fully automatic light-activated chemiluminescence analysis system (LiCA HT) developed by bosa biotechnology (Shanghai) limited and the detection results were outputted, and the specific detection results are shown in table 6 below.

TABLE 6

As is clear from the results in Table 6, the kit of the present application was excellent in both the sensitivity and the upper limit of detection. And the sensitivity and upper limit of detection of the detection using the kit comprising the donor reagent in example 7 are both superior to those of the kit comprising the donor reagent in example 8. It can be seen that the performance of the donor microspheres using surface non-coated polysaccharide is more excellent.

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

Claims

1. A homogeneous chemiluminescent detection kit comprising:

2. The kit according to claim 1, wherein the average particle diameter of the donor microspheres is 100nm to 400nm, the average particle diameter of the acceptor microspheres is 100nm to 350nm, and the average particle diameter ratio of the donor microspheres to the acceptor microspheres is 1.1 to 4.0;

preferably, the average particle diameter of the donor microspheres is 190nm to 250nm, the average particle diameter of the acceptor microspheres is 180nm to 240nm, and the ratio of the average particle diameter of the donor microspheres to the average particle diameter of the acceptor microspheres is 1.2 to 3.0; or alternatively

The average particle diameter of the donor microspheres is 150nm, the average particle diameter of the acceptor microspheres is 100nm, and the ratio of the average particle diameter of the donor microspheres to the average particle diameter of the acceptor microspheres is 1.5.

3. The kit according to claim 1 or 2, wherein the donor microsphere comprises a first carrier, the interior of which is filled with a sensitizer, the surface of which is chemically bound to a label;

preferably, the surface of the first carrier is not coated or attached with a polysaccharide substance, which is directly chemically bound to the label.

4. A kit according to any one of claims 1 to 3, wherein the label is avidin; preferably, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like, preferably from the group consisting of neutravidin or streptavidin; and/or the number of the groups of groups,

the avidin is chemically bonded to the surface of the first carrier by reacting an amino group with an aldehyde group on the surface of the first carrier to form a Schiff base.

5. The kit according to claim 3 or 4, wherein the surface of the first carrier has a bonding functional group for chemically bonding a label to the surface of the first carrier;

preferably, 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;

Preferably, the content of the bonding functional groups on the surface of the first carrier is 100 to 500nmol/mg, preferably 200 to 400nmol/mg.

6. A kit according to claim 3, wherein the surface of the first carrier is coated with a hydrophilic aldehyde dextran, the aldehyde dextran having an aldehyde chemically bonded label.

7. The kit according to any one of claims 1 to 6, wherein the receptor microsphere comprises a second carrier, the interior of the second carrier is filled with a luminescent composition, the surface of the second carrier is coated with at least one polysaccharide layer, and the surface of the polysaccharide layer is connected with biomolecules;

preferably, the surface of the second carrier is coated with hydrophilic carboxydextran.

8. The kit of claim 7, wherein the luminescent composition comprises a europium complex; preferably, the europium complex is MTTA-EU ³⁺ 。

9. Kit according to any one of claims 3 to 8, wherein the first carrier and/or the second carrier is/are 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.

10. The kit according to any one of claims 1 to 9, wherein the donor microspheres have a coefficient of variation C.V value of the particle size distribution in the donor reagent of 5% or more; and/or the number of the groups of groups,

the variation coefficient C.V value of the particle size distribution of the receptor microspheres in the receptor reagent is more than or equal to 5%.

11. Kit according to any one of claims 1 to 10, characterized in that it comprises in particular:

12. Kit according to any one of claims 1 to 10, characterized in that it comprises in particular:

13. Kit according to any one of claims 1 to 10, characterized in that it comprises in particular:

14. A homogeneous chemiluminescent detection method for detecting a test target molecule in a test sample using the kit according to any one of claims 1-13.

15. A homogeneous chemiluminescent detection device for detecting a test target molecule in a test sample using the kit of any one of claims 1-13 or the method of claim 14;

preferably, the device is a POCT instant detection device.