CN116539597A - cTnI homogeneous phase chemiluminescence detection kit, detection method and device - Google Patents

cTnI homogeneous phase chemiluminescence detection kit, detection method and device Download PDF

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CN116539597A
CN116539597A CN202310486930.1A CN202310486930A CN116539597A CN 116539597 A CN116539597 A CN 116539597A CN 202310486930 A CN202310486930 A CN 202310486930A CN 116539597 A CN116539597 A CN 116539597A
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donor
microspheres
microsphere
kit
acceptor
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杨阳
刘宇卉
李临
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Kemei Boyang Diagnostic Technology Shanghai Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein

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Abstract

The invention relates to a cTnI homogeneous chemiluminescent detection kit, a detection method and a detection device, belonging to the technical field of homogeneous chemiluminescent detection. The kit comprises: a donor agent comprising donor microspheres and a first label bound thereto, the donor microspheres being capable of generating reactive oxygen species in an excited state; and, a receptor agent comprising a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal and a first binding unit bound thereto, said first binding unit capable of specifically binding to a first epitope of human cardiac troponin I; wherein the particle size of the donor microsphere is not smaller than the particle size of the acceptor microsphere. The particle size of the donor microsphere in the kit is not smaller than that of the acceptor microsphere, so that the precision and the sensitivity of the kit are improved.

Description

cTnI homogeneous phase chemiluminescence detection kit, detection method and device
The application is a divisional application of Chinese patent application with the application date of 2018, 7 month and 18 days, the application number of 201810790622.7 and the name of cTnI homogeneous chemiluminescence detection kit, detection method and device.
Technical Field
The invention belongs to the technical field of homogeneous chemiluminescence detection, and particularly relates to a cTnI homogeneous chemiluminescence detection kit, a detection method and a detection device.
Background
Currently, the detection of biomacromolecule human cardiac troponin I (cTnI) is commonly performed by ELISA, colloidal gold method, homogeneous chemiluminescence analysis and the like. 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 greatly improved by the methods described in the art, there remains a need to develop a method for further improving the luminescent efficiency of detection of human cardiac troponin I based on the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a cTnI homogeneous chemiluminescence detection kit, and the method for detecting human cardiac troponin I by using the kit has high detection sensitivity.
To this end, the first aspect of the present invention provides a cTnI homogeneous chemiluminescent assay kit comprising:
a donor agent comprising donor microspheres and a first label bound thereto, the donor microspheres being capable of generating reactive oxygen species in an excited state; and, a step of, in the first embodiment,
a receptor reagent comprising a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal and a first binding unit bound thereto, the first binding unit capable of specifically binding to a first epitope of human cardiac troponin I;
wherein the particle size of the donor microsphere is not smaller than the particle size of the acceptor microsphere.
In some embodiments of the invention, the particle size of the donor and acceptor microspheres is selected from 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, most preferably from 150nm to 250nm.
In some preferred embodiments of the invention, the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.
In some preferred embodiments of the invention, the donor and acceptor microspheres each have a particle size of 200nm.
In some preferred embodiments of the invention, the donor microspheres have a particle size greater than the acceptor microspheres.
In some preferred embodiments of the invention, the particle size ratio of the donor microspheres to the acceptor microspheres is from 1.06 to 8.60, preferably from 1.2 to 4.0, more preferably from 1.5 to 2.01.
In some embodiments of the invention, the donor microsphere surface is coated with a hydrophilic aldehyde dextran.
In other embodiments of the invention, the receptor microsphere surface is coated with hydrophilic carboxyglucose.
In some embodiments of the invention, the donor microsphere is filled with a photosensitizer selected from one of methylene blue, rose bengal, porphine, and phthalocyanine.
In other embodiments of the invention, the luminescent microspheres are filled with luminescent compounds; preferably, the luminescent compound is a europium complex;further preferably, the europium complex is MTTA-EU 3+ 。
In some embodiments of the invention, the donor microspheres and the acceptor microspheres are polystyrene microspheres.
In other embodiments of the invention, the active oxygen is singlet oxygen.
In some embodiments of the invention, the kit further comprises a first reagent comprising a specific conjugate of a first label bound thereto and a second binding unit capable of specifically binding to a second epitope of human cardiac troponin I that is an epitope of a different binding property or an epitope of the same binding property of a different location of human cardiac troponin I.
In some preferred embodiments of the invention, the first binding unit and the second binding unit are each independently selected from a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably from a polyclonal antibody and/or a monoclonal antibody, having binding specificity for human cardiac troponin I.
In some preferred embodiments of the invention, the first binding unit and/or the second binding unit independently comprises at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specificity to epitopes of different binding properties of human cardiac troponin I or to epitopes of the same binding properties at different positions.
In some embodiments of the invention, the first label is selected from avidin and/or streptavidin.
In some preferred embodiments of the invention, the concentration of the receptor microspheres in the receptor reagent is selected from 20 to 300 μg/mL, preferably 30 to 200 μg/mL, more preferably 40 to 100 μg/mL.
In other preferred embodiments of the invention, the concentration of the donor microspheres in the donor reagent is selected from 0.2 to 10. Mu.g/mL, preferably 0.5 to 8. Mu.g/mL, more preferably 1 to 6. Mu.g/mL.
In a second aspect the invention provides a method for homogeneous chemiluminescent detection of human cardiac troponin I using a kit according to the first aspect of the 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 donor reagent to form a mixture to be tested;
s2, exciting the mixture to be tested to chemiluminescent by using energy or an active compound, and measuring the signal intensity of the chemiluminescent;
wherein the donor agent comprises donor microspheres capable of generating active oxygen in an excited state; the acceptor reagent comprises acceptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the donor microspheres have a particle size not smaller than the acceptor microspheres.
In a third aspect, the present invention provides a chemiluminescent detection device for detecting human cardiac troponin I in a sample to be tested 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 and the detection sensitivity is good when the kit is used for detecting the human cardiac troponin I through chemiluminescence. In addition, the acceptor microsphere and the donor microsphere in the kit are polystyrene microsphere, hydrophilic carboxyl glucose is wrapped on the surface of the acceptor microsphere, hydrophilic aldehyde dextran is wrapped on the surface of the donor microsphere, so that nonspecific adsorption is greatly reduced, influences of other environmental factors such as pH value and electrolyte outside a system are reduced, and further detection accuracy can be improved.
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.
The term "test sample" as used herein refers to a mixture that may contain an analyte, 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.
In the present invention, the "donor microsphere" may be polymer particles filled with a photosensitizer formed by coating a functional group on a substrate, 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 surface of the donor microsphere is provided with hydrophilic aldehyde dextran, and the inside of the donor microsphere is 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 microsphere surface 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 a polymer microparticle filled with a light-emitting compound formed by coating a functional group on a substrate, and may be referred to as a light-emitting microsphere or a light-emitting microparticle. The surface of the luminescent microsphere acceptor microsphere is provided with hydrophilic carboxyl glucan, and the inside of the luminescent microsphere acceptor microsphere is filled with a chemiluminescent agent which can react with active oxygen (such as singlet oxygen). In some embodiments of the inventionThe chemiluminescent agent undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that may decompose with or subsequent to luminescence. In some preferred embodiments of the invention, the luminescent compound is a europium complex; further preferably, the europium complex is MTTA-EU 3+ 。
The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods well known to those skilled in the art.
The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins derived from more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.
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.
II. Detailed description of the preferred embodiments
The present invention will be described in more detail below.
The cTnI homogeneous chemiluminescent detection kit related to the first aspect of the invention comprises the following components:
a donor agent comprising donor microspheres and a first label bound thereto, the donor microspheres being capable of generating reactive oxygen species in an excited state; and, a step of, in the first embodiment,
a receptor reagent comprising a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal and a first binding unit bound thereto, the first binding unit capable of specifically binding to a first epitope of human cardiac troponin I;
wherein the particle size of the donor microsphere is not smaller than the particle size of the acceptor microsphere.
According to the invention, by controlling the relation between the particle sizes of the donor microsphere and the acceptor microsphere in the kit, when the kit is used for detecting the human cardiac troponin I through chemiluminescence, the luminous efficiency of detection is improved, and the kit has good detection sensitivity.
In some embodiments of the invention, the particle size of the donor and acceptor microspheres is selected from 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, most preferably from 150nm to 250nm. For example, in some embodiments of the invention, the particle size of the donor and acceptor microspheres may be 20nm, 50nm, 70nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, and 250nm.
In some preferred embodiments of the invention, the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.
In some preferred embodiments of the invention, the donor and acceptor microspheres each have a particle size of 200nm.
In some preferred embodiments of the invention, the donor microspheres have a particle size greater than the acceptor microspheres.
In some preferred embodiments of the invention, the particle size ratio of the donor microspheres to the acceptor microspheres is from 1.06 to 8.60, preferably from 1.2 to 4.0, more preferably from 1.5 to 2.01. For example, in some embodiments of the invention, the donor microsphere has a particle size of 150nm, the acceptor microsphere has a particle size of 100nm, and the ratio of the donor microsphere to the acceptor microsphere has a particle size of 1.5.
In some embodiments of the invention, the donor microsphere surface is coated with a hydrophilic aldehyde dextran.
In other embodiments of the invention, the receptor microsphere surface is coated with hydrophilic carboxyglucose.
When the microsphere 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 some embodiments of the invention, the donor microsphere is filled with a photosensitizer selected from one of methylene blue, rose bengal, porphine, and phthalocyanine. The loading amount of the photosensitizer in the donor microsphere is not particularly limited, and it may be an amount commonly used in the art.
In other embodiments of the invention, the receptor microsphere is filled with a luminescent compound. In some preferred embodiments of the invention, the luminescent compound is a europium complex. 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 microspheres and the acceptor microspheres are polystyrene microspheres.
In other embodiments of the invention, the active oxygen is singlet oxygen.
In some embodiments of the invention, the kit further comprises a first reagent comprising a specific conjugate of a first label bound thereto and a second binding unit capable of specifically binding to a second epitope of human cardiac troponin I that is an epitope of a different binding property or an epitope of the same binding property of a different location of human cardiac troponin I. The concentration of the first reagent may be selected from 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, 100 μg/mL, 120 μg/mL, 160 μg/mL, 180 μg/mL, or 200 μg/mL.
In some preferred embodiments of the invention, the first binding unit and the second binding unit are each independently selected from a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably from a polyclonal antibody and/or a monoclonal antibody, having binding specificity for human cardiac troponin I.
In some preferred embodiments of the invention, the first binding unit and/or the second binding unit independently comprises at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specificity to epitopes of different binding properties of human cardiac troponin I or to epitopes of the same binding properties at different positions.
In some embodiments of the invention, the first label is selected from avidin and/or streptavidin.
In some preferred embodiments of the invention, the concentration of the receptor microspheres in the receptor reagent is selected from 20 to 300 μg/mL, preferably 30 to 200 μg/mL, more preferably 40 to 100 μg/mL. In some embodiments of the invention, the concentration of the receptor microspheres in the receptor reagent may include, but is not limited to, a concentration selected from 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, 100 μg/mL, 120 μg/mL, 140 μg/mL, 150 μg/mL, 160 μg/mL, 180 μg/mL, or 200 μg/mL.
In other preferred embodiments of the invention, the concentration of the donor microspheres in the donor reagent is selected from 0.2 to 10. Mu.g/mL, preferably 0.5 to 8. Mu.g/mL, more preferably 1 to 6. Mu.g/mL. In some embodiments of the invention, the concentration of donor microspheres in the donor reagent may include, but is not limited to, a concentration selected from 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, or 7 μg/mL.
In some embodiments of the invention, the kit further comprises a diluent for diluting the sample 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 a second aspect the invention provides a method for homogeneous chemiluminescent detection of human cardiac troponin I using a kit according to the first aspect of the 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 donor reagent to form a mixture to be tested;
s2, exciting the mixture to be tested to chemiluminescent by using energy or an active compound, and measuring the signal intensity of the chemiluminescent;
wherein the donor agent comprises donor microspheres capable of generating active oxygen in an excited state; the acceptor reagent comprises acceptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the donor microspheres have a particle size not smaller than the acceptor microspheres.
In some embodiments of the present invention, in step S1, a sample to be tested is first mixed with a acceptor reagent to form a first mixture, and then the first mixture is mixed with a donor reagent to form a mixture to be tested.
In other embodiments of the present invention, in step S2, the mixture to be tested is irradiated with red excitation light of 600-700 nm to excite the mixture to generate chemiluminescence.
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 a third aspect, the present invention provides a chemiluminescent detection device for detecting human cardiac troponin I in a sample to be tested 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.
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 holding an acceptor reagent comprising acceptor microspheres capable of reacting with active oxygen to generate a chemiluminescent signal, the donor microspheres having a particle size not less 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, and the additional reagent hole site is covered with a film to seal 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 human cardiac troponin I 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: cTnI homogeneous phase chemiluminescence 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-cTnI 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 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. Sampling and measuring the concentration, particle size and signal value of the washed conjugated antibody receptor microsphere.
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: dialyzing anti-cTnI antibody II 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 the treated 1mg/mL anti-cTnI antibody II with the prepared biotin solution according to the volume ratio of 10000:54, and rapidly and uniformly mixing. 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 cTnI homogeneous chemiluminescence detection kit.
Example 2: homogeneous chemiluminescence detection method for human cardiac troponin I
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: homogeneous chemiluminescent detection of human cardiac troponin I
Human cardiac troponin I was detected using the kit prepared in example 1, the method described in example 2, and the detection results are shown in table 1.
TABLE 1
As is clear from table 1, when the particle size of the donor microspheres is not smaller than the particle size of the acceptor microspheres in the case where the particle sizes of the acceptor microspheres and the donor microspheres are constant, the detected luminescence signal value is large, and the detection sensitivity is good.
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 cTnI homogeneous chemiluminescence 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 reagent used in the detection:
(1) The receptor microspheres prepared in example 2 (receptor microspheres having a particle size of 200nm and respectively linked to anti-cTnI antibody i;
(2) The first reagent prepared in example 5 (biotin-labeled antibody, antibody anti-cTnI antibody ii);
(3) Example 4 donor microspheres (donor microspheres coated with avidin having different particle sizes (80 nm, 200nm, 300 nm)) were prepared.
The reagents (1) - (3) were matched to form a reagent set 1, a reagent set 2 and a reagent set 3 shown in table 2, cTnI antigens were detected separately (after dilution to a suitable concentration, the reagent sets 1, 2 and 3 detected the same sample simultaneously), 20 wells were repeatedly detected, the detection procedure was as described in example 2, and the detection results were shown in table 3.
TABLE 2
Reagent grouping Reagent set 1 Reagent set 2 Reagent set 3
Receptor microsphere particle size/nm 200 200 200
Donor microsphere particle size/nm 80 200 300
TABLE 3 Table 3
As is clear from Table 3, the light intensities of the reagent sets 2 and 3 are increased as compared with the reagent set 1, and the light intensity detected by this method is increased when the particle size of the donor microspheres is not smaller than the particle size of the acceptor microspheres. Meanwhile, compared with the reagent set 1, the reagent sets 2 and 3 have smaller variation Coefficient (CV), namely, the precision is higher when the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.
Example 6: detection of analysis sensitivity of cTnI homogeneous chemiluminescence detection kit
Sample to be measured: zero value calibration material;
the process comprises the following steps: repeating the detection for 20 times to obtain a light intensity value (RLU)
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 reagent sets 2 and 3 have better sensitivity than the reagent set 1, i.e., the donor microspheres have a particle size not smaller than the acceptor microspheres, which is advantageous for improving the sensitivity of the reagent.
Comparative example 1: preparation of contrast kit
1. Receptor microsphere conjugated antibodies
1. anti-cTnI antibody i was dialyzed to 50mM CB buffer at ph=10, and the concentration was measured to be 1mg/mL.
2. 0.5mL of the receptor microsphere prepared in example 1 and 0.5mL of anti-cTnI antibody I are added into a 2mL centrifuge tube, and 100 mu L of 10mg/mL NaBH is added after uniform mixing 4 Solution (50 mM)CB buffer), 2-8deg.C 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 conjugated antibodies
1. anti-cTnI antibody ii was dialyzed to 50mM CB buffer at ph=10, and the concentration was measured to be 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 microsphere coated with anti-cTnI antibody II with the particle sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.
Comparative example 2: homogeneous chemiluminescence detection of human cardiac troponin I by contrast kit
The test procedure was the same as in example 3, except that the donor and acceptor microspheres used were replaced with the comparative donor and acceptor microspheres 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 when the particle size of the donor microsphere is not smaller than that of the acceptor microsphere in the contrast kit, the detected luminous signal value is not good.
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 (16)

1. A cTnI homogeneous chemiluminescent assay kit comprising:
a donor agent comprising donor microspheres and a first label bound thereto, the donor microspheres being capable of generating reactive oxygen species in an excited state; and, a step of, in the first embodiment,
a receptor reagent comprising a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal and a first binding unit bound thereto, the first binding unit capable of specifically binding to a first epitope of human cardiac troponin I;
Wherein the particle size of the donor microsphere is not smaller than the particle size of the acceptor microsphere.
2. Kit according to claim 1, wherein the particle size of the donor and acceptor microspheres is selected from 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, most preferably from 150nm to 250nm.
3. The kit according to claim 1 or 2, wherein the donor microspheres have a particle size equal to the particle size of the acceptor microspheres; preferably, the particle size of the donor microsphere and the acceptor microsphere is 200nm.
4. The kit of claim 1 or 2, wherein the donor microspheres have a particle size greater than the acceptor microspheres;
preferably, the particle size ratio of the donor microspheres to the acceptor microspheres is from 1.06 to 8.60, preferably from 1.2 to 4.0, more preferably from 1.5 to 2.01.
5. The kit of any one of claims 1-4, wherein the surface of the donor microsphere is coated with hydrophilic aldehyde dextran; and/or the number of the groups of groups,
the surface of the receptor microsphere is wrapped with hydrophilic carboxyl glucose.
6. The kit of any one of claims 1-5, wherein the donor microsphere is filled with a photosensitizer selected from one of methylene blue, rose bengal, porphine, and phthalocyanine.
7. The kit according to any one of claims 1 to 6, wherein the luminescent microspheres are filled with luminescent compounds; preferably, the luminescent compound is a europium complex; further preferably, the europium complex is MTTA-EU 3+ 。
8. The kit of any one of claims 1-7, wherein the donor and acceptor microspheres are polystyrene microspheres; and/or the number of the groups of groups,
the active oxygen is singlet oxygen.
9. The kit of any one of claims 1-8, further comprising a first agent comprising a specific conjugate of a second binding unit capable of specifically binding to a second epitope of human cardiac troponin I, said second epitope being an epitope of a different binding property or an epitope of the same binding property of a different location of human cardiac troponin I, and a first label bound thereto.
10. The kit of claim 9, wherein:
the first binding unit and the second binding unit are each independently selected from a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably from a polyclonal antibody and/or a monoclonal antibody, having binding specificity for human cardiac troponin I.
11. The kit of claim 10, wherein:
the first binding unit and/or the second binding unit independently comprises at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specificity to epitopes of different binding properties of human cardiac troponin I or to epitopes of the same binding properties of different positions.
12. The kit of any one of claims 1-11, wherein: the first label is selected from avidin and/or streptavidin.
13. Kit according to any one of claims 1 to 12, wherein the concentration of the receptor microspheres in the receptor reagent is selected from 20 to 300 μg/mL, preferably 30 to 200 μg/mL, more preferably 40 to 100 μg/mL; and/or the number of the groups of groups,
the concentration of the donor microspheres in the donor reagent is selected from 0.2 to 10. Mu.g/mL, preferably 0.5 to 8. Mu.g/mL, more preferably 1 to 6. Mu.g/mL.
14. A homogeneous chemiluminescent detection method of human cardiac troponin I using the kit of any one of claims 1-13.
15. The method according to claim 14, characterized in that it comprises the steps of:
S1, mixing a sample to be tested with an acceptor reagent and a donor reagent to form a mixture to be tested;
s2, exciting the mixture to be tested to chemiluminescent by using energy or an active compound, and measuring the signal intensity of the chemiluminescent;
wherein the donor agent comprises donor microspheres capable of generating active oxygen in an excited state; the acceptor reagent comprises acceptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the donor microspheres have a particle size not smaller than the acceptor microspheres.
16. A chemiluminescent detection device for detecting human cardiac troponin I in a sample to be tested using the kit according to any one of claims 1-13 or the method according to any one of claims 14-15;
preferably, the device is a POCT instant detection device.
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