CN110736734A

CN110736734A - cTnI homogeneous phase chemiluminescence detection kit, detection method and device

Info

Publication number: CN110736734A
Application number: CN201810790622.7A
Authority: CN
Inventors: 杨阳; 赵卫国; 刘宇卉; 李临
Original assignee: Boyang Biotechnology (Shanghai) Co Ltd
Current assignee: Boyang Biotechnology (Shanghai) Co Ltd
Priority date: 2018-07-18
Filing date: 2018-07-18
Publication date: 2020-01-31
Also published as: CN116539597A

Abstract

The invention relates to a cTnI homogeneous phase chemiluminescence detection kit, a detection method and a device in the technical field of homogeneous phase chemiluminescence detection, wherein the kit comprises a donor reagent and an acceptor reagent, wherein the donor reagent comprises donor microspheres and markers combined with the donor microspheres, the donor microspheres can generate active oxygen in an excited state, the acceptor reagent comprises acceptor microspheres and combination units combined with the acceptor microspheres, the acceptor microspheres can react with the active oxygen to generate detectable chemiluminescence signals, the combination units can be specifically combined with epitope of human cardiac troponin I, the particle size of the donor microspheres is not smaller than that of the acceptor microspheres, the particle size of the donor microspheres in the kit is not smaller than that of the acceptor microspheres, and the precision and the sensitivity of the kit are improved.

Description

cTnI homogeneous phase chemiluminescence detection kit, detection method and device

Technical Field

The invention belongs to the technical field of homogeneous phase chemiluminescence detection, and particularly relates to cTnI homogeneous phase chemiluminescence detection kits, detection methods and devices.

Background

Currently, biomacromolecule human cardiac troponin I (cTnI) is commonly detected by ELISA, colloidal gold, homogeneous chemiluminescence assay, etc. among others, the chemiluminescence assay is methods for detecting light waves emitted by chemiluminescence substances, which are used as labels in nucleic acid detection and immunoassay, for example, molecules in a specific binding pair can be combined with the chemiluminescence substances in various ways to form a luminescent complex, which can react with a detected object (the other molecules in the specific binding pair) in a sample, and the complex is distributed in a solid phase and a liquid phase, and the distribution ratio is related to the amount of the detected object.

To increase the efficiency of the emission of the donor and/or acceptor microspheres, methods to increase the efficiency of the light exposure of the dye in the donor microsphere and/or the efficiency and efficiency of the emission of the light-emitting compound in the acceptor microsphere are commonly used in the art .

Although the detection sensitivity of the chemiluminescence detection method can be greatly improved by using the method in the field, methods for detecting human cardiac troponin I by increasing the luminous efficiency by steps based on the prior art still need to be developed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides cTnI homogeneous phase chemiluminescence detection kits, and the method for detecting human cardiac troponin I by adopting the kits has high detection sensitivity.

To this end, the invention according to the provides cTnI homogeneous chemiluminescent assay kits comprising:

a donor reagent comprising a donor microsphere capable of generating a reactive oxygen species in an excited state and an th label bound thereto,

a receptor reagent comprising a receptor microsphere and th binding unit bound thereto, the receptor microsphere being capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, the th binding unit being capable of specifically binding to the th epitope of human cardiac troponin I;

wherein the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

In embodiments of the present invention, the donor and acceptor microspheres have a particle size selected from the group consisting of 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250 nm.

In preferred embodiments of the present invention, the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.

In preferred embodiments of the present invention, the donor and acceptor microspheres each have a particle size of 200 nm.

In preferred embodiments of the present invention, the donor microspheres have a larger particle size than the acceptor microspheres.

In preferred embodiments of the present invention, the ratio of the particle size of the donor microspheres to that of the acceptor microspheres is 1.06 to 8.60, preferably 1.2 to 4.0, and more preferably 1.5 to 2.01.

In , the donor microspheres are surface coated with hydrophilic aldehyde dextran.

In still other embodiments of the invention, the acceptor microspheres are surface coated with hydrophilic carboxyglucose.

In embodiments of the invention, the donor microspheres are filled with a photosensitizer selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the luminescent microsphere is filled with a luminescent compound, preferably the luminescent compound is a europium complex, and further preferably the europium complex is MTTA-EU³⁺。

In , the donor and acceptor microspheres are polystyrene microspheres.

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

In embodiments of the invention, the kit further comprises a reagent comprising a specific binder of a second binding unit capable of specifically binding to a second epitope of human cardiac troponin I, which second epitope and epitope are epitopes of different binding properties or epitopes of the same binding properties at different positions of human cardiac troponin I, and a marker bound thereto.

In preferred embodiments of the present invention, the th binding unit and the second binding unit are each independently selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably selected from the group consisting of a polyclonal antibody and/or a monoclonal antibody, having binding specificity for human cardiac troponin I.

In preferred embodiments of the invention, the th binding unit and/or the second binding unit independently comprise at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specifically to epitopes of different binding properties or to epitopes of the same binding properties at different positions of human cardiac troponin I.

In embodiments of the invention, the marker is selected from avidin and/or streptavidin.

In preferred embodiments of the present invention, the concentration of the acceptor microsphere in the acceptor reagent is selected from 20-300 μ g/mL, preferably 30-200 μ g/mL, and more preferably 40-100 μ g/mL.

In still other preferred embodiments of the present invention, the concentration of the donor microspheres in the donor reagent is selected from 0.2-10 μ g/mL, preferably 0.5-8 μ g/mL, more preferably 1-6 μ g/mL.

In a second aspect, the invention provides homogeneous chemiluminescent assay methods for human cardiac troponin I using a kit according to aspect for the chemiluminescent assay.

In embodiments of the present invention, the method includes the steps of:

s1, mixing the sample to be tested with the receptor reagent and the donor reagent to form a mixture to be tested;

s2, exciting the mixture to be detected to generate chemiluminescence by using energy or active compounds, and measuring the signal intensity of the chemiluminescence;

wherein the donor reagent comprises donor microspheres capable of generating reactive oxygen species in an excited state; the receptor reagent comprises a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

In a third aspect, the present invention provides chemiluminescent detection devices for detecting human cardiac troponin I in a test sample using the kit according to aspect or the method according to the second aspect of the present invention.

In preferred embodiments of the present invention, the device is a POCT point of care device.

The invention has the beneficial effects that: by controlling the matrix and the grain size of the acceptor microsphere and the donor microsphere, the kit improves the luminous efficiency of detection when being used for chemiluminescence detection of human cardiac troponin I, and has good detection sensitivity. In addition, the acceptor microspheres and the donor microspheres in the kit are polystyrene microspheres, hydrophilic carboxyl glucose is coated on the surfaces of the acceptor microspheres, and hydrophilic aldehyde dextran is coated on the surfaces of the donor microspheres, so that nonspecific adsorption is greatly reduced, the influence of other environmental factors outside the system such as pH value, electrolyte and the like is reduced, and the detection accuracy can be improved.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

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

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

Term (I)

The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

The term "test sample" as used herein refers to mixtures that may contain analytes including, but not limited to, proteins, hormones, antibodies or antigens, typical test samples that may be used in the disclosed methods include body fluids such as whole blood, serum, plasma, saliva, urine, etc. the test sample may be diluted with a diluent as necessary prior to use.

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

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

In the present invention, the "donor microsphere" can be a polymeric microparticle coated on a substrate with a functional group to form a photosensitizer-filled polymer microparticle capable of generating reactive oxygen species (e.g., singlet oxygen) upon photoexcitation, in which case the donor microsphere can also be referred to as a photosensitive microsphere or photosensitive microparticle, the surface of the donor microsphere has hydrophilic aldehyde dextran and the interior of the donor microsphere is filled with a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrin, and phthalocyanine, and derivatives of these compounds having 1-50 atomic substituents that are used to render these compounds more lipophilic or hydrophilic and/or as a linking group to a specific binding partner.

The "acceptor microsphere" may be a polymer particle coated with a functional group on a substrate to form a polymer particle filled with a luminescent compound, which may be referred to as a luminescent microsphere or a luminescent particle, the surface of the acceptor microsphere of the luminescent microsphere may include hydrophilic carboxyl dextran, and the inside of the acceptor microsphere may be filled with a chemiluminescent agent capable of reacting with active oxygen (e.g., singlet oxygen)³⁺。

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted from a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced from or more clones of B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "biotin" as used herein is broadly applicable to animal and plant tissues, which have two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin, the activated biotin can be coupled with almost all known biological macromolecules including proteins, nucleic acids, polysaccharides, lipids, etc., under the mediation of a protein cross-linking agent, and "streptavidin" is a protein secreted by streptomyces, and the "streptavidin" molecule with a molecular weight of 65 kD. consists of 4 identical peptide chains, each of which can bind biotin.

The term "particle size" as used herein refers to the average particle size of the microspheres, as measured by conventional particle sizers.

Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The cTnI homogeneous phase chemiluminescence detection kit according to the aspect of the invention , comprising:

According to the invention, by controlling the relationship between the particle sizes of the donor microsphere and the acceptor microsphere in the reagent kit, when the reagent kit is used for chemiluminescence detection of human cardiac troponin I, the detection luminous efficiency is improved, and the detection sensitivity is good.

In embodiments of the invention, the donor and acceptor microspheres have a particle size selected from 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250nm, e.g., in embodiments of the invention, the donor and acceptor microspheres may have a particle size of 20nm, 50nm, 70nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, and 250 nm.

In preferred embodiments of the present invention, the ratio of the particle size of the donor microspheres to the particle size of the acceptor microspheres is 1.06-8.60, preferably 1.2-4.0, more preferably 1.5-2.01. for example, in preferred embodiments of the present invention, the donor microspheres have a particle size of 150nm, the particle size of the acceptor microspheres is 100nm, and the ratio of the particle size of the donor microspheres to the particle size of the acceptor microspheres is 1.5.

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

In the embodiments of the invention, the donor microspheres are filled with a photosensitizer selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the acceptor microspheres are filled with a luminescent compound, in other preferred embodiments of the present invention, the luminescent compound is an europium complex, the europium complex filled in the polystyrene microspheres interacts with the polystyrene microspheres to further increase the luminescence efficiency of the polystyrene microspheres at , and in other preferred embodiments of the present invention, the europium complex is MTTA-EU³⁺The complex can directly capture singlet oxygen generated by phthalocyanine dye in the 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 has a structural formula shown in a formula I, and the synthesis is referred to CN 200510130851.9.

Europium complex MTTA-EU³⁺The synthesis of the (europium (III) complex) is as follows:

(1) a500 mL three-necked flask was charged with 732mg of MTTA (1mmoL) and 366mg of EuCl₃·6H₂O (1mmoL) was dissolved in 100mL of methanol and refluxed at 70 ℃ for 2 hours with stirring.

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

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

(4) Vacuum drying to obtain 830mg MTTA-EU³⁺。

In , the donor and acceptor microspheres are polystyrene microspheres.

In embodiments of the invention, the kit further includes a reagent comprising a specific binder of 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 of the same binding property at a different location to the epitope and a marker bound thereto, the concentration of the reagent may be selected from 60 μ g/mL, 70 μ g/mL, 80 μ g/mL, 90 μ g/mL, 100 μ g/mL, 120 μ g/mL, 160 μ g/mL, 180 μ g/mL, or 200 μ g/mL.

In preferred embodiments of the invention, the concentration of the receptor microspheres in the receptor reagent is selected from 20-300 μ g/mL, preferably 30-200 μ g/mL, more preferably 40-100 μ g/mL in embodiments of the invention, the concentration of the receptor microspheres in the receptor reagent can include, but is not limited to, a concentration selected from 50 μ g/mL, 60 μ g/mL, 70 μ g/mL, 80 μ g/mL, 90 μ g/mL, 100 μ g/mL, 120 μ g/mL, 140 μ g/mL, 150 μ g/mL, 160 μ g/mL, 180 μ g/mL, or 200 μ g/mL.

In still other preferred embodiments of the present invention, the concentration of the donor microspheres in the donor agent is selected from 0.2-10 μ g/mL, preferably 0.5-8 μ g/mL, more preferably 1-6 μ g/mL in the embodiments of the present invention, the concentration of donor microspheres in the donor agent can include, but is not limited to, a concentration selected from 1 μ g/mL, 2 μ g/mL, 3 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, or 7 μ g/mL.

According to , the kit further comprises a diluent for diluting the sample to be tested, wherein the diluent comprises a buffer solution, a protein, a stabilizer, a preservative and the like, and the diluent has the functions of dilution and buffering, so that the accuracy of the final test result and the stability of the sample to be tested are improved.

In embodiments of the present invention, the method includes the steps of:

In , in step S1, the test sample is first mixed with the acceptor reagent to form a th mixture, and then the th mixture is mixed with the donor reagent to form a test mixture.

In another embodiments of the present invention, in step S2, the mixture to be tested is irradiated by 600-700 nm red excitation light to excite the mixture to be tested to generate chemiluminescence.

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

In 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:

a sample hole site to be detected for containing a sample to be detected containing target molecules to be detected;

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

a second reagent well site for holding an acceptor reagent comprising acceptor microspheres capable of reacting with reactive oxygen species to produce a chemiluminescent signal, the donor microspheres having a particle size not smaller than the acceptor microspheres;

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

c. and the detection mechanism is electrically connected with the sample adding mechanism and is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen.

In other embodiments of the present invention, the sample well to be tested, the donor reagent well and the acceptor reagent well are coated to seal the opening of the well, so as to prevent the contamination of the substances therein.

In order to conveniently identify and read the information of the sample to be tested, the preferable technical scheme is that the side 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, wherein the bar code can be -dimensional or two-dimensional.

Correspondingly, the POCT device also 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 bar code medium (paper or reagent card) printing scanning, and the information reading adopts contact scanning or non-contact scanning in a mode of infrared or radio frequency and the like; the information includes, but is not limited to, assay project name, standard curve, reagent composition, lot number, expiration date, manufacturer information.

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

In , the reagent cup strip is further provided with an additional reagent hole for containing an additional reagent, and the additional reagent hole is covered with a film to close the opening.

In preferred embodiments of the present invention, the sample application mechanism comprises:

a pipetting assembly for aspirating or discharging a liquid;

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

the vertical moving assembly is arranged on the horizontal moving assembly, and the horizontal moving assembly is used for driving the liquid transfer assembly to move horizontally.

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

a base for carrying 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;

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

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

In , the detection wavelength of the chemiluminescent signal generated by the reaction of the acceptor microsphere and active oxygen is 450-650 nm.

In preferred embodiments of the present invention, the liquid transfer assembly includes a piston mechanism, a connector and a pipette sequentially arranged from top to bottom, the piston mechanism is connected to the connector, the pipette is arranged at the edge of the end face of the base, when liquid transfer is required, the connector descends and is connected to the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

In , the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction, wherein the temperature of the reagent cup strip and the contents of the reagent cup strip is 20-50 ℃ by means of a metal bath, water bath or oil bath.

In still other embodiments of the present invention, the sample well site to be tested, the donor reagent well site, and the acceptor reagent well site have cross-sections with different shapes.

The using process of the device comprises the steps of respectively containing a sample to be detected, a donor reagent hole site and a receptor reagent hole site, placing the reagent card in the POCT analyzer, taking the sample to be detected with the corresponding volume by using a sample adding mechanism, adding the sample to be detected into the th reagent hole site, reacting for times, continuously taking volumes of mixed liquid, adding the mixed liquid into the second reagent hole site, irradiating laser to the second reagent hole site by using an exciter in the detection assembly, reacting for times, detecting a chemiluminescent signal generated by the reaction of receptor microspheres and active oxygen by using the detection mechanism, and calculating the concentration of the human cardiac troponin I in the sample to be detected.

Example III

In order that the invention may be more readily understood, the invention is now described in further detail at with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.

Example 1: cTnI homogeneous phase chemiluminescence detection kit

preparation of acceptor microspheres

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

2. A100 mL three-necked flask was prepared, 10mL 95% ethanol, 10mL water and 10mL 10% polystyrene microspheres 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 and dropwise adding the europium (III) complex solution in the step 1 into the three-neck flask in the step 2, reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling to obtain the emulsion.

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

5. The same method is used for preparing acceptor microsphere solutions with the grain diameters of 100nm, 150nm, 250nm, 300nm and 350nm respectively.

II, receptor microsphere coupling antibody

1. Weighing 10mg of receptor microsphere coated with carboxyl dextran hydrogel in a centrifugal tube according to the preparation amount, and centrifuging at 10000rpm for 60 min.

2. The supernatant was discarded, and 2mg of anti-cTnI antibody I and 50. mu.L of Tween-20(50mg/mL) were added to the pellet, followed by volumes of 0.05M MES at pH 6.0 to give a final concentration of 10mg/mL of acceptor microspheres.

3. And (5) quickly mixing by ultrasound.

4. Add 50. mu.L of NaBH to the centrifuge tube₃CN (50mg/mL, 0.05M MES pH 6.0) was mixed well and reacted in a rotary mixer at 37 ℃ for 36-48 h.

5. And (3) sealing: 1mL of BSA (50mg/mL, 0.05M MES pH 6.0) was added and reacted in a rotary mixer at 37 ℃ for 12-16 h.

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

7. And sampling and measuring the concentration, the grain diameter and the signal value of the washed receptor microsphere of the coupled antibody.

Preparation of donor microspheres

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

2. Preparing a 100mL three-neck flask, adding 10mL 95% ethanol, 10mL water and 10mL polystyrene microspheres which are 10% in concentration and 200nm in particle size and coated with aldehyde dextran hydrogel, magnetically stirring, and heating in a water bath to 70 ℃.

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

4. The emulsion was centrifuged for 1 hour at 30000g, and after centrifugation the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugal washing 3 times, the mixture was resuspended in 50mM CB buffer solution 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. The same method is used to prepare donor microsphere solutions with the particle sizes of 80nm, 100nm, 150nm, 250nm, 300nm and 350nm respectively.

Four, donor microsphere coupling avidin

1. And (3) processing the donor microsphere suspension, namely sucking quantitative donor microspheres into a high-speed refrigerated centrifuge for centrifugation, removing the supernatant, adding quantitative MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruptor until the particles are resuspended, and adding MES buffer solution to adjust the concentration of the donor microspheres to 100 mg/mL.

2. Avidin solution preparation-weighing quantitative avidin (streptavidin or neutralized avidin is also available), adding MES buffer to dissolve to 8 mg/mL.

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

4. Reaction: preparing 25mg/mL NaBH by MES buffer solution₃Adding CN solution according to the volume ratio of 1:25 to the reaction solution, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.

5. And (3) sealing: MES buffer solution is prepared into 75mg/mL Gly solution and 25mg/mL NaBH₃Adding CN solution into the solution according to the volume ratio of 2:1:10 of the reaction solution, mixing uniformly, and carrying out rotary reaction for 2 hours at 37 ℃. Then, 200mg/mL BSA solution (MES buffer) was added thereto at a volume ratio of 5:8, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.

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

Fifth, preparation of Biotin-labeled antibody (reagent )

1. Antibody treatment: dialyzing anti-cTnI antibody II against 0.1M NaHCO₃Solution, antibody concentration was determined and adjusted to 1 mg/mL.

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

3. Marking: mixing the treated 1mg/mL anti-cTnI antibody II and the prepared biotin solution according to the volume ratio of 10000:54, and quickly and uniformly mixing. Standing and reacting for 12-16 hours at the temperature of 2-8 ℃.

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

5. Dialyzed biotinylated antibody was aspirated and transferred to a clean centrifuge tube, and samples were taken to determine antibody concentration. The concentration of the biotin labeled antibody which is qualified for quality inspection is adjusted to 0.5 mg/mL.

Sixthly, assembling

And assembling the prepared reagents to obtain the cTnI homogeneous phase chemiluminescence detection kit.

Example 2: homogeneous phase chemiluminescence detection method for human cardiac troponin I

Adding 25 muL of sample to be detected, 25 muL of biotin-labeled antibody, 175 muL of donor reagent and 25 muL of acceptor reagent into a hole site of a sample to be detected, an additional reagent hole site, reagent hole site and a second reagent hole site of a reagent cup strip respectively, placing the reagent cup strip into a POCT analyzer developed by Boyang biotechnology (Shanghai) Inc., adding a sample to be detected with a corresponding volume by a sample adding mechanism into the additional reagent hole site, vibrating the sample, incubating the sample at 37 ℃ for 10 minutes, adding liquid incubated in the additional reagent hole site into a reagent hole site, vibrating the liquid, incubating the liquid at 37 ℃ for 10 minutes to form a mixture to be detected, continuously adding the mixed liquid into the second reagent hole site, vibrating the hole site, incubating the mixed liquid at 37 ℃ for 10 minutes to form a mixture to be detected, irradiating the second reagent hole site by laser emitted by an exciter in the detection assembly, reacting the hole site for hours, detecting a chemiluminescent signal generated by the reaction of the acceptor microspheres and active oxygen by the detection mechanism, and calculating the concentration of target molecules to be detected in the sample.

Example 3: homogeneous chemiluminescence detection of human cardiac troponin I

Human cardiac troponin I was detected using the kit prepared in example 1 and the method described in example 2, and the results are shown in table 1.

TABLE 1

As can be seen from table 1, when the particle size of the donor microsphere is not smaller than the particle size of the acceptor microsphere, the detected luminescence signal value is larger and the detection sensitivity is better in the case where the particle size of the acceptor microsphere and the donor microsphere is determined to be .

Example 4: preparation of quality control product and calibrator

1. Preparation of quality control product

And (3) taking the newborn bovine serum as a diluent, and respectively diluting the pure antigen products into 2 working solutions with different concentrations, wherein the working solutions are quality control products Q1 and Q2. And taking the quality control products Q1 and Q2 to be detected to perform three times of repeated calibration on the instrument system of the company. And measuring 10 holes each time, and calculating the overall mean value and SD, wherein the mean value +/-3 SD is the allowable range of the concentration measurement of the quality control substance.

2. Preparation of calibrator

The pure antigen is diluted into a series of concentrations by calf serum (containing preservative), and is frozen and stored for later use after being calibrated by national standard for immunoassay. The shelf life is 2 years when the product is stored 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; while the two dose-response curves did not deviate significantly from parallelism (t-test); the ratio of the measured titer of the calibrator to the calibrated titer is 0.90-1.10 by taking the national standard as a standard.

Example 5: detection of batch-to-batch precision of cTnI homogeneous phase chemiluminescence detection kit

A sample to be detected: quality control Q1 prepared in example 4;

the process is as follows: repeating the detection 20 times to obtain a light intensity value (RLU)

Criterion for outlier determination: not less than 3SD

Reagents used in the detection:

(1) the receptor reagent comprises receptor microspheres (the particle size of the receptor microspheres is 200nm, and the receptor microspheres are respectively connected with the anti-PCT antibody I;

(2) reagent (biotin labeled antibody, anti-PCT antibody II);

(3) donor reagents including donor microspheres (avidin coated donor microspheres of different particle size (80nm, 200nm, 300 nm)).

The reagents (1) - (3) were combined to form a reagent set 1, a reagent set 2, and a reagent set 3 shown in table 2, the cTnI antigens were detected separately (after diluted to an appropriate concentration, the same samples were detected simultaneously by the reagent sets 1, 2, and 3), and the detection was repeated for 20 wells, the detection process 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
				Particle size/nm of acceptor microspheres	200	200	200
Donor microsphere particle size/nm	80	200	300

Table 3:

as is clear from Table 3, when the light intensity of reagent groups 2 and 3 is increased as compared with that of reagent group 1, the light intensity detected by this method is increased when the particle size of the donor microsphere is not smaller than that of the acceptor microsphere. Meanwhile, compared with the reagent group 1, the reagent groups 2 and 3 have smaller Coefficient of Variation (CV), that is, when the particle size of the donor microsphere is not smaller than that of the acceptor microsphere, the precision is higher.

Example 6: detection of analytical sensitivity of cTnI homogeneous phase chemiluminescence detection kit

A sample to be detected: a zero value calibrator;

Sensitivity: RLU substitution calibration curve

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

Table 4:

as can be seen from table 4, the sensitivity of detection was better in reagent groups 2 and 3 than in reagent group 1, i.e., when the particle size of the donor microspheres was not smaller than that of the acceptor microspheres, it was advantageous to improve the sensitivity of the reagent.

Comparative example 1: preparation of comparative kit

acceptor microsphere coupling antibody

1. The anti-cTnI antibody I was dialyzed into 50mM CB buffer at pH 10 to give a concentration of 1 mg/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 mixing₄The solution (50mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

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

4. After completion of the reaction, the reaction mixture was centrifuged at 30000g for 45min, and the supernatant was discarded after centrifugation, followed by resuspension in 50mM MES buffer. And repeating the centrifugal washing for 4 times, and diluting to a final concentration of 100 mu g/mL to obtain the anti-PCT antibody I-coated receptor microspheres with the particle sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

Two, donor microsphere coupled antibody

1. The anti-cTnI antibody II was dialyzed into 50mM CB buffer at pH 10 to give a concentration of 1 mg/mL.

2. Adding 0.5mL of photosensitive microsphere and 0.5mL of conjugated antibody II into a 2mL centrifuge tube, uniformly mixing, and adding 100 mu L of 10mg/mL of NaBH₄The solution (50mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

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

4. After completion of the reaction, the reaction mixture was centrifuged at 30000g for 45min, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated 4 times and diluted to a final concentration of 100. mu.g/mL. Obtaining the donor microspheres coated with anti-cTnI antibodies 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 procedure of the assay was the same as in example 3 except that the donor microspheres and the acceptor microspheres used were replaced with the comparative donor microspheres and acceptor microspheres prepared in comparative example 1, and the assay results are shown in Table 5.

TABLE 5

As is clear from table 5, the luminescence signal amount of the comparative kit was significantly reduced, and the detection sensitivity was significantly reduced. And when the donor microsphere is not smaller than the particle size of the acceptor microsphere in the contrast kit, the detected luminescence signal value is not better.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1, homogeneous chemiluminescent assay kit of cTnI comprising:

2. The kit according to claim 1, wherein the particle size of the donor and acceptor microspheres is selected from the group consisting of 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250 nm.

3. The kit of claim 2, wherein the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.

4. The kit of claim 3, wherein the donor microspheres and the acceptor microspheres each have a particle size of 200 nm.

5. The kit of claim 2, wherein the donor microspheres have a particle size larger than the particle size of the acceptor microspheres.

6. The kit according to claim 5, wherein the ratio of the particle sizes of the donor microspheres to the acceptor microspheres is 1.06-8.60, preferably 1.2-4.0, more preferably 1.5-2.01.

7. The kit of any of , wherein the donor microspheres are surface-coated with hydrophilic aldehyde dextran.

8. The kit of any of of claims 1-7, wherein the receptor microspheres are surface-coated with hydrophilic carboxyglucose.

9. The kit of any of claims 1-8, wherein the donor microspheres are filled with a photosensitizer selected from the group consisting of of methylene blue, rose bengal, porphyrin and phthalocyanine.

10. The kit of any of claims 1-9, wherein the luminescent microsphere is filled with a luminescent compound, preferably the luminescent compound is an europium complex, and further preferably the europium complex is MTTA-EU³⁺。

11. The kit of any of , wherein the donor and acceptor microspheres are polystyrene microspheres.

12. The kit of any of claims 1-11, wherein the reactive oxygen species is singlet oxygen.

13. The kit of any , further comprising a reagent comprising a specific binder for 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 the same binding property at a different location as the epitope, and a marker bound thereto.

14. The kit of claim 13, wherein:

the th binding unit and the second binding unit are each independently selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably from the group consisting of a polyclonal antibody and/or a monoclonal antibody, having binding specificity for human cardiac troponin I.

15. The kit of claim 14, wherein:

said th binding unit and/or said second binding unit independently comprise at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specifically to epitopes of different binding properties or epitopes of the same binding properties at different positions of human cardiac troponin I.

16. The kit of any of , wherein the marker is selected from the group consisting of avidin and/or streptavidin.

17. The kit of any of in claims 1-16, wherein the concentration of the acceptor microspheres in the acceptor reagent is selected from the group consisting of 20-300 μ g/mL, preferably 30-200 μ g/mL, more preferably 40-100 μ g/mL.

18. The kit of any of claims 1-17, wherein the concentration of the donor microspheres in the donor reagent is selected from 0.2-10 μ g/mL, preferably 0.5-8 μ g/mL, more preferably 1-6 μ g/mL.

19, homogeneous chemiluminescent assay for human cardiac troponin I using the kit of any one of claims 1-18 for chemiluminescent assay.

20. The method according to claim 19, characterized in that it comprises the steps of:

21, chemiluminescent assay device for detecting human cardiac troponin I in a test sample using the kit of any one of claims 1 to 18 or the method of claim 19 or 20.

22. The apparatus of claim 21, wherein the apparatus is a POCT point-of-care testing apparatus.