WO2023098135A1 - Light-initiated chemiluminescence immunoassay kit containing magnetic luminescent microspheres, and use thereof - Google Patents

Abstract

The present invention relates to a light-initiated chemiluminescence immunoassay kit, which comprises: magnetic luminescent microspheres which are labeled with first antibodies; second antibodies which are labeled with first binding moieties and target different epitopes of the same antigen; non-magnetic photosensitive microspheres which are labeled with second binding moieties, the second binding moieties capable of binding with the first binding moieties. The present invention also relates to a use of the kit for realizing target object detection.

Description

Kit for photochemiluminescent immunoassay comprising magnetic luminescent microspheres and application thereof technical field

The invention relates to the field of photochemiluminescence immunoassay, in particular to a photochemiluminescence immunoassay kit containing magnetic luminescent microspheres and an application thereof.

Background technique

Photochemiluminescence immunoassay is a typical homogeneous immunoassay method. It is characterized by "double spheres", that is, "luminescent microspheres" and "photosensitive microspheres", based on the antigen or antibody coated on the surface of these two microspheres, which is coupled with the target in the liquid phase to form an immune complex. Bring the two microspheres closer together. Under the action of excitation light, the "photosensitive microspheres" with photosensitive function can convert oxygen molecules in the surrounding environment into singlet oxygen, and transfer them to the "luminescent microspheres" with luminescent function, thereby inducing the formation of oxygen molecules on the luminescent microspheres. Partial chemiluminescent reaction produces high-energy red light. The number of photons is converted to the target concentration by single photon counter and mathematical fitting. However, when the sample to be tested does not contain the target object, the two types of microspheres cannot form immune complexes, and the distance between them will exceed the transmission range of singlet oxygen, and no high-energy red light signal will be generated.

Generally speaking, this homogeneous immunoassay method directly measures the sample to be tested and the relevant reagents in the reaction system after mixing and reacting during the determination process, without redundant separation or cleaning steps, so it has rapid, separation-free and Cleaning, high sensitivity and easy operation are featured. However, due to the lack of the sample separation process, in some samples, due to the large interference of the sample matrix, the recognition of antigens and antibodies will be greatly affected in some cases. In addition, in some assays that require high sensitivity, the sensitivity of this homogeneous immunoassay method may not meet the requirements due to the low concentration of microsphere complexes (or called microclusters) caused by the low concentration of target substances.

Therefore, there is a need in the art for an improved light-activated chemiluminescence immunoassay that can address matrix interference issues due to lack of separation and washing of samples and sensitivity due to low microsphere complexes when required low problem.

Contents of the invention

The purpose of the present invention is to provide an improved photochemiluminescent immunoassay method. Specifically, the present invention provides a kit containing magnetic luminescent microspheres and its application for photo-activated chemiluminescence immunoassay.

The inventors of the present application found that adding magnetic cores to the "luminescent microspheres" in the double-spheres of light-induced chemiluminescence immunoassay makes the luminescent microspheres magnetic, and the movement of the luminescent microspheres can be controlled by magnetism during the reaction process , so that the luminescent microspheres that capture the target can be separated from the reaction system and cleaned before the luminescent microspheres and photosensitive microspheres form a microsphere complex, thereby reducing background interference in subsequent detection, improving luminescent signal, and improving detection The flexibility and adjustability of the method can realize various possibilities in methodology.

Therefore, in the first aspect of the present invention, there is provided a kit for immunodetection of a target by photoactivated chemiluminescence, comprising:

- magnetic luminescent microspheres, the magnetic luminescent microspheres are labeled with the first antibody;

- a second antibody labeled with a first binding moiety and specific for a different epitope of the same antigen as said first antibody;

- Non-magnetic photosensitive microspheres labeled with a second binding moiety capable of binding to the first binding moiety.

In a second aspect, there is provided a method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit described in the first aspect, and the method includes the following steps:

a) mixing the magnetic luminescent microspheres and the sample to be tested in the first reaction system and incubating at a temperature range of 30° C. to 42° C. for 3 minutes to 30 minutes;

b) the magnetic luminescent microspheres after magnetic separation incubation;

c) adding the separated magnetic luminescent microspheres and the second antibody to the second reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

d) adding the non-magnetic photosensitive microspheres to the second reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

e) detecting the luminous intensity of the magnetic luminescent microspheres.

In a third aspect, there is provided a method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit according to the first aspect, the method comprising the following steps:

a) Mix the magnetic luminescent microspheres, the sample to be tested and the second antibody in the first reaction system, and incubate at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

b) the magnetic luminescent microspheres after magnetic separation incubation;

c) adding the separated magnetic luminescent microspheres to the second reaction system, and adding the non-magnetic photosensitive microspheres to the second reaction system, and incubating at a temperature range from 30°C to 42°C for 3 minutes to 30 minutes;

d) detecting the luminous intensity of the magnetic luminescent microspheres.

The beneficial effects of the present invention are as follows: the luminescent microspheres (luminescent microsphere-target complexes) that capture the target on the antibody can be separated from the reaction system by using magnetism, and the complex is purified by the cleaning process, thereby reducing subsequent detection. Background interference in the medium; in the detection that requires high sensitivity, the complex can be transferred to a smaller reaction system using magnetism, thereby increasing the concentration of the complex, and then making singlet oxygen between the photosensitive microspheres and the luminescent microspheres The transmission can be more efficient, thereby improving the detection signal; the complex can be transferred in different reaction systems by using magnetism, so as to realize the process of binding and detection in different systems, and improve the flexibility and adjustability of detection.

Description of drawings

The technical solutions and benefits of the present invention will become apparent to those skilled in the art thanks to the following detailed description and with reference to the accompanying drawings.

Figure 1 shows the particle size distribution of magnetoluminescent microspheres prepared according to one embodiment of the present invention.

Fig. 2 shows the linear correlation curve between the low concentration range of 0 pg/ml to 150 pg/ml and the luminescence amount of gastrin releasing peptide precursor obtained by using non-magnetic luminescent microspheres.

Figure 3 shows the linear correlation curve between the full concentration range of the gastrin releasing peptide precursor from 0 pg/ml to 8000 pg/ml and the luminescence amount obtained by using non-magnetic luminescent microspheres.

Fig. 4 shows the linear correlation curve between the low concentration range of 0 pg/ml to 150 pg/ml and the luminescence amount of the gastrin releasing peptide precursor obtained by using the magnetic luminescent microspheres of the present invention.

Fig. 5 shows the linear correlation curve of the gastrin releasing peptide precursor in the full concentration range from 0 pg/ml to 8000 pg/ml and the luminescence amount obtained by using the magnetic luminescent microspheres of the present invention.

Fig. 6 shows the linear correlation curve between the concentration of procalcitonin in the range of 0 ng/ml to 30 ng/ml and the amount of luminescence obtained by using non-magnetic luminescent microspheres.

Fig. 7 shows the linear correlation curve between the concentration range of procalcitonin from 0 ng/ml to 30 ng/ml and the amount of luminescence obtained by using magnetic luminescent microspheres.

Detailed ways

The present invention is described in detail below. It should be understood that the following description illustrates the present invention by way of example only, and is not intended to limit the scope of the present invention, and the protection scope of the present invention shall prevail in the appended claims. Moreover, those skilled in the art understand that the technical solutions of the present invention can be modified without departing from the spirit and gist of the present invention.

In a first aspect, the present application provides a kit for immunodetection of a target by photoactivated chemiluminescence, which includes:

- magnetic luminescent microspheres, the magnetic luminescent microspheres are labeled with the first antibody;

- a second antibody labeled with a first binding moiety and specific for a different epitope of the same antigen as said first antibody;

- Non-magnetic photosensitive microspheres labeled with a second binding moiety capable of binding to the first binding moiety.

In a further specific embodiment, the first binding moiety and the second binding moiety are selected from a pair of substances capable of specifically binding to each other, such as ligands, oligonucleotides, oligonucleotide binding proteins, Lectin, hapten, antigen, immunoglobulin binding protein, avidin, avidin or biotin.

In a preferred embodiment, the first binding moiety is one of avidin and biotin, and the second binding moiety is the other of avidin and biotin. As an example, the avidin may be, for example, avidin, vitellavidin, streptavidin, neutravidin, or avidin-like, but not limited thereto.

In a more preferred embodiment, the first binding moiety is one of streptavidin and biotin, and the second binding moiety is the other of streptavidin and biotin. In a specific embodiment, said first binding moiety is biotin and said second binding moiety is streptavidin.

Those skilled in the art can clearly know from the contents of the present invention that the combination between the first binding part and the second binding part can make the second antibody bind to the non-magnetic photosensitive microsphere, and the second antibody can bind to the magnetic luminescent microsphere. The first antibody bound to the target forms a double-antibody sandwich structure, thereby shortening the distance between the magnetic luminescent microspheres and the non-magnetic photosensitive microspheres, so that the chemiluminescent reaction can occur under the condition of light excitation. Therefore, those skilled in the art can also select appropriate first binding moieties and second binding moieties to label the second antibody and non-magnetic photosensitive microspheres respectively according to needs.

As used herein, the term "binding" in the context of the present invention has a broad meaning as understood by those skilled in the art, and specifically refers to binding due to, for example, covalent coupling, coordination, electrostatic, hydrophobic, ionic and/or hydrogen bonding A direct association between two molecules caused by an equal interaction.

In yet another specific embodiment, the target can be a disease-related marker, for example, a tumor marker, such as gastrin releasing peptide precursor, alpha-fetoprotein, carbohydrate antigen, etc.; an inflammatory disease marker, Such as procalcitonin, interleukin, C-reactive protein, etc.; virus-associated antigens, such as African swine fever, bovine foot-and-mouth disease, bovine viral diarrhea virus, etc.

In yet another specific embodiment, the target substance can also be a drug and its metabolites, such as antibacterial drugs, antifungal drugs, antiviral drugs, antitumor agents, steroids, hormones, etc. for humans or animals and its metabolites.

In yet another specific embodiment, the magnetic luminescent microspheres are prepared by the following method:

- Preparation of Fe ₃ O ₄ magnetic beads;

- using a polymer as a carrier to coat the Fe ₃ O ₄ magnetic beads to obtain magnetic polymer microspheres;

- vortex-coating the magnetic polymer microspheres with a luminescent composition to obtain magnetic luminescent microspheres, wherein the luminescent composition contains an olefin compound and a metal chelate;

- Linkage of primary antibody against target.

In a further specific embodiment, the Fe ₃ O ₄ magnetic beads are prepared from ferric chloride or its hydrate.

The magnetic luminescent microspheres use _Fe3O4 magnetic beads as the core, and the _surface is coated with polymers with active groups such as carboxyl groups, amino groups, aldehyde groups, epoxy groups, azo groups, olefins, and alkynes, such as Polystyrene, polycaprolactone, agarose, silica, etc. This reactive group can be used to conjugate antibodies.

In a further specific embodiment, said luminescent composition comprises an alkene compound and a metal chelate. The olefinic compound may be 2-phenyloxathiene and its derivatives. In a further specific embodiment, the metal of the metal chelate may be a fluorescent rare earth metal, preferably selected from yttrium, europium, gadolinium, lanthanum, cerium, terbium, ytterbium, samarium, etc., more preferably europium. For example, the metal chelate is a europium (Eu) complex, such as (1,10-phenanthroline) tris[4,4,4-trifluoro-1-(2-thienyl)-1,3 - butanedione] europium(III).

In yet another specific embodiment, the particle size of the magnetic luminescent microspheres is 40nm to 800nm. In a further preferred embodiment, the particle size of the magnetic luminescent microspheres is 100 nm to 300 nm.

In yet another specific embodiment, the particle size of the non-magnetic photosensitive microspheres may be 40 nm to 800 nm. In a preferred embodiment, the non-magnetic photosensitive microspheres have a particle diameter of 100 nm to 300 nm. Non-magnetic photosensitive particles are polymer particles filled with photosensitive compounds. The photosensitive compound may be, for example, a phthalocyanine dye, a porphyrin derivative, or other compounds that can receive light and generate active oxygen. The non-magnetic photosensitive microparticles can be obtained commercially, for example, from PerkinElmer Co., Ltd. Those skilled in the art can select non-magnetic photosensitive particles suitable for the present invention according to actual needs. Before being used in the present invention, those skilled in the art can use conventional means in the art to label the second binding moiety on commercially available non-magnetic photosensitive microspheres.

In a second aspect, there is provided a method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit according to the first aspect, the method comprising the following steps:

a) mixing the magnetic luminescent microspheres and the sample to be tested in the first reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

b) the magnetic luminescent microspheres after magnetic separation incubation;

c) adding the separated magnetic luminescent microspheres and the second antibody to the second reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

d) adding the non-magnetic photosensitive microspheres to the second reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

e) detecting the luminous intensity of the magnetic luminescent microspheres.

As used herein, the term "sample to be tested" refers to a sample to be tested that contains or is suspected to contain the target substance to be tested. Samples to be tested that can be used in the present invention include body fluids, such as human or animal serum, plasma, urine, sputum, milk, saliva; solvents; food samples such as vegetables and fruits; environmental samples such as soil or water samples; plant materials; Cells; Bacteria; Viruses; Fungi, etc.

In a specific embodiment, the concentration of the magnetic luminescent microspheres in the first reaction system or the second reaction system is 0.001 mg/mL to 5 mg/mL; The concentration in the system or the second reaction system is 0.001 mg/mL to 5 mg/mL; and the concentration of the non-magnetic photosensitive microspheres in the second reaction system is 0.001 mg/mL to 5 mg/mL.

In a preferred embodiment, the concentration of the magnetic luminescent microspheres in the first reaction system or the second reaction system is 0.04 mg/mL to 0.4 mg/mL; The concentration in the reaction system or the second reaction system is 0.04 mg/mL to 0.4 mg/mL; and the concentration of the non-magnetic photosensitive microspheres in the second reaction system is 0.04 mg/mL to 0.4 mg/mL.

In a preferred embodiment, step a) comprises incubating at a temperature ranging from 35°C to 40°C for 5 minutes to 20 minutes, for example at 37°C for 15 minutes.

In a specific embodiment, the magnetic separation in step b) may include using tools well known in the art such as a magnetic rod, a magnetic plate, etc., without any special limitation.

In yet another specific embodiment, before step c), the separated magnetic luminescent microspheres are transferred to a washing solution for washing. Those skilled in the art can select a cleaning solution according to the actual needs of the detection system, such as HEPES buffer, PBS buffer, TRIS-HCl and the like. Washing can separate the target captured by the first antibody on the magnetic luminescent microspheres from the first reaction system, and the magnetic luminescent microsphere-target complex can be purified by the washing process to reduce background interference.

In a preferred embodiment, step c) comprises incubating at a temperature ranging from 35°C to 40°C for 5 minutes to 15 minutes, for example at 37°C for 15 minutes.

In yet another preferred embodiment, step d) comprises incubating at a temperature ranging from 35°C to 40°C for 5 minutes to 15 minutes, for example at 37°C for 15 minutes.

If the sample to be tested contains the target object, after the incubation in step a), the magnetic luminescent microspheres labeled with the first antibody will be coupled to the target object through the first antibody; after the incubation in step c), the second The second antibody is coupled to the target; after the incubation in step d), the second antibody and the non-magnetic photosensitive microspheres are combined through the first binding part and the second binding part; and, the first antibody and the second Different binding sites of antibodies on the target are coupled to the target.

In further specific embodiments, said first reaction system and said second reaction system are the same or different. The microsphere complex after magnetic separation can be transferred to a system different from the first reaction system, so that the binding and detection process between microspheres, target objects and antibodies can be carried out in different systems, thereby improving the flexibility of detection and adjustability. For example, the second reaction system may have a different solvent, pH, surface activity concentration, etc. from the first reaction system, so that the combination of the second antibody or the photosensitive microsphere and the microsphere complex can be promoted, and the detection efficiency can be reduced. background noise etc.

In yet another specific embodiment, the solution volume of the first reaction system and the solution volume of the second reaction system may be the same or different.

In the detection that requires high sensitivity, the microsphere complexes after magnetic separation can be transferred to the second reaction system with a smaller volume, thereby increasing the concentration of the microsphere complexes, making the microsphere complexes closer to each other, and making The transfer of reactive oxygen species between photosensitive microspheres and luminescent microspheres is more efficient, thereby improving the detection signal. Therefore, in a preferred embodiment, the solution volume of the second reaction system is smaller than the solution volume of the first reaction system.

In a further specific embodiment, methods well known in the art can be used to detect the luminous intensity in step e), for example, firstly, laser light is used, such as using 600nm to 700nm light to excite non-magnetic photosensitive particles, and oxygen in the air can be Molecules are converted into singlet oxygen. When the distance between the non-magnetic photosensitive particles and the magnetic luminescent particles is close enough, the singlet oxygen can be transferred to the magnetic luminescent particles, react with the luminescent compounds in the luminescent particles, and excite the metal chelate in them. metals, eventually producing short-wavelength photons such as 520nm to 620nm. Then, a commercial microplate reader can be used to detect the luminescence intensity of the magnetic luminescent microspheres. The detected luminescence intensity determines whether the target substance is contained in the sample to be tested and the content of the target substance.

In a third aspect, there is provided a method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit according to the first aspect, the method comprising the following steps:

a) Mix the magnetic luminescent microspheres, the sample to be tested and the second antibody in the first reaction system, and incubate at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes;

b) the magnetic luminescent microspheres after magnetic separation incubation;

c) adding the separated magnetic luminescent microspheres to the second reaction system, and adding the non-magnetic photosensitive microspheres to the second reaction system, and incubating at a temperature range from 30°C to 42°C for 3 minutes to 30 minutes;

d) detecting the luminous intensity of the magnetic luminescent microspheres.

In a specific embodiment, the concentration of the magnetic luminescent microspheres in the first reaction system or the second reaction system is 0.001 mg/mL to 5 mg/mL; The concentration in the system or the second reaction system is 0.001 mg/mL to 5 mg/mL; and the concentration of the non-magnetic photosensitive microspheres in the second reaction system is 0.001 mg/mL to 5 mg/mL.

In a preferred embodiment, the concentration of the magnetic luminescent microspheres in the first reaction system or the second reaction system is 0.04 mg/mL to 0.4 mg/mL; The concentration in the reaction system or the second reaction system is 0.04 mg/mL to 0.4 mg/mL, and the concentration of the non-magnetic photosensitive microspheres in the second reaction system is 0.04 mg/mL to 0.4 mg/mL.

In a preferred embodiment, step a) comprises incubating at a temperature ranging from 35°C to 40°C for 5 minutes to 20 minutes, for example at 37°C for 15 minutes.

In yet another specific embodiment, the magnetic separation in step b) may include using tools known in the art such as a magnetic rod, a magnetic plate, etc., and there is no special limitation.

In yet another specific embodiment, before step c), the separated magnetic luminescent microspheres are transferred to a washing solution for washing. Those skilled in the art can select a cleaning solution according to the actual needs of the detection system, such as HEPES buffer, PBS buffer, TRIS-HCl and the like. Washing can separate the target captured by the first antibody on the magnetic luminescent microspheres from the first reaction system, and the magnetic luminescent microsphere-target complex can be purified by the washing process to reduce background interference.

In a particular embodiment, step c) comprises incubating at a temperature in the range of 35°C to 40°C for 5 minutes to 15 minutes, for example at 37°C for 15 minutes.

If the sample to be tested contains a target, after the incubation in step a), the magnetic luminescent microspheres labeled with the first antibody will be coupled to the target through the first antibody, and the second antibody will also be coupled to the target. target object coupling; and, the different binding sites of the first antibody and the second antibody on the target object are coupled with the target object; after step c) incubation, the second antibody and the non-magnetic photosensitive microsphere Binding is via said first binding moiety and second binding moiety.

The inventors of the present application found that whether the photosensitive microsphere-target complex is separated from the system before coupling with the second antibody and then coupled with the second antibody, or the second antibody is coupled to the photosensitive microsphere- After the target complex forms a microsphere complex and is separated from the system, the technical effects of reducing background interference, improving detection signal, and improving detection flexibility and adjustability can be achieved. In a preferred embodiment, the separation is performed after the second antibody is coupled to the photosensitive microsphere-target complex to form a microsphere complex, which is useful for reducing background interference, improving detection signal, and improving detection flexibility and reliability. Tonality is better.

In a further specific embodiment, said first reaction system and said second reaction system are the same or different. The microsphere complex after magnetic separation can be transferred to a system different from the first reaction system, so that the binding and detection process between microspheres, target objects and antibodies can be carried out in different systems, thereby improving the flexibility of detection and adjustability. For example, the second reaction system may have a different solvent, pH, surface activity concentration, etc. from the first reaction system, so that the combination of the second antibody or the photosensitive microsphere and the microsphere complex can be promoted, and the detection efficiency can be reduced. background noise etc.

In yet another specific embodiment, the solution volume of the first reaction system and the solution volume of the second reaction system may be the same or different.

Example

Hereinafter, the present invention will be described in more detail with reference to examples and drawings. However, the specific embodiments disclosed herein are intended for purposes of illustration only and should not be construed as intended to interpret the scope of the invention.

All the chemical reagents can be purchased commercially, for example, from Shanghai Aladdin Biochemical Technology Co., Ltd., and the photosensitive microspheres are purchased from PerkinElmer Co., Ltd.

Example 1. Preparation of magnetic luminescent microspheres without antibody labeling

Preparation of Fe ₃ O ₄ magnetic core: Add 16.2g of ferric chloride hexahydrate, 500ml of polyethylene glycol and 1g of sodium acetate into a 1000ml three-necked flask, vacuumize and nitrogen, 300rpm magnetic stirring, reflux at 200°C for 48 hours, ultrapure water Wash three times to get the magnetic core.

Preparation of magnetic polymer microspheres: Get 1g of the magnetic core obtained above and add 30ml absolute ethanol, add 0.5ml methacrylic acid, 0.25mL polystyrene (average molecular weight 26000), import into 250ml 75 ethanol with 450rpm mechanical Add 5 ml of ethanol and 0.1 mM azobisisobutyronitrile into the stirred three-necked flask, blow nitrogen to remove oxygen, heat to 75° C., and stir overnight to obtain magnetic polymer microspheres.

Preparation of magnetic luminescent microspheres: Take 60 mg of the above-mentioned magnetic polymer microspheres, set the volume to 30 mL, vortex-coat to prepare luminescent microspheres, add 2.85 mL of dichloroethane, add Eu complex (1,10-phenanthroline ) Tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione]europium(III) 20mg, 1-xylylenediamino, 2-phenyloxathia 10 mg of cyclohexene was vortexed for 2 hours, then evaporated in a rotary evaporator at 40° C. for 30 minutes, and then washed 3 times with absolute ethanol to obtain magnetic luminescent microspheres.

Example 2. Characterization of Magnetic Luminescent Microspheres Without Antibody Labeling

Particle size characterization: Take 10 μL of the magnetic luminescent microspheres prepared in Example 1, disperse them in ultrapure water, control the concentration in the range of 0.1-1 mg/mL, and use a laser particle size analyzer to test, the results are shown in Figure 1. It can be seen that the average particle diameter of the magnetic luminescent microspheres prepared according to Example 1 of the present invention is 182 nm, and the PDI is 0.044.

Characterization of carboxyl group content: Take 100 mg of the magnetic luminescent microspheres prepared in Example 1, disperse them in 100 mL of ultrapure water, and use 0.1 M sodium hydroxide solution to titrate with a potentiometric titrator, and calculate the carboxyl group content to be 86 μmol/g. In terms of experimental operability, magnetic luminescent microspheres with a carboxyl content in the range of 60 μmol/g to 200 μmol/g are usually selected.

Example 3: Labeling Magnetic Luminescent Microspheres with Primary Antibody

The steps of labeling the magnetic luminescent microspheres with the antibody of gastrin releasing peptide precursor (anti-ProGRP antibody) are as follows:

Activation: Take 100 μL of 10 mg/mL magnetic luminescent microspheres (ie 1 mg), centrifuge at 10,000 rpm for 15 minutes, remove the supernatant, add 200 μL of pH7.0 100mM HEPES buffer, and resuspend by ultrasonication.

Now take a certain amount of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), with pH7.0 100mM HEPES The buffer solution was dissolved and prepared as 50mg/ml EDC and 50mg/ml NHS respectively. Add 10 μL of NHS to the above-mentioned system containing magnetic luminescent microspheres, then add 5 μL of EDC, and then activate at 37 °C for 90 min.

Coupling: After the activation step, add 100 μL of HEPES buffer to the system, and centrifuge again at 10,000 rpm, 37°C for 15 minutes, remove the supernatant, first add HEPES buffer, resuspend by ultrasonication, and then add antibody (anti-ProGRP antibody) , coupled overnight at 37°C.

Blocking: After the coupling step, add 100 μL blocking agent, then block overnight at 37°C; replace with HEPES solution after overnight.

Example 4: Linear correlation between target concentration and luminous intensity

Add 25 μL of magnetic luminescent microspheres labeled with antibody I of gastrin-releasing peptide precursor, 10 μL of serum samples to be tested at different concentrations ranging from 0 pg to 8000 pg, 25 μl Biotinylated antibody II was incubated at 37°C for 15 minutes; then, it was taken out with a magnetic rod, and placed in a pH 6.5 100mM HEPES solution for 30 seconds, then dispersed in 60μL containing 1% BSA, 0.05% Tween 80, 2% sucrose pH 6.5 100mM HEPES solution, then add 75 μL photosensitive microspheres labeled with streptavidin and incubate at 37°C for 15 minutes, then test the luminous intensity.

Use non-magnetic luminescent microspheres as a control, add 25 μL of non-magnetic luminescent microspheres labeled with antibody I of gastrin-releasing peptide precursor to the microtiter plate, and 10 μL of gastrin-releasing peptide precursor at a concentration ranging from 0 pg to 8000 pg Serum samples of different concentrations to be tested and 25 μL of biotinylated antibody II were incubated at 37°C for 15 minutes; then 75 μL of streptavidin-labeled photosensitive microspheres were added to it and incubated at 37°C for 15 minutes, and then the luminescence intensity was measured.

Compare the concentration of gastrin-releasing peptide precursor in the serum sample to be tested with the corresponding luminescence intensity results obtained by detection and the value of the Roche chemiluminescence test, and make a low concentration range (0pg/mL to 150pg/mL) And the linear correlation curve of the full concentration range (0pg/mL to 8000pg/mL), the results are shown in Figure 2-Figure 5.

It was found that in the full concentration range, especially at high concentrations (higher than 150pg/mL), the results of magnetic luminescent microspheres and non-magnetic luminescent microspheres are very close, and the linearity has reached 0.99, as shown in Figure 3 and Figure 5 Show. This shows that in the case of high concentration, the step of separating the target has little effect on the linear dependence of the concentration. When the concentration of the target substance is lower than 150pg/mL, the linear correlation of magnetic luminescent microspheres is significantly better, reaching 0.95, as shown in Figure 4, while the linear correlation of non-magnetic luminescent microspheres is poor, only 0.867, as shown in Figure 2. This shows that for low concentrations of target substances, the addition of magnetism or the introduction of a separation step has a significant impact on the improvement of the linear correlation. It can be understood that the low concentration is generally the interval most affected by impurities, and the step of separating the target by magnetic luminescent microspheres can significantly improve the linear correlation between concentration and luminous intensity.

Example 5: Testing of particle size and luminous intensity of magnetic luminescent microspheres

Add 25 μL of magnetic luminescent microspheres (five different particle sizes: 278nm, 205nm, 173nm, 127nm and 97nm) labeled with antibody I of gastrin releasing peptide precursor (in pH6.8 100mM HEPES solution) ), 50 μL of serum samples to be tested (three different concentrations of 0.2ng/mL, 0.067ng/mL and 0.022ng/mL) containing gastrin-releasing peptide precursor, and 25 μL of biotinylated antibody II, incubated at 37°C 15 minutes; then, 75 μL of streptavidin-labeled photosensitive microspheres were added thereto and incubated at 37° C. for 15 minutes, and then the luminous intensity was tested, and the results are shown in Table 1 below.

Table 1: Luminescence intensity of samples with different concentrations (ng/mL) under magnetic luminescent microspheres with different particle sizes (nm)

It can be seen from Table 1 that the luminous intensity of the magnetic luminescent microspheres with a particle diameter greater than 100 nm is much higher than that of the magnetic luminescent microspheres with a particle diameter slightly lower than 100 nm.

Example 6: Detection of procalcitonin

Add 25 μL of magnetic luminescent microspheres labeled with procalcitonin antibody I, 25 μL of serum samples to be tested with different concentrations of procalcitonin, and 25 μL of biotinylated procalcitonin antibody II to the microtiter plate. Incubate for 15 minutes; then, take it out with a magnetic rod, place it in a pH 6.5 100mM HEPES solution and stir for 30 seconds, and then disperse it in 75 μL of a pH 6.5 solution containing 1% BSA, 0.05% Tween 80, and 2% sucrose. 100mM HEPES solution, and then add 140 μL photosensitive microspheres labeled with streptavidin, incubate at 37°C for 15 minutes, and then test the luminous intensity.

Add 25 μL of non-magnetic luminescent microspheres labeled with procalcitonin antibody I, 25 μL of serum samples to be tested with different concentrations of procalcitonin, and 25 μL of biotinylated procalcitonin antibody II to the microtiter plate. ℃ for 15 minutes; then, 140 μL of photosensitive microspheres labeled with streptavidin were added thereto, incubated at 37 ℃ for 15 minutes, and then the luminescence intensity was measured.

The concentration of procalcitonin in the serum sample to be tested was compared with the luminescent results obtained by the magnetic luminescent microspheres and non-magnetic luminescent microspheres respectively, and the results are shown in Figures 6-7 and Table 2 below.

It can be seen from Figure 6 and Figure 7 that although from the perspective of linear correlation, the detection of magnetic luminescent microspheres and non-magnetic luminescent microspheres has obtained good results, but from the perspective of slope, the detection of magnetic luminescent microspheres The slope obtained by the spheres is twice that of the non-magnetic luminescent microspheres, which indicates that the magnetic luminescent microspheres can better distinguish samples with low concentrations.

The same conclusion can also be drawn from the data in Table 2: when non-magnetic luminescent microspheres are used to detect procalcitonin in serum samples, the two concentrations of 0 ng/mL and 0.04 ng/mL cannot be clearly distinguished, indicating that non-magnetic The detection limit of magnetic luminescent microspheres is at least greater than 0.04ng/mL. When magnetic luminescent microspheres are used, the two concentrations of 0 ng/mL and 0.008 ng/mL can be clearly distinguished, indicating that the detection limit of magnetic luminescent microspheres is at least 0.008 ng/mL. Due to the use of serum samples, the serum matrix will interfere with the detection of luminescence intensity. This matrix interference is especially evident in the detection of low-concentration samples. For example, the signal difference when the concentration is 0 may be the influence of the serum matrix, which comes from non-specific adsorption in the matrix. It can be seen from Table 2 that this matrix effect makes it impossible for non-magnetic luminescent microspheres to distinguish the three concentrations of 0ng/mL, 0.008ng/mL and 0.04ng/mL, while magnetic luminescent microspheres avoid Influence of matrix effect. It can be seen that the detection limit using magnetic luminescent microspheres reduces the detection limit. At the same time, from the perspective of luminous intensity, the luminous intensity value of using magnetic luminescent microspheres is at least 2 times that of non-magnetic luminescent microspheres, which is obviously more conducive to the distinction of concentration. That is to say, the detection using magnetic luminescent microspheres is beneficial to the distinction of concentration, with reduced detection limit and improved accuracy.

Table 2: Luminescence intensities detected by magnetic and non-magnetic luminescent microspheres

Example 7: Detection of Gastrin-releasing Peptide Precursor

Add 25 μL of magnetic luminescent microspheres labeled with gastrin-releasing peptide precursor antibody I, 25 μL of samples to be tested with different concentrations of gastrin-releasing peptide precursor, and 25 μL of biotinylated antibody II in the microtiter plate. Incubate at ℃ for 15 minutes; then, take it out with a magnetic rod, place it in a pH 6.5 100mM HEPES solution and stir for 30 seconds, and then disperse it in 75 μL of a pH 6.5 solution containing 1% BSA, 0.05% Tween 80, and 2% sucrose. 5 100mM HEPES solution, and then add 140μL photosensitive microspheres labeled with streptavidin, incubate at 37°C for 15 minutes, and then test the luminous intensity.

Add 25 μL of magnetic luminescent microspheres labeled with gastrin-releasing peptide precursor antibody I, 25 μL of serum samples to be tested with different concentrations of gastrin-releasing peptide precursor, and 25 μL of biotinylated antibody II in the microtiter plate. Incubate at 37° C. for 15 minutes; then, add 140 μL of photosensitive microspheres labeled with streptavidin, incubate at 37° C. for 15 minutes, and then measure the luminescence intensity.

The concentration of the gastrin-releasing peptide precursor in the serum sample to be tested was compared with the luminescent results obtained by the magnetic luminescent microspheres and non-magnetic luminescent microspheres respectively, and the results are shown in Table 3 below. It can be seen from the data in the table that although the two concentrations of 0ng/mL and 0.0016ng/mL can be clearly distinguished by using non-magnetic luminescent microspheres and magnetic luminescent The luminescence intensity obtained by detecting the concentration of 0.0016ng/mL is 3 times that of 0ng/mL, while the luminescence intensity obtained by detecting the concentration of 0.0016ng/mL by using the ratio of non-magnetic luminescent particles is less than 2 times that of detecting the concentration of 0ng/mL. This shows from the side that the detection limit of detection using magnetic luminescent particles is significantly lower, which is more conducive to the distinction of concentration and the improvement of detection accuracy.

Table 3: Luminescence intensities detected by magnetic and non-magnetic luminescent microspheres

Similarly, since the sample to be tested uses a standard solution without impurities, the use of magnetic luminescent particles cannot reflect the advantage of removing impurities, so the increase in the detection limit can be attributed to the increase in the concentration of microclusters.

Embodiment 8: Change the detection of reaction system

Mix 25 μL of magnetic luminescent microspheres labeled with African swine fever antibody I at a concentration of 1 mg/mL, 25 μL of 100 mM Tris-HCl pH6.0, and 25 μL of pig serum samples (positive for VP72 protein), incubate at 37 degrees for 15 minutes, and then use Transfer the magnetic stick to 100mM pH7.0 PBS buffer containing 0.1% Tween 80 for washing once, release it into 75 μL of 100 mM pH7.0 PBS buffer containing 0.1% Tween 80, and then add 25 μL biological Primed African swine fever antibody II, incubated at 37 degrees for 15 minutes. Add 15 μL of commercially available photosensitive microspheres labeled with streptavidin at 1 mg/mL, incubate at 37 degrees for 15 minutes, and then test the luminescence intensity of the magnetic luminescent microspheres. The results are shown in Table 4 below.

Table 4: Luminescence intensity of magnetic luminescent microspheres in different reaction systems

From the data in Table 4, it can be seen that when the magnetic luminescent microspheres were transferred from the reaction system Tris-HCl pH6.0 to 100mM pH7.0PBS buffer containing 0.1% Tween 80, the luminescence intensity increased by an order of magnitude, which is obviously more It is beneficial to distinguish the concentration and improve the accuracy of detection.

Claims (10)

1. A kit for immunodetection of a target by light-activated chemiluminescence, comprising:

   - magnetic luminescent microspheres, the magnetic luminescent microspheres are labeled with the first antibody;

   - a second antibody labeled with a first binding moiety and specific for a different epitope of the same antigen as said first antibody;

   - non-magnetic photosensitive microspheres labeled with a second binding moiety capable of binding to the first binding moiety, for example by covalent coupling, coordination, electrostatic, Hydrophobic, ionic, and/or hydrogen bonding interactions.
2. The kit according to claim 1, wherein the first binding moiety and the second binding moiety are selected from a pair of substances capable of specifically binding to each other, such as ligands, oligonucleotides, oligonucleotides binding protein, lectin, hapten, antigen, immunoglobulin binding protein, avidin, avidin or biotin; preferably, the first binding moiety is one of avidin and biotin, The second binding moiety is the other of avidin and biotin; the avidin is, for example, avidin, vitellavidin, streptavidin, neutravidin, or the like Avidin, preferably streptavidin.
3. The kit according to claim 1 or 2, wherein the target substance is a disease-related marker or drug and its metabolites, for example, a tumor marker (such as gastrin releasing peptide precursor, alpha-fetoprotein, sugar Antigens), inflammatory disease markers (such as procalcitonin, interleukin, C-reactive protein), virus (such as African swine fever, bovine foot-and-mouth disease, bovine viral diarrhea virus) related antigens, antibacterial drugs, antifungal drugs , antiviral drugs, antineoplastic agents, steroids, hormones and their metabolites.
4. The kit according to any one of claims 1-3, wherein the magnetic luminescent microspheres are prepared by the following method:

   - Preparation of Fe ₃ O ₄ magnetic beads;

   - using a polymer such as polystyrene as a carrier to coat the Fe ₃ O ₄ magnetic beads to obtain magnetic polymer microspheres;

   - vortex-coating the magnetic polymer microspheres with a luminescent composition to obtain magnetic luminescent microspheres, wherein the luminescent composition contains an olefin compound and a metal chelate;

   - Linkage of primary antibody against target.
5. The kit according to any one of claims 1-4, wherein the particle size of the magnetic luminescent microspheres is 40nm to 800nm, preferably 100nm to 300nm; and wherein the particle size of the non-magnetic photosensitive microspheres is The diameter is 40nm to 800nm, preferably 100nm to 300nm.
6. A method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit according to any one of claims 1-5, said method comprising the following steps:

   a) Mix the magnetic luminescent microspheres and the sample to be tested in the first reaction system, and incubate at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes, preferably at a temperature range of 35°C to 40°C for 5 minutes to 20 minutes;

   b) the magnetic luminescent microspheres after magnetic separation incubation;

   c) Add the separated magnetic luminescent microspheres and the second antibody to the second reaction system, and incubate at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes, preferably at a temperature range of 35°C to 40°C for 5 minutes minutes to 15 minutes;

   d) adding the non-magnetic photosensitive microspheres to the second reaction system, and incubating at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes, preferably at a temperature range of 35°C to 40°C for 5 minutes to 15 minutes;

   e) detecting the luminous intensity of the magnetic luminescent microspheres.
7. A method for immunodetection of a target by photoactivated chemiluminescence, which uses the kit according to any one of claims 1-5, said method comprising the following steps:

   a) Mix the magnetic luminescent microspheres, the sample to be tested and the second antibody in the first reaction system and incubate at a temperature range of 30°C to 42°C for 3 minutes to 30 minutes, preferably at a temperature range of 35°C to 40°C 10 minutes to 20 minutes;

   b) the magnetic luminescent microspheres after magnetic separation incubation;

   c) adding the separated magnetic luminescent microspheres to the second reaction system, and adding the non-magnetic photosensitive microspheres to the second reaction system and incubating at a temperature ranging from 30°C to 42°C for 3 minutes to 30 minutes, preferably at a temperature range of 35°C to 40°C for 5 minutes to 15 minutes;

   d) detecting the luminous intensity of the magnetic luminescent microspheres.
8. The method according to claim 6 or 7, wherein the concentration of the magnetic luminescent microspheres in the first reaction system or the second reaction system is 0.001 mg/mL to 5 mg/mL, preferably 0.04 mg/mL to 0.4 mg/mL; the concentration of the second antibody in the first reaction system or the second reaction system is 0.001 mg/mL to 5 mg/mL, preferably 0.04-0.4 mg/mL; and the nonmagnetic The concentration of photosensitive microspheres in the second reaction system is 0.001 mg/mL to 5 mg/mL, preferably 0.04 mg/mL to 0.4 mg/mL.
9. The method according to claim 6 or 7, wherein the first reaction system and the second reaction system are the same or different.
10. The method according to claim 9, wherein, the solution volume of the first reaction system and the solution volume of the second reaction system are the same or different; preferably, the solution volume of the second reaction system is less than the Describe the solution volume of the first reaction system.

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