CN114062665B - Trace particle marked target molecule and preparation method and application thereof - Google Patents

Trace particle marked target molecule and preparation method and application thereof Download PDF

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Publication number
CN114062665B
CN114062665B CN202010780643.8A CN202010780643A CN114062665B CN 114062665 B CN114062665 B CN 114062665B CN 202010780643 A CN202010780643 A CN 202010780643A CN 114062665 B CN114062665 B CN 114062665B
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antigen
target molecule
virus antigen
virus
preparation
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CN114062665A (en
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郑锦龙
李瑞净
魏钟杰
黄荣生
岑静
覃丽芳
詹佩文
丁正辉
张耀东
陈一尘
庞土玲
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Fapon Biotech Inc
Guangdong Fapon Biotech Co Ltd
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Fapon Biotech Inc
Guangdong Fapon Biotech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the field of biological detection, and in particular provides a trace particle marked target molecule, a preparation method and application thereof. According to the preparation method of the tracer particle marked target molecule, the target molecule is mixed with casein and then combined with the tracer particle for reaction, so that the tracer particle marked target molecule is obtained. The method can improve the labeling state, and enhance the binding capacity and stability of target molecules and trace particles, so that the activity and specificity in immune reaction are improved.

Description

Trace particle marked target molecule and preparation method and application thereof
Technical Field
The invention relates to the field of biological detection, in particular to a trace particle marked target molecule, a preparation method and application thereof.
Background
The immunochromatography technology is a rapid detection technology for qualitatively, semi-quantitatively or quantitatively detecting the content of an antigen or an antibody in a sample by utilizing an adsorption carrier and by tracing particle color development according to an immunological principle. Taking an antibody in a detection sample as an example, an antigen is firstly fixed on a certain area of an adsorption carrier such as a nitrocellulose membrane, after one end of the dried nitrocellulose is immersed into the sample (such as urine, saliva and serum), the sample moves forward along the membrane due to capillary action, and when the sample moves to the area where the antigen is fixed, the corresponding antibody in the sample specifically binds with the antigen, wherein the antigen is marked by a tracer particle, and when an antigen-antibody binding reaction occurs, a certain color is displayed, so that specific immunodetection is realized. Commonly used trace particles include colloidal gold, latex, fluorescent microspheres, and the like.
In the preparation process of the immunochromatography diagnostic reagent, the tracer particles are required to be combined with target molecules, namely, the labeling of the tracer particles of the target molecules is realized. In the practical application process, such as latex, colloidal gold, fluorescent microspheres and the like, the binding force between the trace particles and the target molecules is relatively weak, and the problem of easy breakage is likely to occur. In the immune reaction, if there is higher affinity between the target molecule and the specific receptor/ligand in the sample, the target molecule and the trace particle will be broken, which results in the problems of reduced immune reaction activity and poor specificity, such as dead gold in colloidal gold immunochromatography.
In view of this, the present invention has been made.
Disclosure of Invention
The first aim of the invention is to provide a method for preparing a tracer particle labeled target molecule.
A second object of the present invention is to provide a tracer particle for labelling a target molecule.
A third object of the present invention is to provide the use of a tracer particle to label a target molecule.
The fourth object of the present invention is to provide an immunochromatographic test strip.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the preparation method of the tracer particle marked target molecule comprises the steps of mixing the target molecule with casein, and then carrying out a binding reaction with the tracer particle to obtain the tracer particle marked target molecule;
the trace particles comprise colloidal gold, colloidal selenium, colloidal silver, latex or fluorescent microspheres.
Further, the mass ratio of the target molecule to casein is 1: (0.5-5);
optionally, the mass ratio of target molecule to casein is 1: (0.8-4);
optionally, the mass ratio of target molecule to casein is 1: (1-3.5);
alternatively, the mass ratio of target molecule to casein is 1.5: (1.5-5).
Further, the target molecule is an antigen or an antibody;
further, the antigen or antibody is selected from the following immunodetection item-related antigens or antibodies: immunodetection of infectious diseases, immunodetection of cardiac markers or immunodetection of tumor-associated markers.
Further, the infectious disease includes a viral infection disease, a bacterial infection disease, a fungal infection disease, a chlamydia infection disease, a mycoplasma infection disease or a parasitic infection disease;
alternatively, the relevant antigen of the infectious disease includes an hiv antigen, a hepatitis a virus antigen, a hepatitis b virus antigen, a hepatitis c virus antigen, a hepatitis b virus antigen, a hepatitis e virus antigen, a hepatitis g virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T lymphocyte leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a west nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an echovirus antigen, a coxsackie virus antigen, a encephalitis virus antigen, an EB virus antigen, a mumps virus antigen, a treponema pallidum antigen, a borrelia chlamydia antigen, a trachomatis antigen, a chlamydia pneumoniae antigen, a ureaplasma urealyticum antigen, a helicobacter pylori antigen, a gonococcus antigen, a plasmodium antigen, a trypanosoma antigen, or a trypanosoma antigen;
alternatively, the cardiac markers include troponin, myoglobin, creatine kinase isozymes, N-terminal brain natriuretic peptide precursors, cardiac fatty acid binding proteins, D-dimers, lipoprotein-associated phospholipase A2, myeloperoxidase, or growth-stimulatory expressed gene 2 proteins;
optionally, the tumor-associated marker comprises: gastrin-releasing peptide precursor, carbohydrate antigen, cytokeratin 19, epididymal protein 4, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen, squamous cell carcinoma antigen or human prostate specific gene 1.
Further, the method further comprises a step of sealing after the target molecules are combined with the tracer particles, and then the tracer particles are obtained to mark the target molecules.
The tracer particles prepared by the preparation method label target molecules.
The application of the tracer particle labeled target molecule in immunodetection or preparation of an immunodetection reagent.
An immunochromatography detection test paper comprises the trace particle labeled target molecules.
Further, the immunochromatography detection test paper comprises latex immunochromatography detection test paper, colloidal gold immunochromatography detection test paper, colloidal selenium immunochromatography detection test paper, colloidal silver immunochromatography detection test paper or fluorescence immunochromatography detection test paper.
Compared with the prior art, the invention has the beneficial effects that:
according to the preparation method of the tracer particle marked target molecule, the target molecule is mixed with casein and then combined with the tracer particle for reaction, so that the tracer particle marked target molecule is obtained. In conventional wisdom, the addition of unrelated proteins prior to labeling the tracer particles with target molecules tends to compete for binding sites, resulting in decreased sensitivity. However, the inventors have found that the binding reaction between the target molecule and the trace particle is performed in an environment where casein exists, and the binding ability and stability of the target molecule and the trace particle can be enhanced, so that the activity and specificity in the immune reaction, such as the disappearance of dead gold phenomenon in the colloidal gold chromatography reaction, etc., are improved, and unexpected technical effects are obtained. In particular, in the prior art, the scheme that the target molecule and the trace particle are difficult to combine and need indirect labeling exists, and the method for combining and reacting the target molecule and casein with the trace particle can achieve better effect than the indirect labeling.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.
The invention provides a preparation method of a tracer particle-marked target molecule, which is characterized in that the target molecule is mixed with casein and then combined with the tracer particle for reaction to obtain the tracer particle-marked target molecule, wherein the tracer particle comprises colloidal gold, colloidal selenium, colloidal silver, latex or fluorescent microspheres.
In conventional wisdom, the addition of unrelated proteins prior to labeling the tracer particles with target molecules tends to compete for binding sites, resulting in decreased sensitivity. However, the inventors have found that the binding reaction between the target molecule and the trace particle is performed in an environment where casein exists, and thus the labeling state can be improved, and the binding ability and stability between the target molecule and the trace particle can be enhanced, so that the activity and specificity in the immune reaction, for example, the disappearance of dead gold phenomenon in the colloidal gold chromatography reaction, etc., can be improved, and unexpected technical effects can be obtained. In particular, in the prior art, the scheme that the target molecule and the trace particle are difficult to combine and need indirect labeling exists, and the method for combining and reacting the target molecule and casein with the trace particle can achieve better effect than the indirect labeling.
The binding reaction of the target molecule and the trace particle in the present invention may be a conventional preparation process in the art, and is not particularly limited herein, and may be merely that casein is mixed into the target molecule, and then the mixture of the target molecule and casein is bound to the trace particle.
The colloidal gold is gold particles formed by polymerizing chloroauric acid into a certain size under the action of reducing agents such as sodium citrate, white phosphorus, ascorbic acid, tannic acid and the like, and becomes a stable colloidal state due to the action of static electricity. The colloidal gold labeling is a process in which a polymer such as a protein is adsorbed onto the surface of colloidal gold particles. The adsorption mechanism may be that the surface of the colloidal gold particles has negative charges and the positive charge groups of the proteins are combined due to electrostatic adsorption. In addition, colloidal selenium and colloidal silver are also commonly used trace particles, and the principle of protein labeling is also the adsorption of colloidal metals.
The latex is a particle, and can be combined with amino groups of protein substances under the action of a catalyst such as EDC through special groups such as carboxyl on the surface of the particle, so that the latex marking is realized.
The fluorescent microsphere is a trace particle formed by adsorbing or embedding fluorescent dye into particles by a physical adsorption method, a self-assembly method, a chemical bonding method, a copolymerization method, an embedding method and the like, wherein the diameter of the trace particle is in a range from nanometer to micrometer, and the excited light source can emit fluorescence under excitation.
It should be noted that the labeling of the target molecule by the tracer particles in the present invention corresponds to the labeling of the end in rapid diagnosis.
In a preferred embodiment, the mass ratio of target molecule to casein is: 1: (0.5-5), preferably 1: (0.8-4), further preferably 1: (1-3.5), further preferably 1.5: (1.5-5), specifically, the mass ratio may be, but is not limited to, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. The ratio of the target molecule to the trace particles may be any amount conventionally used in the art, and is not particularly limited herein.
In the present invention, the target molecule may be a substance labeled with a trace particle, which is conventional in the art, and may be an antigen or an antibody. The antigen or antibody may be an antigen or antibody associated with an immunoassay format common in the art.
For example, the immunoassay item may be an infectious disease immunoassay, a cardiac marker immunoassay, or a tumor-associated marker immunoassay.
The infectious disease may be a viral infection disease, a bacterial infection disease, a fungal infection disease, a chlamydia infection disease, a mycoplasma infection disease or a parasitic infection disease.
The infectious disease-associated antigen may be an hiv antigen, a hepatitis a virus antigen, a hepatitis b virus antigen, a hepatitis c virus antigen, a hepatitis b virus antigen, a hepatitis e virus antigen, a hepatitis g virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T lymphocytic leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a west nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an echovirus antigen, a coxsackie virus antigen, a encephalitis b virus antigen, a coxsackie virus antigen, an EB virus antigen, a mumps virus antigen, a treponema pallidum antigen, a c spirochete antigen, a chlamydia trachomatis antigen, a chlamydia pneumoniae antigen, a helicobacter pylori antigen, a gonococcus antigen, a plasmodium antigen, a toxoplasma antigen, or a toxoplasma antigen.
The cardiac markers may be troponin, myoglobin, creatine kinase isozymes, N-terminal brain natriuretic peptide precursors, cardiac type fatty acid binding proteins, D-dimers, lipoprotein-associated phospholipase A2, myeloperoxidase or growth-stimulated expressed gene 2 proteins.
The tumor-associated marker may be a gastrin releasing peptide precursor, a carbohydrate antigen, cytokeratin 19, epididymal protein 4, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen, squamous cell carcinoma antigen or human prostate specific gene 1.
In a preferred embodiment, the binding of the target molecule to the tracer particle is followed by a blocking step, which yields a label of the target molecule by the tracer particle. The blocking may be performed by a conventional blocking method in the art, and is not particularly limited, and may be performed by using an inert protein, for example, bovine serum albumin or the like.
The invention also protects the tracer particle marked target molecule prepared by the preparation method, and the tracer particle marked target molecule has good stability, high specificity and sensitivity for subsequent detection. It should be noted that the tracer particle labeled target molecule provided by the invention is used as a labeling end in specific applications such as rapid diagnosis technology. According to the difference between the trace particles and the target molecules, the method can be widely applied to various fields, for example, when the target molecules are marked by adopting colloidal gold, the method can be applied to specific detection of various substances such as electron microscope, optical microscope, flow cytometry, agglutination detection, immunoblotting, immunochromatography and the like.
The invention also protects the application of the trace particle marked target molecule in immunodetection or preparation of an immunodetection reagent.
The invention finally protects immunochromatography detection test paper which comprises the trace particle labeled target molecule. Specifically, the test paper can be latex immunochromatography test paper, colloidal gold immunochromatography test paper or fluorescence immunochromatography test paper, and the like.
The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.
Example 1 Indirect labelling procedure
1. Preparing 40nm colloidal gold with a concentration of 4 OD;
2. sucking 1ml of colloidal gold into a 4 ml-specification glass bottle;
3. adding 0.2. 0.2M K 2 CO 3 Shaking up for 1min to adjust the pH of the colloidal gold;
4. adding 10 mug GST monoclonal antibody and shaking uniformly for 1min;
5. adding 10 μl of 10% BSA, blocking, and shaking for 1min;
6. transferring the uniformly mixed immune gold solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in an ultracentrifuge at 1000rpm for 7min;
7. carefully sucking the supernatant solution with a pipette, and retaining the precipitate;
8. re-dissolving and precipitating to obtain 100 mu L of concentrated gold;
9. and adding 0.5 mug of coupling protein (TP protein is coupled with GST) into the concentrated gold, and incubating for 15min to obtain the TP antigen (treponema pallidum antigen) of the colloidal gold.
Example 2 direct labelling procedure
1. Preparing 40nm colloidal gold with a concentration of 4 OD;
2. sucking 1ml of colloidal gold into a 4 ml-specification glass bottle;
3. adding 0.2. 0.2M K 2 CO 3 Shaking up for 1min to adjust the pH of the colloidal gold;
4. adding 10 μg target protein (TP protein) and shaking uniformly for 1min;
5. adding 10 μl of 10% BSA, blocking, and shaking for 1min;
6. transferring the uniformly mixed immune gold solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in an ultracentrifuge at 9000rpm for 8min;
7. carefully sucking the supernatant solution with a pipette, and retaining the precipitate;
8. and (3) re-dissolving and precipitating to obtain 100 mu L of concentrated gold, and obtaining the TP antigen marked by colloidal gold.
EXAMPLE 3 direct labeling Casein-adding Process
1. Preparing 40nm colloidal gold with the concentration of 4 OD;
2. sucking 1ml of colloidal gold into a 4 ml-specification glass bottle;
3. adding 0.2. 0.2M K 2 CO 3 Shaking up for 1min to adjust the pH of the colloidal gold;
4. mixing target protein (TP protein) and casein (C) according to a mass ratio of 1.5:1.5 (scheme A), 1.5:3.5 (scheme B) and 1.5:5 (scheme C), respectively;
5. adding 10 mug of the mixed target protein (TP+C) and shaking uniformly for 1min;
6. adding 10 μl of 10% BSA, blocking, and shaking for 1min;
7. transferring the uniformly mixed immune gold solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in an ultracentrifuge at 9000rpm for 8min;
8. carefully sucking the supernatant solution with a pipette, and retaining the precipitate;
9. and (3) re-dissolving and precipitating to obtain 100 mu L of concentrated gold, and obtaining the TP antigen marked by colloidal gold.
Test example 1
Preparing a test strip by using the colloidal gold-labeled TP antigen of examples 1-3, and diluting the colloidal gold-labeled TP antigen obtained in examples 1-3 at 15%; mu.l of the mixture is sucked and evenly spread on a glass fiber film with the thickness of 0.8cm multiplied by 30 cm; lyophilizing with a lyophilizing machine for 2 hr to obtain gold pad; the gold pad was assembled with other components (PVC base plate, NC film, absorbent paper, sample pad) and cut into 2.5mm x 6cm test strips with a slitter; add 35. Mu.l of sample to the test strip and read; and recording an interpretation result.
The following samples were tested:
1. the test samples were 200 negative samples, and the statistical activity and the number of positive tests were counted, and the activity was expressed as a decrease in order from C1 to C9, and the results were shown in the following table:
Figure BDA0002618480310000091
2. the detection sample is a positive quality control product: TP 2/TP 20/TP 60 (i.e. 2-fold, 20-fold, 60-fold dilution of the quality control product, respectively). The results are shown in the following table:
Figure BDA0002618480310000092
the main stream of the current TP antigen item is marked by indirect marking, namely, the marking state of the protein is more stable by coupling target protein to monoclonal antibody marking, compared with the prior art, the marking state is poor, sedimentation and dead gold phenomena occur, color development is ultraviolet, the activity and the specificity are both inferior to those of indirect marking, but casein and target protein are mixed according to a certain proportion and then marked, the marking state can be improved, the original cloudy and ultraviolet marking state is changed into clear red, the specificity and the activity are improved compared with the indirect marking, the strong positive TP of quality control product is higher than the indirect marking 1C, the medium positive TP of quality control product is higher than the indirect marking 1C, and the weak positive TP of quality control product is equal to the indirect marking.
The randomized clinical test results showed that the group of direct labeling + casein was comparable to the detection specificity results of the randomized clinical serum of the indirectly labeled group.
Test example 2
1. Test strips were prepared using HIV antigen (aids antigen) as the target protein, and positive quality control HIV x 20 (20-fold dilution of quality control) was tested according to the preparation procedure of examples 1-3, as shown in the following table:
HIV-colloidal gold item Marking status Quality control HIV 20 color development
Indirect marking Clear red dead gold C4
Direct marking Solution euviolet centrifugal dead gold C5
Direct label + casein (1.5:3.5) Clear red dead gold C4
2. Test strips were prepared using HCV antigen as the target protein, according to the preparation procedure of examples 1-3, and positive control HCV 20 (20-fold dilution of quality control) was detected, with the results shown in the following table:
HCV-colloidal gold item Marking status Quality control HCV x 20 color development
Indirect marking Clear red dead gold C5
Direct marking Solution euviolet centrifugal dead gold C6
Direct label + casein (1.5:3.5) Clear red dead gold C4
Example 4
The latex particles are used as the marking particles, and the marking method is briefly as follows:
1. 100. Mu.L of latex particles were prepared, 900. Mu.L of 0.1M borate buffer pH8.5 was added, and the suspension was washed and resuspended;
2. 0.15mg EDC was added to activate the latex;
3. mixing target protein (HCV protein, i.e. hepatitis C antigen) and casein according to a mass ratio of 1.5:3.5;
4. 10. Mu.g of the mixed target protein (HCV+C) was added and stirred overnight;
5. adding ethanolamine for sealing for 30min, centrifuging, and then re-suspending and concentrating to 100 mu L to obtain latex-marked HCV antigen;
test strips prepared using the above latex-labeled HCV antigens detect positive detection numbers of positive quality control HCV x 20 (20-fold dilution of quality control) and 50 negative samples, activity being expressed as sequential enhancement from L1-L10, with the following table:
HCV-latex project Specificity (specificity) Quality control HCV x 20 color development
Indirect marking 1/50 L7
Direct marking 2/50 L6
Direct label + casein (1.5:3.5) 0/50 L8
Example 5
Fluorescent microspheres are used as marking particles, and the marking method is briefly as follows:
1. preparing 50 mu L fluorescent microspheres (carboxyl polystyrene microspheres containing high-brightness rare earth dye), adding into 1mL 0.01M MES buffer solution, and cleaning and resuspension;
2. adding 250 mu L of EDC, activating for 2 hours in a dark place, cleaning and resuspension;
3. mixing target protein (TP protein) and casein according to a mass ratio of 1.5:3.5;
4. adding 10 mug of the mixed target protein (TP+C), and reacting for 2 hours in a dark place;
5. adding glycine and BSA respectively, blocking for 30min in dark, centrifuging, and concentrating to 100 μl to obtain fluorescent microsphere marked TP antigen;
the test strips prepared by using the above fluorescent microsphere marked TP antigen detect positive detection numbers of positive quality control TP 20 (20 times dilution of quality control) and 50 negative samples, and the results are shown in the following table:
TP-fluorescent microsphere project Specificity (specificity) Quality control TP 20 color development
Indirect marking 1/50 8813
Direct marking 1/50 7744
Direct label + casein (1.5:3.5) 0/50 9892
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (15)

1. The preparation method of the tracer particle directly marked target molecule is characterized in that the target molecule is mixed with casein and then combined with the tracer particle for reaction, so as to obtain the tracer particle marked target molecule;
the trace particles comprise colloidal gold, colloidal selenium, colloidal silver, latex or fluorescent microspheres;
the target molecule is an antigen.
2. The preparation method according to claim 1, wherein the mass ratio of the target molecule to casein is 1: (0.5-5).
3. The preparation method according to claim 2, wherein the mass ratio of the target molecule to casein is 1: (0.8-4).
4. The preparation method according to claim 2, wherein the mass ratio of the target molecule to casein is 1: (1-3.5).
5. The preparation method according to claim 2, wherein the mass ratio of the target molecule to casein is 1.5: (1.5-5).
6. The method of claim 1, wherein the antigen is selected from the group consisting of the following immunodetection item-associated antigens: immunodetection of infectious diseases, immunodetection of cardiac markers or immunodetection of tumor-associated markers.
7. The method of claim 6, wherein the infectious disease comprises a viral infection disease, a bacterial infection disease, a fungal infection disease, a chlamydia infection disease, a mycoplasma infection disease, or a parasitic infection disease.
8. The method according to claim 6, wherein the infectious disease-associated antigen comprises an HIV antigen, a hepatitis A virus antigen, a hepatitis B virus antigen, a hepatitis C virus antigen, a hepatitis B virus antigen, a hepatitis E virus antigen, a hepatitis G virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T lymphoblastic leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a West Nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an Izodiac virus antigen, a Japanese encephalitis virus antigen, a Coxsackie virus antigen, an EB virus antigen, a treponema pallidum antigen, a Czochralski antigen, a Chlamydia trachomatis antigen, a chlamydia antigen, a Mycoplasma antigen, a tuberculosis antigen, a helicobacter pylori antigen, a coccoid antigen, a cone antigen, a Toxoplasma antigen or a Toxoplasma antigen.
9. The method of claim 6, wherein the cardiac markers comprise troponin, myoglobin, creatine kinase isozymes, N-terminal brain natriuretic peptide precursors, cardiac fatty acid binding proteins, D-dimers, lipoprotein-associated phospholipase A2, myeloperoxidase, or growth-stimulatory expressed gene 2 proteins.
10. The method of preparation of claim 6, wherein the tumor-associated marker comprises: gastrin-releasing peptide precursor, carbohydrate antigen, cytokeratin 19, epididymal protein 4, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen, squamous cell carcinoma antigen or human prostate specific gene 1.
11. The method of any one of claims 1 to 10, further comprising a step of blocking after binding of the target molecule to the tracer particle, whereby the tracer particle is obtained to directly label the target molecule.
12. The tracer particles prepared by the method of any one of claims 1-11 directly label target molecules.
13. Use of the tracer particle of claim 12 for directly labelling a target molecule for immunodetection or for preparing an immunodetection reagent.
14. An immunochromatographic test strip comprising the tracer particle of claim 12 directly labeled with a target molecule.
15. The immunochromatographic test strip according to claim 14, which comprises a latex immunochromatographic test strip, a colloidal gold immunochromatographic test strip, a colloidal selenium immunochromatographic test strip, a colloidal silver immunochromatographic test strip or a fluorescent immunochromatographic test strip.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001215228A (en) * 1997-08-04 2001-08-10 Sentan Seimei Kagaku Kenkyusho:Kk Method of detecting or measuring virus
CN101363848A (en) * 2008-09-24 2009-02-11 深圳市菲鹏生物股份有限公司 Sandwich method for detecting double antigen by antibody indirectly marked with nanometer granule and kit thereof
CN103703375A (en) * 2011-06-29 2014-04-02 塞尔雷斯蒂斯有限公司 A cell mediated immune response assay with enhanced sensitivity
CN108700584A (en) * 2017-01-20 2018-10-23 深圳市新产业生物医学工程股份有限公司 Labeled complex and preparation method thereof, kit, application and detecting system
CN111426830A (en) * 2020-03-19 2020-07-17 杭州奥泰生物技术股份有限公司 Colloidal gold immunochromatography detection test paper for combined diagnosis of COVID-19 and mycoplasma pneumoniae and preparation method thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002316512A1 (en) * 2001-06-29 2003-03-03 Subsidiary No. 3 Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US20070134814A1 (en) * 2005-12-09 2007-06-14 Kajander E O Methods and compositions for the detection of calcifying nano-particles, identification and quantification of associated proteins thereon, and correlation to disease
JP7117244B2 (en) * 2016-02-12 2022-08-12 ザ・ユニバーシティ・オブ・シカゴ Compositions and methods related to antibodies that neutralize coagulase activity during Staphylococcus aureus disease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001215228A (en) * 1997-08-04 2001-08-10 Sentan Seimei Kagaku Kenkyusho:Kk Method of detecting or measuring virus
CN101363848A (en) * 2008-09-24 2009-02-11 深圳市菲鹏生物股份有限公司 Sandwich method for detecting double antigen by antibody indirectly marked with nanometer granule and kit thereof
CN103703375A (en) * 2011-06-29 2014-04-02 塞尔雷斯蒂斯有限公司 A cell mediated immune response assay with enhanced sensitivity
CN108700584A (en) * 2017-01-20 2018-10-23 深圳市新产业生物医学工程股份有限公司 Labeled complex and preparation method thereof, kit, application and detecting system
CN111426830A (en) * 2020-03-19 2020-07-17 杭州奥泰生物技术股份有限公司 Colloidal gold immunochromatography detection test paper for combined diagnosis of COVID-19 and mycoplasma pneumoniae and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
宋翔 ; 王云贵 ; 黄登宇 ; .硝苯地平免疫胶体金探针的制备.食品安全质量检测学报.2016,(第08期),第3350-3354页. *
朱文钏 ; 孔繁德 ; 林祥梅 ; 徐淑菲 ; 吴德峰 ; 韩雪清 ; 吴绍强 ; .免疫胶体金技术的应用及展望.生物技术通报.2010,(第04期),第81-87页. *
赵丹丹 ; 杨国平 ; 刁有祥 ; 陈浩 ; 提金凤 ; 张璐 ; 张英 ; 李川川 ; .鸭瘟病毒单抗的制备及胶体金试纸条检测方法的建立.中国农业科学.2016,(第14期),第2796-2804页. *

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