CN113049806B - Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method - Google Patents
Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method Download PDFInfo
- Publication number
- CN113049806B CN113049806B CN201911384038.2A CN201911384038A CN113049806B CN 113049806 B CN113049806 B CN 113049806B CN 201911384038 A CN201911384038 A CN 201911384038A CN 113049806 B CN113049806 B CN 113049806B
- Authority
- CN
- China
- Prior art keywords
- fluorescent
- concentration
- fluorescence
- fluorescent tracer
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The application relates to the technical field of in-vitro diagnostic reagents, and discloses a biomarker preserving fluid, a fluorescent tracer reagent and an immunodetection method, wherein the biomarker preserving fluid comprises: at least one anti-fluorescence quenching agent, wherein the concentration of the anti-fluorescence quenching agent is 2-6mmol/L; the water-soluble protein protective agent, the addition amount of the water-soluble protein protective agent is 0.01-2% of the total mass of the preservation solution; the concentration of the buffer solution is 0.01-0.5mol/L. Through the mode, this application can let the fluorescence tracer reagent of preserving the preservative fluid at the biomarker preserve 30 days at least under the condition of not keeping away from light, and simultaneously, the preparation process of fluorescence tracer reagent is convenient and controllable.
Description
Technical Field
The application relates to the technical field of in-vitro diagnostic reagents, in particular to a preservation solution of a biomarker, a fluorescent tracer reagent and an immunoassay method.
Background
In immunodetection techniques (flow cytometry, flow-fluorescent multiplex detection and fluorescent western blotting, etc.), it is necessary to use fluorescein/fluorescent protein-labeled antibodies/antigens, and therefore the preservation of these fluorescently labeled products risks quenching.
To prevent quenching of fluorescence by light irradiation, we usually physically circumvent the risk: the production and preparation are carried out in a dark environment, and dark reagent bottles (brown and brown) are used for subpackage and storage. These physical means are usually very effective in preventing fluorescence quenching caused by light irradiation, but also bring about many new problems, for example, the whole course of light shielding during reagent preparation makes the manual operation difficult; in addition, direct observation of the reagent state (clarification to turbidity) cannot be achieved by using a dark reagent bottle, and finally, the inspection process becomes more complicated and error-prone.
Disclosure of Invention
The application provides a biomarker preserving fluid, a fluorescent tracer reagent and an immunoassay method, which can preserve the fluorescent tracer reagent for at least 30 days under the condition of not keeping out of the sun, and meanwhile, the preparation process of the fluorescent tracer reagent is convenient and controllable.
In one aspect, the present application provides a preservation solution for biomarkers, comprising: at least one anti-fluorescence quenching agent, wherein the concentration of the anti-fluorescence quenching agent is 2-6mmol/L; water-soluble protein protectant (BSA), the addition amount of which is 0.01-2% of the total mass of the preservation solution; the concentration of the buffer solution is 0.01-0.5mol/L.
In order to solve the technical problem, the application adopts a technical scheme that: there is provided a fluorescent tracer reagent comprising a fluorescent tracer and a preservation fluid as hereinbefore described.
In order to solve the above technical problem, another technical solution adopted by the present application is: an immunoassay method is provided, the method comprising the steps of: preparing a fluorescent tracer composition, wherein the fluorescent tracer composition comprises a fluorescent tracer and a preserving fluid as described above, adding the capture immune composition and the detection immune composition to a test system, and mixing a sample to be tested with the capture immune composition and the detection immune composition; after incubation for a preset time, carrying out magnetic separation and cleaning to obtain a plurality of immune complexes, mixing the immune complexes with a fluorescent tracer composition, and obtaining a complex to be detected; and detecting the fluorescence intensity values of a plurality of complexes to be detected.
The beneficial effect of this application is: different from the situation of the prior art, the preservation solution of the biomarker comprises the anti-fluorescence quencher with the concentration of 2-6mmol/L, and when the preservation solution is used for a fluorescent tracer reagent, the anti-quenching capability of the fluorescent tracer can be enhanced, so that the preparation process of the fluorescent tracer reagent can be operated without avoiding light within a certain time, the preparation process of the fluorescent tracer reagent is convenient and controllable, and the working efficiency of a user is improved. Meanwhile, the fluorescent tracer reagent containing the preservation solution of the biomarker can be preserved for at least 30 days without being protected from light.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic flow chart of an embodiment of the immunoassay method of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.
The term "fluorescent tracer" as used herein, refers to a compound that can generate a fluorescent signal and can be conjugated to a biological material, including, but not limited to, small molecule fluorescein, fluorescent proteins, fluorescent dyes, rare earth ions and their chelators, semiconductor nanocrystals, and the like.
Fluorescence quenching (quenching) refers to the irreversible destruction of fluorescent molecules by both internal and external factors. The internal factor is mainly the energy released by the molecules in the form of non-radiative transitions from the excited state back to the ground state. External factors include many aspects, mainly:
1) Light irradiation is the most common cause of fluorescence quenching, and the generation of fluorescence requires light irradiation, but the light irradiation can promote the interaction of excited molecules and other molecules to cause collision so as to quench the fluorescence;
2) Molecules of the fluorescent substance form a non-fluorescent compound with external molecules (or ions);
3) Fluorescence Resonance Energy Transfer (FRET); the excitation of the donor fluorescent molecule induces the acceptor molecule to fluoresce, while the fluorescence intensity of the donor fluorescent molecule itself decays, i.e., the donor molecule in FRET is quenched.
4) pH value and temperature. Halogen ions, heavy metal ions, oxygen molecules, and nitro compounds, diazo compounds, carboxyl and carbonyl compounds are all common fluorescence extinguishants.
In immunodetection techniques (flow cytometry, flow-fluorescent multiplex detection and fluorescent western blotting, etc.), it is necessary to use fluorescein/fluorescent protein-labeled antibodies/antigens, and therefore the preservation of these fluorescently labeled products risks quenching.
To prevent quenching of fluorescence by light irradiation, we usually physically circumvent the risk: the production and preparation are carried out in a dark environment, and dark reagent bottles (brown and brown) are used for subpackage and storage. These physical means are usually very effective in preventing fluorescence quenching caused by light irradiation, but also bring about many new problems, for example, the whole course of light shielding during reagent preparation makes the manual operation difficult; in addition, direct observation of the reagent state (clarification to turbidity) cannot be achieved by using a dark reagent bottle, and finally, the inspection process becomes more complicated and error-prone.
The application provides a preservation solution for a biomarker, which comprises: at least one anti-fluorescence quenching agent, a water-soluble protein protective agent and a buffer solution. Wherein, the concentration of the anti-fluorescence quenching agent is 2-6mmol/L, the addition amount of the water-soluble protein protective agent is 0.01-2% of the total mass of the preservation solution, and the concentration of the buffer solution is 0.01-0.5mol/L.
Wherein the anti-fluorescence quencher is selected from: at least one of glycerol solution containing P-phenylenediamine (PPD) with the molar concentration of 2-7mmol/L, glycerol solution containing n-propyl gallate (NPG) with the molar concentration of 3-9mmol/L, and glycerol solution containing triethylene diamine (1, 4-diazocyelo (2, 2) -octane, DABCO) with the mass percent concentration of 0.1-4%.
Wherein, PPD has good anti-quenching effect and very good protection capability to various fluorescence, and the glycerol solution of PPD comprises: PBS buffer solution with the mass percent concentration of 10%, glycerol with the mass percent concentration of 90% and PPD with the molar concentration of 2-7 mmol/L. Preferably, the molar concentration of PPD is 4mmol/L PPD. The glycerol solution of PPD was stored at-20 deg.C or-70 deg.C.
DABCO is a non-ionizing, stable anti-fluorescence quencher, inexpensive and easy to use. The anti-quenching effect is slightly worse than PPD, but the toxicity is lower, the storage is easy, and the DABCO can be stored at room temperature, and the glycerol solution of the DABCO comprises: 10 percent of PBS buffer solution, 90 percent of glycerol and 0.1 to 1 percent of DABCO. Preferably, the mass percentage concentration of DABCO is 0.1%.
NPG is an anti-fluorescence quencher that is nontoxic and stable to light and heat, but less effective against bleaching than PPD, and can be used for in vivo studies.
Wherein the water-soluble protein protectant is at least one selected from bovine serum albumin, casein, gelatin and fish skin gel. Wherein, the water-soluble protein protective agent can reduce the degradation and inactivation of the coupling compound to a certain extent. Preferably, the water-soluble protein protectant is bovine serum albumin, which acts as a protein protectant to mitigate adverse environmental effects such as heat, surface tension, and chemical factors that may cause protein denaturation.
The buffer solution is phosphate buffer solution with pH value of 7.4. Wherein the phosphate buffer solution with pH 7.4 is prepared by adding 0.2 g of potassium dihydrogen phosphate, 2.9 g of disodium hydrogen phosphate dodecahydrate, 8.0 g of sodium chloride, 0.2 g of potassium chloride, and 0.5mL of Tween-20 into 1L of distilled water. In other embodiments, phosphate buffer with pH 7.4 can be prepared according to the above proportion.
Further, the preservation solution also contains at least one antioxidant and preservative. Wherein the concentration of the antioxidant is 1-20mmol/L, and the addition amount of the preservative is 0.04-0.06% of the total mass of the preservation solution.
Preferably, the antioxidant is selected from sodium edetate and the preservative is selected from Proclin300.
Different from the situation of the prior art, the preservation solution of the biomarker comprises an anti-fluorescence quencher with the concentration of 2-6mmol/L, and when the preservation solution is used for a fluorescent tracer reagent, the anti-quenching capability of the fluorescent tracer can be enhanced, so that the fluorescent tracer reagent can be prepared in a light-proof manner within a certain time, the preparation process of the fluorescent tracer reagent is convenient and controllable, and the working efficiency of a user is improved. Meanwhile, the fluorescent tracer reagent containing the preservation solution of the biomarker can be preserved for at least 30 days without being protected from light.
The present application also provides a fluorescent tracer reagent comprising a fluorescent tracer and a preservation solution as in the above examples.
Wherein the fluorescent tracer is selected from small molecule fluorescein, fluorescent protein or tandem fluorescent dye.
Specifically, the fluorescent protein is at least one selected from phycoerythrin, phycocyanin, phycoerythrocyanin, and allophycocyanin. Wherein phycobiliprotein (phycobiliprolein) is a functional component of a photosynthesis light-capturing compound of blue algae and red algae. According to the absorption spectrum and fluorescence spectrum characteristics, phycobiliproteins can be classified into phycoerythrin (CPE), phycoerythrocyanin (PEC), phycocyanin (CPC) and Allophycocyanin (APC). CPE, PEC, CPC and APC contain alpha and beta subunits, phycobilin (phycobilin) in each subunit is covalently combined with a sulfhydryl group of cysteine residue of phycobiliprotein apoprotein (apo-phycobilinotein) through thioether bond, and the type and the interaction with the apoprotein determine the spectral property of the phycobiliprotein. Phycobilichrome is covalently bound with apoprotein to form a specific conformation, so that CPE mainly absorbs visible light of about 560nm and emits fluorescence of about 580 nm; the PEC absorbs visible light at about 570nm and emits fluorescence at about 630 nm; CPC absorbs visible light at about 620nm and emits fluorescence at about 640 nm; APC absorbs visible light of about 650-660nm and emits fluorescence of about 660-670 nm. The prosthetic group pigment combined with CPE is phycoerythrobilin (PEB for short); the prosthetic group pigment combined by the alpha subunit of the PEC is phycoviolubilin (PVB for short); the prosthetic group pigment combined with the beta subunit of the PEC is phycocyanobilin (PCB for short); the prosthetic group pigment combined by CPC and APC is phycocyanobilin PCB.
The small molecule fluorescein is at least one selected from FITC, cy5, cy3, cy5.5, alexa flow series fluorescein, dylight series fluorescein, pacific blue, FAM, TRITC, TAMRA and rhodamine.
The tandem fluorescent dye is streptavidin-phycoerythrin (SAPE).
The present application also provides an immunoassay method, wherein the immunoassay method may include at least one of chemiluminescence immunoassay, electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), enzymatic immunoassay, biotin-avidin system assay, radioimmunoassay, immunofluorescence assay. Among them, ELISA is based on immobilization of an antigen or an antibody and enzyme labeling of the antigen or the antibody. It will be appreciated that the composition of the capture immunization composition includes, but is not limited to, a plurality of antibodies and/or antigens, which may vary in composition depending on the detection methodology. The immunoassay method comprises the following steps:
s11: a fluorescent tracer composition is prepared which includes a fluorescent tracer and a preservative fluid for the biomarker as in the examples above.
Wherein the fluorescent tracer is selected from small molecule fluorescein, fluorescent protein or tandem fluorescent dye.
Preferably, the fluorescent tracer is streptavidin-phycoerythrin (SAPE).
S12: adding the capture immune composition and the detection immune composition to a test system, and mixing the test sample with the capture immune composition and the detection immune composition.
Wherein the capture immunization composition comprises: a solid support, a capture ligand attached to the solid support, and a diluent. Wherein the capture ligand is capable of specifically binding to the corresponding ligand to be detected.
The solid phase carrier can couple the capture ligand on the surface thereof in a covalent bond through the coupling group on the surface, and the capture ligand comprises: antigens, antibodies, hormone receptors, enzymes, nucleic acids, oligonucleotides, haptens, and the like. The coupling group comprises at least one of carboxyl, hydroxyl, amino, tosyl, chloromethyl, sulfydryl, aldehyde group, hydrazide, silicon hydroxyl, succinimidyl ester and epoxy. And is not limited thereto. The solid phase carrier is at least one of a microporous plate, a glass sheet, a microfluidic chip, latex, magnetic or non-magnetic polymer microspheres, metal alloy nanoparticles, inorganic microspheres, inorganic/organic hybrid microspheres, fluorescent magnetic spheres, magnetic beads and multifunctional fluorescent magnetic beads. Preferably, the solid support is a fluorescent microsphere with a fluorescent code.
The detection immune composition comprises: NHS biotin, a detector ligand linked to NHS biotin, and a diluent. Wherein each capture ligand, each detection ligand can specifically bind to the corresponding ligand to be detected to form an immune complex.
The above dilution includes 10mmol/L PBS, 0.1% BSA, 1% mannitol, 0.01% casein, 5% sucrose, 0.05% Tween20 and 0.1% preservative.
S13: after incubation for a preset time, carrying out magnetic separation and washing to obtain the immune complex.
Specifically, the mixing of the sample to be tested, the capture antibody composition and the detection antibody composition may be performed simultaneously or sequentially. The simultaneous mode is as follows: and mixing the sample to be detected, the capture antibody composition and the detection antibody composition at the same time, incubating for a certain time, carrying out magnetic separation and washing, and then carrying out detection on a fluorescence signal. The sequential mode is as follows: mixing a sample to be detected and the capture antibody composition, incubating for a certain time, adding the detection antibody composition, incubating for a certain time, carrying out magnetic separation and cleaning, and then carrying out detection on a fluorescence signal.
S14: and mixing the immune complex and the fluorescent tracer composition to obtain a complex to be detected.
Incubating a sample of the subject with the capture immune composition and the detection immune composition of the detection kit to form an immune complex: solid phase carrier-capture ligand-ligand to be detected-detection ligand-streptavidin-phycoerythrin.
S15: and detecting the fluorescence intensity value of the complex to be detected.
The detection instrument is a flow cytometer Novocyte 2040R;
the detection parameters are set to the FSC threshold: 10000, end conditions are aspirate volume: 20 mu L of the solution;
the detection data parameter is mean (PE-H).
The present application is further described below with reference to examples:
example 1
A preservation solution for biomarkers comprising:
glycerol solution of P-phenylenediamine, wherein the concentration of the P-phenylenediamine is 4mmol/L;
bovine serum albumin 0.1% (mass percent);
0.02mol/L phosphate buffer solution with the pH value of 7.4;
0.05 percent of ethylene diamine tetraacetic acid sodium salt (mass percentage);
proclin 300.05% (mass percentage).
Example 2
A preservation solution for biomarkers comprising:
glycerol solution of P-phenylenediamine, wherein the concentration of the P-phenylenediamine is 4mmol/L;
bovine serum albumin 1% (mass%);
0.02mol/L phosphate buffer solution with the pH value of 7.4;
0.05 percent of ethylene diamine tetraacetic acid sodium salt (mass percentage);
proclin 300.05% (mass percentage).
Example 3
A preservation solution for biomarkers comprising:
glycerol solution of n-propyl gallate, wherein the concentration of n-propyl gallate is 4mmol/L;
bovine serum albumin 0.1% (mass percent);
0.02mol/L phosphate buffer solution with the pH value of 7.4;
0.05 percent of ethylene diamine tetraacetic acid sodium salt (mass percentage);
proclin 300.05% (mass percent).
Example 4
A biomarker preservation solution comprising:
glycerol solution of triethylene diamine, wherein the mass percent concentration of the triethylene diamine is 2%;
bovine serum albumin 0.1% (mass percent);
0.02mol/L of phosphate buffer solution with the pH value of 7.4;
0.05 percent of ethylene diamine tetraacetic acid sodium salt (mass percentage);
proclin 300.05% (mass percentage).
Example 5
A fluorescent tracer reagent comprising:
glycerol solution of P-phenylenediamine, the concentration of P-phenylenediamine is 4mmol/L;
bovine serum albumin 1% (mass%);
0.02mol/L of phosphate buffer solution with the pH value of 7.4;
0.05 percent of ethylene diamine tetraacetic acid sodium salt (mass percentage);
proclin 300.05% (mass percent);
SAPE 1μg/mL。
example 6
Immunodetection method
Exemplified by the Procalcitonin (PCT) program, wherein the kit comprises: (1) ra reagent: coating PCT antibody on magnetic beads; (2) an Rb reagent: biotin-labeled PCT antibody; (3) re reagent: the fluorescent tracer stored in the storage solution prepared in examples 1-4 was SAPE at 1. Mu.g/mL.
The sample to be tested is: 100ng/mL of PCT antigen, and the preparation method of the sample to be detected comprises the following steps: PCT native antigen stock (5. Mu.g/mL) was diluted 50-fold with antigen diluent to prepare 100ng/mL of PCT antigen.
A detection step:
1) 50 μ L of Ra reagent was incubated with 50 μ L of PCT antigen (100 ng/mL) for 6min at 37 ℃ and washed once;
2) Adding 50 μ L Rb reagent, incubating at 37 deg.C for 4min, and washing once;
3) Adding 50 μ L Re reagent, incubating at 37 deg.C for 2min, and washing once;
4) Adding 60 μ L of cleaning solution, mixing, and detecting.
5) The detection mode is as follows: and detecting the fluorescence intensity values of a plurality of complexes to be detected.
Wherein, the detecting instrument: flow cytometer NovoCyte 2040R;
setting parameters: FSC threshold value: 10000, end conditions are aspirate volume: 20 mu L of the solution;
data parameters: mean (PE-H).
The fluorescence signal results are given in table 1 below:
TABLE 1 results of fluorescence intensity values of complexes to be tested for SAPE in different treatment modes
| Treatment method | 0h | 2h | 5h | 10h | 24h | 48h |
| Example 1 | 174936 | 173623 | 175647 | 162150 | 152213 | 147565 |
| Example 2 | 175962 | 175536 | 174652 | 172231 | 166765 | 158846 |
| Example 3 | 173257 | 171956 | 173961 | 160593 | 150752 | 146148 |
| Example 4 | 165048 | 163809 | 165719 | 152985 | 143609 | 139224 |
As can be seen from the above table, the quenching prevention effect of PPD of example 1 is significantly better than that of NPG of example 3 and DABCO of example 4; the quenching prevention effect of the embodiment 2 added with more water-soluble protein protective agent is obviously better than that of the PPD of the embodiment 1, which shows that the bovine serum albumin as the protein protective agent can reduce protein denaturation caused by some adverse environments such as heating, surface tension, chemical factors and the like, and is more beneficial to storing fluorescent tracers.
Example 7
Immunodetection method
Exemplified by the Procalcitonin (PCT) program, wherein the kit comprises: (1) ra reagent: coating PCT antibody on magnetic beads; (2) an Rb reagent: labeling a PCT antibody with biotin; (3) re reagent: SAPE (fluorescent tracer reagent of example 5, stored in, not protected from light) in different treatment modes.
The sample to be tested is: 100ng/mL of PCT antigen, and the preparation method of the sample to be detected comprises the following steps: PCT native antigen stock (5. Mu.g/mL) was diluted 50-fold with antigen diluent to prepare 100ng/mL of PCT antigen.
A detection step:
1) 50 μ L of Ra reagent was incubated with 50 μ L of PCT antigen (100 ng/mL) at 37 ℃ for 6min and washed once;
2) Adding 50 μ L Rb reagent, incubating at 37 deg.C for 4min, and washing once;
3) Adding 50 μ L Re reagent, incubating at 37 deg.C for 2min, and washing once;
4) Adding 60 μ L of cleaning solution, mixing, and detecting.
5) The detection mode is as follows: and detecting the fluorescence intensity values of a plurality of complexes to be detected.
Wherein, the detecting instrument: flow cytometer NovoCyte 2040R;
setting parameters: FSC threshold value: 10000, end conditions are aspirate volume: 20 mu L of the solution;
data parameters: mean (PE-H).
The fluorescence signal results are given in table 1 below:
TABLE 1 results of fluorescence intensity values of complexes to be tested for SAPE in different treatment modes
| Method of treatment | 0h | 2h | 5h | 10h | 24h | 48h |
| Light-resistant | 176936 | 176523 | 177647 | 176350 | 175895 | 177565 |
| Not to avoid light | 176486 | 174645 | 171322 | 166874 | 152399 | 126533 |
| Non-photophobic + anti-quencher | 175962 | 175536 | 174652 | 172231 | 166765 | 158846 |
As can be seen from the above table, the preservation solution of the biomarker of the present application includes the anti-fluorescence quencher with a concentration of 2-6mmol/L, and when the preservation solution is used in the fluorescent tracer reagent, the anti-quenching capability of the fluorescent tracer can be enhanced, so that in the process of preparing the fluorescent tracer reagent, the fluorescence intensity of the non-light shielding operation within 10 hours is equivalent to that of the light shielding operation, and the fluorescence intensity of the non-light shielding operation within 48 hours is much stronger than that of the non-light shielding operation.
The above description is only an embodiment of the present application, and is not intended to limit the scope of the present application, and all equivalent structures or equivalent processes performed by the present application and the contents of the attached drawings, which are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.
Claims (6)
1. A biomarker preservation solution, comprising:
at least one anti-fluorescence quenching agent, wherein the concentration of the anti-fluorescence quenching agent is 2-6mmol/L;
the water-soluble protein protective agent is selected from bovine serum albumin, and the addition amount of the water-soluble protein protective agent is 0.1-2% of the total mass of the preservation solution;
the concentration of the buffer solution is 0.01-0.5mol/L, and the buffer solution is phosphate buffer solution with the pH value of 7.4;
the antioxidant is selected from ethylene diamine tetraacetic acid sodium salt, and the concentration of the antioxidant is 1-20 mmol/L;
the preservative is selected from Proclin300, and the addition amount of the preservative is 0.04-0.06% of the total mass of the preservation solution;
wherein the anti-fluorescence quencher is selected from the group consisting of: at least one of a glycerol solution containing P-phenylenediamine (PPD) with the concentration of 2-7mmol/L, a glycerol solution containing n-propyl gallate (NPG) with the concentration of 3-9mmol/L, and a glycerol solution containing triethylene diamine (1, 4-diazobicyclocyclo (2, 2) -octane, DABCO) with the concentration of 0.1-4% by mass.
2. The preservation solution according to claim 1, wherein the phosphate buffer solution having a pH value of 7.4 is prepared by adding 0.2 g of potassium dihydrogen phosphate, 2.9 g of disodium hydrogen phosphate dodecahydrate, 8.0 g of sodium chloride, 0.2 g of potassium chloride, and 0.5mL of Tween-20 to 1L of distilled water.
3. A fluorescent tracer reagent comprising a fluorescent label and the preservation solution of any of claims 1-2.
4. The reagent according to claim 3,
the fluorescent tracer is selected from small molecule fluorescein, fluorescent protein or tandem fluorescent dye.
5. The reagent according to claim 4,
the fluorescent protein is selected from at least one of phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin;
the small molecule fluorescein is at least one selected from FITC, cy5, cy3, cy5.5, alexa flours series fluorescein, dylight series fluorescein, pacific blue, FAM, TRITC, TAMRA and rhodamine;
the tandem fluorescent dye is streptavidin-phycoerythrin (SAPE).
6. An immunoassay method, comprising the steps of:
preparing a fluorescent tracer composition comprising a fluorescent tracer and a preservation fluid according to any one of claims 1-2;
adding a capture immune composition and a detection immune composition to a test system, and mixing a sample to be tested with the capture immune composition and the detection immune composition;
after incubation for a preset time, carrying out magnetic separation and cleaning to obtain a plurality of immune complexes;
mixing the immune complex with the fluorescent tracer composition to obtain a complex to be detected;
and detecting the fluorescence intensity values of a plurality of complexes to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911384038.2A CN113049806B (en) | 2019-12-28 | 2019-12-28 | Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911384038.2A CN113049806B (en) | 2019-12-28 | 2019-12-28 | Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113049806A CN113049806A (en) | 2021-06-29 |
| CN113049806B true CN113049806B (en) | 2023-03-28 |
Family
ID=76507410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911384038.2A Active CN113049806B (en) | 2019-12-28 | 2019-12-28 | Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113049806B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846145A (en) * | 2021-08-20 | 2021-12-28 | 李彦萍 | Fluorescent dye stabilizer, kit containing same and related application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4622291A (en) * | 1984-06-11 | 1986-11-11 | The United States Of America As Represented By The Department Of Health And Human Services | Method and device for quantitative end point determination in immunofluorescence using microfluorophotometry |
| CN104459147A (en) * | 2013-09-18 | 2015-03-25 | 深圳迈瑞生物医疗电子股份有限公司 | Biomarker preserving liquid, biomarker reagent and method |
| WO2015042593A1 (en) * | 2013-09-23 | 2015-03-26 | Assaypro, Llc | Immunoassays using over-labeled fluorescent probes |
| CN104634962A (en) * | 2013-11-11 | 2015-05-20 | 中国计量科学研究院 | Antinuclear antibody quantitative detection kit and use method thereof |
-
2019
- 2019-12-28 CN CN201911384038.2A patent/CN113049806B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113049806A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10851212B2 (en) | Polymeric BODIPY dyes and methods for using the same | |
| US11977078B2 (en) | Ultraviolet absorbing polymeric dyes and methods for using the same | |
| CN104459147A (en) | Biomarker preserving liquid, biomarker reagent and method | |
| CN119306927A (en) | Water-solvated polymer dyes that can excite blue | |
| CN104169710A (en) | Method and apparatus for time-resolved fluorescence immunoassay assays | |
| EP3732254A1 (en) | Deep ultraviolet-excitable water-solvated polymeric dyes | |
| CN113049806B (en) | Biomarker preserving solution, fluorescent tracer reagent, and immunoassay method | |
| CN111351924A (en) | A near-infrared fluorescence immunoassay kit and detection method based on enzyme-induced phosphate ion activation | |
| JP2006234824A (en) | Phycobilisomes, derivative and uses therefor | |
| CN113049835A (en) | Combined detection kit, detection method and immunoassay system | |
| CN101294903A (en) | Method for detecting the expression of the same antigen on the same specimen | |
| CN109298178A (en) | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit | |
| CN107043762A (en) | A kind of preserving stabilizer and its store method for improving horseradish peroxidase storage stability | |
| CN110779912A (en) | Microsphere-free homogeneous chemiluminescence system of biotin-avidin or streptavidin | |
| CN118307662A (en) | Bifunctional protein and detection kit containing same | |
| CN110100180B (en) | Homogeneous phase detection method | |
| CN117890582B (en) | A herpes simplex virus detection kit and detection method thereof | |
| US20230324374A1 (en) | Tandem Dyes Having Internally Positioned Sensors, and Methods for Making and Using the Same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |