CN115078711A - Cleaning fluid for flow-type fluorescence detection - Google Patents
Cleaning fluid for flow-type fluorescence detection Download PDFInfo
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- CN115078711A CN115078711A CN202210682331.2A CN202210682331A CN115078711A CN 115078711 A CN115078711 A CN 115078711A CN 202210682331 A CN202210682331 A CN 202210682331A CN 115078711 A CN115078711 A CN 115078711A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/667—Neutral esters, e.g. sorbitan esters
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/72—Ethers of polyoxyalkylene glycols
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/74—Carboxylates or sulfonates esters of polyoxyalkylene glycols
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/825—Mixtures of compounds all of which are non-ionic
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/02—Inorganic compounds ; Elemental compounds
- C11D3/04—Water-soluble compounds
- C11D3/046—Salts
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/02—Inorganic compounds ; Elemental compounds
- C11D3/04—Water-soluble compounds
- C11D3/06—Phosphates, including polyphosphates
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/20—Organic compounds containing oxygen
- C11D3/2003—Alcohols; Phenols
- C11D3/2065—Polyhydric alcohols
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/37—Polymers
- C11D3/3703—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C11D3/3707—Polyethers, e.g. polyalkyleneoxides
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/37—Polymers
- C11D3/3746—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C11D3/3753—Polyvinylalcohol; Ethers or esters thereof
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/37—Polymers
- C11D3/3746—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C11D3/3769—(Co)polymerised monomers containing nitrogen, e.g. carbonamides, nitriles or amines
- C11D3/3776—Heterocyclic compounds, e.g. lactam
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/384—Animal products
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention relates to the field of medicines, in particular to a cleaning solution for flow-type fluorescence detection, which comprises a basic cleaning solution, proteins and alcohols, wherein the basic cleaning solution comprises buffer salt and a surfactant, the solvent of the basic cleaning solution is water, and the buffer salt in the basic cleaning solution is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride; the surfactant is a nonionic surfactant. The cleaning solution provided by the invention can be used for satisfying the cleaning effect of the flow type fluorescence light-emitting platform, stabilizing the detection signal, reducing the microsphere sedimentation, considering the background signal and avoiding non-specific adsorption.
Description
Technical Field
The invention relates to the field of medicines, in particular to a cleaning solution for flow-type fluorescence detection.
Background
The flow type fluorescence immunoassay method takes an encoding microsphere as a solid phase carrier, connects an antibody (or an antigen) to the encoding microsphere through a chemical bond, captures a target antigen (or the antibody) in serum or plasma, and reacts with the antibody (or the antigen) marked with a luminescent substance. After the completion of the immunoreaction, the reaction product is usually washed with a washing solution to remove unreacted substances. After washing, the microsphere enters an instrument through a sample suction pipe, PE fluorescence on the microsphere is excited by laser, the PE fluorescence is input into a computer after photoelectric conversion, and the PE fluorescence is analyzed and processed by software, so that the detection of the substance to be detected is realized.
At present, many technicians research cleaning solutions, for example, the prior art discloses an electrochemiluminescence cleaning solution, the main components of which comprise Tris buffer solution and the like, and the electrochemiluminescence cleaning solution is mainly applied to a beckmann full-automatic immunoassay analyzer. In addition, the prior art discloses a chemiluminescent cleaning solution, which mainly comprises phosphate buffer solution and the like, and is a cleaning solution developed for an yapei chemiluminescence immunoassay analyzer. Also, the prior art discloses a cleaning solution, which mainly comprises phosphate buffer solution, inorganic salt and the like, and can be suitable for enzymatic chemiluminescence and direct chemiluminescence systems.
However, the above cleaning solutions have the defects of unstable detection signal, abnormal background signal in some experiments, microsphere sedimentation and the like in the flow fluorescence method, and therefore improvements in these aspects are urgently needed.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a cleaning solution for flow-type fluorescence detection, which solves the problems of the prior art.
In order to achieve the above and other related objects, the present invention provides a cleaning solution for flow-type fluorescence detection, the cleaning solution comprising a base cleaning solution, proteins and alcohols, the base cleaning solution comprising a buffer salt and a surfactant, and a solvent of the base cleaning solution being water.
The buffer salt in the basic lotion is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride.
And the final concentration of the buffer salt is 5-10g/L based on the total volume of the basic washing liquid.
The surfactant is a nonionic surfactant. The non-ionic surfactant is one or more of Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305.
The basic lotion also comprises a preservative. The preservative is KroVinn series preservative or ProClin series preservative.
The pH value of the cleaning solution is 5.5-8.5.
In some embodiments, the proteinaceous agent is selected from one or more of BSA, casein, ovalbumin, or serum. The serum is selected from one or more of bovine serum, sheep serum and chicken serum.
When the protein is BSA, casein or ovalbumin, the final concentration of the protein in the cleaning solution is 1-50g/L based on the total volume of the cleaning solution.
The alcohol is selected from one or more of glycerol, mannitol, PVP, PVA and PEG.
When the alcohol is mannitol, PVP, PVA and PEG, the final concentration of the alcohol is 0.1-50 g/L.
The invention also provides the application of the cleaning solution in flow fluorescence detection.
As described above, the cleaning solution for flow-type fluorescence detection according to the present invention has the following advantageous effects: the cleaning solution provided by the invention can be used for satisfying the cleaning effect of the flow type fluorescence light-emitting platform, stabilizing the detection signal, reducing the microsphere sedimentation, considering the background signal and avoiding non-specific adsorption.
Detailed Description
The invention provides a cleaning solution for flow-type fluorescence detection, which comprises a basic cleaning solution, proteins and alcohols, wherein the basic cleaning solution comprises buffer salt and a surfactant.
In certain embodiments of the invention, the buffer salt in the base wash is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride.
In certain embodiments of the present invention, the buffer salt is present in a final concentration of 5 to 10g/L, such as 5 to 6g/L, 6 to 7g/L, 7 to 8g/L, 8 to 9g/L, 9 to 10g/L, based on the total volume of the base wash solution.
In certain embodiments of the present invention, the surfactant is a nonionic surfactant. The non-ionic surfactant is one or more of Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305. The surfactant may be present in a final concentration of 0.05% to 0.5% v/v, for example 0.05 to 0.1% v/v, 0.1 to 0.2% v/v, 0.2 to 0.3% v/v, 0.3 to 0.4% v/v, 0.4 to 0.5% v/v, based on the total volume of the base wash liquor.
In certain embodiments of the invention, the base wash solution further comprises a preservative. The preservative is KroVinn series preservative or ProClin series preservative.
The KroVin series preservative is, for example, KroVin100, KroVin 400, KroVin 500, or KroVin 750; the ProClin series preservative is, for example, ProClin50, ProClin150, ProClin200, ProClin300, ProClin950, or ProClin 5000.
The final concentration of the preservative is 0.05% to 0.2% v/v, for example 0.05% to 0.1% v/v, 0.1% to 0.15% v/v, 0.15% to 0.2% v/v, based on the total volume of the base lotion.
The solvent of the basic lotion is water.
The buffer salts, surfactants and preservatives in the base wash are to ensure basic cleaning and system stability. Only the basic washing liquid is used as the washing liquid of the flow type fluorescence luminescence method, and for different projects, the stability, the accuracy, the microsphere sedimentation and other aspects of the detection result are all insufficient.
The pH value of the cleaning solution is 5.5-8.5. Preferably, the pH value of the cleaning liquid is 6.0-8.0.
In some embodiments, the proteinaceous agent is selected from one or more of BSA, casein, ovalbumin, or serum. The serum is selected from one or more of bovine serum, sheep serum and chicken serum.
When the protein is BSA, casein or ovalbumin, the final concentration of the protein in the cleaning solution is 1-50g/L based on the total volume of the cleaning solution. In a preferred embodiment, the final concentration of the protein in the wash solution is 5-40g/L, such as 5-10g/L, 10-20g/L, 20-30g/L or 30-40 g/L.
When the protein is serum, the final concentration of the protein in the wash solution is 0.1% to 5% v/v, for example, 0.1 to 1% v/v, 1 to 1.5% v/v, 1.5 to 2% v/v, 2 to 2.5% v/v, 2.5 to 3% v/v, 3 to 3.5% v/v, 3.5 to 4% v/v, 4 to 4.5% v/v, 4.5 to 5% v/v.
In one embodiment, the alcohol is selected from one or more of glycerol, mannitol, PVP, PVA, PEG. Specifically, the PEG is selected from PEG200, PEG1000, PEG1500, PEG2000, PEG6000 and PEG 8000.
When the alcohol is glycerol, the final concentration of the glycerol is 0.05-0.5% v/v. The final concentration of glycerol is for example 0.05-0.1% v/v, 0.1-0.2% v/v, 0.2-0.3% v/v, 0.3-0.4% v/v or 0.4-0.5% v/v.
When the alcohol is mannitol, PVP, PVA and PEG, the final concentration of the alcohol is 0.1-50 g/L. The final concentration of the alcohol is, for example, 0.1-1g/L, 1-10g/L, 10-20g/L, 20-30g/L, 30-40g/L, or 40-50 g/L.
The invention also provides the application of the cleaning solution in flow fluorescence detection.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
The lotion of the invention consists of basic lotion, proteins and alcohols; wherein, in 1L of basic lotion, the adding amount of protein substances is 1-50g/L according to the final concentration, and the adding amount of alcohols is 0.1-50g/L according to the final concentration; the pH of the lotion is 5.5-8.5.
The base wash formulation used in the following examples was as follows:
preparing a basic washing solution by taking purified water as a solvent according to the following weight and volume, wherein the total volume is 1L after the preparation is completed:
| Na 2 HPO 4 | 1.1g |
| NaCl | 5.0g |
| KH 2 PO 4 | 0.5g |
| KCl | 1.4g |
| TritonX-100 | 1mL |
| ProClin300 | 1mL |
the pH of the base lotion prepared as above is 5.5-8.5, and the prepared base lotion is filtered through a 0.22 μm filter membrane and stored at room temperature.
Example 1A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| bovine serum | 5mL |
| BSA | 20g |
| Basic lotion | Constant volume to 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 2A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| sheep serum | 1mL |
| Casein protein | 10g |
| Basic lotion | Constant volume is 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2 to 8 ℃.
Example 3A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| BSA | 25g |
| ovalbumin | 5g |
| Basic lotion | Constant volume is 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 4A washing solution for flow-type fluorescence luminescence method
The preparation method of the washing liquid comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| glycerol | 2mL |
| PEG6000 | 10g |
| Basic lotion | Constant volume is 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 5A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| PVA | 5g |
| PEG200 | 30g |
| basic lotion | Constant volume is 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 6A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 5.5-8.5 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| PVP | 2g |
| PEG2000 | 20g |
| basic lotion | Constant volume is 1L |
The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 7A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 6.0-6.6 after the preparation is finished, and setting the total volume of the washing liquid to be 1L:
the washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 8A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to be 6.6-7.3 after the preparation is finished, and enabling the total volume of the washing liquid to be 1L:
| ovalbumin | 10g |
| Bovine serum | 2mL |
| PEG1000 | 25g |
| Mannitol | 5g |
| Basic lotion | Constant volume is 1L |
Example 9A washing solution for flow-type fluorescence luminescence method
The washing solution preparation method comprises the following steps: using basic washing liquid as a solvent, preparing washing liquid according to the following weight and volume, adjusting the pH value to 7.3-8.0 after the preparation is finished, and setting the total volume of the washing liquid to 1L:
| sheep serum | 5mL |
| Ovalbumin | 5g |
| PVA | 1g |
| PVP | 2g |
| Glycerol | 3mL |
| Basic lotion | Constant volume is 1L |
Example 10
And (3) testing the experimental result: flow-through fluorescent immunoassay assays were performed using examples 1-9 and the base wash, and the following experiments were performed:
10.1 cleaning Effect verification
The basic wash solution and the wash solutions of examples 1 to 9 were measured using a beckmann coulter flow cytometer Dxflex, and in the table, reagent a, reagent B, and reagent C were all reagents of a saccharide antigen 125 quantitative detection kit, a Myoglobin (MYO) detection kit, and an anti-double-stranded DNA antibody (dsDNA) detection kit, and the measurement steps were performed according to the instructions, and were roughly as follows:
the measurement items were carbohydrate antigen 125(CA125), Myoglobin (MYO), and anti-double-stranded DNA antibody (dsDNA), and the samples were CA125, MYO, and dsDNA calibrator 20 samples, and the average value of the detection signal of each sample is shown in table 1:
table 1: background wash and examples 1-9 wash background Signal detection results
| Background signal (RLU) | CA125 | MYO | dsDNA |
| Basic lotion | 2293 | 2192 | 1916 |
| Example 1 | 1213 | 1052 | 664 |
| Example 2 | 1256 | 1024 | 865 |
| Example 3 | 1125 | 995 | 542 |
| Example 4 | 1610 | 1332 | 1033 |
| Example 5 | 1584 | 1361 | 1201 |
| Example 6 | 1609 | 1488 | 994 |
| Example 7 | 1027 | 894 | 655 |
| Example 8 | 939 | 912 | 571 |
| Example 9 | 1028 | 985 | 601 |
The results in Table 1 show that the washing liquid of the invention has good washing effect and low background signal of each item.
10.2 System Signal stability
The same set of samples, which were respective calibrators of CY211, dsDNA, CK-MB, were measured at different times for the base wash and the washes of examples 1-9, respectively, using a beckman coulter flow cytometer Dxflex, after the first measurement was left at room temperature for 60min, and the signal values were again read using the beckman coulter flow cytometer Dxflex, and the results of the detection signals are shown in table 2:
table 2: base lotion and detection results of Signal stability of lotion systems of examples 1 to 9
The results in Table 2 show that the wash solutions of the invention perform better than the base wash solutions in terms of system signal stability.
10.3 sedimentation test
The working solutions of the antigen/antibody-coated magnetic beads were prepared using the base wash solution and examples 7 to 9, respectively, and mixed well to give 4 aliquots, each of which was 1 mL. Taking 200 mu L of supernatant at intervals, reading the number of spheres by a Beckmann Coulter flow cytometer (Dxflex), repeating the detection for 3 times, taking the average value, and taking the average value into the result statistics, wherein the detection results are shown in Table 3:
table 3: measurement of the Natural sedimentation Rate of the base lotion and the lotions of examples 7 to 9
The results in Table 3 show that the antigen/antibody coated magnetic beads settled faster in the base wash than in the examples 7-9 washes, and already at 20min there was a clear difference. In conclusion, it is believed that the wash solution of the present invention does not readily allow the microspheres to settle under natural gravity relative to the base wash solution.
10.4 accuracy experiment
The base wash and the washes of examples 7-9 were each assayed using a Beckmann Coulter flow cytometer (Dxflex) to obtain 20 samples of the same group, which were calibrators of CA125, CEA, NSE, CY211, and ProGRP, and the results of the average detection signal values are shown in Table 4:
table 4: accuracy test results for base washes and examples 7-9 washes
As can be seen from Table 4, the measured values of the 5 indexes are close to each other, and the difference between the two groups of data is not statistically significant (p) by t-test>0.05), and R) 2 The values are all larger than 0.990, and the high positive correlation is shown. The results show that the cleaning solution has good test accuracy, and the added components do not influence the test accuracy.
10.5 precision test
Three batches of cleaning solution were prepared, batch 1, batch 2 and batch 3, respectively, according to examples 7-9. An AFP sample with the concentration of 40ng/ml is taken, a Beckmann Coulter flow cytometer Dxflex is adopted to respectively measure the detection results of three batches of examples 7-9, each batch of cleaning solution is repeatedly detected for 20 times, the intra-batch and inter-batch variation coefficients are calculated, and the detection results are shown in a table 5:
table 5: examples 7-9 results of measuring precision of washing solutions
The results in Table 5 show that the wash solutions of the present invention have a variation coefficient in batch (CV) < 10% and a variation coefficient in batch (CV) < 15%, which meet the requirement that the variation coefficient in batch of general diagnostic reagents is less than 10% and the variation coefficient in batch is less than 15%. The above results show that the precision and repeatability of the detection of the washing solution of the invention are good.
10.6 lotion storage stability
1.4L of the cleaning solution was prepared according to examples 7 to 9, and the solution was divided into 7 bottles of about 200mL each, and left at 2 to 8 ℃ for 0 day for 2 months, 4 months, 6 months, 9 months, 12 months and 15 months, respectively, and then examined. CEA samples with concentration of 5 + -0.5 ng/mL were used for each 20-time repeat test, and the coefficient of variation was calculated, and the test results are shown in Table 5:
table 5: base lotion and storage stability test results of lotions of examples 7 to 9
The results in Table 5 show that the CV values of samples were within 5% when the washes prepared in examples 7 to 9 were stored at 2 ℃ to 8 ℃ for 15 months. From the reagent state, the washing liquid of 3 examples is clear and transparent, and no precipitation occurs, which indicates that the washing liquid of the invention has good storage stability.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. The cleaning solution for flow-type fluorescence detection is characterized by comprising a basic cleaning solution, proteins and alcohols, wherein the basic cleaning solution comprises buffer salt and a surfactant, and the solvent of the basic cleaning solution is water.
2. The washing solution for flow-type fluorescence detection according to claim 1, wherein the buffer salt in the base washing solution is one or more selected from the group consisting of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride; preferably, the final concentration of the buffer salt is 5-10g/L based on the total volume of the basic washing solution.
3. The cleaning solution for flow fluorescence detection according to claim 1, wherein the surfactant is a nonionic surfactant; preferably, the non-ionic surfactant is one or more of Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305; more preferably, the surfactant is present at a final concentration of from 0.05% to 0.5% v/v based on the total volume of the base wash solution.
4. The washing solution for flow fluorescence detection according to claim 1, wherein the base washing solution further comprises a preservative; preferably, the preservative is a KroVin preservative or ProClin preservative; more preferably, the final concentration of the preservative is from 0.05% to 0.2% v/v based on the total volume of the base wash.
5. The cleaning solution for flow fluorescence detection according to claim 1, wherein the pH of the cleaning solution is 5.5 to 8.5.
6. The washing solution for flow fluorescence detection according to claim 1, wherein the protein is one or more selected from BSA, casein, ovalbumin, and serum; preferably, the serum is selected from any one or more of bovine serum, sheep serum and chicken serum.
7. The washing solution for flow-type fluorescence detection according to claim 6, wherein when the protein is BSA, casein or ovalbumin, the final concentration of the protein in the washing solution is 1 to 50g/L based on the total volume of the washing solution;
and/or the presence of a gas in the gas,
when the protein is serum, the final concentration of the protein in the cleaning solution is 0.1-5% v/v based on the total volume of the cleaning solution.
8. The washing solution for flow-type fluorescence detection according to claim 1, wherein the alcohol is one or more selected from glycerol, mannitol, PVP, PVA, and PEG.
9. The washing solution for flow-type fluorescence detection according to claim 1, wherein when the alcohol is glycerol, the final concentration of glycerol is 0.05% to 0.5% v/v based on the total volume of the washing solution;
and/or the presence of a gas in the gas,
when the alcohol is mannitol, PVP, PVA and PEG, the final concentration of the alcohol is 0.1-50g/L based on the total volume of the cleaning solution.
10. Use of the cleaning solution for flow fluorescence detection according to any one of claims 1 to 9 in flow fluorescence detection.
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| CN117129668A (en) * | 2023-10-27 | 2023-11-28 | 江西赛基生物技术有限公司 | Cleaning solution for chemiluminescence immunoassay and preparation method and application thereof |
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| CN117129668A (en) * | 2023-10-27 | 2023-11-28 | 江西赛基生物技术有限公司 | Cleaning solution for chemiluminescence immunoassay and preparation method and application thereof |
| CN117129668B (en) * | 2023-10-27 | 2024-01-09 | 江西赛基生物技术有限公司 | Cleaning solution for chemiluminescence immunoassay and preparation method and application thereof |
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