CN113138270A - Multi-target object detection method for blood sample - Google Patents
Multi-target object detection method for blood sample Download PDFInfo
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- CN113138270A CN113138270A CN202010065819.1A CN202010065819A CN113138270A CN 113138270 A CN113138270 A CN 113138270A CN 202010065819 A CN202010065819 A CN 202010065819A CN 113138270 A CN113138270 A CN 113138270A
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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
- G01N33/54326—Magnetic particles
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention relates to a method for detecting multiple targets in a blood sample, which comprises the following steps: 1) preparing a reagent used for reaction, wherein the reagent comprises a coded magnetic microsphere series, and the surface of the coded magnetic microsphere is connected with a specific capture antibody; the coded magnetic microsphere series comprises magnetic microspheres with different particle sizes, and the particle sizes of the magnetic microspheres can be distinguished by signals of a flow analyzer and are used for identifying the types of the microspheres; 2) reacting and processing a blood sample to be detected with a reagent, removing interference components, and forming a microsphere-based fluorescence-labeled reaction compound; 3) and (3) sending the reaction compound into a flow type optical detection system for analysis, obtaining characteristic signals, identifying the types of the microspheres, and simultaneously detecting the multiple target substances in the blood sample. Compared with the prior art, the invention has the advantages of stable characteristics of the coding microspheres, use of a whole blood sample, realization of multiple detection and the like.
Description
Technical Field
The invention relates to the field of biomolecule analysis and detection, in particular to a multi-target detection method for a blood sample.
Background
Currently, biomolecule analysis, such as protein, nucleic acid, etc., which is performed in large quantities in clinical diagnosis, is generally performed on devices such as biochemical analyzers, chemiluminescence immune analyzers, etc., using serum or plasma samples, but preparing serum or plasma requires coagulation and centrifugation operations, consuming much time and labor.
For acute diseases, a whole blood sample such as a specific protein analyzer is used for rapid detection of characteristic proteins, but the specific protein analyzer generally uses an immunoturbidimetry method such as an immunotransmission or an immunoscattering turbidimetry method, and due to the limitation of the detection principle, the detection sensitivity and precision of the turbidimetry method are not high.
The objects detected by the flow analyzer are cells and microparticles suspended in a liquid in an independent state. A flow cytometry analyzer, which uses hydrodynamic focusing principle (hydrodynamic focusing) to arrange sample cells or microspheres to be analyzed into a line by sheath fluid in an optical flow chamber, and the sample cells or microspheres rapidly flow through detection laser beams one by one; the high-precision optical system collects and detects the excited multi-angle scattered light and the fluorescence with multiple wavelengths; through complex electronic signal processing and computer data analysis, the information of a plurality of physical structural characteristics and biological expression characteristics of tens of thousands of cells or microspheres can be obtained in a short time. The flow cytometry can be equipped with multi-color laser, and multi-path scattered light and fluorescence detector to realize powerful cell and microsphere analysis function.
The scattered light at different angles reflects the physical structural characteristics of the cells or the microspheres, and for example, the forward scattered light and the side scattered light reflect the information of the size, the internal structure and the like of the microspheres.
Lateral fluorescence detection at different wavelengths can reflect the characteristics of biomolecule expression. For example, in order to detect the expression of a specific protein molecule, a suitable fluorescent marker is used for labeling, such as PE fluorescent dye under the excitation of 532nm laser and the detection of emitted fluorescence at 585nm wavelength, so that quantitative information of the specific protein molecule can be obtained, and an important basis is provided for the diagnosis of diseases.
The flow analyzer can analyze not only cells but also microparticles, and one typical application is microsphere-based soluble protein molecular analysis, i.e., flow liquid phase multiple protein quantification.
The special microspheres have characteristics or markers for identifying species, such as fluorescent dyes with various intensities, are respectively coated with specific antigens, antibodies or nucleic acid probes, react with a blood sample, are specifically combined with soluble protein molecules to be detected in the sample, and are connected with fluorescent markers.
After the fluorochrome on the microsphere is excited by a light source with a specific wavelength, the type of the microsphere can be identified by detecting a characteristic signal, such as fluorescence with a specific wavelength or specific wavelengths, namely the type of the soluble protein molecule to be detected. And the other light source excites a fluorescent marker connected with the protein molecule to be detected, such as PE fluorescent dye, and fluorescence detection is carried out in a corresponding fluorescence channel. Because the number of the connected fluorescent labels is in direct proportion to the number of the soluble protein molecules to be detected combined on the microspheres, the fluorescence intensity excited by the fluorescent labels is detected and used for quantitative analysis of specific proteins.
In the flow-type fluorescence analysis system, various soluble proteins, cytokines, autoantibodies, specific nucleic acid sequences, and the like can be quantitatively analyzed and detected by a multicolor light source and a plurality of scattered light and fluorescence detections.
In a typical flow-type liquid-phase multiplex detection system, as shown in fig. 1, a sheath fluid is arranged in a column of sample cells or microspheres to be analyzed in an optical flow cell 21, and rapidly flows through a detection laser beam one by one, and the system is provided with two laser light sources, wherein one laser light source 12 and corresponding lateral fluorescence detectors 42 and 43 are used for identifying the type of the microsphere, i.e., the type of an object to be detected, and the other laser light source 11 and corresponding lateral fluorescence detector 51 are used for detecting the intensity of labeled fluorescence, which is proportional to the number of the object to be detected.
By means of the microspheres and specific markers, dozens of protein molecules can be simultaneously detected in a micro sample (25-50ul), such as multiple characteristic proteins related to immunity, and compared with other immunoassay methods, the used sample amount is greatly reduced. Moreover, the sensitivity and precision of flow-type fluorescence detection are higher than those of the immuno-transmission or immuno-scattering turbidimetry.
The microspheres carry a label for identifying the species, such as a fluorescent dye, which is now commonly used to include organic fluorescent dyes or quantum dots. Generally, the microspheres have the same size, and the emission wavelength and the intensity of the fluorescent dye carried on each microsphere are different, so that one-dimensional or two-dimensional coding of the microspheres is realized, as shown in fig. 3; after the fluorescent dye is excited by laser, the type of the microsphere can be identified by detecting the characteristic signal, for example, after a special fluorescent dye is excited by 635nm laser, the fluorescence with the wavelength of 670nm and/or 780nm is detected for identifying the type of the microsphere.
The fluorescent dye is used for identifying the types of the microspheres, the coding mode has the advantages that the coding types are various, and the types of the microspheres can reach dozens to one hundred by combining two-dimensional coding;
but the disadvantages are:
1. the cost of the fluorescent microsphere is high, the manufacturing process is fine and complex, and the fluorescence stability is poor;
2. the cost of the instrument is high, the optical adjustment is difficult, and the electronic and software processing is complex:
the optical system usually needs two laser light sources, one laser is used for identifying the type of the microsphere, namely the type of the target object to be detected, and the other laser is used for detecting the intensity of the marked fluorescence, wherein the fluorescence intensity is in direct proportion to the number of the target object to be detected; in addition, each light source needs to be correspondingly provided with a plurality of high-sensitivity photodetectors, such as PMT or APD;
the optical adjustment is difficult, the distance between two detection points focused by laser is about 100 micrometers, and the two detection points need to be stable for a long time;
the electronic and software processing is complex, and the alignment, identification and the like of signals generated by two laser detection points are required;
3. the types of the microspheres in the multiple detection reagent are too many, and if the types of the microspheres can reach dozens, the risks of nonspecific binding and cross interference of detection items are large, the requirements on the specificity of a detection antibody are high, and the difficulty in development and verification is large.
Currently, new needs in clinical diagnostics are emerging:
requiring the use of whole blood samples, rapid detection of characteristic proteins can be performed for acute conditions, such as point-of-care testing (POCT);
the sample amount is required to be small, so that the pain of a patient is reduced, and particularly, the patient with difficulty in taking blood for infants is suffered;
high sensitivity and low system cost are also required.
Therefore, the technical problems to be solved are:
the difficulty and challenge of using whole blood samples, which contain a large amount of blood cells and various impurities, causes nonspecific interference compared to serum samples, and the size of the blood cells is close to that of microspheres, which causes great interference in the flow optical detection for the identification of the microspheres.
The requirement of using a small amount of samples, being capable of carrying out multiple detections of a single sample, needs a stable and economic microsphere coding mode, reduces the cost of the microspheres and a detection system, and improves the reliability.
Disclosure of Invention
The present invention is directed to a method for detecting multiple targets in a blood sample, which overcomes the above-mentioned drawbacks of the prior art.
The purpose of the invention can be realized by the following technical scheme:
a method for detecting multiple targets in a blood sample, comprising the steps of:
1) preparing a reagent used for reaction, wherein the reagent comprises a coded magnetic microsphere series, and the surface of the coded magnetic microsphere is connected with a specific capture antibody; the coded magnetic microsphere series comprises magnetic microspheres with different particle sizes, and the particle sizes of the magnetic microspheres can be distinguished by signals of a flow analyzer and are used for identifying the types of the microspheres, namely identifying the types of the target objects to be detected.
2) Reacting and processing a blood sample to be detected with a reagent, removing interference components, and forming a microsphere-based fluorescence-labeled reaction compound;
3) and (3) sending the reaction compound into a flow type optical detection system for analysis, obtaining characteristic signals, identifying the types of the microspheres, and simultaneously detecting the multiple target substances in the blood sample.
The blood sample to be detected is whole blood, and is diluted in the reaction and treatment of the whole blood sample, and a component capable of inhibiting non-specific reaction is added, and the magnetic microspheres are combined with a magnet and washed to remove blood cells and non-specific interference.
In the step 3), the flow optical detection system distinguishes the particle size of the microspheres through detection of scattered light signals, is used for identifying the types of the microspheres, detects the fluorescence intensity excited by the fluorescent label, and is used for quantitative analysis of the target object.
The flow type optical detection system is a minimum detection system consisting of a laser light source, a scattered light detection channel and a fluorescence detection channel, wherein the scattered light detection channel is used for distinguishing the particle size of the microspheres and identifying the types of the microspheres, and the fluorescence detection channel is used for detecting the fluorescence intensity of the fluorescence label excited by the same laser light source and quantitatively analyzing the target object.
In the step 3), the flow type optical detection system distinguishes the particle size of the microspheres through electrical impedance signal detection, is used for identifying the types of the microspheres, detects the fluorescence intensity excited by the fluorescence label, and is used for quantitative analysis of the target object.
In the step 2), the interference components are removed through dilution, capture, labeling and washing/separation, so as to obtain the microsphere-based fluorescence labeled reaction complex.
The particle size range of the coded magnetic microspheres is 0.5-20 microns.
The particle size of the coded magnetic microspheres is 2-10.
The blood sample to be detected is serum or plasma.
The flow optical detection system adopts side scattering light to detect and distinguish the particle size of the microspheres and is used for identifying the types of the microspheres.
Compared with the prior art, the invention has the following advantages:
1. compared with the existing fluorescent coding microspheres, the particle size coding microspheres adopted by the invention have stable characteristics and low cost.
2. And the flow-type fluorescence analysis is adopted to realize multiple detection, so that the sample usage amount is greatly reduced.
3. The complexity and cost of the detection system are greatly reduced, the optical system only comprises a laser light source, a scattered light detection channel and a fluorescence detection channel, a minimum detection system can be formed, the complexity of electronic signal processing and software processing is greatly reduced, the side scattered light detection is adopted to detect the particle size of the microsphere, and the higher distinguishing sensitivity can be achieved.
4. The invention applies magnetic microspheres, is similar to sample treatment of chemiluminescence immunoassay, washes and separates target detection objects, removes blood cells and nonspecific interference, improves specificity, simplifies microsphere coding, and is suitable for whole blood sample detection.
5. The number of types which can be coded is moderate, 2-10 types, and the reagent development difficulty is economic and controllable.
Drawings
FIG. 1 is a schematic diagram of a typical flow-type liquid phase multiplex detection system.
Fig. 2 is a schematic diagram of a flow optical detection system according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of a conventional fluorescence-encoded microsphere.
FIG. 4 is a schematic diagram of a series of encoded microspheres of the present invention.
FIG. 5 is a detection scattergram of the present invention.
FIG. 6 is a flow chart of a method of the present invention.
FIG. 7 is a schematic of the structure of a microsphere-based fluorescently labeled reaction complex.
Description of the drawings
11. Laser light source, 12, laser light source, 21, optical flow cell, 31, forward scattered light FSC _1 detector, 51, side scattered light SSC _2 detector, 52, side fluorescent light FL1-A detector, 32, forward scattered light FSC _2 detector, 41, side scattered light SSC _2 detector, 42, side fluorescent light FL2-B detector, 43, side fluorescent light FL2-C detector.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Examples
As shown in fig. 6, the present invention provides a method for detecting multiple targets in a blood sample, which specifically comprises the following steps:
1) preparing reagents used for reaction, wherein the reagents comprise a series of mixed solution of coded magnetic microspheres, a buffer solution, a mixed solution of PE fluorescent labeled antibodies, a washing solution and the like, wherein the surfaces of the coded magnetic microspheres are respectively connected with specific capture antibodies;
the coded magnetic microsphere series comprises a plurality of superparamagnetic microspheres with different particle sizes, such as 7 magnetic microspheres with diameters of 1, 2 and 3 … … 7um respectively, as shown in fig. 4, the particle sizes can be distinguished by scattered light signals of a flow analyzer, and the coded magnetic microsphere series is used for identifying the types of the microspheres, namely the types of biomolecules to be detected.
The surface of each particle size magnetic microsphere is coated with different specific antigen, antibody or nucleic acid probe, such as CRP, PCT, IL-6 and other capture antibodies, for specific binding with soluble protein molecules to be detected in the sample when reacting with blood sample to be detected.
2) The blood sample to be tested is reacted and processed with a reagent, in this case whole blood is used as the blood sample, and the processes include dilution, capture, labeling, washing/separation, and the like.
Diluting: pretreating the whole blood sample by using a buffer solution, diluting the blood sample, wherein the buffer solution contains a component capable of inhibiting non-specific reaction;
capturing: the coded magnetic microspheres react with a blood sample, and specific capture antibodies on the coded microspheres and soluble protein molecules in the sample respectively perform specific binding reaction;
marking: adding a detection antibody and a fluorescent label, such as PE fluorescent dye, to connect with the protein molecules captured on the microsphere to generate a microsphere-based fluorescent-labeled reaction complex, as shown in FIG. 7;
washing/separation, applying a magnet to the reactant, attracting and fixing the magnetic microspheres, and sucking and discharging other solutions for removing blood cells, impurities, and nonspecific interference. Washing and separation are applied for a plurality of times during the reaction process according to requirements.
The blood sample to be detected is whole blood, the magnetic microspheres are combined with the magnet and washed, blood cells and impurities in the whole blood sample are separated and removed, and interference on flow type optical detection of the microspheres is avoided.
In this case, the blood sample to be tested may also be serum or plasma, and the magnetic microspheres are combined with a magnet and washed to remove impurities and non-specific interference.
3) And (4) flow optical detection.
The reaction compound is sent into a flow optical detection system for analysis, characteristic signals are obtained, and the simultaneous analysis of multiple targets in the blood sample, namely multiple detection, is carried out.
As shown in FIG. 2, the flow-type optical detection system used in this example uses a laser light source, such as a 532nm laser, and the characteristic signal is a scattered light signal, including forward scattered light or side scattered light, to detect and distinguish the particle size of the microspheres for identifying the type of the microspheres.
The flow-type optical detection system uses the same laser light source to excite a fluorescent marker connected with a protein molecule to be detected, such as PE fluorescent dye, and performs fluorescent detection in a corresponding fluorescent channel. Because the number of the connected fluorescent labels is in direct proportion to the number of the soluble protein molecules to be detected bound on the microspheres, the fluorescence intensity of the excited fluorescent labels is detected for quantitative analysis of specific proteins, for example, the PE fluorescent dye is excited by 532nm laser, the fluorescence intensity is detected at a wavelength of about 585nm, the number of the protein molecules to be detected is reflected, and a flow optical detection scatter diagram is shown in FIG. 5.
In addition, in the field of blood cell detection, electrical impedance detection (coulter method) is commonly used for counting and classifying cells. In an electrolyte environment, a high-voltage direct-current electric field is established through a small hole, and when cells or particles flow through the small hole, the change of an electric signal is caused by the change of the electric impedance, so that the high-voltage direct-current electric field can be used for cell counting; meanwhile, the amplitude of the electric signal change is related to the size of the particles, so that the cell particle size can be detected and distinguished for cell classification.
In the flow type optical detection system, an optical flow chamber is used as a small hole, so that electrical impedance detection can be established and realized, and characteristic signals are electrical impedance signals to detect and distinguish the particle size of microspheres and identify the types of the microspheres; a light source is used to excite the fluorescent label connected with the protein molecule to be detected, such as PE fluorescent dye, and the fluorescence detection is carried out in the corresponding fluorescent channel. Because the number of the connected fluorescent labels is in direct proportion to the number of the soluble protein molecules to be detected combined on the microspheres, the fluorescence intensity of the excited fluorescent labels is detected and used for quantitative analysis of specific protein, for example, PE fluorescent dye is excited by a 530nm light source, the fluorescence intensity is detected at a wavelength of about 580nm, and the number of the protein molecules to be detected is reflected.
Claims (10)
1. A method for detecting multiple targets in a blood sample, comprising the steps of:
1) preparing a reagent used for reaction, wherein the reagent comprises a coded magnetic microsphere series, and the surface of the coded magnetic microsphere is connected with a specific capture antibody; the coded magnetic microsphere series comprises magnetic microspheres with different particle sizes, and the particle sizes of the magnetic microspheres can be distinguished by signals of a flow analyzer and are used for identifying the types of the microspheres, namely identifying the types of the target objects to be detected.
2) Reacting and processing a blood sample to be detected with a reagent, removing interference components, and forming a microsphere-based fluorescence-labeled reaction compound;
3) and (3) sending the reaction compound into a flow type optical detection system for analysis, obtaining characteristic signals, identifying the types of the microspheres, and simultaneously detecting the multiple target substances in the blood sample.
2. The method of claim 1, wherein the blood sample is whole blood, and wherein the whole blood sample is diluted during the reaction and treatment, and wherein a component capable of suppressing the non-specific reaction is added, and wherein the magnetic microspheres are bound to a magnet and washed to remove blood cells and non-specific interference.
3. The method as claimed in claim 1, wherein in step 3), the flow optical detection system distinguishes the particle size of the microspheres by detecting the scattered light signals, so as to identify the types of the microspheres, and detects the fluorescence intensity excited by the fluorescent label for quantitative analysis of the target.
4. The method of claim 3, wherein the flow-based optical detection system is a minimum detection system comprising a laser source, a scattered light detection channel and a fluorescence detection channel, the scattered light detection channel distinguishes the particle size of the microspheres for identifying the type of the microspheres, and the fluorescence detection channel detects the fluorescence intensity of the fluorescence label excited by the same laser source for quantitative analysis of the target.
5. The method as claimed in claim 1, wherein in step 3), the flow optical detection system distinguishes the particle size of the microspheres by electrical impedance signal detection for identifying the type of the microspheres, and detects the fluorescence intensity excited by the fluorescent label for quantitative analysis of the target.
6. The method for detecting multiple targets in a blood sample according to claim 1, wherein in step 2), the interfering components are removed by dilution, capture, labeling and washing/separation to obtain the microsphere-based fluorescently labeled reaction complex.
7. The method of claim 1, wherein the encoded magnetic microspheres have a particle size in the range of 0.5-20 microns.
8. The method of claim 1, wherein the encoded magnetic microspheres have a particle size of 2-10 types.
9. The method of claim 1, wherein the blood sample is serum or plasma.
10. The method of claim 4, wherein the flow optical detection system employs side scatter detection to distinguish the microsphere particle size for identification of the type of microsphere.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114088678A (en) * | 2021-12-03 | 2022-02-25 | 嘉兴市唯真生物科技有限公司 | Flow type fluorescence detection method |
| CN114280014A (en) * | 2021-11-30 | 2022-04-05 | 杭州迪英加科技有限公司 | Independent accounting reagent for AI interpretation |
| CN114544472A (en) * | 2022-01-28 | 2022-05-27 | 苏州才博医学科技有限公司 | Method for controlling cross reaction in flow type dot matrix instrument detection |
| WO2023138162A1 (en) * | 2022-01-18 | 2023-07-27 | 北京流荧生物科技有限公司 | Sample detection method, apparatus and system, and electronic device and computer-readable medium |
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2020
- 2020-01-20 CN CN202010065819.1A patent/CN113138270A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114280014A (en) * | 2021-11-30 | 2022-04-05 | 杭州迪英加科技有限公司 | Independent accounting reagent for AI interpretation |
| CN114088678A (en) * | 2021-12-03 | 2022-02-25 | 嘉兴市唯真生物科技有限公司 | Flow type fluorescence detection method |
| WO2023138162A1 (en) * | 2022-01-18 | 2023-07-27 | 北京流荧生物科技有限公司 | Sample detection method, apparatus and system, and electronic device and computer-readable medium |
| CN114544472A (en) * | 2022-01-28 | 2022-05-27 | 苏州才博医学科技有限公司 | Method for controlling cross reaction in flow type dot matrix instrument detection |
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Application publication date: 20210720 |