CN115792219A - Application of quantum dot labeled antibody reagent in cell detection - Google Patents
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Abstract
The invention provides a quantum dot labeled antibody reagent, which is a CdX prepared by taking a monometallic isotope of Cd as a unique Cd source; wherein X = S, se or Te. Cd in the quantum dot CdSe/ZnS is metal element cadmium or isotope thereof: cd-106, cd-108, cd-110, cd-111, cd-112, cd-113, cd-114 or Cd-116. The quantum dot labeled antibody reagent is prepared by coupling quantum dots and an antibody. The quantum dot labeled antibody reagent can be used for fluorescence flow detection or mass spectrometry flow detection. The problem that the matched reagent in the prior art cannot be universally used in a flow system and a mass spectrum flow system of fluorescence analysis is solved. The method is convenient for analyzing multiple protein levels of single cells, has the characteristics of multiple channels, high analysis speed and high sensitivity, and has great clinical application value.
Description
Technical Field
The invention relates to a medical appliance, in particular to an application of a quantum dot labeled antibody reagent in cell detection, in particular to an application of the quantum dot labeled antibody reagent in the cell detection
Background
Quantum Dots (QDs) generally refer to semiconductor nanocrystals having radii smaller than or close to the exciton bohr radius. The quantum dot belongs to one of a large class of new materials, namely solution nanocrystalline. Solution nanocrystals have the dual properties of crystals and solutions, unlike other nanocrystal materials, quantum dots are based on semiconductor crystals. The grain diameter is between 1 and 100 nanometers, and each grain is single crystal. The name of quantum dots is derived from the quantum confinement effect or quantum size effect of semiconductor nanocrystals. When the semiconductor crystal is small to the nanometer scale, different sizes can emit light with different colors. Such as cadmium selenide (CdSe), emits blue light at 2 nm, red light at 8 nm, green yellow orange at the middle, and so on. The compounds have unique fluorescent nano-effect, such as wide and continuous distribution of excitation spectrum, narrow and symmetrical emission spectrum, high fluorescence intensity, slow bleaching rate and high sensitivity, and the light-emitting wavelength can be regulated and controlled by changing particle size and composition, so that the compounds have wide application prospects in the aspects of luminescent materials, photocatalysis, photosensitive sensors, fluorescent probe labeling and the like. Especially, the quantum dots composed of IIB group and VIA group elements, which are called II-VI type quantum dots for short (among which, cdX (x = S, se, te) is mainly studied, such as CdSe cadmium selenide), have important values in biomedical fluorescent probe markers and sensors due to the special excellent fluorescence emission properties in visible and near infrared spectrum regions. The CdSe quantum dots and the CdSe quantum dots with core-shell structures such as CdSe/ZnS and CdSe/CdS quantum dots are very suitable for visual observation due to the fact that the emission wavelength covers the whole visible light region range, and therefore special attention is paid to the CdSe quantum dots and the CdSe quantum dots with core-shell structures.
Flow cytometry is a widely used cell analysis technique in the biomedical field. At present, flow detection systems are mainly divided into two categories, one is a typical flow cytometry detection system based on an optical detection system, the detection principle is to dye cells through fluorescein labeled antibodies, and the optical detection system is used for analyzing the multiple protein levels of a single cell through analyzing different fluorescein excitation light spectrums. The other type is a novel flow system based on a mass spectrum detection system, and the detection principle is that cells are stained through an antibody marked by a metal element, and the content of the metal element is analyzed through a mass spectrum detector so as to analyze multiple protein levels of a single cell. Both the two flow detection systems have the characteristics of multiple channels, high analysis speed and high sensitivity. Because of different principles, the existing matching reagents cannot be used universally, and the development of the detection field is limited.
Quantum Dots immunofluorescence chemistry (QD-IHC), also called Quantum Dots immunofluorescence chemistry, is a technique for detecting antigenic substances in tissues or cells by using Quantum Dots to mark specific antibodies as probes according to the principle of antigen-antibody specific binding. The flow-type fluorescence detection method is different from the traditional fluorescence flow-type fluorescence detection method in that quantum dots replace fluorescent dyes (such as FITC, PE and the like) to be used as marker molecules, and cells or particles which can be marked by the fluorescent molecules can be detected by a flow cytometer, so that the quantum dots or quantum dot coding microspheres are widely applied to flow cytometry, such as cytobiology analysis (quantitative analysis of cell cycle and sorting of cells in different cell cycle phases; analyzing the relationship between biomacromolecules such as DNA, RNA, antigen, oncogene expression products and the like and the cell proliferation cycle, carrying out chromosome karyotype analysis), oncology analysis (the relationship between the cell cycle or DNA ploid and cell surface receptor and antigen expression is researched by utilizing an immune sandwich reaction generated by the tumor marker and a specific antibody coupled with quantum dots, and the fluorescence yield of the immune quantum dots of a detection reactant can represent the content of the tumor marker), immunologic analysis (the relationship between the cell cycle or DNA ploid and cell surface receptor and antigen expression is researched, the typing and purification of immunocompetent cells are carried out, the relationship between lymphocyte subsets and diseases is analyzed, the diagnosis of immunodeficiency diseases such as AIDS (acquired immune deficiency syndrome), the immunologic monitoring after organ transplantation and the like) and microbial detection (the multi-parameter analysis of bacteria) and the like.
Different from fluorescence flow cytometry, the mass flow technology uses a stable heavy metal isotope (mainly lanthanide) to replace a fluorescent group to label an antibody, and at present, no report that quantum dots are used as a labeling molecule to be applied to mass flow detection exists.
The quantum dot antibody is obtained by coupling quantum dots and antibody molecules; the quantum dots can also be coupled with the antibody by modifying carboxyl functional groups on the surface of the microsphere formed by macromolecular self-assembly. Due to the unique fluorescence effect of the quantum dot antibody, the antibody can be used for fluorescence flow cytometry; due to the characteristics that the quantum dot antibody contains Cd metal and the content of the Cd metal in biological cells is very low, the inventor tries to apply the quantum dot antibody to mass cytometry and succeeds in mass cytometry. Therefore, the development of new application of the quantum dot antibody has important significance.
Disclosure of Invention
The invention aims to overcome the defects and research and design the application of the quantum dot antibody reagent in fluorescence flow detection and mass spectrometry flow detection.
The quantum dot antibody is obtained by coupling CdSe/ZnS (cadmium selenide/zinc sulfide) quantum dots with antibody molecules; the quantum dots can also be coupled with the antibody by modifying carboxyl functional groups on the surface of the microsphere formed by macromolecular self-assembly.
The invention provides a quantum dot labeled antibody reagent and application thereof in cell detection.
Specifically, the invention provides a quantum dot labeled antibody reagent, which is prepared by coupling CdX (X = S, se, te) quantum dots (sold or sold by a third party) and an antibody (sold in the market) which are prepared by taking a single metal isotope of Cd as a unique Cd source.
In the quantum dots CdSe/ZnS used in the invention, cd (cadmium) is a metal element cadmium (Cd) and an isotope thereof: cd-106, cd-108, cd-110, cd-111, cd-112, cd-113, cd-114 and Cd-116. The antibody used is an antibody capable of specifically binding to an intracellular protein, an extracellular protein, an intracellular nucleic acid, or the like, and the antibody may be an antibody of any species, for example, human, murine, monkey, horse, alpaca, or the like, and the source of the antibody is not limited, and the type of the antibody may be a monoclonal antibody or a polyclonal antibody. Preferably human anti-CD 3 antibody, human anti-CD 45 antibody or mouse anti-CD 3e antibody.
The coupling of the quantum dot and the antibody is prepared by the following method: the method comprises the following steps:
the method refers to a quantum dot nanosphere labeled fluorescence immunoblotting detection method which is disclosed in patent No. CN 108663345A.
Adding 0.5-5 mg of quantum dot nanospheres (CdX (X = S, se, te) quantum dot nanospheres, kunlun organisms) and 0.1-1 mmol/L of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) solution into phosphate buffer solution (pH4.0-6.0 sodium dihydrogen phosphate solution), uniformly mixing and activating for thirty minutes, adding 50-500 ug/ml of antibody after activation, centrifuging for 5-60 minutes at 5000-20000 rpm after reaction for thirty minutes, discarding supernatant, adding 1-10 mg/ml of bovine serum albumin, sealing for 2 hours, and storing at 4 ℃ to obtain the quantum dot labeled antibody reagent Qdot-CD quantum dot-antibody conjugate.
The invention also provides a detection method of the quantum dot-antibody reagent for fluorescence flow detection.
The detection method of the quantum dot labeled antibody reagent for fluorescence flow detection comprises the following steps:
(1) Centrifuging and filtering a human peripheral blood sample, and then resuspending the human peripheral blood sample into a single cell suspension by using 1ml of phosphate buffer solution;
(2) Adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or reversing and uniformly mixing, incubating for 15 minutes, centrifuging for 10 minutes at 4 ℃ by 450g (the centrifugation condition is 450 times of gravity acceleration) to precipitate white blood cells, sucking and removing supernatant, and resuspending the cells by 1mL of phosphate buffer;
(3) Adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating for 10 minutes at room temperature (25 ℃) to obtain a quantum dot labeled antibody reagent Qdots-CD;
(4) Adding the prepared quantum dot labeled antibody Qdots-CD into the single cell suspension according to the needs by using 1-5 ug of quantum dot labeled antibody for each 3E6 cell, carrying out vortex oscillation for 10 seconds, uniformly mixing, and incubating for 30 minutes at the room temperature of 25 ℃;
(5) 500g (the centrifugal condition is 500 times of the gravity acceleration), centrifuging for 5 minutes at 4 ℃, discarding the supernatant, adding 2 ml of PBS to wash the cells, and repeating for 3 times;
(6) Adding 100 microliters of 16% formaldehyde solution into the Cell suspension, fixing at the room temperature of 25 ℃ for 10 minutes, adding 2 milliliters of Staining Buffer (chening in an biological Cell stabilizing Buffer) to wash the cells, repeating the process for 3 times, and resuspending 1 milliliter of Staining Buffer to prepare for detection;
(7) And detecting the marked cell sample by a fluorescence Flow detection system (Beckman Dx Flex Flow Cytometer), wherein an original data signal output by the fluorescence Flow detection system is a fluorescence intensity signal of a fluorescence label, and the fluorescence intensity signal is converted into expression abundance information of a corresponding target protein in the cell according to fluorescence excitation wavelength and emission wavelength of quantum dots on the quantum dot marked antibody and the fluorescence excitation wavelength and the emission wavelength of the quantum dots on the quantum dot marked antibody.
The invention also provides a detection method of the quantum dot labeled antibody reagent for mass spectrometry flow detection, which comprises the following steps:
(1) Centrifuging and filtering a human peripheral blood sample, and then resuspending the human peripheral blood sample by using 1ml of phosphate buffer solution (PBS-phosphate buffer solution) to prepare a single cell suspension;
(2) Adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or reversing and uniformly mixing, incubating for 15 minutes at room temperature (25 ℃), centrifuging for 10 minutes at 4 ℃ for precipitating white blood cells at 450g (the centrifugation condition is 450 times of the gravity acceleration), carefully sucking and removing supernatant, and resuspending cells by 1mL of phosphate buffer;
(3) Adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating for 10 minutes at room temperature (25 ℃) to obtain a quantum dot labeled antibody reagent Qdots-CD;
(4) Adding the prepared quantum dot labeled antibody Qdots-CD into the single cell suspension by using 1-5 ug of quantum dot labeled antibody per 3E6 cells, performing vortex oscillation for 10 seconds, mixing uniformly, and incubating for 30 minutes at room temperature (25 ℃);
(5) 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer solution to wash the cells, and repeating for 3 times;
(6) The cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, and the cells were washed with 2 ml of staining buffer, repeated 3 times, and resuspended in 1ml of staining buffer, and prepared for detection.
(7) And detecting the marked cell sample by a mass flow system, wherein an original data signal output by the mass flow detection system is a metal atom abundance signal of a metal element label, and converting the original data signal into expression abundance information of a corresponding target protein in the cell according to the metal label information carried by the corresponding antibody.
The invention provides a quantum dot labeled antibody reagent, which is prepared by coupling CdX (X = S, se, te) quantum dots (sold or customized by a third party) and an antibody (sold) which are prepared by taking a single metal isotope of Cd as a unique Cd source.
The antibody reagent can be used for flow cytometry detection and can also be used for novel mass cytometry flow cytometry detection. Flow cytometers include classical fluorescence flow cytometers and novel mass cytometry flow cytometers. The label of the antibody reagent for detection is metal element cadmium (Cd) and isotope thereof: cd-106, cd-108, cd-110, cd-111, cd-112, cd-113, cd-114 or Cd-116.
The antibody reagent package of the present invention constitutes a kit comprising: the quantum dot-antibody reagent of the invention; containers such as a flow tube, a centrifuge tube, a penicillin bottle and the like for containing the quantum dot-antibody reagent; and instructions for use.
The quantum dot labeled antibody reagent can be used for fluorescence flow detection or mass spectrometry flow detection. The problem that a matched reagent in the prior art cannot be universally used in a flow system and a mass spectrum flow system of fluorescence analysis is solved. The method is convenient to analyze multiple protein levels of single cells, has the characteristics of multiple channels, high analysis speed and high sensitivity, and has great clinical application value.
Drawings
FIG. 1, example 1, the Cd (cadmium) signal carried by the quantum dots, i.e., the CD3 positive signal binding to the cell surface, can be detected by mass spectrometry flow system.
Fig. 2, example 2 the fluorescence signal of the quantum dots, i.e. a CD3 positive signal indicating binding to the cell surface, can be detected by a fluorescent flow system.
FIG. 3, example 4 the signal of Cd (cadmium) 106 carried by quantum dots can be detected by mass flow system, i.e. CD3 positive signal binding to cell surface is illustrated
FIG. 4 example 5 CdS-CD4 flow antibody reagent using Cd106 monoisotope for murine peripheral blood lymphocytes in fluorescence flow detection and mass spectrometry flow detection
The left panel shows the fluorescence flow results and the right panel shows the mass flow results
FIG. 5 example 6: the results show that the left picture of the CdS-CD3e flow-type antibody reagent using Cd106 monoisotope for mouse peripheral blood lymphocytes in fluorescence flow detection and mass spectrometry flow detection is the fluorescence flow result, and the right picture is the mass spectrometry flow result
Detailed Description
The reagents used in this example are commercially available unless otherwise indicated.
Example 1: this example illustrates the use of quantum dot conjugated antibodies in CdS-CD3 flow antibody reagents for human peripheral blood lymphocytes.
Adding 1 mg of quantum dot nanosphere (source: kunlou organism) and 6 microliters of 20mg/mL EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) solution into 200 microliters of phosphate buffer (25mM, pH 6.0), uniformly mixing and activating for thirty minutes, centrifuging at a high speed at 15000rpm for 25 minutes after activation is completed, discarding the supernatant, resuspending with 100 microliters of phosphate buffer (25mM, pH 6.0), adding 100 micrograms of CD3 antibody (source: biolegend), adjusting the reaction volume to 350 microliters, centrifuging at 8000rpm for 5 minutes after reaction for thirty minutes, discarding the supernatant, adding 200 microliters of blocking agent (1-2% BSA or casein) for 2 hours, and storing at 4 ℃.
Example 2
The embodiment is application of a quantum dot CdS-CD3 flow antibody reagent in mass spectrometry flow detection.
Centrifuging and filtering a human peripheral blood sample (2 mL), then resuspending the human peripheral blood sample into a single cell suspension by using 1mL of staining buffer (light safety cell staining buffer), adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or turning over the mixture, standing the mixture on ice for 15 minutes, gently swirling and mixing the mixture twice, centrifuging the mixture at 450g4 ℃ for 10 minutes to precipitate white blood cells, carefully sucking supernatant, abandoning 1mL of staining buffer (light safety cell staining buffer) to resuspend the cells, adding 50 microliters of Fc receptor blocking solution into the cell suspension, and incubating the cell suspension at room temperature (25 ℃) for 10 minutes; adding 1ug CdS-CD3 into the single cell suspension, vortex shaking for 10 seconds, mixing, and incubating at room temperature (25 deg.C) for 30 min; 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer (chen an biological cell staining buffer) to wash the cells, and repeating for 3 times; the Cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, 2 ml of staining Buffer (chen ' an biological Cell stabilizing Buffer) was added to wash the cells, 3 times were repeated, 1ml of machine Buffer (chen ' an biological Cell Acquisition Buffer) was resuspended, the labeled Cell sample was detected by mass flow system (chen ' an biological Starion), and the result was shown in FIG. 1, and the quantum dot-CD 3 signal was detected by mass flow system.
Example 3: this example is the application of quantum dot CdS-CD3 flow antibody reagent in fluorescence flow detection.
Centrifuging and filtering a human peripheral blood sample (2 mL), then resuspending the human peripheral blood sample into a single-cell suspension by using 1mL of staining buffer (chen 'an biological cell staining buffer), adding 3 mL of erythrocyte lysate into the single-cell suspension, gently swirling or reversely mixing the mixture, standing the mixture on ice for 15 minutes, gently swirling and mixing the mixture twice, centrifuging the mixture at 450g4 ℃ for 10 minutes to precipitate white blood cells, carefully sucking supernatant, resuspending the cells by using 1mL of staining buffer (chen' an biological cell staining buffer), adding 50 microliters of Fc receptor blocking solution into the cell suspension, and incubating the mixture at room temperature (25 ℃) for 10 minutes; adding 1ug CdS-CD3 into the single cell suspension, vortex shaking for 10 seconds, mixing, and incubating at room temperature (25 deg.C) for 30 min; 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer (chen an biological cell staining buffer) to wash the cells, and repeating for 3 times; the Cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, 2 ml of staining Buffer (chen 'an biological Cell stabilizing Buffer) was added to wash the cells, 3 times were repeated, 1ml of on-machine Buffer (chen' an biological Cell Acquisition Buffer) was resuspended, the labeled Cell sample was detected by a fluorescence Flow system (Beckman Dx Flex Flow Cytometer), and the result is shown in FIG. 2, and the quantum dot-CD 3 signal was detected by the fluorescence Flow system.
Example 4: this example illustrates the use of CdS-Cd45 flow antibody reagents using Cd106 monoisotopes for peripheral blood lymphocytes in mass spectrometry flow assays.
Centrifuging and filtering a human peripheral blood sample (2 mL), then resuspending the human peripheral blood sample into a single cell suspension by using 1mL of phosphate buffer solution, adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or evenly mixing the mixture in a reverse mode, standing the mixture on ice for 15 minutes, gently swirling and evenly mixing the mixture twice in the process, centrifuging the mixture at 450g for 10 minutes at 4 ℃ to precipitate white blood cells, carefully sucking and discarding supernatant, resuspending the cells by using 1mL of phosphate buffer solution, adding 50 microliters of Fc receptor blocking solution into the cell suspension, and incubating the cell suspension at room temperature (25 ℃) for 10 minutes; adding 1ug CdS-CD45 into the single cell suspension, vortex shaking for 10 seconds, mixing, and incubating at room temperature (25 deg.C) for 30 min; 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer solution to wash the cells, and repeating for 3 times; the Cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, 2 ml of staining Buffer was added to wash the cells, 3 times were repeated, 1ml of loading Buffer was resuspended in organism Cell Acquisition Buffer, the labeled Cell sample was detected by mass flow system (organism Starion was programmed in and out), and the result is shown in FIG. 3, and the quantum dot-CD 45 signal was detected by mass flow system.
Example 5: this example illustrates the use of CdS-Cd4 flow antibody reagents using Cd106 monoisotopes for murine peripheral blood lymphocytes in fluorescence flow and mass spectrometry flow assays.
Centrifuging and filtering a mouse peripheral blood sample (2 mL), then re-suspending the mouse peripheral blood sample by using 1mL of phosphate buffer solution to prepare single cell suspension, adding 3 mL of erythrocyte lysate into the single cell suspension, slightly swirling or inverting the mixture to mix the mixture evenly, standing the mixture on ice for 15 minutes, slightly swirling the mixture evenly twice, centrifuging the mixture at 450g for 10 minutes at 4 ℃ to precipitate white blood cells, carefully sucking and discarding supernatant, re-suspending the cells by using 1mL of phosphate buffer solution, adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating the cell suspension for 10 minutes at room temperature (25 ℃); adding 1ug CdS-CD4 into the single cell suspension, vortex shaking for 10 seconds, mixing, and incubating at room temperature (25 deg.C) for 30 min; 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer solution to wash the cells, and repeating for 3 times; the Cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, 2 ml of staining Buffer was added to wash the cells, 3 times were repeated, 1ml of loading Buffer was resuspended in organism Cell Acquisition Buffer, the labeled Cell sample was detected by mass flow system (organism Starion was programmed in and out), and the result is shown in FIG. 4, and the quantum dot-CD 45 signal was detected by mass flow system.
The labeled cell sample was detected by a fluorescence Flow system (Beckman Dx Flex Flow Cytometer), and the result is shown in fig. 4, and a quantum dot-CD 4 signal was detected by the fluorescence Flow system.
Example 6: this example illustrates the use of CdS-Cd3e flow antibody reagents using Cd106 monoisotope for murine peripheral blood lymphocytes in fluorescence flow detection and mass spectrometry flow detection.
Centrifuging and filtering a mouse peripheral blood sample (2 mL), then resuspending the mouse peripheral blood sample into a single cell suspension by using 1mL of phosphate buffer solution, adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or evenly mixing the mixture in a reverse mode, standing the mixture on ice for 15 minutes, gently swirling and evenly mixing the mixture twice, centrifuging the mixture at 450g4 ℃ for 10 minutes to precipitate white blood cells, carefully sucking and discarding supernatant, resuspending the cells by using 1mL of phosphate buffer solution, adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating the cell suspension at room temperature (25 ℃) for 10 minutes; adding 1ug CdS-CD3e into the single cell suspension, vortex shaking for 10 seconds, mixing, and incubating at room temperature (25 deg.C) for 30 min; 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer solution to wash the cells, and repeating for 3 times; the Cell suspension was fixed in 100. Mu.l of 16% formaldehyde solution at room temperature (25 ℃) for 10 minutes, 2 ml of staining Buffer was added to wash the cells, 3 times were repeated, 1ml of loading Buffer was resuspended in organism Cell Acquisition Buffer, the labeled Cell sample was detected by mass flow system (organism Starion programmed in and installed), and the result is shown in FIG. 5, and the quantum dot-CD 3e signal was detected by mass flow system.
The labeled cell sample was detected by a fluorescence Flow system (Beckman Dx Flex Flow Cytometer), and the quantum dot-CD 3e signal was detected by the fluorescence Flow system as shown in fig. 5.
Claims (8)
1. A quantum dot labeled antibody reagent is characterized in that the quantum dot labeled antibody reagent is CdX prepared by taking a single metal isotope of Cd as a unique Cd source; wherein X is S, se or Te.
2. The reagent of the quantum dot labeled antibody of claim 1, wherein Cd in the quantum dot CdSe/ZnS is metal element cadmium or isotope thereof: cd-106, cd-108, cd-110, cd-111, cd-112, cd-113, cd-114 or Cd-116.
3. The quantum dot labeled antibody reagent of claim 1, wherein the antibody is selected from the group consisting of human anti-CD 3 antibody, human anti-CD 45 antibody, murine anti-CD 4 antibody, and murine anti-CD 3e antibody.
4. The reagent for a quantum dot labeled antibody according to claim 1,
the quantum dot labeled antibody reagent is prepared by coupling quantum dots and an antibody, and the method comprises the following steps:
taking 0.5-5 mg of quantum dot nanosphere CdX, wherein X is S, se or Te quantum dot nanospheres, adding 0.1-1 mmol/L of EDC solution into phosphate buffer solution pH4.0-6.0 sodium dihydrogen phosphate solution, uniformly mixing and activating for thirty minutes, adding an antibody with the concentration of 50-500 ug/ml after activation, reacting for thirty minutes, centrifuging for 5-60 minutes per minute at 5000-20000 revolutions, discarding supernatant, adding bovine serum albumin with the concentration of 1-10 mg/ml, sealing for 2 hours, and storing at 4 ℃ to obtain the quantum dot labeled antibody reagent Qdot-CD.
5. The application of the quantum dot labeled antibody reagent of claim 1 in cell detection is application in fluorescence flow cytometry or mass spectrometry flow cytometry.
6. The application of the quantum dot labeled antibody reagent in cell detection according to claim 5, wherein the flow cytometer comprises a typical fluorescence flow cytometer and a mass spectrometer; the label of the antibody reagent for detection is metal element Cd and the isotope thereof: cd-106, cd-108, cd-110, cd-111, cd-112, cd-113, cd-114 or Cd-116.
7. The application of the quantum dot labeled antibody reagent in cell detection according to claim 5, wherein the detection method of the quantum dot labeled antibody reagent for fluorescence flow detection comprises the following steps:
(1) Centrifuging and filtering a human peripheral blood sample, and then resuspending the human peripheral blood sample into a single cell suspension by using 1ml of phosphate buffer solution;
(2) Adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or reversing and uniformly mixing, incubating for 15 minutes, centrifuging for 10 minutes at 450g and 4 ℃ to precipitate white blood cells, sucking and removing supernatant, and resuspending the cells by 1mL of phosphate buffer;
(3) Adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating for 10 minutes at the room temperature of 25 ℃ to obtain a quantum dot labeled antibody reagent Qdots-CD;
(4) Adding the prepared quantum dot labeled antibody reagent Qdots-CD into the single cell suspension by using 1-5 ug of quantum dot labeled antibody per 3E6 cells, carrying out vortex oscillation for 10 seconds, uniformly mixing, and incubating at room temperature of 25 ℃ for 30 minutes;
(5) 500g, centrifuging at 4 ℃ for 5 minutes, removing supernatant, adding 2 ml of PBS to wash cells, and repeating for 3 times;
(6) Adding 100 microliters of 16% formaldehyde solution into the cell suspension, fixing at room temperature of 25 ℃ for 10 minutes, adding 2 milliliters of staining buffer solution to wash the cells, repeating for 3 times, resuspending 1 milliliter of staining buffer solution, and preparing for detection;
(7) And (3) detecting the marked cell sample by a fluorescence flow system, wherein an original data signal output by the fluorescence flow system is a fluorescence intensity signal of a fluorescence label, and converting the fluorescence intensity signal into expression abundance information of a corresponding target protein in the cell according to fluorescence excitation wavelength and emission wavelength of quantum dots on the quantum dot labeled antibody according to fluorescence label information carried by the corresponding antibody.
8. The application of the quantum dot labeled antibody reagent in cell detection according to claim 5, wherein the detection method of the quantum dot labeled antibody reagent for mass spectrometry flow detection comprises the following steps:
(1) Centrifuging and filtering a human peripheral blood sample, and then resuspending the human peripheral blood sample by using 1ml of phosphate buffer solution to prepare single cell suspension;
(2) Adding 3 mL of erythrocyte lysate into the single cell suspension, gently swirling or reversing and mixing uniformly, incubating for 15 minutes at the room temperature of 25 ℃, centrifuging for 10 minutes at 450g4 ℃ to precipitate white blood cells, carefully sucking and removing supernatant, and re-suspending the cells by 1mL of phosphate buffer;
(3) Adding 50 microliters of Fc receptor confining liquid into the cell suspension, and incubating for 10 minutes at the room temperature of 25 ℃ to obtain a quantum dot labeled antibody reagent Qdots-CD;
(4) Adding the prepared quantum dot labeled antibody reagent Qdots-CD into the single cell suspension by using 1-5 ug of quantum dot labeled antibody for each 3E6 cell, carrying out vortex oscillation for 10 seconds, uniformly mixing, and incubating at the room temperature of 25 ℃ for 30 minutes;
(5) 500g, centrifuging at 4 ℃ for 5 minutes, discarding the supernatant, adding 2 ml of staining buffer solution to wash the cells, and repeating for 3 times;
(6) Adding 100 microliters of 16% formaldehyde solution into the cell suspension, fixing at room temperature of 25 ℃ for 10 minutes, adding 2 milliliters of staining buffer solution to wash the cells, repeating for 3 times, resuspending 1 milliliter of staining buffer solution, and preparing for detection;
(7) And detecting the marked cell sample by a mass spectrometry flow detection system, wherein an original data signal output by the mass spectrometry flow detection system is a metal atom abundance signal of a metal element label, and converting the metal atom abundance signal into expression abundance information of a corresponding target protein in the cell according to the metal label information carried by the corresponding antibody.
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