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CN116256513A - Single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof - Google Patents

Single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof Download PDF

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CN116256513A
CN116256513A CN202310268654.1A CN202310268654A CN116256513A CN 116256513 A CN116256513 A CN 116256513A CN 202310268654 A CN202310268654 A CN 202310268654A CN 116256513 A CN116256513 A CN 116256513A
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antibody
reagent tube
freeze
microchip
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陈亚楠
孙双午
王宇翀
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Taizhou Chen'an Biotechnology Co ltd
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Abstract

The invention provides a single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof. The detection reagent tube comprises a reagent tube (1), a freeze-drying micro-core (2) and a sealing membrane (3). The freeze-drying microchip is arranged at the bottom of the reagent tube, and the mouth of the reagent tube is sealed by a sealing film. The invention overcomes the defects of the prior art, simultaneously detects the multi-antigen markers on the sample at the single cell level, simplifies the reagents of experimental operation, achieves the standard antibody usage amount, ensures the accuracy and reliability of the detection result, has good stability especially at normal temperature, does not need cold chain transportation and storage, and reduces the cost and risk. The freeze-dried micro-core reagent tube is convenient and quick to use, shortens the time required by sample treatment, ensures the standardization of sample dyeing treatment, ensures the consistency of results and the accuracy of output results, and has great clinical application value.

Description

Single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof
Technical Field
The invention relates to medical equipment, in particular to a single-person mixed flow type antibody freeze-dried microchip detection reagent tube and preparation and application thereof.
Background
Human immune cells are mainly of two major classes, lymphocytes and myeloid-derived non-lymphocytes. Lymphocytes are one of the white blood cells, and are the most important cellular components involved in the immune response function of the body. Early studies largely classified lymphocytes into T cells, B cells and natural killer cells based on their migration, surface molecules and functions. Wherein the T cells are further classified into cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, etc.; b cells can be further divided into sub-cell populations such as primary B cells, plasma cells, and memory B cells.
With the continued progress of scientific research, the field of immunology has found hundreds of cell surface markers, specific antigens that appear or disappear during differentiation, maturation, and activation of cells of different lineages, called leukocyte differentiation antigens (CD molecules, cluster of Differentiation). These CD molecules are mainly adhesion molecules on the surface of immune cells, cytokine receptors or chemokine receptors, etc., and can be recognized by specific antibodies. These molecules have a high degree of variability in the different types of immune cells, which can be separated into finer sub-cellular taxonomies by analysis of these cell surface markers. For example, T cells can be further classified into helper T cells, helper depleting T cells, helper central memory T cells, helper effector memory T cells, cytotoxic helper T cells, cytotoxic depleting T cells, cytotoxic central memory T cells, and cytotoxic effector memory T cells, etc.
Immune cell typing is intimately involved in a variety of diseases. The absolute quantification of immune cell subpopulations or the relative quantification of various cell populations has great significance for judging pathological processes of various diseases such as autoimmune diseases, immunodeficiency diseases, malignant tumors, type I diabetes, inflammations, virus infections and the like, researching pathogenesis and monitoring the degree of immune system change in the diseases. Absolute counts of cd4+ T lymphocytes, for example, are clinically useful for disease staging in HIV-infected individuals, as well as for complications judgment; expression of HLA-DR on monocyte surfaces is positively correlated with prognosis in post-operative and post-traumatic patients; abnormal elevation of cd4+ T lymphocytes/cd8+ T lymphocytes ratio is common in rheumatoid arthritis, type I diabetes, and significant elevation of cd4+ T lymphocytes/cd8+ T lymphocytes ratio after organ transplantation suggests a higher likelihood of immune rejection; CD4+/CD25+/Foxp3+ T cells are important participants in immune tolerance, and abnormal numbers of the cells can lead to autoimmune diseases and can also be used as tracking before and after bone marrow transplantation.
The current methods for immune cell typing are mainly: immunological enzyme labeling method and flow cytometry method. The immune enzyme labeling method is based on antigen-antibody reaction to detect one or more markers carried by immune cells of a population, but the method cannot detect at single cell level, the number of required sample cells is large, multiple cell markers cannot be detected at the same time, and the method has the defects of narrow detection range, complex operation and the like. Flow cytometry can be used for carrying out fine enough subcellular group typing on immune cells at a single cell level through a plurality of antibody combination modes, and has relatively simple operation and wide application in clinic. The flow cytometry is to quantitatively analyze single cells or other biological ions at the cellular molecular level through monoclonal antibodies in a multi-parameter, high-flux and rapid manner, so that tens of thousands of cells can be analyzed at a high speed, and a plurality of parameters can be measured from one cell at the same time, and the flow cytometry has the advantages of high channel speed, high precision and good accuracy. Flow cytometry can achieve accurate immunophenotyping of cell populations, comprehensive analysis of intracellular signaling networks, analysis of functional relationships between cell subsets, and high throughput multiparameter detection of large numbers of samples. The method has wide application in research in various fields such as hematopoiesis, immunity, stem cells, cancer, drug screening and the like.
At present, the flow detection system is mainly divided into two major types, namely a typical flow cell detection system based on an optical detection system, and the detection principle is that cells are stained by using antibodies marked by fluorescein, and the analysis of different fluorescein excitation light spectrums is performed by using the optical detection system, so that the multiple protein levels of single cells are analyzed. The other type is a novel flow system based on a mass spectrum detection system, and the detection principle is that cells are stained by a metal element marked antibody, and the content of the metal element is analyzed by a mass spectrum detector so as to analyze the levels of various proteins of single cells. Both flow detection systems have the characteristics of multiple channels, high analysis speed and high sensitivity. However, in the existing test schemes, several indexes, even up to forty indexes, are detected simultaneously, which results in that when the sample is dyed, more than forty kinds of single liquid antibodies need to be added into the sample to be detected one by one and in a trace amount, and the process is complex in operation and takes a long time. Due to the long exposure of liquid antibodies in the environment, part of the antibodies can also have repeated freeze thawing, which is detrimental to long-term stable storage of the antibodies and transport. Otherwise, the temperature rise and repeated freezing and thawing in any process can cause the decline of the detection performance of the reagent, and the detection sensitivity is affected, so that the judgment of the result is affected. Thus, the research of the single-person mixed flow antibody freeze-dried microchip reagent tube is of great significance.
Disclosure of Invention
The invention aims to overcome the defects, and designs a single-person mixed flow antibody reagent which is simple to operate, high in detection efficiency and free from cold chain preservation.
The invention provides a single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof.
The invention provides a single-person mixed flow antibody freeze-dried microchip detection reagent tube which comprises a reagent tube 1, a freeze-dried microchip 2 and a sealing film 3 (see figure 1). The freeze-drying microchip is arranged at the bottom of the reagent tube, and the mouth of the reagent tube is sealed by a sealing film. The lyophilized microchip reagent contains a volume of lyophilized excipient equal to the antibody, and the volume of the lyophilized microchip is 5 microliters to 60 microliters, preferably 25 microliters.
The diameter of the reagent tube is 12mm, the height of the reagent tube is 75mm, and the reagent tube is a common consumable for a commercially available conventional matched classical flow cytometer and mass spectrometer.
The reagent tube can be applied to detection of classical flow cytometry and mass flow cytometry.
The invention further aims to provide a preparation method of the single-person mixed flow type antibody freeze-dried microchip detection reagent tube.
The method comprises the following steps:
(1) Mixing the antibody mixture with a lyophilization excipient; the lyophilized microchip reagent contains a lyophilization excipient in the same volume as the antibody;
(2) Dropping the mixed solution into liquid nitrogen through a micro needle to naturally form spherical particles, and freeze-drying to obtain freeze-dried micro core particles; the liquid nitrogen temperature is-196 ℃, and the volume of the spherical particles is 5 microliters to 60 microliters, preferably 25 microliters.
(3) And sub-packaging the freeze-dried micro-cores at the bottom of the flow tube, and sealing the tube mouth with a sealing film to obtain the reagent tube for containing the freeze-dried micro-core reagent.
The reagent tube for containing the freeze-dried micro-core reagent is made of polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), acrylonitrile styrene copolymer (SAN), transparent resin (ABS), polyethylene terephthalate (PET), polycarbonate (PC) or organic glass (PMMA). The diameter of the reagent tube is 12mm, the height is 75mm, and the reagent tube can be suitable for common fluorescence flow cytometry and mass flow cytometry.
The lyophilized micro core reagent includes four to sixty antibodies, the antibody species is not limited, and the antibodies are selected from antibodies which specifically bind to cell membrane surface antigens, extracellular factors, intracellular factors or nuclear factors, preferably antibodies such as CD2 antibodies, CD5 antibodies, CD7 antibodies, CD25 antibodies, CD44 antibodies, CD57 antibodies, CD49d antibodies, CD127 antibodies, CD161 antibodies, CCR7 antibodies, CXCR3 antibodies, CCR4 antibodies, CCR5 antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD8 antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD56 antibodies, CD45RA antibodies, CD45RO antibodies, TCR- γδ antibodies, CD24 antibodies, CD27 antibodies, CD38 antibodies or IgD antibodies.
The antibodies are respectively marked with different labels, and the labels are fluorescein labels, metal element labels or quantum dots, nano particles and the like.
The fluorescein tag may be selected from the group including, but not limited to, fluorescein isothiocyanate FITC, phycoerythrin PE, polymethylchlorophyll protein PerCP or propidium iodide PI, the metal element tag is selected from the group consisting of metal elements or isotopes thereof: preferably yttrium Y, ruthenium Ru, rhodium Rh, palladium Pd, cadmium Cd, indium In, lanthanum La, cerium Ce, praseodymium Pr, neodymium Nd, samarium Sm, europium Eu, gadolinium Gd, terbium Tb, dysprosium Dy, holmium Ho, erbium Er, thulium Tm, ytterbium Yb, lutetium Lu, platinum Pt or bismuth Bi.
The freeze-drying excipient is selected from one or more of glycerin, trehalose, sucrose, mannitol or lactose.
The invention also aims to provide the application of the single-person mixed flow antibody freeze-dried microchip detection reagent tube. The application is in detecting cells.
When the freeze-dried microchip reagent tube containing the flow antibody is used, 100 microliters of single cell suspension to be detected is directly added into the reagent tube, vortex oscillation is carried out for 10 seconds to dissolve, incubation is carried out for 30 minutes at 15-25 ℃,1 milliliter of buffer solution such as phosphate buffer solution, cell staining solution, membrane breaking solution and the like is used, preferably, 500g of cell staining solution is centrifuged for 10 minutes, supernatant is discarded, the supernatant is repeated once, redundant antibody is washed, and the suspension is resuspended according to the required cell concentration by using the buffer solution, so that the suspension can be detected on a machine.
The invention overcomes the defects of the prior art, and avoids the conditions of transportation and preservation of the convection type antibody reagent and the harsh requirements on a cold chain. The single mixed flow antibody freeze-dried microchip detection tube has good stability at normal temperature, high precision, long storage period, no need of cold chain transportation and storage, no need of evaluating the bottle opening validity period, and reduced management cost and risk; the packaging of single reaction components is uniformly dissolved in buffer solution at once, so that the loss is avoided, the use is simple and convenient, the use cost is greatly reduced, and the method is particularly suitable for basic research laboratories, basic medical institutions and inspection institutions with poor cold chain transportation and storage conditions. Moreover, the freeze-dried micro-core reagent tube uses the sealing film to seal the reagent tube, and can be added into a sample to be detected for detection only through mechanical puncture, so that the method is convenient and quick, the workload that a plurality of antibodies are added into the sample to be detected one by one is greatly reduced, the time required by sample treatment is shortened, the standardization of sample dyeing treatment is ensured, the consistency of results is ensured, and the accuracy of output results is ensured. Has great clinical application value.
Drawings
FIG. 1 is a schematic diagram of a single-person mixed flow antibody freeze-dried microchip detection reagent tube structure.
Reagent tube 1, freeze-dried microchip 2 and sealing film 3
Fig. 2: example 5 a single-serving, mixed-flow antibody lyophilized microchip test of the present invention was compared to the consistency of prior art antibody solutions.
The left graph shows the results of cell surface antigen detection by the single-person mixed flow antibody freeze-dried microchip.
The right panel shows the results of single liquid antibody detection of cell surface antigens in the prior art.
Fig. 3: example 6 stability of Single-serve hybrid flow antibody lyophilized Microchips of the invention at different temperatures
Detailed Description
The materials and reagents used in this example were all commercially available unless otherwise indicated.
Example 1
Referring to FIG. 1, the preparation of the single-person mixed flow antibody freeze-dried microchip detection reagent tube of the invention
The single mixed flow type antibody freeze-dried microchip detection reagent tube in the embodiment is a freeze-dried microchip detection reagent tube for lymphocyte TBNK cell typing in human peripheral blood, wherein the antibody amount for preparing the freeze-dried microchip detection reagent is as follows:
identifying target points Antibody name Metal label Antibody content Antibody concentration Antibody volume
CD3 Anti-human CD3 antibodies 154Sm 100 micrograms 500 micrograms/milliliter 200 microliters
CD4 Anti-human CD4 antibodies 173Yb 100 micrograms 500 micrograms/milliliter 200 microliters
CD8 Anti-human CD8 antibodies 168Er 100 micrograms 500 micrograms/milliliter 200 microliters
CD19 Anti-human CD19 antibodies 165Ho 100 micrograms 500 micrograms/milliliter 200 microliters
CD16 Anti-human CD16 antibodies 145Nd 100 micrograms 500 micrograms/milliliter 200 microliters
CD56 Anti-human CD56 antibodies 151Eu 100 micrograms 500 micrograms/milliliter 200 microliters
Fully mixing all antibody components according to the table, wherein the total volume of the mixed antibody is 1200 microliters, taking an equal volume (namely 1200 microliters) of freeze-drying excipient which is a mixture of 20% of trehalose and 8% of mannitol, fully and uniformly mixing the freeze-drying excipient with the mixed antibody solution, uniformly dividing the mixed liquid into 100 parts, wherein the volume of each part is 24 microliters, each part is one reaction system dosage, and dripping the mixture into liquid nitrogen according to each reaction system dosage for shaping; and then freezing and drying; the freeze-dried micro-core reagent for the typing of peripheral blood lymphocytes is obtained, the freeze-dried micro-core reagent is in the form of white spherical particles and has the volume of 24 microliters, and each tube is divided into 5 milliliter flow tubes according to one reaction dosage, namely, one white spherical particle per tube. In order to prevent the reagent from being wetted, the freeze-dried reagent is subjected to nitrogen protection treatment, and then a sealing film is added at the mouth of the flow pipe to carry out sealing treatment. The freeze-dried micro-core reagent tube containing the flow antibody is prepared.
The single-serving mixed flow antibody lyophilized microchip test reagent tube of this example 1 is a lyophilized microchip test reagent tube for lymphocyte immunophenotyping in human peripheral blood, wherein the amounts of antibodies used to make the lyophilized microchip test reagents are listed in the following table:
Figure BDA0004133884210000091
fully mixing all antibody components according to the table, wherein the total volume of the mixed antibody is 850 microliters, taking an equal volume (namely 850 microliters) of freeze-dried excipient, wherein the freeze-dried excipient is a mixture of 20% of trehalose and 8% of mannitol, fully and uniformly mixing the freeze-dried excipient and the mixed antibody solution, uniformly dividing the mixed liquid into 100 parts, wherein the volume of each part is 17 microliters, each part is one reaction system dosage, and dripping the mixture into liquid nitrogen according to each reaction system dosage for shaping; and then freezing and drying; the freeze-dried micro-core reagent for the typing of peripheral blood lymphocytes is obtained, the freeze-dried micro-core reagent is in the form of white spherical particles and has the volume of 17 microliters, and each tube is divided into 5 milliliter flow tubes according to one reaction dosage, namely, one white spherical particle per tube. In order to prevent the reagent from being wetted, the freeze-dried reagent is subjected to nitrogen protection treatment, and then a sealing film is added at the mouth of the flow pipe to carry out sealing treatment. The freeze-dried micro-core reagent tube containing the flow antibody is prepared.
Example 3
The single-serving mixed flow antibody lyophilized microchip detection reagent tube in this embodiment is a lyophilized microchip detection reagent tube for murine spleen lymphocyte immunophenotyping, wherein the amounts of antibodies used to make the lyophilized microchip detection reagent are listed in the following table:
identifying target points Antibody name Metal label Antibody content Antibody concentration Antibody volume
CD45 Anti-murine CD45 antibodies 147Sm 100 micrograms 500 micrograms/milliliter 200 microliters
CD11b Anti-murine CD11b antibodies 148Nd 100 micrograms 500 micrograms/milliliter 200 microliters
CD3e Anti-murine CD3e antibodies 152Sm 100 micrograms 500 micrograms/milliliter 200 microliters
CD8a Anti-murine CD8a antibodies 168Er 100 micrograms 500 micrograms/milliliter 200 microliters
CD4 Anti-murine CD4 antibodies 172Yb 100 micrograms 500 micrograms/milliliter 200 microliters
CD45R Anti-murine CD45R antibodies 176Yb 100 micrograms 500 micrograms/milliliter 200 microliters
Fully mixing all antibody components according to the table, wherein the total volume of the mixed antibody is 1200 microliters, taking an equal volume (namely 1200 microliters) of freeze-drying excipient which is a mixture of 20% of trehalose and 8% of mannitol, fully and uniformly mixing the freeze-drying excipient with the mixed antibody solution, uniformly dividing the mixed liquid into 100 parts, wherein the volume of each part is 24 microliters, each part is one reaction system dosage, and dripping the mixture into liquid nitrogen according to each reaction system dosage for shaping; and then freezing and drying; the freeze-dried micro-core reagent for the typing of peripheral blood lymphocytes is obtained, the freeze-dried micro-core reagent is in the form of white spherical particles and has the volume of 24 microliters, and each tube is divided into 5 milliliter flow tubes according to one reaction dosage, namely, one white spherical particle per tube. In order to prevent the reagent from being wetted, the freeze-dried reagent is subjected to nitrogen protection treatment, and then a sealing film is added at the mouth of the flow pipe to carry out sealing treatment. The freeze-dried micro-core reagent tube containing the flow antibody is prepared.
EXAMPLE 4 use of the invention in detecting cells
Mixed flow type antibody freeze-dried microchip detection reagent tube for single-person lymphocyte TBNK cell typing in human peripheral blood prepared in example 1
Wherein the following antibodies are included:
identifying target points Antibody name Metal label
CD3 Anti-human CD3 antibodies 154Sm
CD4 Anti-human CD4 antibodies 173Yb
CD8 Anti-human CD8 antibodies 168Er
CD19 Anti-human CD19 antibodies 165Ho
CD16 Anti-human CD16 antibodies 145Nd
CD56 Anti-human CD56 antibodies 151Eu
When the detection is used, the diameter of the reagent tube 1 is 12mm, and the height is 75mm, so that the reagent tube can be suitable for common classical flow cytometry and mass flow cytometry. Removing the sealing film 3, opening a reagent tube containing the freeze-dried microchip, transferring a single cell suspension sample into the reagent tube 1, wherein the sample to be tested comprises cells to be tested and a reaction buffer solution, and the reaction buffer solution can enable the freeze-dried microchip 2 to be quickly redissolved. Vortex shaking for 10 seconds to fully dissolve and mix the freeze-dried microchip 2, and incubating for 30 minutes at room temperature (15-25 ℃);
washing the cells with 2 ml of staining buffer, centrifuging at 4deg.C for 5 min, discarding the supernatant, and repeating for 2 times; the resulting cell pellet was added to 100. Mu.l of 4% formaldehyde solution, fixed at room temperature (15-25 ℃) for 10 minutes, the cells were washed by adding 2 ml of staining buffer, repeated 2 times, and resuspended in 1 ml of staining buffer, ready for detection. Detecting the marked cell sample through a mass spectrometry flow system, wherein an original data signal output by the mass spectrometry flow system is a metal atom abundance signal of a metal element tag, and converting the original data signal into expression abundance information of a corresponding target protein in the cell according to the metal tag information carried by the corresponding antibody. Further analysis gave the antigen expression results of the cells.
Example 5 comparison with the prior art
The invention is used for single-person mixed flow type antibody freeze-dried microchip detection reagent tube detection of lymphocyte immunophenotyping in human peripheral blood
Comparison with the identity of prior art antibody solutions.
Referring to fig. 1, the single-person mixed flow antibody lyophilized microchip test reagent tube of this example 1 is a lyophilized microchip test reagent tube for lymphocyte immunophenotyping in human peripheral blood, which comprises the following antibodies:
identifying target points Antibody name Metal label
CD45 Anti-human CD45 antibodies 142Nd
CD3 Anti-human CD3 antibodies 154Sm
CD4 Anti-human CD4 antibodies 173Yb
CD8 Anti-human CD8 antibodies 168Er
CD25 Anti-human CD25 antibodies 160Gd
CD127 Anti-human CD127 antibodies 139La
CD45RA Anti-human CD45RA antibodies 155Gd
CD45RO Anti-human CD45RO antibodies 164Dy
CCR7 Anti-human CCR7 antibodies 161Dy
TCRγδ Anti-human TCRγδ antibodies 171Yb
CD19 Anti-human CD19 antibodies 159Tb
IgD Anti-human IgD antibodies 169Tm
CD27 Anti-human CD27 antibodies 167Er
CD24 Anti-human CD24 antibodies 174Yb
CD38 Anti-human CD38 antibodies 175Lu
CD16 Anti-human CD16 antibodies 145Nd
CD56 Anti-human CD56 antibodies 149Sm
When in use and detection, a single mixed flow antibody freeze-dried microchip detection reagent tube is taken, the diameter of the reagent tube is 12mm, and the height of the reagent tube is 75mm, so that the reagent tube can be suitable for common classical flow cytometry and mass spectrometry flow cytometry. Removing the sealing film 3, opening a reagent tube containing the freeze-dried microchip, transferring a single cell suspension sample into the reagent tube 1, wherein the sample to be tested comprises cells to be tested and a reaction buffer solution, and the reaction buffer solution can enable the freeze-dried microchip 2 to be quickly redissolved. Vortex shaking for 10 seconds to fully dissolve and mix the freeze-dried microchip 2, and incubating for 30 minutes at room temperature (15-25 ℃); washing the cells with 2 ml of staining buffer, centrifuging at 4deg.C for 5 min, discarding the supernatant, and repeating for 2 times; the resulting cell pellet was added to 100. Mu.l of 4% formaldehyde solution, fixed at room temperature (15-25 ℃) for 10 minutes, the cells were washed by adding 2 ml of staining buffer, repeated 2 times, and resuspended in 1 ml of staining buffer, ready for detection. Detecting the marked cell sample through a mass spectrometry flow system, wherein an original data signal output by the mass spectrometry flow system is a metal atom abundance signal of a metal element tag, and converting the original data signal into expression abundance information of a corresponding target protein in the cell according to the metal tag information carried by the corresponding antibody. Further analysis gave the antigen expression results of the cells.
The prior art uses the following method to detect:
identifying target points Antibody name Metal label
CD45 Anti-human CD45 antibodiesBody 142Nd
CD3 Anti-human CD3 antibodies 154Sm
CD4 Anti-human CD4 antibodies 173Yb
CD8 Anti-human CD8 antibodies 168Er
CD25 Anti-human CD25 antibodies 160Gd
CD127 Anti-human CD127 antibodies 139La
CD45RA Anti-human CD45RA antibodies 155Gd
CD45RO Anti-human CD45RO antibodies 164Dy
CCR7 Anti-human CCR7 antibodies 161Dy
TCRγδ Anti-human TCRγδ antibodies 171Yb
CD19 Anti-human CD19 antibodies 159Tb
IgD Anti-human IgD antibodies 169Tm
CD27 Anti-human CD27 antibodies 167Er
CD24 Anti-human CD24 antibodies 174Yb
CD38 Anti-human CD38 antibodies 175Lu
CD16 Anti-human CD16 antibodies 145Nd
CD56 Anti-human CD56 antibodies 149Sm
In the detection process, adding the liquid mixed antibodies in the table into the sample to be detected one by one according to the dosage, and incubating for 30 minutes at room temperature (15-25 ℃) after fully and uniformly mixing; washing the cells with 2 ml of staining buffer, centrifuging at 4deg.C for 5 min, discarding the supernatant, and repeating for 2 times; the resulting cell pellet was added to 100. Mu.l of 4% formaldehyde solution, fixed at room temperature (15-25 ℃) for 10 minutes, the cells were washed by adding 2 ml of staining buffer, repeated 2 times, and resuspended in 1 ml of staining buffer, ready for detection. Detecting the marked cell sample through a mass spectrometry flow system, wherein an original data signal output by the mass spectrometry flow system is a metal atom abundance signal of a metal element tag, and converting the original data signal into expression abundance information of a corresponding target protein in the cell according to the metal tag information carried by the corresponding antibody. Further analysis gave the antigen expression results of the cells.
From FIG. 2, it can be seen that the single-person mixed flow antibody lyophilized microchip of the present invention shows the same results as the cell antigen expression obtained from the prior art antibody solution. The left graph shows the result of detecting the cell surface antigen by single-person mixed flow antibody freeze-dried microchip detection.
The right panel shows the results of single liquid antibody detection of cell surface antigens in the prior art.
Example 6 stability of Single-serve hybrid flow antibody lyophilized Microchips of the invention at different temperatures
The single-serving mixed flow antibody lyophilized microchip test reagent tube of this example 1 is a lyophilized microchip test reagent tube for lymphocyte TBNK cell typing in human peripheral blood, which comprises the following antibodies:
Figure BDA0004133884210000141
Figure BDA0004133884210000151
a liquid mix assay for the genotyping of lymphocytes TBNK cells in human peripheral blood for comparison comprising the following antibodies:
identifying target points Antibody name Metal label
CD3 Anti-human CD3 antibodies 154Sm
CD4 Anti-human CD4 antibodies 173Yb
CD8 Anti-human CD8 antibodies 168Er
CD19 Anti-human CD19 antibodies 165Ho
CD16 Anti-human CD16 antibodies 145Nd
CD56 Anti-human CD56 antibodies 151Eu
The single-person mixed flow antibody freeze-dried microchip detection reagent tube is stored for 50 days at the temperature of minus 20 ℃,4 ℃,25 ℃ (room temperature) and 37 ℃ respectively, then the same sample is detected by reagents with different storage conditions respectively, and the detected expression conditions of the cell surface CD4 and CD8 antigens are analyzed, and as can be seen from figure 3, the single-person mixed flow antibody freeze-dried microchip detection reagent tube is consistent in the performance and the result of the cell surface CD4 and CD8 antigen detection after being stored for 50 days at the temperature of minus 20 ℃,4 ℃,25 ℃ (room temperature) and 37 ℃ respectively.
The invention has the beneficial effects that:
the single-person mixed flow antibody freeze-dried microchip detection tube has good stability, high precision and long storage period at normal temperature, does not need cold chain transportation and storage, does not need evaluation of the bottle opening validity period, and reduces management cost and risk; the packaging of single reaction components is uniformly dissolved in buffer solution at once, so that the loss is avoided, the use is simple and convenient, the use cost is greatly reduced, and the method is particularly suitable for basic research laboratories, basic medical institutions and inspection institutions with poor cold chain transportation and storage conditions. Moreover, the freeze-dried micro-core reagent tube uses the sealing film to seal the reagent tube, and can be added into a sample to be detected for detection only by mechanical puncture, so that the method is convenient and quick, the workload that a plurality of antibodies are added into the sample to be detected one by one is greatly reduced, the time required by sample treatment is shortened, the standardization of sample dyeing treatment is ensured, the consistency of results is ensured, and the accuracy of output results is ensured.

Claims (10)

1. The single-person mixed flow type antibody freeze-dried microchip detection reagent tube is characterized by comprising a reagent tube 1, a freeze-dried microchip 2 and a sealing film 3.
2. The single-serving mixed flow antibody lyophilized microchip test reagent tube of claim 1, wherein the lyophilized microchip reagent contains a volume of lyophilized excipient equal to the antibody; the freeze-drying excipient is selected from one or more of glycerol, trehalose, sucrose, mannitol or lactose; the volume of the lyophilized microcores is from 5 microliters to 60 microliters, preferably 25 microliters; the diameter of the reagent tube is 12mm, and the height is 75mm.
3. A method of preparing a single-serving, mixed flow antibody lyophilized microchip test reagent tube as defined in claim 1, comprising the steps of:
(1) Mixing the antibody mixture with a lyophilization excipient; the lyophilized microchip reagent contains a lyophilization excipient in the same volume as the antibody;
(2) Dropping the mixed solution into liquid nitrogen through a micro needle to naturally form spherical particles, and freeze-drying to obtain freeze-dried micro core particles; the liquid nitrogen temperature is-196 ℃, and the volume of the spherical particles is 5 microliters to 60 microliters, preferably 25 microliters.
(3) And sub-packaging the freeze-dried micro-cores at the bottom of the flow tube, and sealing the tube mouth with a sealing film to obtain the reagent tube for containing the freeze-dried micro-core reagent.
4. The single-person mixed flow antibody lyophilized micro-core detection reagent tube according to claim 1, wherein the reagent tube for containing the lyophilized micro-core reagent is made of a material selected from the group consisting of polypropylene, polyvinyl chloride, polystyrene, acrylonitrile-styrene copolymer, transparent resin, ethylene terephthalate, polycarbonate and plexiglass.
5. The single-serving mixed flow antibody lyophilized microchip test reagent tube of claim 1, wherein the lyophilized microchip reagent comprises four to sixty antibodies; the antibody is selected from an antibody that specifically binds a cell membrane surface antigen, an extracellular factor, an intracellular factor, or a nuclear factor.
6. The single-serving mixed flow antibody lyophilized microarray assay tube of claim 5, wherein the antibody is a CD2 antibody, a CD5 antibody, a CD7 antibody, a CD25 antibody, a CD44 antibody, a CD57 antibody, a CD49d antibody, a CD127 antibody, a CD161 antibody, a CCR7 antibody, a CXCR3 antibody, a CCR4 antibody, a CCR5 antibody, a CCR7 antibody, a CD3 antibody, a CD4 antibody, a CD8 antibody, a CD16 antibody, a CD19 antibody, a CD20 antibody, a CD56 antibody, a CD45RA antibody, a CD45RO antibody, a TCR- γδ antibody, a CD24 antibody, a CD27 antibody, a CD38 antibody, or an IgD antibody.
7. The single-serving mixed flow antibody lyophilized microchip test reagent tube of claim 5, wherein the antibodies are labeled with different labels, respectively, which are fluorescein labels, metal element labels, or quantum dots or nanoparticles.
8. The single-person mixed flow antibody freeze-dried microchip detection reagent tube according to claim 7, wherein the fluorescein label is selected from fluorescein isothiocyanate, phycoerythrin, polymethylchlorophyll protein or propidium iodide; the metal element tag is selected from metal elements or isotopes thereof: preferably yttrium, ruthenium, rhodium, palladium, cadmium, indium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, platinum Pt or bismuth Bi.
9. Use of the single-serving, mixed-flow antibody lyophilized microchip assay reagent tube of claim 1.
10. Use of a single-serving, mixed flow antibody lyophilized microchip detection reagent tube according to claim 9, characterized in that the use is in detecting cells on a fluorescence flow cytometer or mass spectrometer.
CN202310268654.1A 2023-03-20 2023-03-20 Single-person mixed flow antibody freeze-dried microchip detection reagent tube and preparation and application thereof Pending CN116256513A (en)

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