CN110567861A - kit for screening antigenic peptide with immunogenicity based on mass flow detection technology and detection method - Google Patents
kit for screening antigenic peptide with immunogenicity based on mass flow detection technology and detection method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
the invention provides a kit and a detection method for screening antigenic peptides with immunogenicity based on a mass spectrometry flow detection technology, which can determine whether specific antigenic peptides have the immunogenicity of specifically activating immune cells in a sample or not by using the mass spectrometry flow detection technology aiming at the immune cells in clinical and scientific research tissue samples, improve the depth and precision of sample detection and greatly expand the flux and detection connotation of screening and analysis.
Description
[ technical field ] A method for producing a semiconductor device
the invention relates to the technical field of cell biology, in particular to a kit for screening immunogenic antigen peptides based on a mass spectrometry flow detection technology and a detection method thereof.
[ background of the invention ]
the mass flow detection technology is a technology for detecting a rare metal labeled antibody through a mass spectrometer at a single cell level and performing multi-parameter and high-throughput qualitative analysis on each cell. Compared with the traditional flow cytometry, the technology has the advantages of more detection parameters and higher signal-to-noise ratio, is the most advanced single-cell flow detection technology at present, and is more and more widely applied in the fields of clinical medicine and biological research.
t lymphocytes are important regulatory cells in the response of acquired immune response of an organism, and can carry out immune clearance on exogenous immunogenic antigen substances, so that the stability of the internal environment of the organism is maintained. When the major histocompatibility complex is bound to an antigenic peptide and transported to the surface of a cell membrane, the molecular complex can be recognized by T cells, and then the immune function of the T cells is activated. Therefore, the antigen peptide to be detected presented by the major histocompatibility complex is marked, and then the marked antigen peptide is incubated with a sample to be detected, so that whether the cells in the sample to be detected can specifically recognize the antigen peptide can be determined, and whether the antigen peptide has immunogenicity to the cells of the sample to be detected can be determined.
the tetramer formed by the major histocompatibility complex can have stronger detection precision than the monomer, so the tetramer technology has important significance in immune cell detection. The technology allows researchers to accurately detect the recognition effect of immune cells on various antigens in the pathological processes of infectious diseases, tumors, autoimmune diseases and the like.
at present, the commercial major histocompatibility complex tetramer technology can only evaluate the detection activity of the antigen peptide from the common flow detection level, has few synchronous detection parameters and low detection flux, and cannot well meet the requirements of clinical examination and scientific research of high-throughput antigen substance screening. The detection method for screening the antigenic peptide with the immunogenicity based on the mass flow detection technology can greatly meet the requirements of clinical examination and scientific research on screening the antigenic substances and fill the gap of commercial application in the field at present.
[ summary of the invention ]
the invention provides a kit for screening antigenic peptides with immunogenicity based on a mass flow detection technology, which can screen whether the antigenic peptides to be detected have immunogenicity.
The invention also provides a detection method for screening the antigenic peptide with immunogenicity based on the mass spectrometry flow detection technology.
the technical solution of the invention is as follows:
The kit for screening the immunogenic antigen peptide based on the mass spectrometry flow detection technology is characterized by comprising LD liquid, B liquid, NB liquid, W liquid, P liquid, F liquid, T liquid and BC1-BC10 liquid;
the LD liquid comprises 194Pt and RPMI 1640 basic culture medium;
the solution B is a blocking buffer solution containing human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution for flow cytometry;
The NB fluid is a tetramer complex sealant;
the W liquid is a transparent buffer liquid;
the liquid P is a fixed/transparent concentrated liquid and a fixed/transparent diluent;
the T liquid is tetramer staining incubation liquid;
the F liquid comprises formaldehyde and phosphate buffer;
the solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution;
the BC1-BC10 liquid comprises phosphate buffer and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd 110.
further, LD liquid was 1 part by volume of RPMI 1640 basic medium, 0.01 part by volume of 1mM 194Pt (201194, Fluidigm).
further, the solution B is 0.5 to 2 parts by volume of a human immunoglobulin solution (2mg/mL), 0.5 to 2 parts by volume of a mouse immunoglobulin solution (2mg/mL), 0.5 to 2 parts by volume of a rat immunoglobulin solution (2mg/mL), 0.5 to 2 parts by volume of a hamster immunoglobulin solution (2mg/mL), 0.5 to 2 parts by mass of bovine serum albumin, 100 parts by volume of a phosphate buffer (GNM20012), and the parts by mass and the parts by volume are converted in terms of mass in mg and volume in mL, that is, 1 part by mass/1 part by volume is equal to 1 mg/mL.
further, the NB liquid is a blocking liquid prepared from 10 to 200 parts by mass of N-ethylmaleimide (E3876, Sigma), 0.0001 to 0.001 part by mass of biotin (i.e., vitamin B7) (V900418, Sigma), and 1 part by volume of double distilled water, and the parts by mass and the parts by volume are converted in terms of mass in mg and volume in mL, that is, 1 part by mass/1 part by volume is equal to 1 mg/mL.
further, the W solution was 1 part by volume of 10 Xpermeation solution (00-8333-56, Invitrogen) and 9 parts by volume of water.
further, the solution P was 1 part by volume of a fixed/permeation concentrate (00-5123-43, Invitrogen) and 3 parts by volume of a fixed/permeation diluent (00-5223-56, Invitrogen).
Further, the solution T is a tetramer staining solution prepared from 0.001 to 0.01 parts by mass of a tetramer complex of a specific antigen peptide that can be customized, 0.0005 to 0.002 parts by mass of bovine serum albumin, and 1 part by volume of a phosphate buffer solution (GNM20012), wherein the parts by mass and the parts by volume are converted according to the mass in mg and the volume in mL, that is, 1 part by mass/1 part by volume is equal to 1 mg/mL.
Further, the F solution is 0.9-1.3 parts by volume of 16 wt% formaldehyde solution and 9 parts by volume of phosphate buffer solution (GNM 20012).
Further, the D liquid was 0.005 part by volume of Cell-IDTMIntercalator-Ir (201192B, Fluidigm), 0.9-1.3 parts by volume of 16 wt% formaldehyde solution, 9 parts by volume of phosphate buffer (GNM 20012).
further, the air conditioner is provided with a fan,
BC1 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC2 solution contains 1 volume part of Pd106 (1.5-2. mu.M, Fluidigm), 1 volume part of Pd108 (1.5-2. mu.M, Fluidigm), 100 volume parts of phosphate buffer (GNM 20012);
BC3 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC4 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC5 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd108 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC6 solution contains 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC7 solution contains 1 volume part of Pd104 (1.5-2. mu.M, Fluidigm), 1 volume part of Pd108 (1.5-2. mu.M, Fluidigm), 100 volume parts of phosphate buffer (GNM 20012);
BC8 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC9 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC10 solution contained 1 part by volume of Pd108 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), and 100 parts by volume of phosphate buffer (GNM 20012).
wherein, each sample to be detected selects one of BC (1-10) solution for dyeing, different BC solutions are required to be selected for different samples, and at most 10 different samples to be detected can be detected simultaneously. Parts by volume are measured in mL.
further, LD solution, B solution, NB solution, W solution, P solution, F solution, T solution, and BC1-BC10 solution were prepared and then stored at 2-8 ℃.
the antigen peptide tetramer complex of the kit has specificity on the identification of cells capable of activating immunocompetence, and the tetramer assembled by the antigen peptide incapable of activating the immunocompetence of sample cells cannot identify the sample cells.
the detection method for screening the immunogenic antigen peptide based on the mass spectrometry flow detection technology is characterized in that the kit is used, and comprises the following steps:
Step one, preparing single cell suspension:
collecting digestive cells, and gently blowing and beating the digestive cells by using a pipette gun to obtain a single cell suspension;
Step two, determining cell death and cell viability:
adding 1mL LD solution prepared from 1mL RPMI 1640 basic culture medium and 0.01mL 1mM 194Pt (201194, Fluidigm), gently blowing the cell sediment at the bottom of the tube to resuspend the cells, and incubating in a cell culture box at 37 deg.C, preferably for 5 min;
step three, sealing:
add 50. mu.L of liquid B to each sample to resuspend the cell pellet and incubate on ice, preferably for 20 minutes; the solution B is prepared from 0.5-2mL human immunoglobulin solution (2mg/mL), 0.5-2mL mouse immunoglobulin solution (2mg/mL), 0.5-2mL rat immunoglobulin solution (2mg/mL), 0.5-2mL hamster immunoglobulin solution (2mg/mL), 0.5-2mg bovine serum albumin, and 100mL phosphate buffer (GNM 20012);
step four, blocking the tetramer complex:
Adding 50mL of the solution T to 50mL of the solution NB to obtain a final volume of 100. mu.L; after mixing, the mixture is incubated at room temperature for a certain period of time (preferably 10 minutes). Wherein the T liquid is tetramer staining incubation liquid prepared by 0.001-0.01mg of customizable tetramer compound of specific antigen peptide, 0.0005-0.002mg of bovine serum albumin and 1mL of phosphate buffer solution; NB liquid is prepared from 10-200mg of N-ethylmaleimide (E3876, Sigma), 0.0001-0.001mg of biotin (V900418, Sigma) and 1mL of double distilled water;
Step five, dyeing the tetramer compound:
adding a mixed solution of 100 mu LT solution and NB solution into each cell sample, and incubating for a certain time (preferably 1 hour) at room temperature;
step six, staining the cell surface with antibodies:
Adding bovine serum albumin solution (0.5-2% by mass/volume, dissolved in phosphate buffer) into the cell surface antibody at a proper dilution ratio, mixing with the sample, and incubating on ice for a certain time, preferably 30 minutes;
Step seven, transparent:
adding 100 μ L of P solution to each cell sample to resuspend the cells, wherein the P solution is prepared from 1mL of fixed/permeable concentrated solution (00-5123-43, Invitrogen) and 3mL of fixed/permeable diluted solution (00-5223-56, Invitrogen), and incubating at room temperature for a certain time, preferably 30 minutes;
Step eight, staining intracellular antibodies:
preparing intracellular antibody staining solution, adding an antibody to 100 mu L of W solution according to a proper proportion, adding the mixture into cell sediment, uniformly mixing, and incubating on ice for a certain time, preferably 30 minutes; w solution was prepared from 1mL of 10X permeant solution (00-8333-56, Invitrogen), 9mL of water;
Step nine, cell fixation:
adding 1mL of F solution into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating at room temperature for a certain time, preferably 10 minutes; f II solution is composed of 0.9-1.3mL of 0.16mg/mL formaldehyde solution and 9mL of phosphate buffer solution (GNM 20012);
step ten, DNA staining and fixing:
adding 1mL of solution D into each EP tube, blowing and sucking by a pipette, uniformly mixing, and standing overnight in a refrigerator at 4 ℃; solution D was composed of 0.005mL of Cell-IDTMIntercalator-Ir (201192B, Fluidigm), 0.9-1.3mL of 0.16mg/mL formaldehyde solution and 9mL of phosphate buffer solution (GNM 20012);
Step eleven, labeling cell barcodes:
Adding 100 μ L of a BC solution containing phosphate buffer solution and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd110 into the sample cells, resuspending the cells, and incubating on ice for a certain time, preferably 30 minutes;
Step twelve, rinsing and computer detection:
after rinsing the cells with double distilled water, transferring the cells to a flow tube, counting the cells, and detecting the cells on a machine.
The invention has the following overall beneficial effects:
the invention carries out accurate immune typing on cell populations through a mass flow cytometry technology, carries out comprehensive analysis on an intracellular signal conduction network, analyzes functional connection among cell subsets, and is used for high-throughput multi-parameter detection of a large number of samples by combining with an MHC-Tetramer technology for T cell detection, sorting, activation and amplification of antigen specificity, thereby being used for screening T cell recognition epitopes and detecting specific T cells, realizing specific, effective and high-throughput screening of immunogenic antigen peptides of immune cells in an activated tissue organ sample at a single cell level, greatly improving the depth and precision of sample detection, and greatly expanding the flux and detection connotation of screening and analysis.
[ description of the drawings ]
FIG. 1 is a schematic flow chart of a detection method for screening immunogenic antigenic peptides based on a mass spectrometric detection technique;
FIG. 2 shows the flow detection results of the antigen peptide A tetramer complex and the antigen peptide B tetramer complex of the negative control sample;
FIG. 3 shows the results of antigenic peptide detection using the kit of the present invention;
FIG. 4 shows the results of detection of intracellular signals while detecting A antigen peptide cells using the kit of the present invention.
[ detailed description ] embodiments
the present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
the following examples are not intended to limit the scope of the present invention, nor are the steps described to limit the order in which they are performed, i.e., to aqueous solutions, unless otherwise specified, concentrations differing by unit from one type of concentration, e.g., "mg/mL" to indicate the ratio of solute mass to solution volume, and so on. Modifications of the invention which are obvious to those skilled in the art in view of the prior art are also within the scope of the invention as claimed.
the kit for screening the immunogenic antigen peptide based on the mass spectrometry flow detection technology is characterized by comprising LD liquid, B liquid, NB liquid, W liquid, P liquid, F liquid, T liquid and BC1-BC10 liquid;
the LD liquid comprises 194Pt and RPMI 1640 basic culture medium;
the solution B is a blocking buffer solution containing human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution for flow cytometry;
the NB fluid is a tetramer complex sealant;
the W liquid is a transparent buffer liquid;
The liquid P is a fixed/transparent concentrated liquid and a fixed/transparent diluent;
the T liquid is tetramer staining incubation liquid;
the F liquid comprises formaldehyde and phosphate buffer;
the solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution;
the BC1-BC10 liquid comprises phosphate buffer and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd 110.
based on the kit for screening the immunogenic antigen peptide by the mass flow detection technology, CD45(Biolegend) and Ki67(eBiosciences) are additionally used for detecting extracellular and intracellular target proteins of sample cells, and the specific steps are as follows:
step one, preparing a sample single cell suspension:
1) collecting single cell suspension obtained after digestion, counting cells, wherein the sample cell tested in the example is mouse m33 cell, and dividing the sample cell into a control group and a staining group for experiment;
2) get 107unicellular, centrifugate 400g/5min at room temperature, abandon the supernatant;
3) At 107adding a serum-free RPMI 1640 culture medium preheated at 37 ℃ into the/mL cell density, centrifuging at room temperature for 400g/5min, and discarding the supernatant;
Step two, determining cell death and cell viability:
4) Adding 1mL LD solution prepared from 1mL RPMI 1640 basic culture medium and 0.01mL 1mM 194Pt (201194, Fluidigm), gently blowing the cell precipitate at the bottom of the tube to resuspend the cells, and incubating in a cell culture box at 37 ℃ for 5 min;
5) Adding 5mL of RPMI 1640 medium containing serum preheated at 37 ℃, centrifuging at room temperature for 400g/5min, and discarding the supernatant;
step three, sealing:
6) add 50. mu.L of liquid B to each sample to resuspend the cell pellet and transfer to a clean 1.5mL EP tube, label, incubate for 20 minutes on ice; the solution B is prepared from 0.5-2mL human immunoglobulin solution (2mg/mL), 0.5-2mL mouse immunoglobulin solution (2mg/mL), 0.5-2mL rat immunoglobulin solution (2mg/mL), 0.5-2mL hamster immunoglobulin solution (2mg/mL), 0.5-2mg bovine serum albumin, and 100mL phosphate buffer (GNM 20012);
Step four, blocking the tetramer complex:
7) Preparing a tetramer complex closed mixed solution during the incubation period of the cell sample on ice, adding NB solution into T solution to make the final volume 100 mu L, and incubating at room temperature for 10min after uniform mixing; the solution T is a tetramer staining solution prepared from 0.001-0.01 parts by mass of a customizable tetramer compound of a specific antigen peptide, 0.0005-0.002 parts by mass of bovine serum albumin and 1 part by volume of a phosphate buffer solution; NB liquid is prepared by 10-200 parts by mass of N-ethylmaleimide (E3876, Sigma), 0.001-0.01 part by mass of biotin (V900418, Sigma) and 1 part by volume of double distilled water;
step five, dyeing the tetramer compound:
8) adding 1mL of bovine serum albumin solution (0.5% by mass/volume in phosphate buffer) into each EP tube to wash the cells, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
9) adding a mixed solution of 100 mu LT solution and NB solution into each EP tube, and incubating for 60min at room temperature;
step six, staining the cell surface with antibodies:
10) adding 1mL of bovine serum albumin solution (0.5% by mass/volume in phosphate buffer) into each EP tube to wash the cells, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
11) preparing a mixed solution of cell surface marker antibodies, adding an antibody CD45(1:100 volume ratio) to a bovine serum albumin solution (0.5-2% by mass volume ratio, dissolved in a phosphate buffer solution) according to a proper dilution ratio, adding 100 mu L of the mixed solution to each sample, and preparing 300 mu L of the mixed solution;
12) adding 100 mu L of cell surface marker antibody mixed solution into each EP tube, uniformly mixing, and incubating on ice for 30 min;
Step seven, transparent:
13) adding 1mL of W solution into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
14) adding 100 μ L of P solution into each cell sample, resuspending cells, preparing two horizontal lines from 1mL of fixed/transparent concentrated solution (00-5123-43, Invitrogen) and 3mL of fixed/transparent diluted solution (00-5223-56, Invitrogen), and incubating at room temperature for 30 min;
15) Adding 1mL of W solution into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant for the first time;
16) adding 1mL of W solution into each EP tube, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant for the second time;
Step eight, staining intracellular antibodies:
17) preparing intracellular antibody staining solution, adding an antibody Ki67(1:200 volume ratio) to 100 μ L of W solution in a proper proportion, adding into cell precipitate, mixing, and incubating on ice for 30 min; w solution was prepared from 1mL of 10X permeant solution (00-8333-56, Invitrogen), 9mL of water;
18) adding 1mL of P solution into each EP tube to stop washing cells, wherein the P solution is prepared from 1mL of fixed/transparent concentrated solution (00-5123-43, Invitrogen) and 3mL of fixed/transparent diluent (00-5223-56, Invitrogen), centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant;
19) Adding 1mL of bovine serum albumin solution (0.5% by mass/volume in phosphate buffer) into each EP tube to wash the cells, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
step nine, cell fixation:
20) adding 1mL of F solution into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating for 10min at room temperature; the F solution is composed of 0.9-1.3mL of 16% formaldehyde solution and 9mL of phosphate buffer solution (GNM 20012);
21) centrifuging at 4 deg.C for 800g/5min, and removing supernatant;
step ten, DNA staining and fixing:
22) Adding 1mL of solution D into each EP tube, blowing and sucking by a pipette, uniformly mixing, and standing overnight in a refrigerator at 4 ℃; liquid D was prepared from 0.0005mL of Cell-IDTMIntercalator-Ir (201192B, Fluidigm), 0.09-0.13mL of 16% (mass/volume ratio, mg/mL) formaldehyde solution and 0.9mL of phosphate buffer solution (GNM 20012);
step eleven, labeling cell barcodes:
23) taking out the cell sample from a refrigerator at 4 ℃, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
24) adding 1mL of phosphate buffer solution (GNM20012) into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
25) adding 100 μ L of BC1 solution into the sample cells, wherein BC1 solution contains 1mL of LPd104(1.5-2 μ M, Fluidigm), 1mL of Pd105(1.5-2 μ M, Fluidigm) and 100mL of phosphate buffer (GNM20012), resuspending the cells, and incubating on ice for 30 min;
26) adding 1mL of bovine serum albumin solution (0.5-2 parts by mass of bovine serum albumin, 100 parts by volume of phosphate buffer GNM20012, wherein the parts by mass are mg and the parts by volume are mL) into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
27) Adding 1mL of water into each EP tube to resuspend cells, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant;
28) preparing a flow pipe with a filter membrane filter head, and marking the pipe wall;
Thirteen steps of rinsing and computer detection:
29) after 1mL of water was added to each EP tube to resuspend the cells, the cell suspension was aspirated by pipette gun through the filter head filter and transferred to the corresponding flow tube. Adding 1mL of water again to clean the wall of the EP tube for a plurality of times, absorbing the cleaning solution by using a pipette gun, filtering the same flow tube filter membrane and putting the same flow tube filter membrane into the same flow tube;
30) centrifuging at 4 deg.C for 800g/5min, and removing supernatant;
31) after 1mL of water was added to each flow tube to resuspend the cells, 10. mu.L of each was counted.
32) according to the cell counting result, the cells of the control group and the treated group with the same cell number are taken, after mixing, the cell sample is washed for a plurality of times by water, a proper amount of balance magnetic beads are added, the detection is carried out on a Helios mass spectrometer (Fluidigm) machine, and the two groups of data are processed and analyzed.
we assembled tetrameric complexes using specific antigenic peptide a capable of binding to m33 cell surface T cell receptors, and verified whether the assembled specific antigenic peptide tetrameric complexes could recognize m33 cell surface T cell receptors using mass spectrometry flow techniques. Meanwhile, we selected antigenic peptide B weakly bound to m33 cell surface T cell receptor to assemble into tetrameric complexes as a negative control sample. The flow detection results of the antigen peptide A tetramer complex and the antigen peptide B tetramer complex of the negative control sample are shown in FIG. 2. After the kit for screening the immunogenic antigen peptides based on the mass flow detection technology is used for detecting the antigen peptides, cell populations capable of specifically identifying the A antigen peptides in m33 cells can be successfully detected, and an effective detection result similar to flow cytometry detection is obtained, wherein the detection result is shown in figure 3. In addition, the kit for screening the antigenic peptide with immunogenicity based on the mass spectrometry flow detection technology can simultaneously detect the intracellular signal Ki67 of the sample cell, and the blank staining group has no obvious difference from the staining effect of the A antigenic peptide tetramer, which indicates that the staining effect of the tetramer does not influence the intracellular staining effect of the sample cell, as shown in FIG. 4. The existing using method allows the signal path detection kit based on the mass flow detection technology or the cell cycle detection kit based on the mass flow detection technology and the detection method to be utilized to simultaneously detect the signal path and the cell cycle regulation and control change in immune cells activated by the specific antigen peptide while detecting whether cells responding to the specific antigen peptide to activate the immune reaction exist in sample cells, thereby expanding the application connotation of the kit.
Claims (12)
1. the kit for screening the immunogenic antigen peptide based on the mass spectrometry flow detection technology is characterized by comprising LD liquid, B liquid, NB liquid, W liquid, P liquid, F liquid, T liquid and BC1-BC10 liquid;
the LD liquid comprises 194Pt and RPMI 1640 basic culture medium;
The solution B is a blocking buffer solution containing human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution for flow cytometry;
the NB fluid is a tetramer complex sealant;
The W liquid is a transparent buffer liquid;
the liquid P is a fixed/permeable liquid;
the T liquid is tetramer staining incubation liquid;
the F liquid comprises formaldehyde and phosphate buffer;
The solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution;
the BC1-BC10 liquid comprises phosphate buffer and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd 110.
2. the kit for screening immunogenic antigen peptides based on mass spectrometry flow detection technology of claim 1, wherein the LD solution is 1 volume part of RPMI 1640 basic medium, 0.01 volume part of 1mM 194 Pt.
3. The kit for screening immunogenic antigenic peptides based on mass spectrometry flow detection technology of claim 1, wherein said solution B comprises 0.5-2 volume parts of human immunoglobulin solution with concentration of 2mg/mL, 0.5-2 volume parts of mouse immunoglobulin solution with concentration of 2mg/mL, 0.5-2 volume parts of rat immunoglobulin solution with concentration of 2mg/mL, 0.5-2 volume parts of hamster immunoglobulin solution with concentration of 2mg/mL, 0.5-2 mass parts of bovine serum albumin, and 100 volume parts of phosphate buffer, wherein the mass parts and the volume parts are converted according to mass in mg and volume in mL.
4. The kit for screening immunogenic antigen peptides based on the mass spectrometry flow detection technology as claimed in claim 1, wherein the NB fluid is a blocking fluid prepared from 10-200 parts by mass of N-ethylmaleimide, 0.0001-0.001 part by mass of biotin, and 1 part by volume of double distilled water, and the parts by mass and the parts by volume are converted according to the mass in mg and the volume in mL.
5. the kit for screening antigenic peptides with immunogenicity based on mass spectrometric detection technique of claim 1, characterized in that said W liquid is 1 part by volume of 10 x passing liquid and 9 parts by volume of water.
6. The kit for screening immunogenic antigen peptides based on the mass spectrometry flow detection technology as claimed in claim 1, wherein the liquid P is 1 part by volume of fixed/transparent concentrated liquid and 3 parts by volume of fixed/transparent diluted liquid.
7. The kit for screening immunogenic antigen peptides based on mass spectrometry flow detection technology of claim 1, wherein the solution T is a tetramer staining solution prepared from 0.001-0.01 parts by mass of a tetramer complex of a customizable specific antigen peptide, 0.0005-0.002 parts by mass of bovine serum albumin, and 1 part by volume of phosphate buffer solution, and the parts by mass and the parts by volume are converted according to mass in mg and volume in mL.
8. the kit for screening antigenic peptides with immunogenicity based on mass spectrometric detection technique according to claim 1, characterized in that said solution F is 0.9-1.3 parts by volume of 16 wt% formaldehyde solution and 9 parts by volume of phosphate buffer.
9. The kit for screening immunogenic antigen peptides based on mass spectrometry flow detection technology of claim 1, wherein the D liquid is 0.005 volume part of Cell-IDTMIntercalator-Ir, 0.9-1.3 parts by volume of 16 wt% formaldehyde solution and 9 parts by volume of phosphate buffer solution.
10. the kit for screening antigenic peptides with immunogenicity based on mass spectrometric detection technique according to claim 1,
the BC1 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd105 and 100 volume parts of phosphate buffer solution;
the BC2 solution contains 1 volume part of 1.5-2 mu M Pd106, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
The BC3 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd110 and 100 volume parts of phosphate buffer solution;
The BC4 liquid contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd106 and 100 volume parts of phosphate buffer;
the BC5 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
the BC6 solution contains 1 volume part of 1.5-2 mu M Pd106, 1 volume part of 1.5-2 mu M Pd110 and 100 volume parts of phosphate buffer solution;
The BC7 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
the BC8 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd106 and 100 volume parts of phosphate buffer solution;
the BC9 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd110 and 100 volume parts of phosphate buffer solution;
the BC10 solution contains 1 volume part of 1.5-2 mu M Pd108, 1 volume part of 1.5-2 mu M Pd110, and 100 volume parts of phosphate buffer solution.
11. the kit for screening immunogenic antigen peptides based on mass spectrometry flow detection technology of claim 1, wherein the LD solution, B solution, NB solution, W solution, P solution, F solution, T solution, BC1-BC10 solution are stored at 2-8 ℃ after the preparation is completed.
12. an assay for screening antigenic peptides having immunogenicity based on mass spectrometric detection techniques, characterized in that the kit according to any one of claims 1 to 11 is used, comprising the following steps:
step one, preparing single cell suspension:
collecting digestive cells, and gently blowing and beating the digestive cells by using a pipette gun to obtain a single cell suspension;
Step two, determining cell death and cell viability:
adding 1mL of LD solution, wherein the LD solution is prepared from RPMI 1640 basic culture medium and 194Pt, gently blowing and beating cell sediment at the bottom of a tube to resuspend cells, and incubating in a cell culture box at 37 ℃;
step three, sealing:
add 50. mu.L of liquid B to each sample to resuspend the cell pellet and incubate on ice; the solution B is prepared from human immunoglobulin solution with concentration of 2mg/mL, mouse immunoglobulin solution with concentration of 2mg/mL, rat immunoglobulin solution with concentration of 2mg/mL, hamster immunoglobulin solution with concentration of 2mg/mL, bovine serum albumin and phosphate buffer solution;
step four, blocking the tetramer complex:
Adding NB solution into the solution T to make the final volume 100 μ L, mixing uniformly, and incubating at room temperature; the T liquid is a tetramer staining liquid prepared from a customizable tetramer compound of specific antigen peptide, bovine serum albumin and a phosphate buffer solution; NB liquid is prepared from N-ethylmaleimide, biotin and double distilled water;
step five, dyeing the tetramer compound:
Adding a mixed solution of 100 mu L T liquid and NB liquid into each cell sample, and incubating at room temperature;
step six, staining the cell surface with antibodies:
adding a bovine serum albumin solution which is dissolved in a phosphate buffer solution and has the mass volume ratio of 0.5-2mg/mL into the cell surface antibody according to a proper dilution ratio, uniformly mixing the cell surface antibody with the sample, and incubating the mixture on ice;
step seven, transparent:
adding 100 mu L of P solution into each cell sample to resuspend the cells, wherein the P solution is prepared by 1mL of fixed/transparent concentrated solution and 3mL of fixed/transparent diluent, and incubating at room temperature;
step eight, staining intracellular antibodies:
Preparing intracellular antibody staining solution, adding an antibody to 100 mu L of W solution according to a proper proportion, adding the mixture into cell sediment, uniformly mixing, and incubating on ice; the W liquid is prepared from 1mL of 10 × permeable liquid and 9mL of water;
step nine, cell fixation:
adding 1mL of F solution into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating at room temperature; f II solution consists of 0.9-1.3mL of 16 wt% formaldehyde solution and 9mL of phosphate buffer solution;
Step ten, DNA staining and fixing:
adding 1mL of solution D into each EP tube, blowing and sucking by a pipette, uniformly mixing, and standing overnight in a refrigerator at 4 ℃; liquid D was prepared from 0.0005mL of Cell-IDTMIntercalator-Ir, 0.09-0.13mL of formaldehyde solution with the mass volume ratio of 16 wt% and 0.9mL of phosphate buffer solution;
step eleven, labeling cell barcodes:
Adding 100 mu L of BC solution containing phosphate buffer solution and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd110 into the sample cells, resuspending the cells, and incubating the cells on ice;
step twelve, rinsing and computer detection:
And (4) rinsing the cells with double distilled water, transferring the cells to a flow tube, counting, and detecting on a machine.
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Denomination of invention: A kit and detection method for screening immunogenic antigen peptides based on mass spectrometry flow cytometry technology Granted publication date: 20211221 Pledgee: Guotou Taikang Trust Co.,Ltd. Pledgor: ZHEJIANG PLTTECH HEALTH TECHNOLOGY CO.,LTD. Registration number: Y2024980011422 |
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