CN110498858B - Method for dynamically detecting secretion condition of single-cell exoprotein - Google Patents
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Abstract
The invention discloses a method for dynamically detecting secretion conditions of single-cell exoproteins, which belongs to the technical field of exoprotein detection and comprises the following steps: immobilization of antibodies, blocking, specific capture of biological samples, attachment of nucleic acid-labeled detection antibodies, visualization of fluorescent spots, microscopic cleavage of fluorescent spots, cleavage of nucleic acid fragments, purification and amplification, detection and analysis of nucleic acid fragments. The detection method combines the ELISPOT and the nucleic acid sequence analysis to form the ELISEQ method, has the technical characteristics of high sensitivity, less non-specific combination, high flux, high accuracy and the like, can simultaneously realize detection of tens or hundreds of indexes, is simple and convenient to operate, and has good application prospect and larger market demand.
Description
Technical Field
The invention belongs to the technical field of exocrine protein detection, and particularly relates to a method for dynamically detecting single-cell exocrine protein secretion.
Background
Currently, the methods for detecting exoproteins are as follows: ELISA, ELISPOT, FACS, tetramer, 3H thymidine incorporation method, 51Cr release method. Among them, ELISA, tetramer, 3H thymidine incorporation method, 51Cr release method and the like detect the condition of the exoproteins in a single solution, so that the condition of the cell dynamic exoproteins cannot be detected.
The enzyme-linked immunospot (ELISPOT) combines a cell culture technology and an enzyme-linked immunosorbent technology, displays immune cells in a spot form, and is a main method for dynamically analyzing the type and frequency of immune cell exoproteins. The method integrates the advantages of high sensitivity, high reliability, single cell level, functional detection, low cost and the like, is highly accepted and widely applied in the immunological community at home and abroad, and becomes one of the mainstream immunological detection technologies.
However, in a typical ELISPOT assay, only one cytokine is detected per assay well. If two cytokines are to be detected simultaneously in one ELISPOT well, two antibodies can be coated to capture the two cytokines, and then two colored spots can be visualized with two different enzymes and a chromogenic system (e.g., horseradish peroxidase system and alkaline phosphatase system). However, the dual color ELISPOT has an inherent weakness, namely the problem of dual color spot separation. The mixed color of the speckles is a physical "three primary colors of pigment" problem, and two different colored pigments, if mixed in different proportions, can appear as a series of different apparent colors. The fine difference is difficult to judge by the full-automatic plate reader, so that in the two-color ELISPOT spot recognition, a certain color mixing spot cannot be recognized well, and experimental data are inaccurate.
To solve this problem, a method of detecting ELISPOT by fluorescence, i.e., fluoroSpot, is conceivable. The detection of multi-color cytokine secreting cells is achieved by coating a mixture of monoclonal capture antibodies of different specificities on a PVDF membrane of a 96-well plate, recognizing one secretion by a biotin-labeled detection antibody, and recognizing the other secretion by a Tag-labeled detection antibody. The detection is carried out by amplifying signal spots by conjugate reagent with specific fluorescent groups, and fluorescent lights with different colors are mixed together, so that the target fluorescent light can be accurately filtered out by only adding a light filter, and fluorescent lights with other colors are shielded, the fluorescent lights can not interfere with each other, and the analysis can be carried out by an automatic plate reader. At present, the fluorescence Elispot can detect 4 indexes at most simultaneously, but in the practical application process, the color recognizable by the optical filter is limited due to factors such as fluorescent optical filter materials, manufacturing process, selectable wavelength and the like, and if more antibodies or cytokines are required to be analyzed, the requirements cannot be met.
In addition, watson and Crick in 1953 revealed a double helix structure of DNA, which greatly motivated the exploration of DNA sequences, sequencing was an important experimental technique, and has wide application in biological research. The current DNA sequencing technology has undergone three generations of development so far, and has the characteristics of high flux and low sequencing cost. However, in single-cell sequencing products proposed by many companies engaged in single-cell sequencing, the sequencing technology can detect the expression of single-cell surface proteins and single-cell nucleic acids, but the expression of cell dynamic exoproteins cannot be detected, and still the fluorescent Elispot technology is required to be relied on, so that more methods for detecting the single-cell exoproteins by combining the analysis of the cell surface proteins with the sequencing of genomes or transcriptomes are more complicated to detect.
Therefore, there is a great market demand to develop a detection method combining ELISPOT and nucleic acid technology to accurately determine the expression of single-cell exoproteins.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for dynamically detecting the secretion situation of single-cell exoproteins, which combines ELISPOT with nucleic acid sequence analysis to form an ELISPEQ technology, can be better applied to simultaneous detection of multi-index exoproteins, is simple and convenient to operate, and realizes high-throughput single-cell level analysis.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for dynamically detecting secretion of single-cell exoproteins, comprising the steps of:
(1) coating the monoclonal capture antibody on a solid-phase carrier by antibody immobilization, coating overnight, and washing off redundant uncoated antibody by using a washing solution;
(2) sealing
Adding a blocking solution into the solid phase carrier coated with the capture antibody, washing, beating, and drying at low temperature to obtain a solid phase antibody;
(3) inoculating a cell to be detected into the solid-phase antibody obtained in the step (2) to specifically capture a biological sample, wherein the cell to be detected secretes the exoprotein spontaneously or under the action of a stimulating agent, the capture antibody specifically binds with the exoprotein, and washing off excessive unbound materials by using a washing solution to obtain a capture antibody-exoprotein complex which is fixed on a solid-phase carrier;
(4) connecting the nucleic acid labeled detection antibody, adding the nucleic acid labeled detection antibody-fluorescent labeled secondary antibody or fluorescent nucleic acid double-labeled detection antibody into the 'capture antibody-exoprotein' complex, and washing off excessive unbound materials by using a washing solution to obtain a fluorescent complex fixed on a solid phase carrier;
(5) the solid phase carrier fixed with the fluorescent compound is placed in a laser environment at the spot point of showing the fluorescence, and the fluorescent compound is obtained after the corresponding fluorescent spot is shown;
(6) microdissection of fluorescent spots the fluorescent spots are cut and recovered under a fluorescent microscope to obtain fluorescent complexes, and the fluorescent complexes are transferred into an EP tube;
(7) enzyme digestion, purification and amplification of nucleic acid fragments, adding an enzymolysis reaction solution and restriction enzyme into a fluorescent compound, adding an enzymolysis reaction stopping solution after the nucleic acid marked on the antibody is cut off, purifying after the enzymolysis reaction is stopped to obtain nucleic acid fragments, and transferring the nucleic acid fragments into a PCR tube for amplification to obtain a gene library of the nucleic acid corresponding to the single cell exoprotein;
(8) detecting and analyzing the nucleic acid fragments, determining the molecular weight and the gray value of the nucleic acid fragments of the exoproteins by an electrophoresis method, and judging the type and the content of the single fluorescent spot cytokines; sequencing was performed by gene libraries to analyze the frequency of single cell secreted exoproteins and cell secreted exoproteins in the cell population.
By adopting the technical scheme, the invention combines the Elispot with the nucleic acid sequence analysis, adopts a method of capturing antigen by double antibody sandwiches, overcomes the technical problems that the detection index of fluorescent Elispot is few and single-cell exoprotein can not be detected by a sequencing means, and forms an ELISEQ method. Therefore, when the single-cell exoproteins are detected, the interference among different exoproteins is small, the method has the technical characteristics of high sensitivity, less non-specific binding, high flux, high accuracy and the like, can simultaneously realize detection of tens or hundreds of indexes, is simple and convenient to operate, and has good application prospect and large market demand.
Further, in the step (1), the coating temperature is 2-8 ℃.
By adopting the technical scheme, when the coating temperature is 2-8 ℃, the capture antibody has good activity, so that the capture antibody is effectively coated on the solid-phase carrier, the effective activity of the capture carrier coated on the solid-phase carrier is ensured, and the capture antibody can be conveniently and well specifically combined with the exoprotein in the subsequent operation process.
Further, in the step (1), the solid phase carrier is one of a nylon membrane, a PVDF membrane, an NC membrane and a hydrogel.
By adopting the technical scheme, the nylon membrane, the PVDF membrane, the NC membrane and the hydrogel are all materials easy to cut, and can be better used as the solid phase carrier of the application. The nylon membrane is a traditional solid phase carrier for detecting nucleic acid, and the nucleic acid fragments are fixed mainly through electrostatic adsorption and simple UV crosslinking, so that the adsorption capacity of the nylon membrane is lower than that of the other three types. Although PVDF membrane is the most main carrier used by ELISPOT at present, the PVDF membrane is completely synthesized, and the length and arrangement mode of the fiber can be controlled, so that the surface of the membrane is flat and smooth, and the formed spots are compact. Compared with NC membrane, PVDF membrane has better adsorption capacity to protein, stronger mechanical bearing capacity and chemical corrosion resistance, and becomes the first solid phase carrier of ELISPOT technology. In contrast, hydrogel (Hydrogel) is a new generation of fluorescent plate bottom medium, is gel using water as a dispersion medium, has a three-dimensional reticular cross-linked structure, is not limited on protein adsorption capacity, is more suitable for simultaneous coating of multiple antibodies (3-10 kinds), and can obtain extremely high fluorescence signal to noise ratio due to no fluorescence background, so that the solid phase carrier is further preferably Hydrogel.
Further, in the step (4), the nucleic acid-labeled detection antibody-fluorescent-labeled secondary antibody-linked "capture antibody-exoprotein-solid phase carrier" complex includes the steps of:
a1, marking the Barcode which corresponds to the exoproteins one by one and primer sequences P5/P7 connected to two ends of the Barcode on different detection antibodies to obtain nucleic acid marked antibodies;
b1, mixing the nucleic acid labeled antibodies, and adding the mixture into a reaction system to form a complex of a capture antibody, an exoprotein and the nucleic acid labeled antibody;
and c1, marking a fluorescent group on the secondary antibody, and adding the fluorescent group into a 'capture antibody-exoprotein-nucleic acid marked antibody' complex to form a 'capture antibody-exoprotein-nucleic acid marked antibody-fluorescent marked secondary antibody' complex, namely a fluorescent complex fixed on a solid-phase carrier.
Further, in step (4), the fluorescent nucleic acid double-labeled detection antibody-linked "capture antibody-exoprotein-solid phase carrier" complex comprises the following steps:
a2, marking the Barcode which corresponds to the exoproteins one by one, primer sequences P5/P7 connected to two ends of the Barcode and fluorescent groups on different detection antibodies to obtain fluorescent nucleic acid double-marked antibodies;
b2, mixing the fluorescent nucleic acid double-labeled antibodies, and adding the mixture into a reaction system to form a complex of a capture antibody, an exoprotein and the fluorescent nucleic acid double-labeled antibodies, namely a fluorescent complex fixed on a solid-phase carrier.
By adopting the technical scheme, the primer provides a stimulating effect during nucleotide polymerization, the same primer sequence is convenient for synchronously synthesizing different nucleic acid fragments, the connection efficiency of the nucleic acid labeled detection antibody-fluorescent labeled secondary antibody or fluorescent nucleic acid double-labeled detection antibody and the complex of the capture antibody-exoprotein-solid phase carrier is effectively improved, and the synthesis efficiency of the fluorescent complex-solid phase carrier is further improved.
In addition, as different detection antibodies can be distinguished by different Barcode, for example, nucleic acid with the Barcode sequence of ATCACG is marked on an IL-6 detection antibody, nucleic acid with the Barcode sequence of CGATGT is marked on an IFN-gamma detection antibody, and nucleic acid with the Barcode sequence of TTAGGC is marked on an IL-17 detection antibody; therefore, by adding the nucleotide sequences of the Barcode regions corresponding to the exoproteins one by one, in the detection analysis of the nucleic acid fragments, the detection instrument is helped to accurately identify the nucleic acid sequences corresponding to different exoproteins, so that the condition that single cells secrete cytokines (namely the exoproteins) and the frequency that cell populations secrete the cytokines are conveniently and simultaneously analyzed.
Further, in the step (7), the enzymolysis reaction solution consists of 0.1mol/L, pH =7.4 of Tirs-HCl,1mol/L of NaCl and 0.07mol/L of MgCl 2 Composition is prepared.
By adopting the technical scheme, tirs-HCl is used as a protein buffer solution, and NaCl and MgCl are used as the buffer solution 2 As an activator of the restriction enzyme, under the concentration ratio, the restriction enzyme can rapidly and effectively cut off the exoprotein connected with the capture antibody, so that the nucleic acid fragment has higher enzyme cutting efficiency.
Further, in step (7), the restriction endonuclease is one of EcoR V, alu I, ecoR I, not I, hindIII.
By adopting the technical scheme, ecoR V, alu I, ecoR I, not I and HindIII are convenient to purchase and obtain on the market, compared with other special restriction enzymes, the restriction enzyme disclosed by the invention has wide obtaining channel and relatively low cost, and can better play the enzyme digestion function in the detection method of the invention, so that the restriction enzyme can effectively reduce the detection cost of the invention on the premise of ensuring the enzyme digestion effect.
Further, in the step (7), the enzymolysis reaction stopping solution is one of the following two groups;
1. 0.1mol/L EDTA-2Na+20wt% Ficoll 400+0.25wt% light-resistant orange G;
2. 0.25wt% bromophenol blue+0.25 wt% xylene blue FF or 0.25wt% xylene blue FF+40wt% sucrose aqueous solution or 30wt% glycerol aqueous solution.
By adopting the technical scheme, the EDTA-2Na, the sucrose and the glycerol can inhibit the enzyme activity of the restriction enzyme, so that the degradation of the exoprotein can be effectively reduced, and the stability of the exoprotein is improved; ficoll 400 is usually used as a medium of density gradient centrifugation, is favorable for effectively separating a nucleic acid fragment of an exoprotein from a capture antibody, is light-resistant orange G, bromophenol blue, xylene blue FF and xylene blue FF, is a tracking dye, and is favorable for intuitively observing the reaction progress of enzyme digestion. Therefore, under the concentration ratio, the enzyme digestion reaction of the restriction enzyme can be effectively terminated by the enzyme digestion reaction termination solution, and the integrity of the nucleic acid fragment of the exoprotein is ensured.
Further, in the step (7), the nucleic acid sequence of the exoprotein is purified by one or both of a centrifugal column and magnetic beads.
By adopting the technical scheme, the centrifugal column and the magnetic beads both use the nano technology to improve and surface modify the surface of the superparamagnetic nano particles, so that the superparamagnetic nano particles can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface, and the exocrine proteins in the invention can be better purified, and the method has the characteristics of excellent purification effect and detection accuracy improvement. In addition, the purification of nucleic acid sequences can be achieved in other ways than the centrifugation column and magnetic beads disclosed above.
In summary, the invention has the following beneficial effects:
1. the invention combines the ELISPOT and the nucleic acid sequence analysis to form an ELISEQ method, has the technical characteristics of high sensitivity, less non-specific combination, high flux, high accuracy and the like, is simple and convenient to operate, can realize detection of tens or hundreds of indexes at the same time, and has good application prospect and larger market demand;
2. the invention improves the synthesis efficiency of the fluorescent compound by adding the same primer sequences P5/P7 and the nucleotide sequences of the Barcode regions corresponding to the exoproteins one by one, and is beneficial to a detection instrument to accurately identify the nucleic acid sequences corresponding to different exoproteins, thereby being convenient for simultaneously analyzing the condition of secreting cytokines by single cells and the frequency of secreting cytokines by cell groups;
3. the invention limits the enzymolysis reaction liquid, the restriction endonuclease, the enzymolysis reaction stopping liquid and the purification treatment, and the scheme formed by the combination of the enzymolysis reaction liquid, the restriction endonuclease and the purification treatment effectively reduces the detection cost and has the characteristics of high flux, high accuracy and high sensitivity.
Drawings
FIG. 1 is a schematic diagram of a method for dynamically detecting secretion of single-cell exoproteins;
FIG. 2 is a process diagram of a method for dynamically detecting secretion of single cell exoproteins;
FIG. 3 is a schematic diagram of the ligation of a nucleic acid-labeled detection antibody-fluorescently labeled secondary antibody or a fluorescent nucleic acid double-labeled detection antibody to a "capture antibody-exoprotein" complex;
FIG. 4 shows the expression of cytokines in three examples.
In the figure, 1 in fig. 1 is antibody immobilization and blocking; 2 is a specific capture biological sample; 3 is the formation of a "capture antibody-exoprotein-nucleic acid labeled antibody" complex; 4 is to form a complex of a capture antibody, an exoprotein, a nucleic acid labeled antibody and a fluorescent labeled secondary antibody; 5 is to form a complex of a capture antibody, an exoprotein and a fluorescent nucleic acid double-labeled antibody; 6 is immune cleavage of fluorescent spots; 7 is the enzyme digestion, purification and amplification of the nucleic acid fragment; 8 is an electrophoresis method; 9 is gene sequencing.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1
A method for dynamically detecting secretion of single cell exoproteins, see fig. 1 and 2, comprising the steps of:
(1) antibody immobilization the monoclonal capture antibody was coated on a solid support using 384-well Hydrogel (Hydrogel) at 2 ℃ overnight (16-24 h), and the excess uncoated antibody was washed away with PBS solution containing 0.05% tween, wherein the coating time in this example was 20h;
the solid phase carrier may be a material which is easily cut, such as a nylon membrane, a PVDF membrane, or an NC membrane, in addition to the hydrogel.
(2) Blocking the solid phase carrier coated with the capture antibody, adding blocking solution (200 mu L/well of PBS solution containing 1% BSA) into the solid phase carrier, blocking blank sites on the solid phase carrier by the blocking solution to prevent other proteins from being directly combined with the solid phase carrier, washing the solid phase carrier by the blocking solution, beating the solid phase carrier to dry the solid phase carrier at the temperature of 2-8 ℃ for 3 days, and turning the membrane at the bottom into white to obtain the solid phase antibody.
(3) Specifically capturing peripheral blood mononuclear cells PBMC (purified T cells and cell lines in addition) separated from peripheral blood of a biological sample as test cells, inoculating the test cells to the solid-phase antibody obtained in the step (2), adding T cell epitope peptide (polypeptide, protein and other stimulators in addition), carrying out combined culture overnight (16-20 h, 20h in the embodiment), secreting the exoproteins by the cells under the action of the stimulators, specifically binding the capture antibodies in the solid-phase antibody with the exoproteins, washing off redundant unbound substances by using a PBS solution containing 0.05% Tween, and obtaining the capture antibody-exoprotein complex which is fixed on a solid-phase carrier.
(4) And (3) connecting the nucleic acid labeling detection antibody, adding the nucleic acid labeling detection antibody-fluorescent labeling secondary antibody or fluorescent nucleic acid double labeling detection antibody into the 'capture antibody-exoprotein' complex, and washing off excessive unbound materials by using a PBS solution containing 0.05% Tween to obtain a fluorescent complex.
In this example, a nucleic acid-labeled detection antibody-fluorescent-labeled secondary antibody is used, and, in combination with fig. 3, the steps of linking the secondary antibody to the "capture antibody-exoprotein" complex are as follows:
a1, marking the Barcode which corresponds to the exoproteins one by one and primer sequences P5/P7 connected to two ends of the Barcode on different detection antibodies to obtain nucleic acid marked antibodies;
wherein the detection antibody specifically corresponds to the analyte and the capture antibody, namely, if IL-6, IL-17A and IFN-gamma are required to be analyzed, the detection antibody which is required to be marked is an anti-IL-6 antibody, an anti-IL-17A antibody and an anti-IFN-gamma antibody, and so on;
the primer sequence P5 is:
CCTTGGCACCCGAGAATTCCA；
the primer sequence P7 is:
BAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA * A * A；
b1, mixing the nucleic acid labeled antibodies, and adding the mixture into a reaction system to form a complex of a capture antibody, an exoprotein and the nucleic acid labeled antibody;
and c1, marking a fluorescent group on the secondary antibody, and adding the fluorescent group into a 'capture antibody-exoprotein-nucleic acid marked antibody' complex to form a 'capture antibody-exoprotein-nucleic acid marked antibody-fluorescent marked secondary antibody' complex, namely a fluorescent complex fixed on a solid-phase carrier.
(5) The solid phase carrier fixed with the fluorescent compound is placed in a laser environment (Mabtech SA-550) to correspondingly show the fluorescent spots, namely the fluorescent compound.
(6) Microdissection of fluorescent spots were cut under a fluorescent microscope and recovered to obtain fluorescent complexes, which were transferred to EP tubes.
(7) And designing restriction enzyme cutting sites (EcoRV enzyme cutting sites) in a nucleic acid bridge region, adding an enzymolysis reaction solution and restriction enzyme (EcoRV) into a fluorescent compound, adding an enzymolysis reaction stopping solution after the nucleic acid marked on the antibody is cut, stopping the enzymolysis reaction, purifying by adopting a centrifugal column or magnetic beads to obtain a nucleic acid fragment, transferring the nucleic acid fragment into a PCR tube, and carrying out denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 20s, cyclic 35 times of denaturation, annealing and extension operation and extension at 72 ℃ for 5min, thereby amplifying to obtain a gene library of the nucleic acid corresponding to the single-cell exoprotein.
Wherein the enzymolysis reaction liquid consists of 0.1mol/L, pH =7.4 of Tirs-HCl,1mol/L of NaCl and 0.07mol/L of MgCl 2 Composition;
the restriction endonuclease may be other restriction endonucleases which can be replaced equivalently, such as Alu I, ecoR I, not I, hindIII, etc., in addition to EcoR V;
the enzymolysis reaction stopping solution is as follows: 0.1mol/L EDTA-2Na+20wt% Ficoll 400+0.25wt% light-fast orange G.
(8) Detecting and analyzing the nucleic acid fragments, determining the molecular weight and the gray value of the nucleic acid fragments of the exoproteins by an electrophoresis method, and judging the type and the content of the single fluorescent spot cytokines; sequencing was performed by gene libraries to analyze the frequency of single cell secreted exoproteins and cell secreted exoproteins in the cell population.
The specific analysis is as follows:
1. if the lengths of the nucleic acid sequences of the Barcode of the specific detection antibodies are inconsistent, the molecular weights of the nucleic acids carried by the Barcode are different, the migration speeds are inconsistent during electrophoresis, and whether a single fluorescent spot carries a single band or a plurality of bands can be analyzed according to the number of the bands and the sizes of the bands, wherein the sizes of the bands indicate the corresponding Barcode, so that the corresponding exoproteins are indicated;
2. sequencing the nucleic acid fragments, wherein the sequence of the nucleic acid indicates the sequence of the corresponding barcode, and the sequence of the nucleic acid indicates the corresponding barcode, so that the expression quantity of the corresponding exoprotein is indicated.
The invention mixes anti-IL-2, anti-IL-12, anti-IFN-gamma, anti-TNF-beta, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-10, anti-IL-17, anti-IL-21, anti-IL-22, anti-IL-26 monoclonal capture antibodies as capture antibodies of the invention; the secretion of single cell exoproteins can be measured by taking immune cells Th1/Th2/Th3/Th17 as single cells, i.e., whether the secretion frequency (expressed by the expression level of exoproteins) of each capture antibody and exoproteins is detected. In this embodiment, taking Th1 as an example, the detection results are shown in the following table one and fig. 3.
Example 2
The present embodiment 1 makes the following limitations on the above method:
the solid phase carrier is Hydrogel; coating at 4 ℃ overnight;
and designing an Alu I enzyme cutting site in the nucleic acid bridge region, wherein Alu I is selected as the restriction enzyme.
The results of the test are shown in the following Table I and FIG. 3.
Example 3
The present embodiment makes the following limitations on the above method:
the solid phase carrier is a 96-hole PVDF plate; coating at 8deg.C overnight;
the use of fluorescent nucleic acid double-labeled detection antibodies, see fig. 3, is followed by the steps of ligation to the "capture antibody-exoprotein" complex:
a2, marking the Barcode which corresponds to the exoproteins one by one, primer sequences P5/P7 connected to two ends of the Barcode and FITC fluorescent groups on different detection antibodies to obtain fluorescent nucleic acid double-marked antibodies;
wherein the detection antibody specifically corresponds to the analyte and the capture antibody, namely, if IL-6, IL-17A and IFN-gamma are required to be analyzed, the detection antibody which is required to be marked is an anti-IL-6 antibody, an anti-IL-17A antibody and an anti-IFN-gamma antibody, and so on;
the primer sequence P5 is:
CCTTGGCACCCGAGAATTCCA；
the primer sequence P7 is:
BAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA * A * A；
b2, mixing the fluorescent nucleic acid double-labeled antibodies, and adding the fluorescent nucleic acid double-labeled antibodies into a reaction system to form a 'capture antibody-exoprotein-fluorescent nucleic acid double-labeled antibody' complex, namely a fluorescent complex fixed on a solid phase carrier;
EcoR I enzyme cutting sites are calculated in the nucleic acid bridge region, ecoR I is selected as restriction enzyme, and the enzymolysis reaction stopping solution is: 0.25wt% bromophenol blue+0.25 wt% xylene blue FF or 0.25wt% xylene blue FF+40wt% sucrose aqueous solution or 30wt% glycerol aqueous solution.
The results of the test are shown in the following Table I and FIG. 4.
The fluorescent Elispot method adopts the existing fluorescent Elispot method to measure the expression quantity of the exoproteins of the anti-IL-2, anti-IL-12, anti-IFN-gamma, anti-TNF-beta, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-10, anti-IL-17, anti-IL-21, anti-IL-22 and anti-IL-26 monoclonal capture antibodies one by one, and the detection results are shown in the following table I.
The present application uses the fluorescence Elispot method as a reference, and if the detection result of the present invention is closer to the detection result of the fluorescence Elispot method, the higher the accuracy of the present application is.
TABLE one measurement of secretion of single cell exoproteins by the fluorescent Elispot method using examples 1-3
In conclusion, the ELISEQ method is formed by combining the ELISPOT with the nucleic acid sequence analysis, has the technical characteristics of high sensitivity, less non-specific combination, high flux, high accuracy and the like, can simultaneously realize detection of tens or hundreds of indexes, is simple and convenient to operate, and has good application prospect and larger market demand.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (6)
1. A method for dynamically detecting secretion of single-cell exoproteins, comprising the steps of:
(1) immobilization of antibody
Coating a monoclonal capture antibody on a solid-phase carrier, coating overnight, and washing off redundant uncoated antibodies by using a washing solution;
(2) sealing
Adding a blocking solution into the solid phase carrier coated with the capture antibody, washing, beating, and drying at low temperature to obtain a solid phase antibody;
(3) specifically capturing biological samples
Inoculating the cells to be detected into the solid-phase antibody obtained in the step (2), wherein the cells to be detected secrete the exoproteins spontaneously or under the action of a stimulating agent, and the capture antibody and the exoproteins are specifically combined to form a capture antibody-exoproteins complex which is fixed on a solid-phase carrier;
(4) detection antibody labeled with nucleic acid
Adding a nucleic acid labeled detection antibody-fluorescent labeled secondary antibody or a fluorescent nucleic acid double-labeled detection antibody into the 'capture antibody-exoprotein' complex, and washing off excessive unbound materials by using a washing solution to obtain a fluorescent complex fixed on a solid phase carrier;
the nucleic acid labeled detection antibody-fluorescent labeled secondary antibody linked "capture antibody-exoprotein" complex comprises the following steps:
a1, marking the Barcode which corresponds to the exoproteins one by one and primer sequences P5/P7 connected to two ends of the Barcode on different detection antibodies to obtain nucleic acid marked antibodies;
b1, mixing the nucleic acid labeled antibodies, and adding the mixture into a reaction system to form a complex of a capture antibody, an exoprotein and the nucleic acid labeled antibody;
c1, marking a fluorescent group on a secondary antibody, and adding the fluorescent group into a 'capture antibody-exoprotein-nucleic acid labeled antibody' complex to form a 'capture antibody-exoprotein-nucleic acid labeled antibody-fluorescent labeled secondary antibody' complex, namely a fluorescent complex fixed on a solid phase carrier;
the fluorescent nucleic acid double-labeled detection antibody connection 'capture antibody-exoprotein' complex comprises the following steps:
a2, marking the Barcode which corresponds to the exoproteins one by one, primer sequences P5/P7 connected to two ends of the Barcode and fluorescent groups on different detection antibodies to obtain fluorescent nucleic acid double-marked antibodies;
b2, mixing the fluorescent nucleic acid double-labeled antibodies, and adding the fluorescent nucleic acid double-labeled antibodies into a reaction system to form a 'capture antibody-exoprotein-fluorescent nucleic acid double-labeled antibody' complex, namely a fluorescent complex fixed on a solid phase carrier;
the primer sequence P5 is as follows: CCTTGGCACCCGAGAATTCCA;
the primer sequence P7 is as follows: baaaaaaaaaaaaaaaaaaaaax a;
(5) appear fluorescent spot
Placing the solid phase carrier fixed with the fluorescent compound in a laser environment, and correspondingly displaying fluorescent spots, namely the fluorescent compound;
(6) microdissection of fluorescent spots
Cutting and recycling the fluorescent spots under a fluorescent microscope to obtain a fluorescent compound, and transferring the fluorescent compound into an EP tube;
(7) cleavage, purification and amplification of nucleic acid fragments
Adding an enzymolysis reaction solution and a restriction enzyme into the fluorescent compound, adding an enzymolysis reaction stopping solution after the nucleic acid marked on the antibody is cut off, purifying after the enzymolysis reaction is stopped to obtain a nucleic acid fragment, and transferring the nucleic acid fragment into a PCR tube for amplification to obtain a gene library of the nucleic acid corresponding to the single cell exoprotein;
the enzymolysis reaction stopping solution is one of the following two groups;
1. 0.1mol/L EDTA-2Na+20wt% Ficoll 400+0.25wt% light-resistant orange G;
2. 0.25wt% bromophenol blue+0.25 wt% xylene blue FF or 0.25wt% xylene blue ff+40wt% sucrose aqueous solution or 30wt% glycerol aqueous solution;
(8) detection and analysis of nucleic acid fragments
Determining the molecular weight and gray value of the nucleic acid fragment of the exoprotein by an electrophoresis method, and judging the type and content of the single fluorescent spot cytokine; sequencing was performed by gene libraries to analyze the frequency of single cell secreted exoproteins and cell secreted exoproteins in the cell population.
2. The method according to claim 1, wherein in the step (1), the coating temperature is 2-8 ℃.
3. The method of claim 1, wherein the solid support is one of a nylon membrane, a PVDF membrane, an NC membrane, and a hydrogel.
4. The method according to claim 1, wherein in the step (7), the enzymatic hydrolysis reaction solution comprises 0.1mol/L, pH =7.4 of Tirs-HCl,1mol/L of NaCl and 0.07mol/L of MgCl 2 Composition is prepared.
5. The method according to claim 1, wherein in the step (7), the restriction endonuclease is one of EcoR V, alu I, ecoR I, not I, hindIII.
6. The method according to claim 1, wherein in the step (7), the nucleic acid sequence of the single cell exoprotein is purified by one or both of a centrifugal column and magnetic beads.
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