CN112816686B - Multiple signal amplification system and application thereof in detection by immuno-spot method - Google Patents
Multiple signal amplification system and application thereof in detection by immuno-spot method Download PDFInfo
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Abstract
The invention relates to a signal amplification system and application thereof in detection by an immune spot method. The invention aims to provide a method for detecting an immune spot method by using a multiple signal amplification system, which comprises the following steps: (1) the solid phase substrate is fixedly coated with an antibody or an antigen; (2) adding cells to be detected and cell stimulators or directly adding cells to be detected which have acted with the stimulators; (3) adding antibody-DNA/RNA connecting chain or adding antigen-DNA/RNA connecting chain, acting for a certain time; (4) adding DNA/RNA amplification chain, acting for a certain time; (5) adding DNA/RNA detection chain, reacting for a certain time; (6) washing, and detecting the intensity of a signal to be detected on a DNA/RNA detection chain; (7) quantitatively analyzing the number of the immuno-spots with detection signals formed on the solid phase matrix. The detection method has the advantages of multiple signal amplification, low detection limit, higher sensitivity, capability of simultaneously detecting various target objects, capability of performing multiple rounds of detection, simplicity and convenience in operation and the like.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a DNA/RNA complementary pairing-based multiple signal amplification system and detection of an immune spot method by using the same.
Background
The immunoblotting method is an immunological detection method in which an antibody is bound to a solid phase carrier such as PVDF or a multiwell aldehydic plate, and then a substance such as a cytokine secreted from a detection cell is specifically bound by an antigen-antibody. Enzyme Linked Immunospot (ELISPOT) is the most commonly used immunospot, and through a color reaction, clearly distinguishable spots are displayed at corresponding positions of substances such as soluble protein secreted by cells to be detected, the spots can be manually counted directly under an instrument or counted through an ELISPOT analysis system, 1 spot represents 1 active cell, and thus the number of the cells secreting specific substances is calculated. The traditional ELISPOT detection method is limited by the chromogenic types of enzyme and substrate, and only one antigen can be detected in one experiment. The fluorescent immuno spot method (FluoroSpot) can detect two antigens at a time, but can only perform one round of detection and has low sensitivity due to the lack of an amplification system. In order to realize simultaneous detection of multiple antigen or antibody signals and multi-round detection, the invention adopts a DNA complementary pairing technology to prepare a multistage multiplex signal amplification system with high detection sensitivity, and applies the multistage multiplex signal amplification system to the detection of an immune spot method.
Disclosure of Invention
The invention aims to provide a method for detecting an immune spot method by using a multiple signal amplification system, which comprises the following steps:
(1) the solid phase substrate is fixedly coated with an antibody or an antigen;
(2) adding cells to be detected and cell stimulators or directly adding cells to be detected which have acted with the stimulators or directly adding cells to be detected which are not stimulated by the stimulators and then acting for a period of time;
(3) removing the cells and/or cell irritants and washing; adding antibody-DNA/RNA connecting chain or adding antigen-DNA/RNA connecting chain, acting for a certain time;
(4) adding DNA/RNA amplification chain, acting for a certain time;
(5) adding a DNA/RNA detection chain, and reacting for a certain time;
(6) washing, and detecting the intensity of a signal to be detected on a DNA/RNA detection chain;
(7) quantitatively analyzing the number of the immuno-spots with detection signals formed on the solid phase matrix.
In a preferred technical scheme of the invention, in the step (1), after the solid phase matrix is coated with the antibody or the antigen, a sealing solution is added for sealing.
In a preferred embodiment of the present invention, in step (2), the cells to be detected include, but are not limited to, any one or more of human, mouse, sheep, camel, rabbit, bacteria, virus, and any other source.
In a preferred embodiment of the present invention, in step (2), the cell stimulator may be any substance that can make the cell secrete any antigen or antibody after interacting with the cell.
In a preferred embodiment of the present invention, in step (3), when the antibody or antigen is linked to the DNA/RNA connecting chain via the linking intermediate, any one or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, an alkenyl group, or any other feasible linking group is used. In a preferred embodiment of the present invention, in step (3), each antibody/antigen molecule in the antibody/antigen-DNA/RNA linkage chain is linked to one or more linking intermediates.
In a preferred embodiment of the present invention, the antibody or antigen is linked to the linking intermediate using any one or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, an alkenyl group, or any other feasible linking group.
In a preferred embodiment of the present invention, any one or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, an alkenyl group, or any other feasible linking group is used for linking the DNA/RNA connecting strand to the linking intermediate.
In a preferred embodiment of the present invention, in step (3), 1 or more DNA/RNA connecting strands are connected to each connecting intermediate in the antibody/antigen-DNA/RNA connecting strand.
In a preferred embodiment of the present invention, the ratio of the two functional groups in the intermediate for linking the antibody or antigen to the DNA/RNA linking strand is one-to-one or one-to-many.
In a preferred embodiment of the present invention, the two functional groups are not the same functional group at the same time.
In the preferred technical scheme of the invention, in the step (4), the primary DNA/RNA amplification strand can be connected firstly, and then the secondary DNA/RNA amplification strand is added after connection, and more levels of DNA/RNA amplification strands are connected according to the requirement.
In the preferred technical scheme of the invention, in the step (6), the first round of DNA/RNA detection strand can be firstly carried out for detection; then, dissociating, and adding a second round of DNA/RNA detection chain for detection; then, dissociating, and adding a third round of DNA/RNA detection chain for detection; and carrying out multiple rounds of detection as required.
In the preferred technical scheme of the invention, the detection is not less than 1 round, preferably 2 to 6 rounds in the detection process; 1 or more than 1 antigen or antibody is detected simultaneously in each detection round.
In a preferred technical scheme of the invention, one-round or multi-round detection can be performed between the connecting chain and the amplifying chain of the multiple signal amplifying system, between the amplifying chain and the amplifying chain, between the amplifying chain and the detecting chain, and between the connecting chain and the detecting chain.
In a preferred embodiment of the present invention, the position of dissociation elution before the next round of detection after each round of detection may be a position where the detection strand is complementarily paired with the amplification strand, a position where the amplification strand is complementarily paired with the linker strand, or a position where the linker strand is complementarily paired with the detection strand.
In a preferred embodiment of the present invention, dissociation at different positions is achieved by adjusting the concentration of the eluent.
In a preferred embodiment of the present invention, the detection signal can be linked to any position of the DNA/RNA detection strand, and can be at the 5 'end, the 3' end, or any position in the middle of the detection strand.
In a preferred embodiment of the present invention, the detection signal includes, but is not limited to, any one or a combination of fluorescence, phosphorescence, chemiluminescence, electromagnetic signal, nuclear magnetic signal, and radioactive signal.
In the preferable technical scheme of the invention, after the substances in each step are added for reaction, redundant substances are removed, and then washing operation is carried out.
In a preferred embodiment of the present invention, the DNA/RNA connecting strand, the DNA/RNA amplifying strand, and the DNA/RNA detecting strand may contain a base repeating unit.
In a preferred embodiment of the invention, the DNA/RNA amplification strand includes, but is not limited to, the following sequences (5 'to 3'):
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A(40≤n≤60)、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A(40≤n≤60)、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A(40≤n≤60)、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A(40≤n≤60)、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A(40≤n≤60)、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A(40≤n≤60)、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A(40≤n≤60)、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A(40≤n≤60)、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A(40≤n≤60)。
in a preferred embodiment of the present invention, the DNA/RNA connecting strand includes, but is not limited to, the following sequences (5 'to 3'):
AAAUUCCUCUACCACCUACA、AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC。
in a preferred embodiment of the present invention, the DNA/RNA detection strand includes, but is not limited to, the following sequences (5 'to 3'):
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
And detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected.
The invention also aims to provide a detection system for carrying out detection by an immunospot method by using a multiple signal amplification system, which comprises a solid phase substrate capable of being combined with a coating antibody or a coating antigen, the coating antibody or the coating antigen capable of being combined with the solid phase substrate, a cell capable of secreting a specific substance by stimulation or without stimulation, an antibody/antigen-DNA/RNA connecting chain, a DNA/RNA amplifying chain and a DNA/RNA detection chain connected with a signal to be detected, wherein the multiple signal amplification system comprises the antibody/antigen-DNA/RNA connecting chain, the DNA/RNA amplifying chain and the DNA/RNA detection chain connected with the signal to be detected, and is used for amplifying the signal secreted by the cell and then carrying out detection.
In a preferred technical scheme of the invention, the coated antibody or the coated antigen, the specific substance secreted by the cell and the antibody/antigen-DNA/RNA connecting chain can form a double-antibody sandwich or double-antigen sandwich structure.
In a preferred embodiment of the present invention, the solid phase matrix is selected from any solid phase matrix capable of adsorbing and coating the antibody/antigen or coating the antibody/antigen by chemical bonds, such as a multi-well plate, a PVDF membrane, and an aldehyde-based solid phase matrix.
In a preferred embodiment of the present invention, the coated antibody or antigen is selected from any antibody or antigen that can bind to the corresponding antigen or antibody and can be coated on a solid phase substrate.
In a preferred embodiment of the present invention, the antibody or antigen may be coated on the solid phase matrix by chemical bonding, adsorption, or the like.
In a preferred embodiment of the present invention, the cells include, but are not limited to, any one or more of human, mouse, sheep, camel, rabbit, bacteria, virus, and any other source.
In a preferred embodiment of the present invention, the cell stimulator may be any substance that can make the cell secrete any antigen or antibody after interacting with the cell.
In a preferred embodiment of the present invention, the signal to be detected can be linked to any position of the DNA/RNA detection strand, and can be at the 5 'end, the 3' end, or any position in the middle of the detection strand.
In a preferred embodiment of the present invention, the DNA/RNA connecting strand, the DNA/RNA amplifying strand, and the DNA/RNA detecting strand may contain a base repeating unit.
In a preferred embodiment of the invention, the DNA/RNA amplification strand includes, but is not limited to, the following sequences (5 'to 3'):
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A(40≤n≤60)、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A(40≤n≤60)、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A(40≤n≤60)、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A(40≤n≤60)、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A(40≤n≤60)、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A(40≤n≤60)、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A(40≤n≤60)、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A(40≤n≤60)、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A(40≤n≤60)。
in a preferred embodiment of the present invention, the DNA/RNA connecting strand includes, but is not limited to, the following sequences (5 'to 3'):
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC。
in a preferred embodiment of the present invention, the DNA/RNA detection strand includes, but is not limited to, the following sequences (5 'to 3'):
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
And detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected.
Another object of the present invention is to provide a multiplex signal amplification system, which comprises a DNA/RNA detection strand to which a signal to be detected is ligated, a DNA/RNA amplification strand, a DNA/RNA junction strand, an antibody or antigen molecule, and a junction intermediate for connecting the DNA/RNA junction strand to the antibody or antigen, wherein the DNA/RNA detection strand and the DNA/RNA junction strand are ligated after complementary pairing, or the DNA/RNA detection strand is ligated to the DNA/RNA junction strand through the DNA/RNA amplification strand, wherein bases between the strands are ligated to each other by complementary pairing.
In a preferred embodiment of the invention, one intermediate of the antibody or antigen may be linked to one or more DNA/RNA strands.
In a preferred embodiment of the present invention, the DNA/RNA detection strand and the DNA/RNA junction strand are directly complementarily paired and ligated, and the base of the DNA/RNA detection strand can be directly complementarily paired with the base of the DNA/RNA junction strand.
In a preferred embodiment of the present invention, when the DNA/RNA detection strand is directly complementarily paired with the DNA/RNA junction strand, the base length of the complementary pairing moiety is 5 to 180 base pairs, preferably 7 to 140, more preferably 8 to 100, still more preferably 9 to 70, and still more preferably 10 to 50.
In a preferred embodiment of the present invention, when the DNA/RNA detection strand is connected to the DNA/RNA connecting strand through the DNA/RNA amplification strand, the DNA/RNA connecting strand is complementarily base-paired with a portion of the one or more DNA/RNA amplification strands, and simultaneously, a portion of the DNA/RNA amplification strand is complementarily base-paired with the one or more DNA/RNA detection strands.
In a preferred embodiment of the present invention, when the DNA/RNA detection strand is linked to the DNA/RNA connecting strand via the DNA/RNA amplification strand, the DNA/RNA connecting strand is base-complementarily paired with one or more DNA/RNA amplification strand partial fragments, and then the DNA/RNA amplification strand is further base-complementarily paired with one or more DNA/RNA amplification strand partial fragments, and the latter DNA/RNA amplification strand partial fragments are base-complementarily paired with one or more DNA/RNA detection strands.
In a preferred embodiment of the present invention, when the DNA/RNA detection strand is connected to the DNA/RNA connecting strand through the DNA/RNA amplification strand, the DNA/RNA connecting strand is complementarily base-paired with a portion of the primary DNA/RNA amplification strand, the primary DNA/RNA amplification strand is complementarily base-paired with a portion of the one or more secondary DNA/RNA amplification strands, and the secondary DNA/RNA amplification strand is complementarily base-paired with the one or more DNA/RNA detection strands.
In the preferred technical scheme of the invention, the second-stage amplification chain can be connected with the third-stage amplification chain as required, and the third-stage amplification chain is connected with the fourth-stage amplification chain and sequentially connected to realize multi-stage amplification.
In a preferred technical scheme of the invention, the plurality of amplification chains are two or more amplification chains.
In the preferred technical scheme of the invention, the multistage amplification is two-stage or more amplification.
In a preferred embodiment of the present invention, all bases of the DNA/RNA connecting strand or bases of a partial fragment thereof may be connected to the DNA/RNA amplifying strand or the DNA/RNA detecting strand by base complementary pairing.
In a preferred embodiment of the present invention, all bases of the DNA/RNA detection strand or bases of a partial fragment of the DNA/RNA detection strand may be linked to the DNA/RNA amplification strand or the DNA/RNA junction strand by base complementary pairing.
In a preferred embodiment of the present invention, when the DNA/RNA detection strand is complementarily matched with the DNA/RNA amplification strand portion fragment, the base length of the matching portion is 5 to 180 base pairs, preferably 7 to 140, more preferably 8 to 100, still more preferably 9 to 70, and yet more preferably 10 to 50.
In a preferred embodiment of the present invention, when one amplified DNA/RNA strand is complementarily matched with a portion of the fragments of other amplified strands, the base length of the matched portion is 5 to 180 base pairs, preferably 7 to 140, more preferably 8 to 100, still more preferably 9 to 70, and still more preferably 10 to 50.
In a preferred embodiment of the present invention, when complementary pairing is performed between different DNA/RNA amplification strands (e.g., when primary is connected to secondary, when secondary is connected to tertiary, and so on), the base length of the pairing moiety is 5 to 180 base pairs, preferably 7 to 140, more preferably 8 to 100, still more preferably 9 to 70, and still more preferably 10 to 50.
In a preferred embodiment of the present invention, when the amplified DNA/RNA strand is complementarily paired with the ligated DNA/RNA strand, the base length of the pairing moiety is 5 to 180 base pairs, preferably 7 to 140, more preferably 8 to 100, still more preferably 9 to 70, and yet more preferably 10 to 50.
In a preferred embodiment of the present invention, the base length of the base of the DNA/RNA junction strand, the same-order DNA/RNA amplification strand (e.g., the pairing between the primary strands, the pairing between the secondary strands), and the DNA/RNA detection strand is 0 to 5 base pairs.
In a preferred technical scheme of the invention, one or more rounds of detection can be performed between the connecting chain and the amplifying chain, between the amplifying chain and the detecting chain, and between the connecting chain and the detecting chain of the multiple signal amplifying system.
In a preferred embodiment of the present invention, the position dissociated and eluted by the eluent before the next detection after each detection can be a complementary pairing position of the detection strand and the amplification strand, a complementary pairing position of the amplification strand and the connecting strand, or a complementary pairing position of the detection strand and the connecting strand.
In a preferred embodiment of the present invention, dissociation at different positions is achieved by adjusting the concentration of the eluent.
In a preferred embodiment of the present invention, the DNA/RNA connecting strand, the DNA/RNA amplifying strand, and the DNA/RNA detecting strand may contain a base repeating unit.
In a preferred embodiment of the present invention, the DNA/RNA amplification strand includes, but is not limited to, the following sequences (5 'to 3'):
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A(40≤n≤60)、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A(40≤n≤60)、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A(40≤n≤60)、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A(40≤n≤60)、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A(40≤n≤60)、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A(40≤n≤60)、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A(40≤n≤60)、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A(40≤n≤60)、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A(40≤n≤60)。
in a preferred embodiment of the present invention, the DNA/RNA connecting strand includes, but is not limited to, the following sequences (5 'to 3'):
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC。
in a preferred embodiment of the present invention, the DNA/RNA detection strand includes, but is not limited to, the following sequences (5 'to 3'):
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
And detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected.
In a preferred embodiment of the present invention, the intermediate is bifunctional at any position of two ends or in the middle, one functional group is linked to a corresponding functional group on the antibody or antigen, the other functional group is linked to a corresponding functional group on the DNA/RNA linkage strand, and the ratio of the two functional groups in the bifunctional group of the intermediate is one to one or more than one.
In a preferred embodiment of the present invention, the functional group on the antibody, antigen, DNA or RNA for reacting with or linking to the linking intermediate includes, but is not limited to, any one or more of amino, carboxyl, hydroxyl, thiol, hydrazone, alkynyl, azide, and alkenyl.
In a preferred embodiment of the present invention, one of the bifunctional groups of the linking intermediate is reacted with any one or more functional groups of the antigen or the antibody, including, but not limited to, amino, carboxyl, hydroxyl, thiol, hydrazone, alkynyl, azide, and alkenyl groups.
In a preferred embodiment of the present invention, another functional group of the bifunctional groups on the linking intermediate may be reacted with any one or more functional groups on DNA/RNA including, but not limited to, amino, carboxyl, hydroxyl, thiol, hydrazone, alkynyl, azide, and alkenyl groups.
In a preferred embodiment of the present invention, the antibody or antigen molecule linked to the linking intermediate may be linked to one or more linking intermediates simultaneously.
In a preferred embodiment of the present invention, the DNA/RNA connecting strand connected to the connecting intermediate may be connected to one or more DNA/RNA connecting strands simultaneously.
In a preferred technical scheme of the invention, the detection chain is connected with a signal to be detected.
In a preferred embodiment of the present invention, the signal to be detected can be linked to any position of the DNA/RNA detection strand, and can be at the 5 'end, the 3' end, or any position in the middle of the detection strand.
In a preferred embodiment of the present invention, the signal to be detected is selected from any one or a combination of, but not limited to, fluorescence, phosphorescence, chemiluminescence, electromagnetic signal, nuclear magnetic signal, and radioactive signal.
In a preferred technical scheme of the invention, the multiple signal amplification system can be applied to detection of an immune spot method.
It is another object of the present invention to provide a multiple signal amplification system, wherein the multiple signal amplification system employs a DNA/RNA amplification strand, wherein the DNA/RNA amplification strand may be one or more.
In a preferred embodiment of the present invention, the DNA/RNA amplification strand may be amplified in one stage or in multiple stages.
In a preferred embodiment of the present invention, when there are one or more amplified DNA/RNA strands, a portion of the amplified DNA/RNA strands is base-complementarily paired to the linked DNA/RNA strand, while a portion of the amplified DNA/RNA strands is base-complementarily paired to one or more test DNA/RNA strands.
In a preferred embodiment of the present invention, when the number of the DNA/RNA amplification strands is plural, one DNA/RNA amplification strand partial fragment is base-complementarily paired with the DNA/RNA detection strand, and is also complementarily paired with other plural DNA/RNA amplification strand partial fragments.
In a preferred embodiment of the present invention, when the DNA/RNA amplification strand is multiple, the DNA/RNA connecting strand is base-complementarily paired with one or more partial fragments of the DNA/RNA amplification strand, and then the DNA/RNA amplification strand is further base-complementarily paired with one or more partial fragments of the DNA/RNA amplification strand, and the latter partial fragments of the DNA/RNA amplification strand are base-complementarily paired with one or more detection strands of DNA/RNA.
In a preferred embodiment of the present invention, when the DNA/RNA amplification strand is amplified in multiple stages, the DNA/RNA connecting strand is complementarily base-paired with a portion of the primary DNA/RNA amplification strand, the primary DNA/RNA amplification strand is complementarily base-paired with a portion of the one or more secondary DNA/RNA amplification strands, and the secondary DNA/RNA amplification strand is complementarily base-paired with one or more DNA/RNA detection strands.
In a preferred embodiment of the present invention, all bases of the DNA/RNA connecting strand or bases of a partial fragment thereof may be connected to the DNA/RNA amplifying strand or the DNA/RNA detecting strand by base complementary pairing.
In a preferred embodiment of the present invention, all bases of the DNA/RNA detection strand or bases of a partial fragment of the DNA/RNA detection strand may be linked to the DNA/RNA amplification strand or the DNA/RNA junction strand by base complementary pairing.
In the preferred technical scheme of the invention, the second-stage amplification chain can be connected with the third-stage amplification chain as required, and the third-stage amplification chain is connected with the fourth-stage amplification chain in sequence to realize multi-stage amplification.
In a preferred technical scheme of the invention, the plurality of amplification chains are two or more amplification chains.
In the preferred technical scheme of the invention, the multistage amplification is two-stage or more amplification.
It is another object of the present invention to provide a DNA/RNA amplification strand, which includes, but is not limited to, the following sequences (5 'to 3' sequences):
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A(40≤n≤60)、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A(40≤n≤60)、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A(40≤n≤60)、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A(40≤n≤60)、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A(40≤n≤60)、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A(40≤n≤60)、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A(40≤n≤60)、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A(40≤n≤60)、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A(40≤n≤60)。
it is another object of the present invention to provide a DNA/RNA junction strand, which includes but is not limited to the following sequences (5 'to 3' sequences):
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC。
it is another object of the present invention to provide a DNA/RNA detection strand, which includes but is not limited to the following sequences (5 'to 3' sequences):
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
And detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected.
Another objective of the present invention is to provide a method for preparing a multiplex signal amplification system, which comprises preparing an antigen or antibody-DNA/RNA connecting strand, and preparing a DNA/RNA amplifying strand.
In a preferred technical scheme of the invention, the antigen or the antibody-DNA/RNA connecting chain is prepared by connecting the antibody or the antigen with the DNA/RNA connecting chain through a connecting intermediate, wherein the connecting intermediate has double functional groups, one functional group is connected with the antibody or the antigen, and the other functional group is connected with the DNA/RNA connecting chain.
In a preferred embodiment of the present invention, the functional group on the antibody or antigen or DNA/RNA connecting chain for connecting to the connecting intermediate includes, but is not limited to, any one or more of a hydrazone group, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an alkynyl group, an azide group, and an alkenyl group.
In the preferable technical scheme of the invention, one functional group in the bifunctional group of the connecting intermediate can be reacted and connected with amino, and the other functional group can be reacted and connected with carboxyl; or one functional group may be reactive linked to an amino group and another functional group may be reactive linked to a hydroxyl group; or one functional group can be reacted with an amino group for attachment and the other functional group can be reacted with a thiol group for attachment; or one functional group may be bonded to a carboxyl group and the other functional group may be bonded to a hydroxyl group; or one functional group can be reacted with carboxyl group for connection, and the other functional group can be reacted with sulfhydryl group for connection; or one functional group can be reacted with a hydroxyl group for linking, and the other functional group can be reacted with a sulfhydryl group for linking; or any other feasible connection scheme.
In a preferred embodiment of the invention, the amino group of the antibody or antigen is reactive linked to one functional group of the linking intermediate, and the thiol group of the DNA/RNA linking strand is reactive linked to another functional group of the linking intermediate.
In a preferred embodiment of the present invention, the preparation of the antigen or antibody-DNA/RNA connecting strand comprises the following steps:
(1) mixing the antigen or antibody and the connecting intermediate according to the molar ratio of 1:0.01-1:10000, and reacting at 0-50 ℃ to form the antigen or antibody-connecting intermediate;
(2) mixing the antigen or antibody-connecting intermediate and the DNA/RNA connecting chain according to the molar ratio of 1:0.01-1:10000, and reacting at 0-50 ℃ to form the antigen or antibody-DNA/RNA connecting chain.
In a preferred embodiment of the present invention, in step (1), the molar ratio of the antibody or antigen to the intermediate is 1:0.05 to 1:1000, preferably 1:0.1 to 1: 100.
In the preferred technical scheme of the invention, in the step (1), the reaction temperature is 1-25 ℃, preferably 2-8 ℃.
In a preferred embodiment of the present invention, in step (1), the antigen or antibody-linked intermediate can be purified by desalting, centrifugation column, ultrafiltration, or dialysis.
In a preferred embodiment of the present invention, in step (2), the molar ratio of the antigen or antibody-linking intermediate to the DNA/RNA-linked strand is 1:0.05 to 1:1000, preferably 1:0.1 to 1: 100.
In the preferred technical scheme of the invention, in the step (2), the reaction temperature is 1-25 ℃, preferably 2-8 ℃.
In a preferred embodiment of the present invention, in step (2), the antigen or antibody-DNA/RNA connecting strand can be purified by ultrafiltration, centrifugation or dialysis.
In a preferred embodiment of the present invention, the method for verifying the success of the antigen or antibody-DNA/RNA connecting chain connection comprises verifying by mass spectrometry.
In a preferred technical scheme of the invention, the mass spectrum is selected from any one of matrix assisted laser desorption time of flight mass spectrum (MALDI-TOF) and electrospray ionization mass spectrum (ESI-MS) or a combination thereof.
In a preferred embodiment of the invention, the DNA/RNA amplification strand includes, but is not limited to, the following sequences (5 'to 3'):
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A(40≤n≤60)、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A(40≤n≤60)、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A(40≤n≤60)、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A(40≤n≤60)、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A(40≤n≤60)、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A(40≤n≤60)、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A(40≤n≤60)、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A(40≤n≤60)、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A(40≤n≤60)。
in a preferred embodiment of the present invention, the method for preparing the DNA/RNA amplified strand comprises the following steps:
(1) mixing polymerase, corresponding primer, hairpin structure and substrate in a certain proportion and reacting for a certain time;
(2) after the reaction is finished, inactivating polymerase to prepare a DNA/RNA amplification chain reaction product;
(3) the resulting amplified DNA/RNA strand is used as it is or after purification.
In a preferred embodiment of the present invention, the amplified DNA/RNA strand is used as it is without being purified after synthesis. In a preferred embodiment of the present invention, the purification method used in the purification after the synthesis of the amplified DNA/RNA strand includes, but is not limited to, one or more of gel recovery, high performance liquid chromatography, gel permeation chromatography, ion exchange chromatography, ultrafiltration centrifugation, dialysis, precipitation, crystallization, and the like.
In a preferred embodiment of the present invention, the DNA/RNA connecting strand includes, but is not limited to, the following sequences (5 'to 3'):
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC。
the invention also aims to provide the application of the multiple signal amplification system in the detection of the immune spot method.
The invention also aims at the application of the multiple signal amplification detection system in the detection of the immunospot method.
In the present invention, "DNA/RNA" refers to DNA or RNA.
In the present invention, "antibody/antigen" refers to an antibody or an antigen.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the single-stage multiple amplification of signals is realized by connecting the DNA/RNA connecting chain with one or more DNA/RNA amplifying chains and connecting the DNA/RNA amplifying chains with a plurality of DNA/RNA detecting chains; even, the DNA/RNA amplification strand can be connected with a plurality of DNA/RNA amplification strands to realize multiple amplification of two or more stages, so that the invention not only realizes the detection of the single cell level after the cells to be detected are activated, but also greatly enhances the detection sensitivity. Under the condition of low target molecule density, signal amplification can be realized, and the cost is saved.
2. The invention can realize one or more rounds of simultaneous detection of multiple antigens or antibodies secreted by activated cells.
3. The invention can realize multi-round detection of cells to be detected secreting various antibodies or antigens, has wider applicability and reduces time cost and material cost.
4. The invention realizes gradient elution and multi-round detection by detecting the difference of the binding force caused by the length difference of the complementary pairing part base pairs of the strand, the amplification strand and the connecting strand, and only the complementary pairing base groups with the length less than a specific length can be dissociated and eluted when eluent with a certain concentration is used for elution. The position of dissociation of the complementary pair strand may be a position at which the DNA/RNA connecting strand is complementarily paired with the amplification strand, a position at which complementary pairing between multistage amplification strands occurs, a position at which the amplification strand is complementarily paired with the detection strand, and a position at which the connecting strand is complementarily paired with the detection strand.
5. The present invention allows quantification of the number of DNA/RNA linked strands coupled to each antigen or antibody.
6. The immunospot assay of the present invention can detect the number of cells that secrete two or more different antigens or antibodies simultaneously.
7. The preparation method of the amplification chain has universality, and can be used for designing and preparing the amplification chain with any required length.
8. The immune spot method can detect at the level of single cell, has high detection sensitivity, and can simultaneously detect different antigens or antibodies secreted by the same cell.
Drawings
Tables 1 to 6 show sequence information of the DNA/RNA connecting strand, the DNA/RNA amplifying strand and the DNA/RNA detecting strand in the examples of the present invention.
FIG. 1 ligation of antibody to DNA/RNA connecting strand. (1) Reacting and linking the antibody with the linking intermediate; (2) the antibody-linked intermediate conjugate is reacted with a DNA/RNA connecting strand to bind; (3) the antibody is linked to the DNA/RNA connecting strand to form an antibody-DNA/RNA connecting strand.
Fig. 2 shows the principle mechanism of signal amplification of the detection system according to the invention. Wherein, the diagram a shows a schematic diagram of a primary multiple signal amplifying system, and the diagram b shows a schematic diagram of a secondary multiple signal amplifying system. (1) Continuously circulating the primer Z-A, the hairpin structure and the substrate under the catalysis of enzyme to generate a long-chain compound 1, Z-AAAA … AA; (2) long-chain complex 1 binds to DNA/RNA connecting strand Z on the antibody; (3) detecting chain A is combined with long chain compound 1 connected to antibody and emits light to realize single multiple signal amplification; (4) in the same way as the step 1, the primers are continuously cycled to generate a long-chain complex 2A-BBBB … BB under the catalysis of A-B and the clamping of the enzyme; (5) long-chain complex 2 binds to long-chain complex 1 which has been linked to an antibody; (6) the detection chain B x B binds to the long chain complex 2 already attached to the antibody and emits light, enabling 2 multiple signal amplifications.
FIG. 3 is a flow chart of the antigen detection by the immuno-spot method of the primary multiple amplification system. (1) Coating the antibody a on a solid phase matrix; (2) adding cells, and allowing the cells to secrete the antigen and combine with the antibody a; (3) adding an antibody b-DNA/RNA connecting chain to form a double-antibody sandwich structure with a conjugate of the antigen and the antibody a; (4) adding a DNA primary amplification chain for signal amplification; (5) adding two detection chains and carrying out a first round of detection; (6) washing off two detection chains in the previous step, adding the other two detection chains, and performing a second round of detection; (7) and (4) washing off the two detection chains in the previous step, and adding the other two detection chains to perform a third round of detection.
FIG. 4 shows a process of antigen detection by the immuno-spot method of the two-stage multiplex amplification system. (1) Coating the antibody on a solid phase substrate; (2) adding cells, and allowing them to secrete antigen and bind to the antibody; (3) adding antibody b-DNA/RNA connecting chain; (4) adding a DNA primary amplification chain for primary signal amplification; (5) adding a DNA second-stage amplification chain for secondary signal amplification; (6) adding two detection chains and carrying out a first round of detection; (7) washing off two detection chains in the previous step, adding the other two detection chains, and performing a second round of detection; (8) and (4) washing off the two detection chains in the previous step, and adding the other two detection chains to perform a third round of detection.
FIG. 5A schematic representation of the gel electrophoresis results of the amplified strands prepared in example 1.
FIG. 6 Experimental results for example 1. The ordinate represents the number of spots formed by cells which can secrete specific antigens after being activated by the stimulators, and one spot represents one cell; the abscissa is the antigen to be detected secreted by the cells after activation by the stimulus. Wherein IL-2& TGF-beta indicates that IL-2 and TGF-beta can be secreted simultaneously after being activated; IL-6& IL-10 indicates that IL-6 and IL-10 can be secreted simultaneously after being activated; TNF-alpha & IFN-gamma indicates that TNF-alpha and IFN-gamma can be secreted simultaneously when activated.
FIG. 7 Experimental results for example 2. The ordinate represents the number of spots formed by cells which can secrete specific antigens after being activated by the stimulators, and one spot represents one cell; the abscissa is the antigen to be detected secreted by the cells after activation by the stimulus. Wherein IL-2& TGF-beta indicates that IL-2 and TGF-beta can be secreted simultaneously after being activated; IL-6& IL-10 indicates that IL-6 and IL-10 can be secreted simultaneously after being activated; TNF-alpha & IFN-gamma indicates that TNF-alpha and IFN-gamma can be secreted simultaneously after being activated.
Detailed Description
The present invention is illustrated by the following examples, which should be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
The embodiment of the invention detects the antigen in the process of the detection by the immune spot method and forms a double-antibody sandwich structure, and can detect the antibody in the process of practical application and form the double-antigen sandwich structure.
Comparative example 1 detection of TGF-. beta.by Using conventional ELISPOT method
(1) The coated antibody a (Anti-human TGF-. beta.capture antibody) was diluted to 10. mu.g/mL with PBS buffer and coated onto 96-well plates overnight at 4 ℃.
(2) The coated antibody a was removed and washed 3 times with PBS buffer, blocking solution (1% BSA in PBS) was added, and blocking was performed at 37 ℃ for 2 hours.
(3) Removing the blocking solution, and diluting Human Peripheral Blood Mononuclear Cells (HPBMC) to 5X 10 with RPMI-1640 medium5mL 100. mu.L of diluted HPBMC mixed with stimulators (20. mu.g/mL PHA and 20. mu.g/mL LPS) and added to corresponding wells containing 5% CO at 37 ℃2Incubate under conditions overnight.
(4) The cells were removed and washed 6 times with washing buffer (0.1% tween-20in PBS), detection antibody b (Anti-human TGF- β detection antibody) was added, the detection antibody was diluted to 2 μ g/mL with blocking solution, and added to the corresponding wells followed by incubation at room temperature for 2 hours.
(5) The detection antibody was removed and washed 6 times with washing buffer, while streptavidin was diluted 1:1000 in PBS buffer, and the diluted streptavidin was added to the corresponding wells and incubated for 45 minutes at room temperature.
(6) Streptavidin was removed and washed 3 times with wash buffer and 3 times with PBS buffer, and BCIP/NBT plus substrate (one piece diluted in 10mL of ultra pure water) was added to the corresponding wells and incubated for 15 minutes at room temperature.
(7) The substrate was removed, the 96-well plate was rinsed with water, and the 96-well plate was air-dried in the dark for detection analysis. The results of the experiment are shown in FIG. 6.
Comparative example 2 TNF-alpha detection Using the conventional ELISPOT method
(1) The coated antibody a (Anti-human TNF-. alpha.capture antibody) was diluted to 10. mu.g/mL with PBS buffer, coated onto a 96-well plate, and left overnight at 4 ℃.
(2) The coated antibody a was removed and washed 3 times with PBS buffer, blocking solution (1% BSA in PBS) was added, and blocking was performed at 37 ℃ for 2 hours.
(3) Removing the blocking solution, and diluting Human Peripheral Blood Mononuclear Cell (HPBMC) to 5 × 10 with RPMI-1640 culture medium5mL, 100. mu.L of diluted HPBMC was mixed with stimulators (20. mu.g/mL PHA and 20. mu.g/mL LPS) and added to the corresponding wells at 37 ℃ with 5% CO2Incubate under conditions overnight.
(4) The cells were removed and washed 6 times with washing buffer (0.1% tween-20in PBS), detection antibody b (Anti-human TNF- α detection antibody) was added, the detection antibody was diluted to 2 μ g/mL with blocking solution and added to the corresponding wells followed by incubation at room temperature for 2 hours.
(5) The detection antibody was removed and washed 6 times with washing buffer while streptavidin was diluted 1:1000 in PBS buffer and the diluted streptavidin was added to the corresponding wells and incubated for 45 minutes at room temperature.
(6) Streptavidin was removed and washed 3 times with wash buffer and 3 times with PBS buffer, and BCIP/NBTplus substrate (one piece diluted in 10mL of ultra pure water) was added to the corresponding wells and incubated for 15 minutes at room temperature.
(7) The substrate was removed, the 96-well plate was rinsed with water, and the 96-well plate was air-dried in the dark for detection analysis. The results of the experiment are shown in FIG. 6.
Example 1 detection of antigens Using a Primary multiplex amplification System and Immunopotentiating method
In this example, the primary multiplex amplification system was applied to the immunoblot method to simultaneously detect 6 antigens secreted by activated cells, the 6 antigens being IL-10, TGF- β, IFN- γ, TNF- α, IL-2 and IL-6, respectively.
First, preparation of antibody and DNA linker (exemplified by ligation of Anti-human IL-10antibody and DNA linker 1)
(1) The antibody (Anti-human IL-10antibody) was concentrated to 2mg/mL by ultrafiltration centrifuge tubes (100kDa MWCO) to remove azide and other preservatives.
(2) The antibody was exchanged into PBS buffer (pH7.4) by desalting spin column (7000Da MWCO).
(3) The antibody was reacted with LC-SMCC (succinimidyl-4- [ N-maleimidomethyl ] -cyclohexane-1-carboxy- [ 6-aminocaproate ]) (molar ratio 1:20) at 4 ℃ for 3 hours.
(4) Excess ligation intermediate was removed by desalting spin column (7000Da MWCO).
(5) The antibody having been linked to the linking intermediate was mixed with the purified DNA linking strand (AAATTCCTCTACCACCTACATCAC, molar ratio 1:15) and reacted at 4 ℃ for 12 hours.
(6) The antibody-DNA linkage was purified and concentrated by ultrafiltration centrifuge tubes (100kDa MWCO).
(7) Verification of successful antibody ligation to DNA linker: matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF) was used to verify the successful coupling of DNA links to the antibodies and to quantify the amount of DNA coupled to each antibody. The results showed that 1 DNA linker chain was attached to each antibody.
Second, preparation of DNA first-order amplification strand (preparation of C.1 amplification strand as an example)
(1) 10mM MgSO4, 80units/ml polymerase, 600. mu.M dATP/dCTP/dTTP, 10. mu.M hairpin structure (h.1.1) and 10. mu.M corresponding primer (p.1) were added to 100. mu.L of PBS reaction solution, respectively, and the reaction solution was left to react at 37 ℃ for 9 hours.
(2) After completion of the reaction, the reaction mixture was left at 80 ℃ for 20 minutes to inactivate the enzyme.
(3) The reaction product was mixed with a protein loading buffer (containing 30mM ethylenediaminetetraacetic acid, 50% glycerol, 0.25% xylenesulene cyan, 0.25% bromophenol blue) at a volume ratio of 9:1, and reacted at 95 ℃ for 5 minutes.
(4) A6% TBE-UREA PAGE gel (optionally a 1% agarose gel) was prepared using gelred as the dye.
(5) The gel was placed in the electrophoresis solution, the voltage was adjusted to 75V and run for 30 minutes, then the sample and DL 1000DNA marker were added to the lane, the voltage was adjusted to 200V and run for 25 minutes, and the results are shown in FIG. 5.
(6) The target DNA band was cut from the gel, purified and recovered by a DNA recovery kit, and the recovered DNA was lyophilized and stored at-20 ℃.
Thirdly, detecting various antigens by adopting an immune spot method
(1) 6 different coating antibodies a (Anti-human IL-2capture antibody/Anti-human IL-6capture antibody/Anti-human IL-10capture antibody/Anti-human IFN-. gamma.capture antibody/Anti-human TNF-. alpha.capture antibody/Anti-human TGF-. beta.capture antibody) were mixed, each coating antibody concentration was diluted to 10. mu.g/mL with PBS buffer, and the mixture was coated in 96-well plates overnight at 4 ℃.
(2) 6 coated antibodies a were removed and washed 3 times with PBS buffer, blocking solution (1% BSA in PBS) was added and blocked at 37 ℃ for 2 hours.
(3) Removing the blocking solution, and diluting Human Peripheral Blood Mononuclear Cells (HPBMC) to 5X 10 with RPMI-1640 medium5mL 100. mu.L of diluted HPBMC mixed with stimulators (20. mu.l PHA and 20. mu.l LPS) and added to corresponding wells with 5% CO at 37 ℃2Incubate under conditions overnight.
(4) Removing cells and washing 6 times with washing buffer (0.1% tween-20in PBS), adding 6detection antibodies b (Anti-human IL-2detection antibody/Anti-human IL-6detection antibody/Anti-human IL-10detection antibody/Anti-human IFN-gamma detection antibody/Anti-human TNF-alpha detection antibody/Anti-human TGF-beta detection antibody) -DNA linker, (IL-2 antibody linked DNA linker 1, TGF-beta antibody linked DNA linker 2, IFN-gamma antibody linked DNA linker 3, TNF-alpha antibody linked DNA linker 4, IL-10antibody linked DNA linker 5, IL-6 antibody linked DNA linker 6), diluting each detection antibody to 2 μ g/mL with blocking solution, and added to the corresponding wells and incubated at room temperature for 2 hours.
(5) Each detection antibody was removed and washed 6 times with washing buffer, 1 time with hybridization washing buffer (15% formamide, 1mM EDTA), and 6 DNA amplification strands C.1, C.2, C.3, C.4, C.5, C.6(60nM-150nM) were added and incubated overnight at 37 ℃.
(6) The primary amplification strand of DNA was removed and washed 3 times with hybridization wash buffer, 1 time with detection buffer (500mM NaCl in 1mM Tris-HCl), the first round of two DNA detection strands i.1X and i.2X (1. mu.M) were added and the reaction was carried out at room temperature for 1 hour, i.1X detection strand was linked with a green fluorescent probe (FAM, excitation wavelength 485nm, emission wavelength 528nm), i.2X detection strand was linked with a Red fluorescent probe (Texas Red, excitation wavelength 579nm, emission wavelength 620 nm).
(7) The first round of DNA detection strands was removed and washed 2 times with detection buffer for imaging.
(8) The dissociation buffer (50% formamide) was added and the reaction was carried out at room temperature for 20 minutes.
(9) The dissociation buffer was removed, washed 2 times with PBS buffer, 2 times with assay buffer, and two assay strands i.3 and i.4 (1 μ M) from the second round of assay were added and reacted at room temperature for 1 hour, i.3 with a green fluorescent probe (FAM) and i.4 with a Red fluorescent probe (Texas Red).
(10) The second round of DNA detection strands was removed and washed 2 times with detection buffer for imaging.
(11) The dissociation buffer (50% formamide) was added and the reaction was carried out at room temperature for 20 minutes.
(12) The dissociation buffer was removed, washed 2 times with PBS buffer, 2 times with assay buffer, and a third round of assay of two assay strands i.5 and i.6 (1 μ M) was added and reacted at room temperature for 1 hour, i.5 with a green fluorescent signal (FAM) and i.6 with a Red fluorescent signal (Texas Red).
(13) The third round of DNA detection strand was removed and washed 2 times with detection buffer for imaging.
Fourth, experimental results
The results are shown in FIG. 6. 2 different antigens (cytokines) secreted after the cells are activated can be detected at one time by combining the amplification system with an immune spot method; and can detect 6 antigens in three rounds at the same time of detecting two antigens in each round. In addition, the number of cells secreting two different antigens simultaneously can be measured in each round of detection, for example, the number of cells secreting IL-2 and TGF-. beta.simultaneously is about 90 in this example. Compared with the traditional enzyme-linked immunospot (ELISPOT) method, the immunospot method can detect more spots with higher sensitivity under the same condition, for example, cells which can secrete TGF-beta after being stimulated by a stimulant can not be detected by the traditional ELISPOT method, and about 350 cells which can secrete TGF-beta after being stimulated can be detected by combining the amplification system with the immunospot method.
Example 2 antigen detection Using two-stage multiplex amplification System and Immunopotential Spot method
In this example, a two-stage multiplex amplification system was applied to the immunoblotch method to simultaneously detect 6 antigens secreted from cells, the 6 antigens being IL-10, TGF- β, IFN- γ, TNF- α, IL-2, and IL-6, respectively.
Preparation of antibodies and DNA linkers
In this example, amino groups on antibody protein are reacted with GMBS (N- [ gamma-maleimidobutyryloxy ] succinimide ester), and then GMBS is linked to thiol groups on DNA linking chains to link the antibody to a DNA linker, and the linker intermediate used has bifunctional groups, one end of which can be linked to the antibody and the other end of which can be linked to DNA. The specific experimental procedure was the same as in example 1.
Second, preparation of primary DNA amplification chain in signal amplification system
The procedure for preparing the primary amplified DNA strand and the specific steps are the same as those in example 1.
Third, preparation of the second-order DNA amplification strand in the Signal amplification System (for example, for the C.1 amplification strand, preparation of the C.7 amplification strand, hereinafter referred to as C.7 amplification strand)
(1) 10mM MgSO4, 80units/ml polymerase, 600. mu.M dATP/dCTP/dTTP, 10. mu.M hairpin structure (h.7.7) and 10. mu.M corresponding primer (p.7) were added to 100. mu.L of PBS reaction solution, respectively, and the reaction solution was left at 37 ℃ for 9 hours.
(2) After the reaction was completed, the reaction solution was left at 80 ℃ for 20 minutes to inactivate the enzyme.
(3) The reaction product was mixed with a protein Loading buffer (Loading buffer) at a volume ratio of 9:1 to prepare a sample, and reacted at 95 ℃ for 5 minutes.
(4) A6% TBE-UREA PAGE gel (1% agarose gel may also be used) was prepared using gelred as the dye.
(5) The gel was placed in the electrophoresis solution and the voltage was adjusted to 75V for 30 minutes, after which the sample and DL 1000DNA marker were added to the lane and the voltage was adjusted to 200V for 25 minutes.
(6) The target DNA band was cut from the gel, purified and recovered by a DNA recovery kit, and the recovered DNA was lyophilized and stored at-20 ℃.
Fourthly, detecting various antigens by adopting an amplification system and an immune spot method
(1) 6 different coating antibodies a (Anti-human IL-2capture antibody/Anti-human IL-6capture antibody/Anti-human IL-10capture antibody/Anti-human IFN-. gamma.capture antibody/Anti-human TNF-. alpha.capture antibody/Anti-human TGF-. beta.capture antibody) were mixed, each coating antibody concentration was diluted to 10. mu.g/mL with PBS buffer, and the mixture was coated in 96-well plates overnight at 4 ℃.
(2) 6 coated antibodies a were removed and washed 3 times with PBS buffer, blocking solution (1% BSA in PBS) was added and blocked at 37 ℃ for 2 hours.
(3) Removing the blocking solution, and diluting Human Peripheral Blood Mononuclear Cells (HPBMC) to 5X 10 with RPMI-1640 medium5mL 100. mu.L of diluted HPBMC mixed with stimulators (20. mu.l PHA and 20. mu.l LPS) and added to corresponding wells with 5% CO at 37 ℃2Incubate under conditions overnight.
(4) Removing cells and washing 6 times with washing buffer (0.1% tween-20in PBS), adding 6detection antibodies b (anti-human IL-2detection antibody/anti-human TGF- β detection antibody/anti-human TNF- α detection antibody/anti-human IFN- γ detection antibody/anti-human IL-6detection antibody/anti-human IL-10detection antibody) -DNA linker, (IL-2 antibody linked DNA linker 1, TGF- β antibody linked DNA linker 2, IFN- γ antibody linked DNA linker 3, TNF- α antibody linked DNA linker 4, IL-10antibody linked DNA linker 5, IL-6 antibody linked DNA linker 6), diluting each detection antibody to 2 μ g/mL with blocking solution, and added to the corresponding wells and incubated at room temperature for 2 hours.
(5) Each detection antibody was removed and washed 6 times with washing buffer, 1 time with hybridization washing buffer (15% formamide, 1mM EDTA), and 6 primary DNA amplification strands C.1, C.2, C.3, C.4, C.5, C.6(60nM-150nM) were added and incubated overnight at 37 ℃.
(6) The primary DNA amplification strand was removed, washed 4 times with hybridization washes, and then 4 secondary DNA amplification strands C.7, C.8, C.9, C.10, C.11, C.12(60nM-150nM) partially complementary to the primary DNA amplification strand were added and incubated overnight at 37 ℃.
(7) The secondary DNA strands were removed and washed 3 times with hybridization wash buffer, 1 time with assay buffer (500mM NaCl in 1mM Tris-HCl), and the first round of two DNA strands i.7 and i.8 (1. mu.M) were added and reacted at room temperature for 1 hour, i.7 with a green fluorescence signal (FAM) attached to the strands and i.8 with a Red fluorescence signal (Texas Red).
(8) The first round of DNA detection strands was removed and washed 2 times with detection buffer for imaging.
(9) The dissociation buffer (50% formamide) was added and the reaction was carried out at room temperature for 20 minutes.
(10) The dissociation buffer was removed, washed 2 times with PBS buffer, 2 times with assay buffer, and two assay strands i.9 and i.10 (1 μ M) from the second round of assay were added and reacted at room temperature for 1 hour, i.9 with a green fluorescence signal (FAM) and i.10 with a Red fluorescence signal (Texas Red).
(11) The second round of DNA detection strands was removed and washed 2 times with detection buffer for imaging.
(12) Dissociation buffer (50% formamide) was added and the reaction was carried out at room temperature for 20 minutes.
(13) The dissociation buffer was removed, washed 2 times with PBS buffer, 2 times with assay buffer, and the third round of two assay strands, i.11 x and i.12 x (1 μ M), with a green fluorescent signal (FAM) attached to the i.11 x strand and a Red fluorescent signal (Texas Red) attached to the i.12 x strand were added and reacted at room temperature for 1 hour.
(14) The third round of DNA detection strand was removed and washed 2 times with detection buffer for imaging.
Fifth, experimental results
The results are shown in FIG. 7. 2 different antigens (cytokines) secreted after cell activation can be detected at one time by combining the multistage multiple amplification system with an immune spot method; and can detect 6 antigens in three rounds at the same time of detecting two antigens in each round. In addition, the number of cells secreting two different antigens simultaneously can be measured in each round of detection, for example, the number of cells secreting IL-2 and TGF-. beta.simultaneously in this example is about 2100.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications without departing from the spirit of the present invention, which should fall within the scope of the appended claims.
Sequence listing
<110> Ikefa (Beijing) Biotechnology Ltd
<120> signal amplification system and application thereof in detection by immunospot assay
<150> 2020112536617
<151> 2020-11-11
<150> 2020112536246
<151> 2020-11-11
<150> 2020112536299
<151> 2020-11-11
<150> 2020112562382
<151> 2020-11-11
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<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (43)..(43)
<223> 3' terminal, reverse dT modification
<400> 41
attattcact gggccttttg gcccagtgaa taatagtgaa taa 43
<210> 42
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (43)..(43)
<223> 3' terminal, reverse dT modification
<400> 42
atcattactt gggccttttg gcccaagtaa tgataagtaa tga 43
Claims (76)
1. A method for carrying out an immunospot assay by using a multiplex signal amplification system is characterized by comprising the following steps:
(1) the solid phase substrate is fixedly coated with an antibody or an antigen;
(2) adding cells to be detected and cell stimulators or directly adding cells to be detected which have acted with the stimulators or directly adding cells to be detected which are not stimulated by the stimulators and then acting for a period of time;
(3) removing the cells and/or cell irritants and washing; adding antibody-DNA/RNA connecting chain or adding antigen-DNA/RNA connecting chain, acting for a certain time;
(4) adding DNA/RNA amplification chain, acting for a certain time;
(5) adding DNA/RNA detection chain, reacting for a certain time;
(6) washing, and detecting the intensity of a signal to be detected on a DNA/RNA detection chain;
(7) quantitatively analyzing the number of the immuno-spots with detection signals formed on the solid matrix;
the DNA/RNA amplification strand comprises the following sequences:
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A；
the DNA/RNA connecting chain comprises the following sequences:
AAAUUCCUCUACCACCUACA、AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC；
the DNA/RNA detection strand comprises the following sequences:
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
Detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected;
n is within the range of 40-60;
the sequence is from left to right 5 'to 3' of the sequence.
2. The detection method according to claim 1, wherein in the step (1), the antibody or the antigen is coated on the solid phase substrate, and then a blocking solution is added to perform blocking.
3. The detection method according to claim 1, wherein in the step (2), the cells to be detected comprise any one or more of human, mouse, sheep, camel, rabbit, bacteria and virus.
4. The method according to claim 1, wherein in the step (2), the cell stimulant is a substance which can cause the cell to secrete any antigen or antibody after reacting with the cell.
5. The detection method according to claim 1, wherein in the step (3), any one of or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, and an alkenyl group is used for the connection of the antibody or the antigen to the DNA/RNA connecting chain via the intermediate for connection.
6. The detection method according to claim 5, wherein in the step (3), one or more linking intermediates are linked to each antibody/antigen molecule in the antibody/antigen-DNA/RNA linking chain.
7. The detection method according to claim 5, wherein the antibody or antigen is linked to the linking intermediate using any one or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, and an alkenyl group.
8. The detection method according to claim 5, wherein any one or a combination of a thiol group, an amino group, a carboxyl group, a hydroxyl group, a hydrazone group, an alkynyl group, an azide group, and an alkenyl group is used for the linkage of the DNA/RNA connecting strand to the linking intermediate.
9. The detection method according to claim 5, wherein in the step (3), 1 or more DNA/RNA connecting strands are connected to each of the antibody/antigen-DNA/RNA connecting strands.
10. The detection method according to claim 5, wherein the ratio of the two functional groups in the linking intermediate for linking the antibody or antigen and the DNA/RNA linking strand is one to one or one to more.
11. The detection method according to claim 10, wherein the two functional groups are not the same functional group at the same time.
12. The detection method according to claim 1, wherein in the step (4), the primary DNA/RNA amplification strand is ligated, and then the secondary DNA/RNA amplification strand is added, and further levels of DNA/RNA amplification strands are ligated.
13. The detection method according to claim 1, wherein in step (6), a first DNA/RNA detection strand is added for detection; then, dissociating, and adding a second round of DNA/RNA detection chain for detection; then dissociation is carried out, and then a third round of DNA/RNA detection chain is added for carrying out a third round of detection or a plurality of rounds of detection.
14. The detection method according to claim 1, wherein no less than 1 round of detection is performed during the detection; 1 or more than 1 antigen or antibody is detected simultaneously in each detection round.
15. The inspection method according to claim 14, wherein the inspection of not less than 1 round in the inspection process is 2 to 6 rounds; 1 or more than 1 antigen or antibody is detected simultaneously in each round of detection.
16. The detection method according to claim 1, wherein one or more rounds of detection can be performed between the connection chain and the amplification chain, between the amplification chain and the detection chain, and between the connection chain and the detection chain of the multiple signal amplification system.
17. The method of claim 16, wherein the position of the dissociation elution before the next round of detection after each round of detection is a position where the detection strand is complementarily paired with the amplification strand, a position where the amplification strand is complementarily paired with the linker strand, and a position where the linker strand is complementarily paired with the detection strand.
18. The detection method according to claim 13, wherein the dissociation at different positions is achieved by adjusting the concentration of the eluent.
19. The method of claim 1, wherein the detection signal is linked to the 5 'end, the 3' end of the DNA/RNA detection strand, or anywhere in the middle of the detection strand.
20. The detection method according to claim 1, wherein the detection signal comprises any one of fluorescence, phosphorescence, chemiluminescence, electromagnetic signal, nuclear magnetic signal, radioactive signal, or a combination thereof.
21. The detection method according to claim 1, wherein after the addition of the substances in each step, the reaction is carried out, excess substances are removed, and then a washing operation is carried out.
22. The detection method according to claim 1, wherein the DNA/RNA connecting strand, the DNA/RNA amplifying strand, and the DNA/RNA detection strand contain a base repeating unit.
23. A detection system for carrying out detection by an immune spot method by using a multiple signal amplification system is characterized by comprising a solid phase substrate which can be combined with a coating antibody or a coating antigen, the coating antibody or the coating antigen which can be combined with the solid phase substrate, a cell which can secrete a specific substance by stimulation or without any stimulation, an antibody/antigen-DNA/RNA connecting chain, a DNA/RNA amplifying chain and a DNA/RNA detection chain connected with a signal to be detected, wherein the multiple signal amplification system comprises the antibody/antigen-DNA/RNA connecting chain, the DNA/RNA amplifying chain and the DNA/RNA detection chain connected with the signal to be detected, is used for amplifying the signal secreted by the cell and then carrying out detection,
the DNA/RNA amplification strand comprises the following sequence:
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A；
the DNA/RNA connecting strand comprises the following sequences:
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC；
the DNA/RNA detection strand comprises the following sequences:
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
Detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected;
n is within the range of 40-60;
the sequence is from left to right from the 5 'end to the 3' end of the sequence.
24. The detection system according to claim 23, wherein the coated antibody or antigen, the specific substance secreted by the cell, and the antibody/antigen-DNA/RNA connecting chain can form a double antibody sandwich or double antigen sandwich structure.
25. The detection system of claim 23, wherein the solid phase matrix is selected from the group consisting of a multiwell plate, a PVDF membrane, and an aldehydized solid phase matrix.
26. The detection system of claim 23, wherein the coating antibody or antigen is selected from the group consisting of an antibody or antigen that binds to the corresponding antigen or antibody and can be coated on a solid substrate.
27. The detection system of claim 23, wherein the antibody or antigen is coated onto the solid phase matrix by chemical bonding or adsorption.
28. The detection system of claim 23, wherein the cells comprise any one or more of human, mouse, ovine, camelid, rabbit, bacteria, and virus.
29. The test system according to claim 23, wherein the cell stimulant is a substance that causes the cell to secrete an antigen or antibody upon interaction with the cell.
30. The test system according to claim 23, wherein the signal to be detected is linked to the 5 'end, the 3' end of the DNA/RNA detection strand or anywhere in the middle of the detection strand.
31. The test system according to claim 23, wherein the DNA/RNA junction strand, the DNA/RNA amplification strand, and the DNA/RNA test strand comprise base repeat units.
32. A multiple signal amplification system, comprising a DNA/RNA detection strand to which a signal to be detected is ligated, a DNA/RNA amplification strand, a DNA/RNA junction strand, an antibody or antigen molecule, and a junction intermediate for connecting the DNA/RNA junction strand to the antibody or antigen, wherein the DNA/RNA detection strand and the DNA/RNA junction strand are ligated after complementary pairing, or the DNA/RNA detection strand is ligated to the DNA/RNA junction strand through the DNA/RNA amplification strand, wherein bases between the strands are ligated to each other by complementary pairing, and the DNA/RNA amplification strand comprises the following sequence:
GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACCTA-A)n-ATAAACCTA-A、
CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)n-CATCATCAT-A、
GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A、
CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A、
ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAATCAAAA-A)n-CAATCAAAA-A、
GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCAATAATA-A)n-CCAATAATA-A、
TAGGTTTAT-T-TAGGTTTAT-T-TAGGTTTAT-TTT-TTCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A、
ATGATGATG-T-ATGATGATG-T-ATGATGATG-TTT-TTTTCTACC-A-(TTTTCTACC-A)n-TTTTCTACC-A、
GAGAGTATT-T-GAGAGTATT-T-GAGAGTATT-TTT-TCCTTTTAT-A-(TCCTTTTAT-A)n-TCCTTTTAT-A、
GTTAAGTTG-T-GTTAAGTTG-T-GTTAAGTTG-TTT-CCTTCTATT-A-(CCTTCTATT-A)n-CCTTCTATT-A、
GTAAATGAA-T-GTAAATGAA-T-GTAAATGAA-TTT-TTATTCACT-A-TTATTCACT-A-(TTATTCACT-A)n-TTATTCACT-A、
TTTTGATTG-T-TTTTGATTG-T-TTTTGATTGTTT-TCATTACTT-A-TCATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A、
TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TTCTTACTC-A-(TTCTTACTC-A)n-TTCTTACTC-A；
the DNA/RNA connecting strand comprises the following sequences:
AAAUUCCUCUACCACCUACA、
AAATTCCTCTACCACCTACATCAC、
TATTTAGTGTTCGAATAGTT、
TATTTAGTGTTCGAATAGTTCGATCTAG、
AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、
GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、
GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、
CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、
AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、
AAAUUCCUCUACCACCUACAUCAC；
the DNA/RNA detection strand comprises the following sequences:
the signal to be detected-TT-TAGGTTTAT-T-TAGGTTTAT-T,
The signal to be detected-TT-ATGATGATG-T-ATGATGATG-T,
The signal to be detected-TT-GAGAGTATT-T-GAGAGTATT-T,
The signal to be detected-TT-GTTAAGTTG-T-GTTAAGTTG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-TTTTGATTG-T-TTTTGATTG-T,
The signal to be detected-TT-TATTATTGG-T-TATTATTGG-T,
The signal to be detected-TT-GTAAATGAA-T-GTAAATGAA-T,
The signal to be detected-TT-GGTAGAAAA-T-GGTAGAAAA-T,
The signal to be detected-TT-ATAAAAGGA-T-ATAAAAGGA-T,
The signal to be detected-TT-AATGAAAGA-T-AATGAAAGA-T,
The signal to be detected-TT-AGTGAATAA-T-AGTGAATAA-T,
The signal to be detected-TT-AAGTAATGA-T-AAGTAATGA-T,
The signal to be detected-TT-GAGTAAGAA-T-GAGTAAGAA-T,
Detecting a signal-UU-GUAAAUGAA-U-GUAAAUGAA-U to be detected;
n is within the range of 40-60;
the sequence is from left to right from the 5 'end to the 3' end of the sequence.
33. The multiple signal amplification system of claim 32, wherein one linking intermediate of an antibody or antigen is linked to one or more DNA/RNA linking strands.
34. The multiple signal amplification system of claim 32, wherein the DNA/RNA detection strand is linked to the DNA/RNA junction strand in direct complementary pairing such that the bases of the DNA/RNA detection strand are directly complementary paired to the bases of the DNA/RNA junction strand.
35. The multiple signal amplification system of claim 32, wherein when the DNA/RNA detection strand is linked to the DNA/RNA linking strand via the DNA/RNA amplification strand, the DNA/RNA linking strand is base complementary paired with a portion of the one or more DNA/RNA amplification strands, and a portion of the DNA/RNA amplification strand is base complementary paired with a portion of the one or more DNA/RNA detection strands.
36. The multiple signal amplification system of claim 32, wherein when the DNA/RNA detection strand is linked to the DNA/RNA linking strand via the DNA/RNA amplification strand, the DNA/RNA linking strand is base complementary paired with one or more DNA/RNA amplification strand portion fragments, and then the DNA/RNA amplification strand is base complementary paired with one or more DNA/RNA amplification strand portion fragments that are base complementary paired with one or more DNA/RNA detection strand.
37. The multiple signal amplification system of claim 32 wherein when the DNA/RNA detection strand is linked to the DNA/RNA connecting strand via the DNA/RNA amplification strand, the DNA/RNA connecting strand is complementary base paired to a portion of the primary DNA/RNA amplification strand, the primary DNA/RNA amplification strand is complementary base paired to a portion of the one or more secondary DNA/RNA amplification strands, and the secondary DNA/RNA amplification strand is complementary base paired to one or more DNA/RNA detection strands.
38. The multiple signal amplification system of claim 32, wherein the second amplification chain is connected to a third amplification chain, and the third amplification chain is connected to a fourth amplification chain, and the third amplification chain and the fourth amplification chain are connected in sequence to achieve multi-stage amplification.
39. The multiple signal amplification system of claim 38, wherein the plurality of amplification chains is two or more amplification chains.
40. The multiple signal amplification system of claim 38, wherein the multi-stage amplification is two or more stages of amplification.
41. The multiple signal amplification system of claim 32, wherein all bases of the DNA/RNA connecting strand or bases of a partial fragment thereof are connected to the DNA/RNA amplifying strand or the DNA/RNA detecting strand by base complementary pairing.
42. The multiple signal amplification system of claim 32, wherein all bases of the DNA/RNA detection strand or bases of a partial fragment thereof are ligated to the DNA/RNA amplification strand or the DNA/RNA junction strand by base complementary pairing.
43. The multiple signal amplification system of claim 32, wherein one or more rounds of detection are performed between the link chain and the amplification chain, between the amplification chain and the detection chain, and between the link chain and the detection chain.
44. The multiple signal amplification system of claim 32 wherein the positions eluted by dissociation with an eluent after each detection cycle and before the next detection cycle are the position of complementary pairing of the detection strand and the amplification strand, the position of complementary pairing of the amplification strand and the connecting strand, and the position of complementary pairing of the detection strand and the connecting strand.
45. The multiple signal amplification system of claim 44 wherein dissociation at different positions is achieved by adjusting the concentration of the eluent.
46. The multiple signal amplification system of claim 32, wherein the DNA/RNA junction strand, the DNA/RNA amplification strand, and the DNA/RNA detection strand comprise a base repeat unit.
47. The multiple signal amplification system of claim 32 wherein the linking intermediate is bifunctional at either end or any position in the middle, one functional group is linked to a corresponding functional group on the antibody or antigen, the other functional group is linked to a corresponding functional group on the DNA/RNA linking strand, and the ratio of the two functional groups in the bifunctional linking intermediate is one to one or one to more.
48. The multiple signal amplification system of claim 47, wherein the functional group on the antibody, antigen, DNA or RNA for reaction with or attachment to the linking intermediate comprises any one or more of amino, carboxyl, hydroxyl, sulfhydryl, hydrazone, alkynyl, azide, or alkenyl.
49. The multiple signal amplification system of claim 48, wherein one of the bifunctional groups of the linking intermediate is reactive linked to any one or more of the functional groups of the antigen or antibody, including amino, carboxyl, hydroxyl, thiol, hydrazone, alkynyl, azide, or alkenyl groups.
50. The multiple signal amplification system of claim 49, wherein another functional group of the bifunctional groups of the linking intermediate is reacted and linked with any one or more functional groups of the DNA/RNA, including amino, carboxyl, hydroxyl, thiol, hydrazone, alkynyl, azide, or alkenyl groups.
51. The multiple signal amplification system of claim 32, wherein the antibody or antigen molecule linked to the linking intermediate is capable of linking to one or more linking intermediates simultaneously.
52. The multiple signal amplification system of claim 32, wherein the DNA/RNA junction strands are connected to a junction intermediate, and one junction intermediate can connect one or more DNA/RNA junction strands simultaneously.
53. The multiple signal amplification system of claim 32, wherein the signal to be detected is linked to the 5 'end, the 3' end of the DNA/RNA detection strand, or anywhere in the middle of the detection strand.
54. The multiple signal amplification system of claim 32, wherein the signal to be detected is selected from any one of fluorescence, phosphorescence, chemiluminescence, electromagnetic signal, nuclear magnetic signal, radioactive signal, or a combination thereof.
55. The multiple signal amplification system of claim 32, wherein the multiple signal amplification system is used in an immuno-spot assay.
56. The method for preparing a multiplex signal amplification system as defined in any one of claims 32 to 55 comprising preparing an antigen or antibody-DNA/RNA linker, preparing a DNA/RNA amplification strand, wherein the antigen or antibody-DNA/RNA linker is prepared such that the antibody or antigen is linked to the DNA/RNA linker via a linker intermediate, wherein the linker intermediate has bifunctional groups, one of which is linked to the antibody or antigen and the other of which is linked to the DNA/RNA linker.
57. The method of claim 56, wherein the functional group on the antibody or antigen or DNA/RNA connecting chain for connecting to the intermediate comprises one or more of hydrazone group, amino group, carboxyl group, hydroxyl group, mercapto group, alkynyl group, azide group and alkenyl group.
58. The method according to claim 56, wherein one of the bifunctional groups of the intermediate linker is reactive with an amino group and the other of the bifunctional groups is reactive with a carboxyl group; or one functional group may be reactive linked to an amino group and another functional group may be reactive linked to a hydroxyl group; or one functional group can be reacted with an amino group for attachment and the other functional group can be reacted with a thiol group for attachment; or one functional group may be bonded to a carboxyl group and the other functional group may be bonded to a hydroxyl group; or one functional group can be reacted with carboxyl group for connection, and the other functional group can be reacted with sulfhydryl group for connection; or one functional group may be reactive linked to a hydroxyl group and the other functional group may be reactive linked to a thiol group.
59. The method of claim 56, wherein an amino group on the antibody or antigen is reacted with one functional group on the linking intermediate to link, and a thiol group on the DNA/RNA linking strand is reacted with another functional group on the linking intermediate to link.
60. The method of claim 56, wherein the preparation of the antigen or antibody-DNA/RNA linked strand comprises the steps of:
(1) mixing the antigen or antibody and the connecting intermediate according to the molar ratio of 1:0.01-1:10000, and reacting at 0-50 ℃ to form the antigen or antibody-connecting intermediate;
(2) mixing the antigen or antibody-connecting intermediate and the DNA/RNA connecting chain according to the molar ratio of 1:0.01-1:10000, and reacting at 0-50 ℃ to form the antigen or antibody-DNA/RNA connecting chain.
61. The method according to claim 60, wherein in the step (1), the molar ratio of the antibody or antigen to the intermediate is 1:0.05 to 1: 1000.
62. The method according to claim 61, wherein in the step (1), the molar ratio of the antibody or antigen to the intermediate is 1:0.1 to 1: 100.
63. The method according to claim 60, wherein the reaction temperature in the step (1) is 1 to 25 ℃.
64. The method according to claim 63, wherein the reaction temperature in the step (1) is 2 to 8 ℃.
65. The method according to claim 60, wherein in the step (1), the antigen or antibody-linked intermediate is purified by desalting, centrifugation column or ultrafiltration or dialysis.
66. The method according to claim 60, wherein in the step (2), the molar ratio of the antigen or antibody-linked intermediate to the DNA/RNA-linked strand is 1:0.05 to 1: 1000.
67. The method according to claim 66, wherein in the step (2), the molar ratio of the antigen or antibody-linked intermediate to the DNA/RNA-linked strand is 1:0.1 to 1: 100.
68. The method according to claim 60, wherein in the step (2), the reaction temperature is 1 to 25 ℃.
69. The method according to claim 68, wherein in the step (2), the reaction temperature is 2 to 8 ℃.
70. The method of claim 60, wherein in step (2), the antigen or antibody-DNA/RNA linked strand is purified by centrifugation or dialysis.
71. The method of claim 60, wherein the successful verification of the antigen or antibody-DNA/RNA junction comprises verification using mass spectrometry.
72. The method of claim 71, wherein the mass spectrometry is selected from any one or a combination of matrix-assisted laser desorption time-of-flight mass spectrometry, electrospray ionization mass spectrometry.
73. The method of claim 60, wherein the DNA/RNA amplification strand is prepared by a method comprising the steps of:
(1) mixing polymerase, corresponding primer, hairpin structure and substrate in a certain proportion and reacting for a certain time;
(2) after the reaction is finished, inactivating polymerase to prepare a DNA/RNA amplification chain reaction product;
(3) the resulting amplified DNA/RNA strand is used as it is or after purification.
74. The method of claim 73, wherein the purification after the DNA/RNA amplification strand synthesis comprises one or more of gel recovery, high performance liquid chromatography, gel permeation chromatography, ion exchange chromatography, ultrafiltration centrifugation, dialysis, precipitation, and crystallization.
75. Use of a multiplex signal amplification system according to any one of claims 32 to 55 or prepared according to any one of claims 56 to 74 in an immuno-dot assay.
76. Use of the multiplex signal amplification detection system of any one of claims 23 to 31 in an immuno-dot assay.
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