CN114544965A

CN114544965A - Multiple signal amplification system and application thereof in immunoadsorption competition method detection

Info

Publication number: CN114544965A
Application number: CN202011604915.5A
Authority: CN
Inventors: 刘密; 彭作富; 李柏松
Original assignee: Aikefa Beijing Biotechnology Co ltd
Current assignee: Aikefa Beijing Biotechnology Co ltd
Priority date: 2020-11-11
Filing date: 2020-12-30
Publication date: 2022-05-27
Anticipated expiration: 2040-12-30
Also published as: CN113355396B; CN114544966A; CN114544967B; CN114544965B; CN114544937B; CN112816686A; CN114544937A; CN113341132A; CN113355396A; CN114544967A; CN113341132B; CN114544966B; CN112816686B

Abstract

The invention relates to a signal amplification system and application thereof in immunoadsorption competition method detection. A method for carrying out immunoadsorption competition method detection by using a multiple signal amplification system comprises the following steps: (1) coating the antigen on a solid phase matrix to form a solid phase antigen; (2) adding a sample to be detected and an antibody-DNA/RNA connecting chain, and mixing for a certain time; (3) adding DNA amplifying chain for certain time; (4) adding DNA detection chain, acting for a certain time; (5) washing, and detecting the signal intensity on the DNA/RNA detection chain; (6) and drawing a standard curve according to the detection signal intensities of the standard substances with different concentrations, and calculating the concentration of the sample to be detected according to the detection signal intensity of the sample to be detected and the standard curve. The detection method has the advantages of multiple amplification of signals, low detection limit, higher sensitivity, capability of simultaneously detecting a plurality of target objects, capability of performing multi-round detection, simplicity and convenience in operation and the like.

Description

Multiple signal amplification system and application thereof in immunoadsorption competition method detection

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a multiple signal amplification system and application thereof in immunoadsorption competition method detection.

Background

An Enzyme linked immunosorbent assay (abbreviated as ELISA) is a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid phase carrier such as polystyrene and immunoreaction is carried out by utilizing the specific binding of the antigen and the antibody. There are 3 reagents necessary in the traditional ELISA assay: immobilized antigen or antibody, enzyme-labeled antigen or antibody, substrate for enzyme action.

The traditional ELISA detection method is limited by the chromogenic types of enzyme and substrate, and only one antigen or antibody can be detected in one experiment. In order to achieve simultaneous detection of multiple antigen or antibody signals and to achieve multiple rounds of detection, other signal amplification methods have been investigated instead of enzymatic signal amplification.

CN101988920A discloses an antigen detection kit and detection method, which uses DNA oligonucleotide conjugated antibody to replace peroxidase fused antibody for signal amplification by enzyme. RNA oligonucleotides are respectively complementary to DNA oligonucleotides, so that simultaneous detection of multiple antigens in single detection can be realized, but the detection method needs to add RNA enzyme, and has the defects of high detection limit, incapability of realizing multiple rounds of detection, low detection efficiency, high cost, complex operation and the like. Therefore, a new signal amplification system having high detection sensitivity and easy operation needs to be studied.

Disclosure of Invention

The invention aims to provide a method for carrying out immunoadsorption competition method detection by using a multiple signal amplification system, which comprises the following steps:

(1) coating the antigen on a solid phase matrix to form a solid phase antigen;

(2) adding a sample to be detected and an antibody-DNA/RNA connecting chain, and mixing for a certain time;

(3) adding DNA/RNA amplification chain, acting for a certain time;

(4) adding DNA/RNA detection chain, acting for a certain time;

(5) washing, and detecting the signal intensity on the DNA/RNA detection chain;

(6) and drawing a standard curve according to the detection signal intensities of the standard substances with different concentrations, and calculating the concentration of the sample to be detected according to the detection signal intensity of the sample to be detected and the standard curve.

In the preferable technical scheme of the invention, in the step (1), after the antigen is coated, the sealing liquid is added for sealing.

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 coating antigen is selected from any antigen that can bind to the corresponding antibody and can be coated on a solid matrix.

In a preferred embodiment of the present invention, the antigen may be coated on the solid phase matrix by chemical bond, adsorption, or the like.

In a preferred technical scheme of the present invention, in the step (2), the sample to be detected is an antigen to be detected or an antibody to be detected.

In a preferred embodiment of the present invention, the antigen to be detected may be 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 antibody to be detected may be from any one or more of, but not limited to, human, mouse, sheep, camel, rabbit, bacteria, virus, and any other source.

In a preferred embodiment of the present invention, in step (2), one or more linking intermediates are linked to each antibody molecule in the antibody-DNA/RNA linking chain.

In a preferred embodiment of the present invention, the antibody 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, in step (2), one or more DNA/RNA connecting strands are connected to each connecting intermediate in the antibody-DNA/RNA connecting strands.

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 linking intermediate to the DNA/RNA linking chain.

In a preferred embodiment of the present invention, the ratio of the two functional groups contained in the intermediate for linking the antibody to the DNA/RNA connecting strand is one to one or one to more.

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 (3), 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 (5), the first round of DNA/RNA detection chain can be firstly detected, then dissociation is carried out, and then the second round of DNA/RNA detection chain is added 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 8 rounds; 1 or more than 1 antigen is detected 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-ATAAACCT A-A(40≤n≤60)、

CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATCATCAT-A)_n-CATC ATCAT-A(40≤n≤60)、

GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-AATACTCTC-A-(AAT ACTCTC-A)_n-AATACTCTC-A(40≤n≤60)、

CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT-A-CAACTTAAC-A-(C AACTTAAC-A)_n-CAACTTAAC-A(40≤n≤60)、

ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-TTCATTTAC-A-(TTC ATTTAC-A)_n-TTCATTTAC-A(40≤n≤60)、

GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-CAATCAAAA-A-(CAA TCAAAA-A)_n-CAATCAAAA-A(40≤n≤60)、

GACGGTGAATGTACGACTATGCGACGGGATACTACAGGAACT-TT-CCAATAATA-A-(CCA ATAATA-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 objective of the present invention is to provide a method for performing immunoadsorption competitive assay using a multiplex signal amplification system, comprising the following steps:

(1) coating the antibody on a solid phase matrix to form a solid phase antibody;

(2) adding a sample to be detected and an antigen-DNA/RNA connecting chain, and mixing for a certain time;

(3) adding DNA/RNA amplification chain, acting for a certain time;

(4) adding DNA/RNA detection chain, acting for a certain time;

(5) washing, and detecting the signal intensity on the DNA/RNA detection chain;

In a preferred technical scheme of the invention, in the step (1), after the antibody is coated, a blocking solution is added for blocking.

In a preferred embodiment of the present invention, the coating antibody is selected from any antibody that binds to the corresponding antigen and can be coated on a solid phase substrate.

In a preferred embodiment of the present invention, the antibody may be coated on a solid phase substrate by chemical bonding, adsorption, or the like.

In a preferred embodiment of the present invention, in step (2), one or more linking intermediates are linked to each antibody molecule in the antigen-DNA/RNA linking chain.

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 antigen and the linking intermediate.

In a preferred embodiment of the present invention, in step (2), one or more DNA/RNA connecting strands are connected to each connecting intermediate in the antigen-DNA/RNA connecting strand.

In a preferred embodiment of the present invention, the ratio of the two functional groups contained in the intermediate for ligation of the antigen and the DNA/RNA connecting strand is one to one or one to more.

AAAUUCCUCUACCACCUACA、

AAATTCCTCTACCACCTACATCAC、TATTTAGTGTTCGAATAGTT、

TATTTAGTGTTCGAATAGTTCGATCTAG、

AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、

GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、

GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、

CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、

AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、

AAAUUCCUCUACCACCUACAUCAC。

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 performing immunoadsorption competitive assay detection by using a multiple signal amplification system, which comprises a solid phase matrix capable of being combined with an antigen or an antibody, the antigen or the antibody capable of being combined with the solid phase matrix, the antigen or the antibody to be detected, an antibody/antigen-DNA/RNA connecting chain, a DNA/RNA amplification 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 amplification chain and the DNA/RNA detection chain connected with the signal to be detected, and is used for amplifying the signal of the antigen or the antibody to be detected and then detecting the signal.

In a preferred embodiment of the present invention, the solid phase matrix is selected from any solid phase matrix capable of adsorbing protein or coating antigen or antibody by chemical bond, 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 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 matrix.

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 antigen or antibody to be detected may be any one or more of human, mouse, sheep, camel, rabbit, bacteria, virus, and any other antigen.

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.

AAAUUCCUCUACCACCUACA、

AAATTCCTCTACCACCTACATCAC、TATTTAGTGTTCGAATAGTT、

TATTTAGTGTTCGAATAGTTCGATCTAG、

AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、

GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、

GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、

CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、

AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、

AAAUUCCUCUACCACCUACAUCAC。

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 aims to provide a multiple signal amplification system which comprises a DNA/RNA detection chain, a DNA/RNA amplification chain, a DNA/RNA connecting chain, an antibody or antigen molecule and a connecting intermediate body for connecting the DNA/RNA connecting chain and the antibody or antigen, wherein the DNA/RNA detection chain and the DNA/RNA connecting chain can be connected after complementary pairing, or the DNA/RNA detection chain is connected with the DNA/RNA connecting chain through the DNA/RNA amplification chain, and the chains can be connected with each other through base 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 complementary pairing between the DNA/RNA detection strand and the DNA/RNA junction strand is that the base of the DNA/RNA detection strand can be directly complementary 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 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, the DNA/RNA amplification strand includes, but is not limited to, the following sequences (5 'to 3'):

GTGATGTAGGTGGTAGAGGAATTT-TT-ATAAACCTA-A-(ATAAACC TA-A)_n-ATAAACCTA-A(40≤n≤60)、

CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATC ATCAT-A)_n-CATCATCAT-A(40≤n≤60)、

GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-A ATACTCTC-A-(AATACTCTC-A)_n-AATACTCTC-A(40≤n≤60)、

CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT- A-CAACTTAAC-A-(CAACTTAAC-A)_n-CAACTTAAC-A(40≤n≤60)、

ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-T TCATTTAC-A-(TTCATTTAC-A)_n-TTCATTTAC-A(40≤n≤60)、

GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-C AATCAAAA-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-TC ATTACTT-A-(TCATTACTT-A)_n-TCATTACTT-A(40≤n≤60)、

TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TT CTTACTC-A-(TTCTTACTC-A)_n-TTCTTACTC-A(40≤n≤60)。

AAAUUCCUCUACCACCUACA、

AAATTCCTCTACCACCTACATCAC、

TATTTAGTGTTCGAATAGTT、

TATTTAGTGTTCGAATAGTTCGATCTAG、

AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、

GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、

GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、

CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、

AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、

AAAUUCCUCUACCACCUACAUCAC。

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 and linked with any one or more functional groups on the 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 connecting strand connected to the ligation intermediate may be connected to one or more DNA 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 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 the preferred technical scheme of the invention, the multiple signal amplification system can be applied to the detection of an immunoadsorption competition method.

It is another object of the present invention to provide a signal amplification system, wherein the 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 the DNA/RNA amplification strand is one or more, a part of the DNA/RNA amplification strand is base-complementarily paired with the DNA/RNA connecting strand, and a part of the DNA/RNA amplification strand is base-complementarily paired with one or more DNA/RNA detection strands. In a preferred embodiment of the present invention, when the number of the DNA/RNA amplification strands is plural, a partial fragment of one DNA/RNA amplification strand is complementarily paired with the DNA/RNA detection strand, and is complementarily paired with the other plural DNA/RNA amplification strands.

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.

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-(ATAAACC TA-A)n-ATAAACCTA-A(40≤n≤60)、

CTAGATCGAACTATTCGAACACTAAATA-TT-CATCATCAT-A-(CATC ATCAT-A)n-CATCATCAT-A(40≤n≤60)、

GGGTTATTGCGAGGATATAGGGCGTGGCGGTGTCATAGAATT-TT-A ATACTCTC-A-(AATACTCTC-A)n-AATACTCTC-A(40≤n≤60)、

CTGTTGCGCGGGAGAACGACACGGACGCTAAATATAGGAAAC-TT- A-CAACTTAAC-A-(CAACTTAAC-A)n-CAACTTAAC-A(40≤n≤60)、

ATTGAGACGGTACGGTTCACTGCTAACGGACGATTTGGATTC-TT-T TCATTTAC-A-(TTCATTTAC-A)n-TTCATTTAC-A(40≤n≤60)、

GAGTATGCGTCGGAGACCTTGACGGACCTTGGACTAGACTTG-TT-C AATCAAAA-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-TC ATTACTT-A-(TCATTACTT-A)n-TCATTACTT-A(40≤n≤60)、

it is another object of the present invention to provide a DNA/RNA junction strand that 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 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 attached by reaction of an amino group and the other functional group may be attached by reaction of 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 may be reactive linked to a carboxyl group and the other functional group may be reactive linked to a thiol group; 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 may be purified by desalting a centrifugal column or ultrafiltration.

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.

TATTATTGG-T-TATTATTGG-T-TATTATTGG-TTT-TTCTTACTC-A-TT CTTACTC-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 and corresponding primer in certain proportion and reacting for 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.

AAAUUCCUCUACCACCUACA、

AAATTCCTCTACCACCTACATCAC、

TATTTAGTGTTCGAATAGTT、

TATTTAGTGTTCGAATAGTTCGATCTAG、

AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC、

GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG、

GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT、

CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC、

AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC、

AAAUUCCUCUACCACCUACAUCAC。

another objective of the invention is to provide the application of the multiple signal amplification system in the detection of the immunoadsorption competition method.

The invention also aims to provide the application of the multiple signal amplification detection system in the detection of the immunoadsorption competition 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 invention relates to a signal amplification system based on DNA or RNA complementary pairing, which is characterized in that a DNA/RNA connecting chain is connected with a DNA/RNA amplifying chain, and the DNA/RNA amplifying chain is connected with a plurality of DNA/RNA detection chains to realize single-stage multiple amplification of signals; in addition, the DNA/RNA amplification strand can be connected with a plurality of amplification strands to realize two-stage multiple amplification, even more than three-stage amplification. The invention not only realizes the concentration detection of the antigen or antibody to be detected, but also reduces the detection limit and has higher sensitivity.

2. The invention can detect the concentration of a plurality of antigens or antibodies simultaneously in each round.

3. The invention can realize multi-round detection of the antibody or the antigen, can realize selective elution of complementary pairing parts of a detection chain, an amplification chain and a connecting chain, has wider applicability, and reduces time cost and material cost.

4. The number of DNA/RNA connecting strands coupled to each antigen or antibody can be quantified in the present invention.

Drawings

FIG. 1 conjugation process of antibody to DNA/RNA connecting strand. The antibody is bound to an intermediate linker; the antibody is conjugated with an intermediate connector, and the prepared antibody-connecting intermediate conjugate is then combined with a DNA/RNA connecting chain; the antibody is linked to the DNA/RNA to form an antibody-DNA/RNA linked chain.

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) The primers Z and A are continuously cycled to generate a long-chain compound 1: Z-AAAA … AA under the catalysis of enzyme; (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.

Represents an antibody.

Representing the complementary strand. For example, single-stranded DNA or RNA: the a strand represents a sequence of a certain DNA or RNA, and then a denotes a sequence of a complementary strand of the a strand.

Represents a fluorescent group

FIG. 3 is a flow chart of the immunoadsorption competition method for detecting antigen in the first-order multiplex amplification system. (1) Coating the antigen on a solid phase matrix; (2) adding an antibody-DNA connecting chain and an antigen to be detected; (3) adding a DNA amplification chain to perform primary signal amplification; (4) adding 2 detection chains and carrying out a first round of detection; (5) and (4) washing off the 2 detection chains in the previous step, and adding the other 2 detection chains and then carrying out second detection.

Represents an antibody.

Representing the complementary strand. For example, single-stranded DNA or RNA: the a strand represents a sequence of a DNA or RNA fragment, and then a denotes the sequence of the complementary strand of the a strand.

Representative antigen 1

Representative antigen 2

Representative antigen 3

Representative antigen 4

Represents a green fluorescent group a

Represents a red fluorescent group b

FIG. 4 is a flow chart of the immunoadsorption competition method for detecting antibodies in the two-stage multiplex amplification system. (1) Coating the antigen on a solid phase matrix; (2) adding an antibody-DNA connecting chain and an antibody to be detected; (3) adding a primary DNA amplification chain for primary signal amplification; (4) adding a secondary DNA amplification chain for secondary signal amplification; (5) adding 2 detection chains and carrying out a first round of detection; (6) and (4) washing off the 2 detection chains in the previous step, and adding the other 2 detection chains and then carrying out second detection.

Represents an antibody.

Representative antigen 1

Representative antigen 2

Representative antigen 3

Representative antigen 4

Represents a green fluorescent group a

Represents a red fluorescent group b

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.

Example 1 detection of antigens Using a Primary multiplex amplification System and immunoadsorption Competition

In this example, the concentration of 4 antigens, IL-10, TGF- β, IFN- γ and TNF- α, were simultaneously detected by a competition method.

First, preparation of antibody and DNA/RNA connecting chain (for example, connection of human TGF-. beta.and DNA connecting chain 2)

(1) The human TGF- β antibody was concentrated to 2mg/mL by ultrafiltration centrifugation (100kDa MWCO), and the azide and other preservatives were removed.

(2) The antigen reacts with a connecting intermediate SIA (N-succinimidyl ethyl iodide) (the molar ratio is 1:20) at 4 ℃ for 3 hours, the SIA has double functional groups, one functional group can react and connect with an amino group on the antigen, and the other functional group can react and connect with a sulfhydryl group.

(3) Removal of excess SM (PEG) by desalting spin columns (7000Da MWCO)₂。

(4) The thiolated modified DNA linker 2(TATTTAGTGTTCGAATAGTT) was mixed with the linker intermediate modified antibody (molar ratio 15:1) and reacted at 4 ℃ for 12 hours.

(5) The antigen-DNA linker was purified by ultrafiltration centrifuge tubes (100kDa MWCO).

(6) Verification of successful antibody ligation to DNA: matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF) was used to verify the successful coupling of DNA links to antigens and to quantify the amount of DNA coupled to each antigen. The results showed that 1 DNA-ligated strand was ligated to each antigen.

Similarly, IL-10-RNA linker 1, INF-gamma-DNA linker 3, and TNF-alpha-DNA linker 4 were prepared.

Preparation of DNA amplification Strand in Signal amplification System (preparation of C.1 amplification Strand is exemplified)

(1) 10mM MgSO4, 80units/mL polymerase, 600. mu.M dATP/dCTP/dTTP, 10. mu.M hairpin h.1.1 and 10. mu.M primer p.1 were added to 100. mu.L of the reaction mixture, and the reaction mixture 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 (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) 6% TBE-UREA PAGE gel was prepared using Gelred as the dye.

(5) The gel was placed in the electrophoresis solution, the voltage was adjusted to 75V for 30 minutes, the sample and DL 1000DNA marker were added to the lane, the voltage was adjusted to 200V, and the run was carried out for 25 minutes.

(6) The DNA band of interest was excised from the gel, purified and recovered by a DNA recovery kit, and the recovered DNA was lyophilized and stored at-20 ℃.

Thirdly, adopting an immunoadsorption competition method to detect various antigens

(1) 4 different antigens (human IL-10/human IFN-. gamma./human TNF-. alpha./human TGF-. beta.) were coated with coating solution (50mM Na)₂CO₃/NaHCO₃in PBS) to 5. mu.g/mL, the mixture was coated onto 96-well plates at 4 ℃ overnight.

(2) The remaining solution, which was not coated with 4 antigens, was removed and washed 3 times with washing buffer (0.1% tween-20in PBS), blocking solution (1% BSA in PBS) was added and blocked for 2 hours at 37 ℃.

(3) The blocking solution was removed and washed 3 times with washing buffer, 4 antibodies (Anti-human IL-10antibody/Anti-human IFN- γ antibody/Anti-human TNF- α antibody/Anti-human TGF- β antibody) -DNA/RNA linker (each antibody-DNA/RNA linker was diluted to 2 μ g/mL with blocking solution) were added and mixed with samples containing 4 antigens to be detected and added to the corresponding wells to incubate with the coated antigen for 2 hours at room temperature.

(5) Each detection antibody was removed and washed 3 times with washing buffer, 1 time with hybridization washing buffer (15% formamide, 1mM EDTA), 4 DNA primary amplified strands C.1, C.2, C.3, C.4 (60nM-150nM) were added and incubated overnight at 37 ℃.

(6) The amplified DNA strands were removed and washed 3 times with hybridization wash buffer, 1 time with detection buffer (500mM NaCl in 1mM Tris-HCl), and the first round of two DNA strands i.1X and i.2X (1. mu.M) were added and reacted at room temperature for 1 hour, i.1X DNA strands were linked with green fluorescence signal (excitation wavelength 485nm, emission wavelength 528nm), i.2X DNA strands were linked with red fluorescence signal (excitation wavelength 579nm, emission wavelength 620 nm).

(7) The DNA detection strands were removed and washed 2 times with detection buffer, while detecting the intensity of fluorescent signal on both DNA detection strands.

(8) The detection buffer was removed, dissociation buffer (35% formamide) was added, and the reaction was carried out for 30 minutes.

(9) The dissociation buffer was removed, washed 2 times with PBS buffer, then 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 the green fluorescent signal on the DNA assay strand, and i.4 with the red fluorescent signal on the DNA assay strand.

(10) The detection strand was removed and washed 2 times with detection buffer, while detecting the intensity of the fluorescent signal on both DNA detection strands.

(11) And drawing a standard curve according to the antigen standard products with different concentrations, and calculating the concentration of each antigen according to different signal intensities in the antigen sample to be detected and the standard curve.

Fourth, experimental results

In the experiment, the concentrations of the four antigens are known, and the detection result is consistent with the known theoretical concentrations of the four antigens. The known IL-10 concentration is 90.0ng/mL, and the concentration is calculated to be 92.3ng/mL after the detection result is brought into a standard curve equation; knowing that the concentration of TGF-beta is 145.0ng/mL, the detection result is substituted into a standard curve equation, and then the calculated concentration is 142.1 ng/mL; the concentration of INF-gamma is known to be 50.0ng/mL, and the concentration is calculated to be 49.7ng/mL after the detection result is substituted into a standard curve equation; as the concentration of TNF-alpha is known to be 60.0ng/mL, the concentration is calculated to be 58.3ng/mL after the detection result is substituted into a standard curve equation.

Example 2 detection of antibodies Using two-stage multiplex amplification System and immunoadsorption Competition method

Preparation of antibodies and DNA linkers

antibody-DNA/RNA connecting chains were prepared by connecting SIAB (N-succinimidyl [ 4-iodoacetyl ] aminobenzoate) as a connecting intermediate to DNA/RNA connecting chains by the same method as in example 1, and GAD65 antibody-RNA connecting chain 1, IA-2 antibody-DNA connecting chain 2, insulin antibody-DNA connecting chain 3, and ZnT8 antibody-DNA connecting chain 4 were prepared.

Second, preparation of DNA amplification chain in signal amplification system

The preparation method is the same as that of example 1, and C.1-C.4, C.8-C.11 amplification chains are prepared.

Third, competitive method for detecting multiple antibodies

(1) 4 different antigens (human glutamate decarboxylase (GAD 65)/human islet cell tumor associated protein 2 (IA-2)/human insulin/human zinc transporter 8(ZnT8)) were combined with coating solution (50mM Na₂CO₃ /NaHCO₃in PBS) to 10. mu.g/mL the mixture was coated onto 96-well plates overnight at 4 ℃.

(3) The blocking solution was removed and washed 3 times with washing buffer, and 4 kinds of antibody-DNA/RNA connecting chains (GAD65 antibody-RNA connecting chain 1, IA-2 antibody-DNA connecting chain 2, insulin antibody-DNA connecting chain 3, ZnT8 antibody-DNA connecting chain 4, concentration 2. mu.g/mL) and the antibody to be detected (human glutamate decarboxylase (GAD65) antibody/human islet cell tumor-associated protein 2(IA-2) antibody/human insulin antibody/human zinc transporter 8(ZnT8) were mixed and added to the respective wells, followed by incubation at room temperature for 2 hours.

(4) Each antibody was removed and washed 3 times with wash buffer, 1 time with hybridization wash buffer (15% formamide, 1mM EDTA), and 4 primary DNA amplification strands C.1, C.2, C.3, C.4 (60nM-150nM) were added and incubated overnight at 37 ℃.

(5) The primary DNA amplification strand was removed, washed 3 times with hybridization washes, and then 4 secondary DNA amplification strands C.8, C.9, C.10, and C.11 (60nM-150nM) partially complementary to the primary DNA amplification strand were added and incubated at 37 ℃ for 5 hours.

(6) The DNA amplification strand 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.8 and i.9 (1. mu.M) were added and the reaction was carried out at room temperature for 1 hour, i.8X DNA detection strand linked with green fluorescence signal (excitation wavelength 485nm, emission wavelength 528nm), i.9X DNA detection strand linked with red fluorescence signal (excitation wavelength 579nm, emission wavelength 620 nm).

(7) The DNA detection strands were removed and washed 2 times with detection buffer to measure the intensity of fluorescent signal on both DNA detection strands.

(9) The detection buffer was removed, dissociation buffer (40% formamide) was added, and the reaction was carried out for 30 minutes.

(10) The dissociation buffer was removed, washed 2 times with PBS buffer, then 2 times with assay buffer, and two assay strands i.10 and i.11 (1 μ M) from the second round of assay were added and reacted at room temperature for 1 hour, i.10 with the green fluorescent signal attached to the DNA assay strand, and i.11 with the red fluorescent signal attached to the DNA assay strand.

(11) The detection strand was removed and washed 2 times with detection buffer, while detecting the intensity of the fluorescent signal on both DNA detection strands.

(12) And drawing a standard curve according to the antibody standard products with different concentrations, and calculating the concentration of each antibody according to different signal intensities in the antibody sample to be detected and standard curve contrast.

Fourth, experimental results

In the experiment, the concentrations of the four antibodies are known, and the detection result is consistent with the known concentrations of the four antibodies. The concentration of the GAD65 antibody is known to be 120.0ng/mL, and the concentration is calculated to be 118.2ng/mL after the detection result is substituted into a standard curve equation; knowing that the concentration of the IA-2 antibody is 5.0ng/mL, the concentration is calculated to be 5.1ng/mL after the detection result is substituted into a standard curve equation; knowing that the concentration of the insulin antibody is 80.0pg/mL, substituting the detection result into a standard curve equation and calculating the concentration to be 80.5 pg/mL; the concentration of the ZnT8 antibody is known to be 200.0pg/mL, and the concentration is calculated to be 205.3pg/mL after the detection result is substituted into a standard curve equation.

Example 3 detection of antibodies Using two-stage multiplex amplification System and immunoadsorption Competition

In this example, the concentration of 6 antibodies, 6 antigens being human IL-2, IL-7, IL-15, IL-17, TNF-. alpha.and IL-6, respectively, were simultaneously detected by direct assay.

Preparation of antibodies and DNA linkers

The preparation method was the same as in example 1, and IL-2 antibody-DNA linker 7, IL-7 antibody-DNA linker 2, IL-15 antibody-DNA linker 3, IL-17 antibody-DNA linker 4, TNF-. alpha.antibody-DNA linker 5, and IL-6 antibody-DNA linker 6 were prepared.

Second, preparation of DNA amplification chain in signal amplification system

The preparation method is the same as that of example 1, and C.2-C.7, C.9-C.14 amplification chains are prepared.

Third, competitive method for detecting multiple antibodies

(1) 6 different antigens were coated with coating solution (50mM Na)₂CO₃/NaHCO₃in PBS) to 10. mu.g/mL the mixture was coated onto 96-well plates at 4 ℃ overnight.

(2) The remaining solution, which was not coated with 6 antigens, was removed and washed 3 times with washing buffer (0.1% tween-20in PBS), blocking solution (1% BSA in PBS) was added and blocked for 2 hours at 37 ℃.

(3) The blocking solution was removed and washed 3 times with washing buffer, and 6 antibody-DNA links (2. mu.g/mL) and the antibody to be detected were mixed and added to the corresponding wells and incubated at room temperature for 2 hours.

(4) Each antibody was removed and washed 3 times with wash buffer, 1 time with hybridization wash buffer (15% formamide, 1mM EDTA), 6 primary DNA amplification strands C.2-C.7 (60nM-150nM) were added, and incubated overnight at 37 ℃.

(5) The primary DNA amplification strand was removed, washed 3 times with hybridization washes, and then 6 secondary DNA amplification strands C.9-C.14 (60nM-150nM) partially complementary to the primary DNA amplification strand were added and incubated at 37 ℃ for 5 hours.

(6) The DNA amplification strand was removed and washed 3 times with hybridization wash buffer, 1 time with detection buffer (500mM NaCl in 1mM Tris-HCl), and the first round of two DNA detection strands i.14 and i.9 (1. mu.M) were added and reacted at room temperature for 1 hour, with green fluorescence signal (excitation wavelength of 485nm, emission wavelength of 528nm) attached to the i.14 strand and red fluorescence signal (excitation wavelength of 579nm, emission wavelength of 620nm) attached to the i.9 strand.

(9) The detection buffer was removed, dissociation buffer (45% formamide) was added, and the reaction was allowed to proceed for 30 minutes.

(10) The dissociation buffer was removed, washed 2 times with PBS buffer, 2 times with assay buffer, and two assay strands i.10 × and i.11 × (1 μ M) from the second round of assay were added and reacted at room temperature for 1 hour, i.10 × DNA assay strand with green fluorescence signal and i.11 × DNA assay strand with red fluorescence signal.

(12) The detection buffer was removed, dissociation buffer (45% formamide) was added, and the reaction was carried out for 30 minutes.

(13) The dissociation buffer was removed, washed 2 times with PBS buffer, then 2 times with assay buffer, and the third round of two assay strands i.12 × and i.13 × were added (1 μ M) and reacted at room temperature for 1 hour, with green fluorescent signal attached to the i.12 × DNA assay strand and red fluorescent signal attached to the i.13 × DNA assay strand.

(14) The detection strand was removed and washed 2 times with detection buffer, while detecting the intensity of the fluorescent signal on both DNA detection strands.

(15) And drawing a standard curve according to the antibody standard substance with different concentrations, and calculating the concentration of each antibody according to different signal intensities in the antibody sample to be detected and the standard curve control.

Fourth, experimental results

In the experiment, the concentrations of the four antibodies are known, and the detection result is consistent with the known concentrations of the four antibodies. The concentration of the IL-2 antibody is known to be 100.0ng/mL, and the concentration is calculated to be 100.2ng/mL after the detection result is brought into a standard curve equation; the concentration of the IL-7 antibody is known to be 10.0ng/mL, and the concentration is calculated to be 10.3ng/mL after the detection result is brought into a standard curve equation; the concentration of the IL-15 antibody is known to be 200.0pg/mL, and the concentration is calculated to be 201.5pg/mL after the detection result is substituted into a standard curve equation; the concentration of the IL-17 antibody is known to be 200.0pg/mL, and the concentration is calculated to be 202.9pg/mL after the detection result is brought into a standard curve equation; the concentration of the TNF-alpha antibody is known to be 50.0pg/mL, and the concentration is calculated to be 50.7pg/mL after the detection result is substituted into a standard curve equation; the concentration of IL-17 antibody is known to be 200.0pg/mL, and the concentration is calculated to be 204.9 pg/mL after the detection result is substituted into the standard curve equation.

TABLE 1 DNA linker chain sequence (from 5 'to 3') for linking antibodies

DNA/RNA connecting strand sequence numbering	DNA/RNA connecting chain sequence
		RNA connecting strand 1	AAAUUCCUCUACCACCUACA
DNA connecting chain 2	TATTTAGTGTTCGAATAGTT
		DNA connecting chain 3	AATTCTATGACACCGCCACGCCCTATATCCTCGCAATAACCC
DNA connecting chain 4	GTTTCCTATATTTAGCGTCCGTGTCGTTCTCCCGCGCAACAG
		DNA connecting chain 5	GAATCCAAATCGTCCGTTAGCAGTGAACCGTACCGTCTCAAT
DNA connecting chain 6	CAAGTCTAGTCCAAGGTCCGTCAAGGTCTCCGACGCATACTC
		DNA connecting chain 7	AGTTCCTGTAGTATCCCGTCGCATAGTCGTACATTCACCGTC

TABLE 2 primers and hairpin sequences for amplified strand preparation (from 5 'to 3')

TABLE 3 DNA amplification strand sequence (from 5 'end to 3' end)

TABLE 4 DNA detection strand sequence information for fluorescence detection (from 5 'end to 3' end)

Detection chain numbering	DNA detection strand sequence
		i.1*	Fluorophore-UU-UAGGUUUAU-U-UAGGUUUAU-U
i.2*	Fluorophore-TT-ATGATGATG-T-ATGATGATG-T
		i.3*	Fluorophore-TT-GAGAGTATT-T-GAGAGTATT-T
i.4*	Fluorophore-TT-GTTAAGTTG-T-GTTAAGTTG-T
		i.5*	Fluorophore-TT-GTAAATGAA-T-GTAAATGAA-T
i.6*	Fluorophore-TT-TTTTGATTG-T-TTTTGATTG-T
		i.7*	Fluorophore-TT-TATTATTGG-T-TATTATTGG-T
i.8*	Fluorophore-TT-GTAAATGAA-T-GTAAATGAA-T
		i.9*	Fluorophore-TT-GGTAGAAAA-T-GGTAGAAAA-T
i.10*	Fluorophore-TT-ATAAAAGGA-T-ATAAAAGGA-T
		i.11*	Fluorophore-TT-AATGAAAGA-T-AATGAAAGA-T
i.12*	Fluorophore-TT-AGTGAATAA-T-AGTGAATAA-T
		i.13*	Fluorophore-TT-AAGTAATGA-T-AAGTAATGA-T
i.14*	Fluorophore-TT-GAGTAAGAA-T-GAGTAAGAA-T

TABLE 5 linker, amplifier and detector for primary multiplex signal amplification immunoadsorption assay in example 1

Antigen to be detected	DNA/RNA connecting strand	First-order amplification strand of DNA	DNA/RNA detection strand
				IL-10	RNA connecting strand 1	C.1	i.1*
TGF-β	DNA connecting chain 2	C.2	i.2*
				INF-γ	DNA connecting chain 3	C.3	i.3*
TNF-α	DNA connecting chain 4	C.4	i.4*

TABLE 6 linker, amplifier and detector for two-stage multiplex signal amplification immunoadsorption assay in example 2

TABLE 7 antibodies and DNA linker, amplifier and detector chains used in the competitive assay in example 3

The above description of the specific 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 according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.

Sequence listing

<110> Ikefa (Beijing) Biotechnology Ltd

<120> multiple signal amplification system and application thereof in immunoadsorption competition method detection

<150> 2020112536617

<151> 2020-11-11

<150> 2020112536246

<151> 2020-11-11

<150> 2020112536299

<151> 2020-11-11

<150> 2020112562382

<151> 2020-11-11

<160> 63

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aaauuccucu accaccuaca 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tatttagtgt tcgaatagtt 20

<210> 3

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aattctatga caccgccacg ccctatatcc tcgcaataac cc 42

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtttcctata tttagcgtcc gtgtcgttct cccgcgcaac ag 42

<210> 5

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaatccaaat cgtccgttag cagtgaaccg taccgtctca at 42

<210> 6

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caagtctagt ccaaggtccg tcaaggtctc cgacgcatac tc 42

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agttcctgta gtatcccgtc gcatagtcgt acattcaccg tc 42

<210> 50

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

ataaaccta 9

<210> 51

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

catcatcat 9

<210> 52

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

aatactctc 9

<210> 53

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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caacttaac 9

<210> 54

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

ttcatttac 9

<210> 55

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

caatcaaaa 9

<210> 56

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccaataata 9

<210> 57

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ttcatttac 9

<210> 58

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ttttctacc 9

<210> 59

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tccttttat 9

<210> 60

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccttctatt 9

<210> 61

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ttattcact 9

<210> 62

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tcattactt 9

<210> 63

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ttcttactc 9

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

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<210> 10

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

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aaatactctc gggccttttg gcccgagagt atttgagagt att 43

<210> 11

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 11

acaacttaac gggccttttg gcccgttaag ttgtgttaag ttg 43

<210> 12

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 12

attcatttac gggccttttg gcccgtaaat gaatgtaaat gaa 43

<210> 13

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 13

acaatcaaaa gggccttttg gcccttttga ttgtttttga ttg 43

<210> 14

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 14

accaataata gggccttttg gccctattat tggttattat tgg 43

<210> 15

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 15

attcatttac gggccttttg gcccgtaaat gaatgtaaat gaa 43

<210> 16

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 16

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<210> 17

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 17

accaataata gggccttttg gccctattat tggttattat tgg 43

<210> 18

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 18

atccttttat gggccttttg gcccataaaa ggatataaaa gga 43

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 19

attattcact gggccttttg gcccagtgaa taatagtgaa taa 43

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

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atcattactt gggccttttg gcccaagtaa tgataagtaa tga 43

<210> 21

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(43)

<223> 3' terminal, reverse dT modification

<400> 21

attcttactc gggccttttg gcccgagtaa gaatgagtaa gaa 43

<210> 22

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

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<223> repeating unit, repeating number of 40-60 times

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<210> 23

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

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<223> repeating unit, repeating number of 40-60 times

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ctagatcgaa ctattcgaac actaaatatt catcatcata catcatcata catcatcata 60

<210> 24

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (55)..(64)

<223> repeating unit, repeating number of 40-60 times

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gggttattgc gaggatatag ggcgtggcgg tgtcatagaa ttttaatact ctcaaatact 60

ctcaaatact ctca 74

<210> 25

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (56)..(65)

<223> repeating unit, repeating number of 40-60 times

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ctgttgcgcg ggagaacgac acggacgcta aatataggaa acttacaact taacacaact 60

taacacaact taaca 75

<210> 26

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (55)..(64)

<223> repeating unit, repeating number of 40-60 times

<400> 26

attgagacgg tacggttcac tgctaacgga cgatttggat tcttttcatt tacattcatt 60

tacattcatt taca 74

<210> 27

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (55)..(64)

<223> repeating unit, repeating number of 40-60 times

<400> 27

gagtatgcgt cggagacctt gacggacctt ggactagact tgttcaatca aaaacaatca 60

aaaacaatca aaaa 74

<210> 28

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (55)..(64)

<223> repeating unit, repeating number of 40-60 times

<400> 28

gacggtgaat gtacgactat gcgacgggat actacaggaa ctttccaata ataaccaata 60

ataaccaata ataa 74

<210> 29

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(52)

<223> repeating unit, repeating number of 40-60 times

<400> 29

taggtttatt taggtttatt taggtttatt ttttcattta cattcattta cattcattta 60

ca 62

<210> 30

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(52)

<223> repeating unit, repeating number 40-60 times

<400> 30

atgatgatgt atgatgatgt atgatgatgt ttttttctac cattttctac cattttctac 60

ca 62

<210> 31

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(52)

<223> repeating unit, repeating number of 40-60 times

<400> 31

gagagtattt gagagtattt gagagtattt tttcctttta tatcctttta tatcctttta 60

ta 62

<210> 64

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (43)..(52)

<223> repeating unit, repeating number of 40-60 times

<400> 64

gttaagttgt gttaagttgt gttaagttgt ttccttctat taccttctat taccttctat 60

ta 62

<210> 33

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (53)..(62)

<223> repeating unit, repeating number 40-60 times

<400> 33

gtaaatgaat gtaaatgaat gtaaatgaat ttttattcac tattattcac tattattcac 60

tattattcac ta 72

<210> 34

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (53)..(62)

<223> repeating unit, repeating number of 40-60 times

<400> 34

ttttgattgt ttttgattgt ttttgattgt tttcattact tatcattact tatcattact 60

tatcattact ta 72

<210> 35

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (53)..(62)

<223> repeating unit, repeating number of 40-60 times

<400> 35

tattattggt tattattggt tattattggt ttttcttact cattcttact cattcttact 60

cattcttact ca 72

<210> 36

<211> 22

<212> RNA

<213> Fluorophore

<400> 36

uuuagguuua uuuagguuua uu 22

<210> 37

<211> 22

<212> DNA

<213> Fluorophore

<400> 37

ttatgatgat gtatgatgat gt 22

<210> 38

<211> 22

<212> DNA

<213> Fluorophore

<400> 38

ttgagagtat ttgagagtat tt 22

<210> 39

<211> 22

<212> DNA

<213> Fluorophore

<400> 39

ttgttaagtt gtgttaagtt gt 22

<210> 40

<211> 22

<212> DNA

<213> Fluorophore

<400> 40

ttgtaaatga atgtaaatga at 22

<210> 41

<211> 22

<212> DNA

<213> Fluorophore

<400> 41

ttttttgatt gtttttgatt gt 22

<210> 42

<211> 22

<212> DNA

<213> Fluorophore

<400> 42

tttattattg gttattattg gt 22

<210> 43

<211> 22

<212> DNA

<213> Fluorophore

<400> 43

ttgtaaatga atgtaaatga at 22

<210> 44

<211> 22

<212> DNA

<213> Fluorophore

<400> 44

ttggtagaaa atggtagaaa at 22

<210> 45

<211> 22

<212> DNA

<213> Fluorophore

<400> 45

ttataaaagg atataaaagg at 22

<210> 46

<211> 22

<212> DNA

<213> Fluorophore

<400> 46

ttaatgaaag ataatgaaag at 22

<210> 47

<211> 22

<212> DNA

<213> Fluorophore

<400> 47

ttagtgaata atagtgaata at 22

<210> 48

<211> 22

<212> DNA

<213> Fluorophore

<400> 48

ttaagtaatg ataagtaatg at 22

<210> 49

<211> 22

<212> DNA

<213> Fluorophore

<400> 49

ttgagtaaga atgagtaaga at 22

Claims

1. A method for carrying out immunoadsorption competition method detection by using a multiple signal amplification system comprises the following steps:

(1) coating the antigen on a solid phase matrix to form a solid phase antigen;

(3) adding DNA/RNA amplification chain, acting for a certain time;

(4) adding DNA/RNA detection chain, acting for a certain time;

(5) washing, and detecting the signal intensity on the DNA/RNA detection chain;

2. The method according to claim 1, wherein in the step (2), the sample to be detected is an antigen to be detected or an antibody to be detected.

3. The method according to any one of claims 1 to 2, wherein in step (2), one or more linking intermediates are linked to each antibody molecule in the antibody-DNA/RNA linking chain; preferably, one or more DNA/RNA connecting strands are attached to each ligation intermediate.

4. The method according to any one of claims 1 to 3, wherein in the step (3), the primary DNA/RNA amplification strand is ligated first, and then the secondary DNA/RNA amplification strand is added after the ligation, and further DNA/RNA amplification strands are ligated as necessary.

5. The method according to any one of claims 1 to 4, wherein in step (5), the first round of DNA/RNA detection is performed, then dissociation is performed, and then the second round of DNA/RNA detection is performed; then, dissociating, and adding a third round of DNA/RNA detection chain for detection; and carrying out multiple rounds of detection as required.

6. The method of any one of claims 1 to 5, wherein the detection signal includes, but is not limited to, any one of fluorescence, phosphorescence, chemiluminescence, electromagnetic signal, nuclear magnetic signal, radioactive signal, or a combination thereof.

7. A method for preparing a multiplex signal amplification system, comprising preparing an antigen or antibody-DNA/RNA connecting strand, and preparing a DNA/RNA amplifying strand.

8. The method of claim 7, wherein the antigen or the antibody-DNA/RNA connecting chain is prepared by connecting the antibody or the antigen to the DNA/RNA connecting chain via a connecting intermediate, wherein the connecting intermediate has two functional groups, one of which is connected to the antibody or the antigen and the other of which is connected to the DNA/RNA connecting chain.

9. The method of any one of claims 7-8, wherein the preparation of the antigen or antibody-DNA/RNA linked strand comprises the steps of:

10. The method for preparing an amplification strand according to any one of claims 7 to 9, wherein the method for preparing the DNA/RNA amplification strand comprises the steps of: