CN110208538B - Detection kit and detection method for prostate specific antigen and application - Google Patents
Detection kit and detection method for prostate specific antigen and application Download PDFInfo
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Abstract
The invention provides a detection kit, a detection method and application of prostate specific antigen, and relates to the technical field of antigen detection. The kit and the detection method utilize a secondary antibody to modify DNA, are connected with the adjacent position of an antigen to form a Y-shaped antigen-antibody-DNA hybrid chain, and are used as a signal converter to convert the input of the antigen into the output of a DNA signal; simultaneously, the chain substitution reaction and the hybridization chain reaction are combined, so that two-stage amplification of signals is realized; the two adjacent end sequences (hairpin DNAH3 and hairpin DNA H4) on the long-chain DNA are assembled to form magnesium ion DNAase, the enzyme cuts molecular beacons in a solution in the presence of magnesium ions, the disconnected molecular beacons are released and emit fluorescence, the new molecular beacons are combined with the magnesium ion DNAase and cut, and fluorescence signals are circularly amplified, so that the third round of signal amplification is realized, the detection efficiency and sensitivity are greatly improved, and the detection limit reaches 0.73pg mL‑1。
Description
Technical Field
The invention belongs to the technical field of antigen detection, and particularly relates to a detection kit and a detection method for prostate specific antigen and application thereof.
Background
Antigen detection has important reference value in clinical diagnosis. Surface molecules of human and animal cells: including various differentiation antigens on the cell surface (such as CD antigen), allotypic antigens (blood group antigen or MHC antigen), virus-associated antigens, tumor-associated emotional antigens, and the like. The detection of these antigens is of great significance in the classification, differentiation and function studies of various cells, diagnosis of various immune-related diseases and the study of pathogenesis.
At present, the antigen detection methods mainly include traditional enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Fluorescent Immunoassay (FIA), chemiluminescence immunoassay (CLIA), automated immunoassay, and the like. The fluorescence immunoassay has the advantages of strong specificity, high sensitivity, good practicability and the like, so that the fluorescence immunoassay is used for measuring low-content bioactive substances, such as proteins (enzymes and antibodies), hormones (steroids and thyroid hormones) and the like. The performance of the detection technology, such as polymerase chain reaction, rolling circle amplification and the like, can be improved by the DNA-assisted protein detection technology, but the application is limited by the complicated steps, false positive possible to polymerase amplification and other factors.
Disclosure of Invention
In view of the above, the present invention provides a prostate specific antigen detection kit, a prostate specific antigen detection method and applications thereof, wherein the method is based on proximity ligation, strand substitution, hybrid chain reaction and magnesium ion dnase antigen detection, and realizes high-sensitivity and high-specificity detection of a target antigen by specifically binding an antibody modified with DNA to the target antigen, converting an antigen input into a DNA signal output, and combining with the strand substitution, hybrid chain reaction and signal amplification technology of magnesium ion dnase.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection kit for prostate specific antigen, which comprises the following components: PSA antibody, succinimide-4- (N-maleimide) cyclohexane-1-carboxylate, tris (2-carboxyethyl) phosphine, thiol-modified DNA1, thiol-modified DNA2, hairpin DNA H1, hairpin DNA H2, hairpin DNA H3, hairpin DNA H4, beacon MB and Mg2+(ii) a The nucleotide sequence of the sulfhydryl modified DNA1 is shown in SEQ ID NO. 1; the nucleotide sequence of the sulfhydryl modified DNA2 is shown in SEQ ID NO. 2; the nucleotide sequence of the hairpin DNA H1 is shown as SEQ ID NO.3(ii) a The nucleotide sequence of the hairpin DNA H2 is shown as SEQ ID NO. 4; the nucleotide 6 sequence of the hairpin DNA H3 is shown as SEQ ID NO. 5; the nucleotide sequence of the hairpin DNA H4 is shown as SEQ ID NO. 6; the beacon MB comprises an rA base which recognizes the ionic enzyme and is modified with FAM and BHQ groups.
Preferably, the nucleotide sequence of the beacon MB is shown as SEQ ID NO. 7.
The invention also provides a detection method of the prostate specific antigen, which comprises the following steps: (1) coupling reaction is carried out on the PSA antibody and succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester for 2h, and then the treated antibody is obtained by filtration; the molar ratio of the PSA antibody to the succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester is 1: 10-20;
(2) carrying out hydroxyl activation reaction on the sulfydryl modified DNA1 and the sulfydryl modified DNA2 and tris (2-carboxyethyl) phosphine for 1h respectively to obtain treated DNA1 and treated DNA 2; the molar ratio of the sulfhydryl modified DNA1 to the sulfhydryl modified DNA2 to the tri (2-carboxyethyl) phosphine is 1:104；
(3) Incubating the treated antibody with the treated DNA1 and the treated DNA2 at 4 ℃ for 12h to obtain Ab1-DNA1 and Ab2-DNA 2; the volume ratio of the treated antibody to both treated DNA1 and treated DNA2 is 1: 1;
(4) mixing the Ab1-DNA1, Ab2-DNA2, PSA, hairpin DNA H1 and hairpin DNA H2, and reacting at 37 ℃ for 2H to obtain a chain substitution reaction product; the mole ratio of the Ab1-DNA1, the Ab2-DNA2, the PSA, the hairpin DNA H1 and the hairpin DNA H2 is 100:100:100:10-3:10-3；
(5) Mixing the chain substitution reaction product with hairpin DNA H3 and hairpin DNA H4, and reacting at 37 ℃ for 1H to obtain a hybrid chain; the molar ratio of the hairpin DNA H3 to the hairpin DNA H4 is 300:10-3:10-3；
(6) The hybrid chain is linked with molecular beacons MB, Mg2+After mixing uniformly, reacting for 1h at 37 ℃, and performing fluorescence detection;
and (3) no time sequence is limited between the step (1) and the step (2).
Preferably, the reaction in step (1) is carried out in a PBS buffer solution, and the temperature of the reaction is 18-25 ℃.
Preferably, the filtration membrane used for filtration in the step (1) has a molecular weight cut-off of 10000 MW.
Preferably, the temperature of the reaction in step (2) is 37 ℃.
Preferably, after the incubation in step (3), the method further comprises filtering with a filter membrane with a molecular weight cutoff of 100000MW, retaining the substances on the membrane, and washing with PBS buffer solution three times.
Preferably, after the Ab1-DNA1 and Ab2-DNA2 are obtained in the step (3), ultraviolet absorption detection is also included.
Preferably, the fluorescence detection in step (6) is detecting a signal peak under 520nm visible light.
The invention also provides application of the detection kit or the detection method in target antigen signal amplification.
The invention provides a detection kit for prostate specific antigen, which comprises the following components: PSA antibody, succinimide-4- (N-maleimide) cyclohexane-1-carboxylate, tris (2-carboxyethyl) phosphine, thiol-modified DNA1, thiol-modified DNA2, hairpin DNA H1, hairpin DNA H2, hairpin DNA H3, hairpin DNA H4, beacon MB and Mg2+(ii) a The nucleotide sequence of the sulfhydryl modified DNA1 is shown in SEQ ID NO. 1; the nucleotide sequence of the sulfhydryl modified DNA2 is shown in SEQ ID NO. 2; the nucleotide sequence of the hairpin DNA H1 is shown as SEQ ID NO. 3; the nucleotide sequence of the hairpin DNA H2 is shown as SEQ ID NO. 4; the nucleotide 6 sequence of the hairpin DNA H3 is shown as SEQ ID NO. 5; the nucleotide sequence of the hairpin DNA H4 is shown as SEQ ID NO. 6; the beacon MB comprises an rA base which recognizes the ionic enzyme and is modified with FAM and BHQ groups.
Furthermore, when the kit is used for detecting the prostate specific antigen, two DNAs modified by the antibodies of mice to human Prostate Specific Antigen (PSA) are used and are connected in the ortho position of the antigen to form a Y-shaped antigen-antibody-DNA hybrid chain, the tail end of the free DNA can initiate chain replacement reaction, two hairpin DNAs are opened in sequence, and the second hairpin DNA which is hybridized up and down can push down the antigen-antibody-DNA hybrid chain, so that the circulation of the target antigen is realizedAnd (4) a ring. The chain substitution reaction cycle generates a large amount of complementary hybrid chains to initiate a hybrid chain reaction consisting of the circular hybridization of the other two hairpin DNAs to form a large amount of long-chain DNAs, each two adjacent end sequences on the long-chain DNAs can be assembled to form magnesium ion DNase, and finally the magnesium ion DNase can cut molecular beacons in a solution to emit fluorescence in the presence of magnesium ions, so that the multiple signal amplification of the target antigen is realized. By using the kit and the detection method, the detection efficiency and the sensitivity can be greatly improved, and the detection limit reaches 0.73pgmL-1And non-responsive to non-target detection of alpha-fetoprotein (AFP), Bovine Serum Albumin (BSA), and carcinoembryonic antigen (CEA).
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention;
FIG. 2 is a graph showing the ultraviolet absorption of antibody-modified DNA in an example of the present invention;
FIG. 3 is a fluorescence detection diagram according to an embodiment of the present invention;
FIG. 4 is a graph of the response to various proteins in an example of the invention.
Detailed Description
The invention provides a detection kit for prostate specific antigen, which comprises the following components: PSA antibody, succinimide-4- (N-maleimide) cyclohexane-1-carboxylate, tris (2-carboxyethyl) phosphine, thiol-modified DNA1, thiol-modified DNA2, hairpin DNA H1, hairpin DNA H2, hairpin DNA H3, hairpin DNA H4, beacon MB and Mg2+(ii) a The nucleotide sequence of the sulfhydryl modified DNA1 is shown in SEQ ID NO. 1; the nucleotide sequence of the sulfhydryl modified DNA2 is shown in SEQ ID NO. 2; the nucleotide sequence of the hairpin DNA H1 is shown as SEQ ID NO. 3; the nucleotide sequence of the hairpin DNA H2 is shown as SEQ ID NO. 4; the nucleotide 6 sequence of the hairpin DNA H3 is shown as SEQ ID NO. 5; the nucleotide sequence of the hairpin DNA H4 is shown as SEQ ID NO. 6; the beacon MB comprises an rA base which recognizes the ionic enzyme and is modified with FAM and BHQ groups.
In the detection kit of the present invention, the PSA antibody is preferably a murine antibody against human prostate specific antigen. The beacon MB of the present invention comprises an rA group, wherein rA is a ribonucleic acid base and a non-deoxyribonucleic acid base recognized and cut by a magnesium ion enzyme core sequence, and the nucleotide sequence of the rA group is preferably shown as SEQ ID NO. 7. In the detection kit of the present invention, the sequences are shown in table 1:
TABLE 1 sequences in the kit
The invention also provides a detection method of the prostate specific antigen, which comprises the following steps: (1) coupling reaction is carried out on the PSA antibody and succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester for 2h, and then the treated antibody is obtained by filtration; the molar ratio of the PSA antibody to the succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester is 1: 10-20;
(2) carrying out hydroxyl activation reaction on the sulfydryl modified DNA1 and the sulfydryl modified DNA2 and tris (2-carboxyethyl) phosphine for 1h respectively to obtain treated DNA1 and treated DNA 2; the molar ratio of the sulfhydryl modified DNA1 to the sulfhydryl modified DNA2 to the tri (2-carboxyethyl) phosphine is 1:104；
(3) Incubating the treated antibody with the treated DNA1 and the treated DNA2 at 4 ℃ for 12h to obtain Ab1-DNA1 and Ab2-DNA 2; the volume ratio of the treated antibody to both treated DNA1 and treated DNA2 is 1: 1;
(4) mixing the Ab1-DNA1, Ab2-DNA2, PSA, hairpin DNA H1 and hairpin DNA H2, and reacting at 37 ℃ for 2H to obtain a chain substitution reaction product; the mole ratio of the Ab1-DNA1, the Ab2-DNA2, the PSA, the hairpin DNA H1 and the hairpin DNA H2 is 100:100:100:10-3:10-3；
(5) Mixing the chain substitution reaction product with hairpin DNA H3 and hairpin DNA H4, and reacting at 37 ℃ for 1H to obtain a hybrid chain; the molar ratio of the hairpin DNA H3 to the hairpin DNA H4 is 300:10-3:10-3；
(6) Will be provided withThe hybrid chain and molecular beacons MB and Mg2+After mixing uniformly, reacting for 1h at 37 ℃, and performing fluorescence detection;
and (3) no time sequence is limited between the step (1) and the step (2).
In the detection method, a PSA antibody and succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester are subjected to coupling reaction for 2 hours, and then the treated antibody is obtained by filtration; the molar ratio of the PSA antibody to the succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester is 1: 10-20. The reaction is preferably performed in a PBS buffer solution, and the reaction is preferably performed at room temperature, more preferably 18-25 ℃. The invention is filtered after the reaction, preferably purified with a filter (cut-off of 10,000MW) and washed three times with PBS buffer. In the coupling reaction, succinimide-4- (N-maleimide) cyclohexane-1-1 hydroxy acid ester (SMCC) is used as a coupling agent, an NHS ester group at one end of a molecule reacts with primary ammonia of a certain protein molecule to form a stable amido bond, and the other end (one end of a maleimide group) can be specifically crosslinked with a sulfydryl of another protein molecule to promote the formation of a subsequent multi-component compound.
The method comprises the steps of respectively carrying out hydroxyl activation reaction on sulfydryl modified DNA1 and sulfydryl modified DNA2 and tri (2-carboxyethyl) phosphine for 1h to obtain treated DNA1 and treated DNA 2; the molar ratio of the sulfhydryl modified DNA1 to the sulfhydryl modified DNA2 to the tri (2-carboxyethyl) phosphine is 1:104. The temperature of the reaction according to the invention is preferably 37 ℃. In the present example, 200. mu.L of thiol-modified DNA1 (10. mu.M) and thiol-modified DNA2 were added with 2. mu.L of TCEP (100mM), respectively, and reacted at 37 ℃ for 1 hour.
After the treated antibody, the treated DNA1 and the treated DNA2 are obtained, the treated antibody is respectively incubated with the treated DNA1 and the treated DNA2 at 4 ℃ for 12 hours to obtain Ab1-DNA1 and Ab2-DNA 2; the volume ratio of the treated antibody to both treated DNA1 and treated DNA2 was 1: 1. After the incubation according to the present invention, it is preferable to further include filtration using a filter membrane with a molecular weight cutoff of 100000MW, to retain the substance on the membrane, and to wash three times using PBS buffer. After the Ab1-DNA1 and Ab2-DNA2 are obtained, ultraviolet absorption detection is preferably included, and if characteristic peaks of DNA and antibody protein can be detected, the reaction is proved to be good.
After Ab1-DNA1 and Ab2-DNA2 are obtained, Ab1-DNA1, Ab2-DNA2, PSA, hairpin DNA H1 and hairpin DNA H2 are mixed and react for 2 hours at 37 ℃ to obtain a chain substitution reaction product; the mole ratio of the Ab1-DNA1, the Ab2-DNA2, the PSA, the hairpin DNA H1 and the hairpin DNA H2 is 100:100:100:10-3:10-3. In the present example, Ab1-DNA1, Ab2-DNA2, PSA each at 100nM, and hairpin DNA (H1, H2) each at 1. mu.M were mixed and reacted at 37 ℃ for 2 hours.
After obtaining a chain substitution reaction product, mixing the chain substitution reaction product with hairpin DNA H3 and hairpin DNA H4, and reacting at 37 ℃ for 1H to obtain a hybrid chain; the molar ratio of the hairpin DNA H3 to the hairpin DNA H4 is 300:10-3:10-3. In the present example, hairpin DNAs (H3, H4) were added to the above-mentioned strand displacement reaction product at 1. mu.M each, mixed well, and reacted at 37 ℃ for 1 hour. The resulting hairpin DNA (H1, H2, H3, H4) assembled long hybrid chains.
After obtaining the hybrid chain, the invention combines the hybrid chain with molecular beacons MB and Mg2+After mixing evenly, reacting for 1h at 37 ℃, and detecting by fluorescence. In the present example, molecular beacons MB and Mg were added to the hybridization chain reaction product obtained as described above2+100nM of each, mixed and reacted at 37 ℃ for 1 h. The signal peak at 520nm can be seen by fluorescence detection.
In the detection method of the present invention, the detection principle is shown in fig. 1, and the antibody with modified DNA is connected to the adjacent position of the antigen to form a Y-type antigen-antibody-DNA hybrid chain, the free DNA end can initiate a chain substitution reaction, two hairpin DNAs are opened in sequence, and the second hairpin DNA hybridized to the top can push down the antigen-antibody-DNA hybrid chain, thereby realizing the circulation of the target antigen. The chain substitution reaction cycle generates a large amount of complementary hybrid chains to initiate a hybrid chain reaction consisting of the circular hybridization of the other two hairpin DNAs to form a large amount of long-chain DNAs, each two adjacent end sequences on the long-chain DNAs can be assembled to form magnesium ion DNase, and finally the magnesium ion DNase can cut molecular beacons in a solution to emit fluorescence in the presence of magnesium ions, so that the multiple signal amplification of the target antigen is realized.
The invention also provides application of the detection kit or the detection method in target antigen signal amplification. In the present invention, the method for amplifying the target antigen signal is the same as the detection method, and is not described herein again. In the application, the signal amplification comprises three-stage amplification, two mouse anti-human prostate specific antigen monoclonal antibodies are used for modifying DNA, and are connected with the adjacent positions of antigens to form a Y-shaped antigen-antibody-DNA hybrid chain which is used as a signal converter to convert the antigen input into a DNA signal output; the chain substitution reaction and the hybridization chain reaction are combined, so that two-stage amplification of signals is realized. Wherein the reaction comprises the following steps: (1) chain substitution reaction: the Ag-Ab-DNA hybrid chain formed by the Ab1-DNA1 and Ab2-DNA2 is hybridized with a first hairpin DNA (H1) to open the stem end of H1, the exposed stem end of H1 is hybridized with a second hairpin DNA (H2), and the Ag-Ab-DNA hybrid chain is arranged under the top of the hybrid-up H2, and the target antigen returns to the cycle from head to realize the first round of signal amplification; (2) and (3) hybridization chain reaction: the chain replacement reaction initiated by the antigen-antibody-DNA hybrid chain generates a large amount of hybrid chains of H1 and H2, initiates a hybrid chain reaction consisting of the cyclic hybridization of the other two hairpin DNAs (H3 and H4), generates a large amount of long-chain DNA, and realizes the second round of signal amplification. And (3) assembling adjacent two end sequences (H3 and H4) on the long-chain DNA formed after the hybridization chain reaction is finished to form magnesium ion DNase, cutting the molecular beacon in the solution by the enzyme in the presence of magnesium ions, releasing the cut molecular beacon and emitting fluorescence, combining the new molecular beacon with the magnesium ion DNase, cutting, circularly amplifying a fluorescence signal, and realizing the signal amplification of the third round.
The following will explain in detail the detection kit, the detection method and the application of prostate specific antigen provided by the present invention with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
An instrument device: a high-speed refrigerated centrifuge (16R, zhhai ama medical instruments ltd), an ultra-pure water machine (SybergyUV, merck millipore), an ultraviolet-visible spectrophotometer (Cary60, agilent technologies, usa), a fluorescence spectrophotometer (F-4600, hitachi high and new technologies, japan), and an agarose horizontal electrophoresis tank (DYCP-31DN, siyiji, beijing).
The reagents used were: PSA antibody, SMCC (succinimidyl-4- (N-maleimide) cyclohexane-1-carboxylate), TCEP (tris (2-carboxyethyl) phosphine), thiol-modified DNA1, thiol-modified DNA2, MB (DNA is biosynthesized from baoza, MB contains one rA base recognized by an ionozyme and is modified with FAM, BHQ groups).
1. Antibody-modified DNA
Reacting the antibody and SMCC (with an appropriate molar ratio of 1: 10-20) in a PBS (phosphate buffer solution) at room temperature for 2 hours, purifying with a filter membrane (with a molecular weight cut-off of 10,000MW), and washing with the PBS for three times; mu.L of TCEP (100mM) was added to 200. mu.L of the thiol-modified DNA1 (10. mu.M) and thiol-modified DNA2 (10. mu.M), respectively, and reacted at 37 ℃ for 1 hour; the above-mentioned antibodies and DNAs were mixed in an amount of 200. mu.L each, and incubated at 4 ℃ for 12 hours. Unreacted DNA was removed by filtration through a filter (molecular weight cut-off 100,000MW) and washed three times with PBS buffer. Ab1-DNA1 and Ab2-DNA2 were obtained. The characteristic peaks of DNA and antibody protein can be seen by ultraviolet absorption detection (FIG. 2).
2. Sequential self-assembly magnesium ion DNA enzyme and detection of fluorescent signal
Ab1-DNA1, Ab2-DNA2, PSA each at 100nM, hairpin DNA (H1, H2) each at 1. mu.M, mixed well and reacted at 37 ℃ for 2H. Hairpin DNAs (H3, H4) were added to the above-mentioned strand displacement reaction product at 1. mu.M each, mixed well, and reacted at 37 ℃ for 1 hour. The resulting hairpin DNA (H1, H2, H3, H4) assembled long hybrid chains. Adding molecular beacons MB and Mg into the hybridization chain reaction product2+100nM of each, mixed and reacted at 37 ℃ for 1 h. The fluorescence detection can see a 520nm signal peak, and the detection limit reaches 0.73pg mL-1(FIG. 3). And no response to non-target assays for alpha-fetoprotein (AFP), Bovine Serum Albumin (BSA), and carcinoembryonic antigen (CEA) (fig. 4).
The invention provides a detection kit, a detection method and application of prostate specific antigen.A second antibody is used for modifying DNA, and is connected with the adjacent position of an antigen to form a Y-shaped antigen-antibody-DNA hybrid chain which is used as a signal converter to convert the input of the antigen into the output of a DNA signal; simultaneously, the chain substitution reaction and the hybridization chain reaction are combined, so that two-stage amplification of signals is realized; each adjacent on the long-chain DNATwo end sequences (hairpin DNA H3 and hairpin DNA H4) are assembled to form magnesium ion DNase, the enzyme cuts molecular beacons in a solution in the presence of magnesium ions, the disconnected molecular beacons are released and emit fluorescence, new molecular beacons are combined with the magnesium ion DNase and cut, and fluorescence signals are circularly amplified, so that the third round of signal amplification is realized, the detection efficiency and sensitivity are greatly improved, and the detection limit reaches 0.73pgmL-1。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (2)
1. A detection kit for prostate specific antigen is characterized by comprising the following components: PSA antibody, succinimide-4- (N-maleimide) cyclohexane-1-carboxylate, tris (2-carboxyethyl) phosphine, thiol-modified DNA1, thiol-modified DNA2, hairpin DNA H1, hairpin DNA H2, hairpin DNA H3, hairpin DNA H4, Beacon MB and Mg2+(ii) a The nucleotide sequence of the sulfhydryl modified DNA1 is shown in SEQ ID NO. 1; the nucleotide sequence of the sulfhydryl modified DNA2 is shown in SEQ ID NO. 2; the nucleotide sequence of the hairpin DNA H1 is shown as SEQ ID NO. 3; the nucleotide sequence of the hairpin DNAH2 is shown in SEQ ID NO. 4; the nucleotide 6 sequence of the hairpin DNAH3 is shown as SEQ ID NO. 5; the nucleotide sequence of the hairpin DNA H4 is shown as SEQ ID NO. 6; the beacon MB comprises an rA base which recognizes the ionic enzyme and is modified with FAM and BHQ groups.
2. The detection kit of claim 1, wherein the nucleotide sequence of the beacon MB is shown in SEQ ID No. 7.
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