CN103454427A - Method for detecting trace protein in blood by utilizing antibody-nucleic acid combined amplification technology - Google Patents
Method for detecting trace protein in blood by utilizing antibody-nucleic acid combined amplification technology Download PDFInfo
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- CN103454427A CN103454427A CN2012101786648A CN201210178664A CN103454427A CN 103454427 A CN103454427 A CN 103454427A CN 2012101786648 A CN2012101786648 A CN 2012101786648A CN 201210178664 A CN201210178664 A CN 201210178664A CN 103454427 A CN103454427 A CN 103454427A
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Abstract
A method for detecting trace protein in blood by utilizing an antibody-nucleic acid combined amplification technology relates to the technical field of bioscience. The detection method is as below: (1) carrying out an antigen-antibody reaction on antibodies of two coupling nucleic acid sequences and a protein to be measured, so that two nucleic acid sequences connected to the antibody approach to each other, and under the catalysis of a DNA polymerase, 3'-DNA chains extend to form a DNA double chain; and (2) utilizing a TaqMan nucleic acid probe and a primer to carry out quantitative determination on the specific nucleic acid sequence of the antibody, thereby realizing quantitative determination on the concentration of the protein to be measured. The method provided by the invention organically combines the principle of antigen-antibody specific combination and the principle of PCR specific amplification to amplify a specific signal by millions or tens of millions of times, thereby substantially raising detection sensitivity; meanwhile, quantitative determination on trace protein in a detection sample can be realized based on the concentration of a standard substance.
Description
Technical field:
The present invention relates to the bio-science technical field, be specifically related to utilize antibody-nucleic acid associating amplification technique to detect the method for trace protein in blood.
Background technology:
Detecting trace of albumin mark in blood or other sample is clinical important means of carrying out medical diagnosis on disease, the most frequently used method is Enzyme-linked Immunosorbent Assay technology (ELISA) at present, and its principle is to utilize the method for antigen-antibody reaction and chromogenic enzyme substrate to detect specific protein marker.When measuring, add testing sample, make antibody in sample or antigen or the antibody generation association reaction of antigen and surface of solid phase carriers.Then the method by washing makes the antigen antibody complex formed on solid phase carrier separate with other molecules in solution.Add again detectable antigens or the antibody of enzyme labeling, and be combined on solid phase carrier by reaction.In enzyme amount on solid phase carrier and sample, the amount of target molecule to be measured is certain proportionate relationship like this.After adding enzyme reaction substrate, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of target molecule to be measured in sample, therefore can carry out qualitative or quantitative test according to the depth of reaction color.Because the catalytic efficiency of enzyme is very high, can indirectly amplify immunoreactive result, so the ELISA method has higher susceptibility.Although the method can reach the purpose of quantitative test, be subject to the restriction of enzyme-substrate reactions, its detection sensitivity still can't be detected the protein marker of denier in sample, thereby has limited its using value in the disease early diagnosis.In addition, due to the operation steps more complicated, the various factors that therefore affects result is more; Running time and operating process require stricter; The range of linearity is narrower, generally only has the range of linearity of 1 to 2 order of magnitude; Specificity is poor, and in serum, the interference of various materials can affect reaction result; Defining of yin and yang attribute is fuzzyyer, needs repeated experiments; Manual operations is loaded down with trivial details, is difficult to robotization and mass; The reaction plate of different batches differs greatly, and result is difficult to normalization; Sample and reagent consumption are larger, generally need the above volume of 50 microlitres to be reacted, so cost are higher.
Based on ELISA, developed again the Immuno-PCR of being combined with the PCR high-sensitivity characteristic.This technology combines the high sensitivity of the high specific of antigen-antibody reaction and polymerase chain reaction, utilizes the high sensitivity of the specificity of antigen-antibody reaction and pcr amplification reaction and a kind of antigen detection technique of setting up.Its essence is a kind of ELISA method of amplifying the antigen-antibody effect with pcr amplification section of DNA reporter molecules replacement enzyme reaction.Immuno-PCR has very high sensitivity, but its operating process based on ELISA, react, step is very complicated relatively still, in order to guarantee the specificity of result, has multistep to wash the plate step, so still there is the intrinsic limitation of ELISA.
Summary of the invention:
The purpose of this invention is to provide a kind of antibody-nucleic acid associating amplification technique that utilizes and detect the method for trace protein in blood, it utilizes the principle of Ag-Ab specific bond and the principle of quantitative PCR specific amplified, by the two organic assembling, make millions of even up to ten million times of specific signals amplification, thereby greatly improved the sensitivity detected, according to the concentration of standard items, can realize that the trace of albumin to detecting in sample is realized quantitatively detecting simultaneously.
In order to solve the existing problem of background technology, the present invention takes following technical scheme: its detection method is: (1) carries out antigen-antibody reaction by the antibody of two coupling nucleic acid sequences and testing protein in solution, two nucleotide sequences that make to be connected on antibody are adjacent to each other, under the catalysis of archaeal dna polymerase, 3 ' DNA chain extension, form the DNA double chain.
(2) utilize TaqMan nucleic acid probe and primer quantitatively to detect described antibody specific nucleic acid sequence, thereby quantitatively detect the concentration of testing protein.
Described testing protein is trace protein in blood.
Described antibody is for a pair of monoclonal antibody of testing protein or a kind of polyclonal antibody.
Described and nucleotide sequence antibody coupling is the nucleotide sequence that 3 ' end can form complementary double-stranded sequence.
Described primer refers to for carrying out PCR, the nucleotide sequence that particularly real-time fluorescence PCR increases.
Described TaqMan nucleic acid probe refers to the nucleotide sequence of being hybridized with above-mentioned PCR product.
The present invention has following beneficial effect: it utilizes the principle of Ag-Ab specific bond and the principle of quantitative PCR specific amplified, by the two organic assembling, make millions of even up to ten million times of specific signals amplification, thereby greatly improved the sensitivity detected, according to the concentration of standard items, can realize that the trace of albumin to detecting in sample is realized quantitatively detecting simultaneously.
Embodiment:
This embodiment is taked following technical scheme: its detection method is: (1) carries out antigen-antibody reaction by the antibody of two coupling nucleic acid sequences and testing protein in solution, two nucleotide sequences that make to be connected on antibody are adjacent to each other, under the catalysis of archaeal dna polymerase, 3 ' DNA chain extension, form the DNA double chain.
(2) utilize TaqMan nucleic acid probe and primer quantitatively to detect described antibody specific nucleic acid sequence, thereby quantitatively detect the concentration of testing protein.
Described testing protein is trace protein in blood.
Described antibody is for a pair of monoclonal antibody of testing protein or a kind of polyclonal antibody.
Described and nucleotide sequence antibody coupling is the nucleotide sequence that 3 ' end can form complementary double-stranded sequence.
Described primer refers to for carrying out PCR, the nucleotide sequence that particularly real-time fluorescence PCR increases.
Described TaqMan nucleic acid probe refers to the nucleotide sequence of being hybridized with above-mentioned PCR product.
This embodiment is utilized the principle of Ag-Ab specific bond and the principle of quantitative PCR specific amplified, by the two organic assembling, make millions of even up to ten million times of specific signals amplification, thereby greatly improved the sensitivity detected, according to the concentration of standard items, can realize that the trace of albumin to detecting in sample is realized quantitatively detecting simultaneously.
Claims (7)
1. utilize antibody-nucleic acid associating amplification technique to detect the method for trace protein in blood, the detection method that it is characterized in that it is: (1) carries out antigen-antibody reaction by the antibody of two coupling nucleic acid sequences and testing protein in solution, two nucleotide sequences that make to be connected on antibody are adjacent to each other, under the catalysis of archaeal dna polymerase, 3 ' DNA chain extension, form the DNA double chain;
(2) utilize TaqMan nucleic acid probe and primer quantitatively to detect described antibody specific nucleic acid sequence, thereby quantitatively detect the concentration of testing protein.
2. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, it is characterized in that described testing protein is trace protein in blood.
3. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, it is characterized in that described antibody is a pair of monoclonal antibody for testing protein.
4. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, and the method that it is characterized in that described antibody trace protein is a kind of polyclonal antibody for testing protein.
5. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, it is characterized in that described and nucleotide sequence antibody coupling is the nucleotide sequence that 3 ' end can form complementary double-stranded sequence.
6. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, it is characterized in that described primer refers to for carrying out PCR, particularly the nucleotide sequence of real-time fluorescence PCR amplification.
7. antibody-nucleic acid associating the amplification technique that utilizes according to claim 1 detects the method for trace protein in blood, it is characterized in that described TaqMan nucleic acid probe refers to the nucleotide sequence of being hybridized with the PCR product.
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Cited By (5)
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| CN110498858A (en) * | 2019-07-26 | 2019-11-26 | 深圳市达科为生物工程有限公司 | A kind of method of the unicellular excretion protein secretion situation of dynamic detection |
| CN111139285A (en) * | 2018-11-02 | 2020-05-12 | 深圳华大生命科学研究院 | Method for detecting contents of different target proteins in multiple samples to be detected in high-throughput manner and special kit thereof |
| CN111733212A (en) * | 2020-06-22 | 2020-10-02 | 广州水石基因科技有限公司 | Hypersensitive molecule lock-key immune polymerase chain reaction detection method |
| CN112778426A (en) * | 2021-01-06 | 2021-05-11 | 深圳伯生生物传感技术有限公司 | Precise antibody nucleic acid directional connection method |
| CN112986573A (en) * | 2019-12-13 | 2021-06-18 | 深圳汇芯生物医疗科技有限公司 | Quantitative detection method of exosome multigroup chemical marker |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111139285A (en) * | 2018-11-02 | 2020-05-12 | 深圳华大生命科学研究院 | Method for detecting contents of different target proteins in multiple samples to be detected in high-throughput manner and special kit thereof |
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| CN110498858B (en) * | 2019-07-26 | 2024-01-23 | 深圳市达科为生物工程有限公司 | Method for dynamically detecting secretion condition of single-cell exoprotein |
| CN112986573A (en) * | 2019-12-13 | 2021-06-18 | 深圳汇芯生物医疗科技有限公司 | Quantitative detection method of exosome multigroup chemical marker |
| CN111733212A (en) * | 2020-06-22 | 2020-10-02 | 广州水石基因科技有限公司 | Hypersensitive molecule lock-key immune polymerase chain reaction detection method |
| CN112778426A (en) * | 2021-01-06 | 2021-05-11 | 深圳伯生生物传感技术有限公司 | Precise antibody nucleic acid directional connection method |
| CN112778426B (en) * | 2021-01-06 | 2023-12-12 | 深圳伯生生物传感技术有限公司 | Accurate antibody nucleic acid directional connection method |
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