CN1428606A - Antigen detection method and detection device made up by using said method - Google Patents
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Abstract
本发明涉及一种新型抗原检测方法。该方法利用抗体Fc片段特异寡核苷酸配基与抗体直接结合，将抗体信号转换成核酸信号，然后经PCR扩增放大，检测出抗原。利用该方法可以组装成检测试剂盒或开发成为实用蛋白质芯片或其他检测各类抗原的生物芯片。该方法检测抗原具有快速、高灵敏度、特异性强等特点。
The invention relates to a novel antigen detection method. In this method, the specific oligonucleotide ligand of the antibody Fc fragment is directly combined with the antibody, and the antibody signal is converted into a nucleic acid signal, and then amplified by PCR to detect the antigen. The method can be assembled into a detection kit or developed into a practical protein chip or other biochips for detecting various antigens. The method for antigen detection has the characteristics of rapidity, high sensitivity and strong specificity.
Description
Technical field
The present invention relates to a kind of novel antigens detection method, be specifically related to a kind of antigen detection method that utilizes the special oligonucleotides aglucon of antibody Fc fragment to set up.The invention still further relates to the pick-up unit that utilizes this method to make.
Background technology
Detection of antigen is to realize by effective identification of antibody to special determinant on the antigen molecule.The high-sensitivity detection of Detection of antigen, particularly protein to medical research, post genome project finish and clinical examination has important effect.At present, the common technology of specific recognition antigen is ELISA (enzyme linked immunosorbent assay), it is by specific antibody identification antigenic determinant, and finishes identification, the amplification of antigenic information by the enzyme, fluorescent material, the radioactive isotope that are connected on the antibody, thereby realizes testing goal.In recent years, though the ELISA detection method has had very big development,, limited when the test sample amount, antigen concentration is extremely low or antibody titer when not high, just can't detect with traditional elisa technique.
Along with the appearance of pcr amplification technology, Santo etc. set up immune PCR technique (Santo, T., et al.Science.258:120-122) in 1992.The basic process of immune PCR technique and ELISA is similar, just with connecting albumen one section biotin labeled dna molecular that can increase is connected on the antibody Fc fragment, and by round pcr antigen signals is amplified, detected.Connecting albumen is Streptavidin-albumin A compound, and it has two single-minded binding sites: one is to be derived and next biotin site by Streptavidin, and another is to be derived and next Fc fragment binding site by albumin A.Dna fragmentation is biotinylated linear plasmid DNA (pUC19).Antibody is normal antibody.Testing process is to connect albumen and the biotinylated DNA pUC19 mixed in molar ratio with 1: 1, produce chimeric protein-DNApUC19 polymkeric substance, the albumin A site combines with antibody Fc section on the albumen by connecting again, forms the complex of antibody-connection protein-dna fragment.During detection, realize antigen-DNA conversion of signals, by pcr amplification antigen signals is amplified again by antigen and antibodies.Immune PCR technique can detect 580 antigen molecules, and remolding sensitivity has improved 105 times with the serve as a mark traditional E LISA technology of thing of alkaline phosphatase.But there is following problem in immune PCR technique: owing to connect the existence of albumen, produce stronger background signal (because the albumin A group can combine with the Fc fragment of any IgG antibody in the sample) during detection; Connect not commercialization of albumen; Can not carry out polymolecular simultaneously detects.
(et al.Nucleic Acids Res.1995 23:522-529) has improved immune PCR technique to nineteen ninety-five Hendrickson etc. for Hendrickson, ER.It is to have replaced connection albumen with difunctional chemical crosslinking molecule, and the Fc fragment of oligonucleotides and antibody is carried out covalent bond, and detection background is reduced, and sensitivity further improves.Immune RCA technology (Rolling the circleamplification) (Schweitzer that Schweitzer etc. set up in 2000, B, et al.PNAS 2000,97:10113-10119), ultimate principle and Hendrickson etc. nineteen ninety-five improved immune PCR technique basic identical, different is that the oligonucleotides that connects increases in the mode of rolling-circle replication at last, and detection sensitivity is further improved.More than two kinds of improved immune PCR techniques all must use corsslinking moleculars, and need the Fc fragment and the oligonucleotides binding site of activation antibody before crosslinked, therefore operation easier greatly, easily makes the antibody activity forfeiture in testing process; Operation can only be carried out under the condition of high-purity antibody, and all must be connected with dna molecular at each antibody, and is time-consuming, cost is high, and difficulty is all compared in popularization and application.
Summary of the invention
The purpose of this invention is to provide a kind of antigen detection method that utilizes the special oligonucleotides aglucon of antibody Fc fragment to set up.
Another object of the present invention provides a kind of pick-up unit that utilizes this method to make.
Method of the present invention is without any need for connecting albumen or corsslinking molecular, but utilizes the special oligonucleotides aglucon of antibody Fc fragment to combine with the direct of antibody, converts antigen signals to nucleic acid signal, through the pcr amplification amplification, detects novel antigens then.
The alleged special oligonucleotides aglucon of the present invention comprise can with any type of oligonucleotides of antibody Fc fragment specific bond, commonly used have single stranded DNA and a single stranded RNA.
For increasing its stability, the base in the alleged special oligonucleotides aglucon sequence of the present invention can be through chemical modification.Also can increase some bases at aglucon sequence two ends, universal sequence be become carry the diversity aglucons of a large amount of artificial information, to satisfy the needs that detect multiple antigen simultaneously as under the prerequisite that does not change with Fc fragment binding characteristic.
The alleged special oligonucleotides aglucon sequence of the present invention can obtain or obtain by other the method such as the method for molecular biology, bioinformatics through the SELEX technology screening.
The alleged antibody of the present invention can be monoclonal antibody or the polyclonal antibody or the phage single chain antibody of any kind (as people, rabbit, mouse, chicken, sheep etc.), any kind of (as IgG, IgM, IgE etc.), any source (as animal generation, genetic engineering preparation etc.).
The principle and the technology path of method of the present invention are: determined antigen is fixed on the media such as nitrocellulose filter, after the confining liquid sealing, the special oligonucleotides aglucon of the similar antibody Fc fragment that obtains with film and antibody and through the SELEX technology screening is hatched certain hour, with the plain film of the abundant washing the fibre of cleansing solution, unconjugated special oligonucleotides aglucon and free antibodies are removed, utilized the oligonucleotides aglucon on the antibody Fc fragment that round pcr increases with antigen combines.The pcr amplification the primer is marked with fluorescent reagent or isotope simultaneously, realizes that by detecting fluorescence or isotopic intensity antigen signals detects.
The present invention emphasizes is no any molecule that is connected between antibody and the oligonucleotides aglucon in this antigen detection method.
Because the oligonucleotides aglucon of a certain antibody-like Fc fragment is the versatility aglucon of this antibody-like, should be able to discern such all antibody, so pass through said method, with the specific nucleic acid squences at this general aglucon is primer, all antigen signals of such antibody recognition all can be converted to nucleic acid signal and be amplified, detects.
With (is to add that some do not influence its nucleotide sequence in conjunction with activity as terminal people) after the artificial modification of a certain antibody-like Fc fragment general oligonucleotide aglucon nucleotide sequence, combine earlier also by method such as UV-crosslinked and fixed, promptly become at the specific nucleic acid marking antibody of synantigen not with specific antibodies.According to modification sequence length difference, the base sequence difference can make the general oligonucleotide aglucon become and represent the not diversity aglucon of synantigen, can make up microarray or biochip, detects when utilizing above-mentioned principle to finish multiple antigen.
The oligonucleotides of indication of the present invention can be ssDNA, also can be RNA.After filtering out sequence by SELEX, can direct chemical synthetic or obtain by other molecular biological methods.
The present invention utilizes the high affinity oligonucleotides aglucon of SELEX technology screening antibody Fc fragment (comprising single stranded DNA and RNA), to the specific recognition of Fc fragment, directly in conjunction with (middle) and pcr amplification, realize the effective transmission and the amplification of antigenic information by the oligonucleotides aglucon without any need for connecting molecule.
Novel antigens detection method of the present invention is being widely used aspect the various Detection of antigen.For example,
1. utilize this know-why can set up a kind of novel antigens detection method platform;
2. utilize this know-why to be assembled into detection kit, can raising rapidly and efficiently have sensitivity, the specificity that all kinds of antibody mediated immunities detect now;
3. utilize this know-why can develop into a kind of novel practical protein-chip or other kinds Detection of antigen biochip constructing technology;
4. utilize the detection kit of this technology assembling or the biochip of structure can be widely used in fundamental research and clinical detection, and bring considerable economic and social benefit.
Method of the present invention has the advantage of following several respects: 1) high sensitivity: utilize SELEX (Systematic evolution of ligands byexponential enrichment) technology screening to come out with the oligonucleotides of antibody Fc fragment specific bond.The SELEX technology be a kind of new bio that grew up in recent years learn a skill (summary is seen: Sun Lei etc., the new development of exponential enrichment part evolution-combinatorial chemistry technique, foreign medical science pharmacy fascicle, 1999,26:193-197).It is that (storage capacity can reach 10 to the application high capacity
18) the random oligonucleotide library, and in conjunction with the oligonucleotides aglucon (aptamer) of PCR amplification in vitro technology with exponential enrichment and target molecule specific bond.Affinity height (the K that the special oligonucleotides aglucon that filters out with the method combines with target molecule
dBe 0.05～50nmol.L
-1), high specificity, easily form stabilized complex.The present invention utilizes the special oligonucleotides aglucon of Fc fragment to combine and round pcr detection antigen with the direct of antibody, not only kept the advantage that immuno-PCR and immune RCA utilize the oligonucleotides amplification, transmit, amplify antigen signals, and since antibody to the specific recognition of Fc fragment with combine, avoided connecting the existence of molecule, make that the detection background of antigen signals is very low, specificity is stronger, and the sensitivity meeting improves greatly; 2) versatility of antibody identification: because the Fc fragment of antibody is a highly conserved sequence, therefore the special oligonucleotides aglucon that filters out at a certain antibody-like (as mouse IgG, IgE, IgM etc.) the Fc fragment of certain kind, should be able to discern such all antibody, effect is similar to the ELIAS secondary antibody among the ELISA; 3) can carry out multiple antigen ultra micro diversity detects: general oligonucleotides aglucon is through behind the artificial sequence modification, make the oligonucleotides aglucon not only have versatility, also can form simultaneously and discern the not diversity aglucon of synantigen, and can be built into when little battle array or biochip are used for multiple antigen and detect; 4) easy and simple to handle, be easy to popularize: because after special oligonucleotides aglucon that obtains and antibody and antigen hatches certain hour, unconjugated nucleic acid aglucon is removed, can detect by round pcr, do not need the oligonucleotides aglucon is coupled on the antibody protein molecule through chemical method, therefore easy and simple to handle, be convenient to the popularization and application of common laboratory or clinical examination section office.
Description of drawings
Fig. 1 is a novel antigens detection method principle schematic of utilizing the special oligonucleotides aglucon of antibody Fc fragment to set up.Wherein the PCR the primer is the complementary series at the oligonucleotides aglucon.
Fig. 2 be the general special oligonucleotides aglucon sequence of a certain antibody-like Fc fragment through artificial increase some bases after, become diversity aglucon sequence and be used for multiple antigen and detect synoptic diagram simultaneously.In order to increase the diversity of aglucon, artificial sequence can be added in 5 ' end, 3 ' end or 5 ' end and 3 ' end adds that simultaneously length can be various.Wherein the PCR the primer only artificially adds sequence at oligonucleotides aglucon two ends.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
The immune detection of embodiment 1. single antigens
Utilize the special oligonucleotides aglucon of mouse IgG Fc fragment to prepare mouse IgG class monoclonal antibody nucleic acid marking reagent, and as the antigen of such antibody recognition of sensor.Concrete grammar is as follows:
Antigen is fixed on the nitrocellulose filter, after the confining liquid sealing, film and mouse IgG class monoclonal antibody and the special oligonucleotides aglucon of mouse IgG Fc fragment were hatched under 37 mouthfuls 30 minutes jointly, behind cleansing solution washing filter membrane several, filter membrane placed again contain the PCR reactant liquor thin-walled PCR pipe of (comprising primer and Standard PC R reaction system), carry out pcr amplification.Utilize the fluorescent reagent of mark on the primer to detect fluorescence signal, set up the corresponding pcr amplification contrast of (corresponding reagent all exists) of not carrying out simultaneously.Deduction contrast fluorescence signal is the signal of determined antigen.
Embodiment 2. is used for the preparation and the application thereof of the novel immune microarray of primary carcinoma of liver early diagnosis
Concrete steps are as follows:
1. the specific nucleic acid labelled antibody for preparing AFP (alpha-fetoprotein), Ft (ferritin), GGT (gamma glutamyl transpeptidase) and GGT isoenzymes: because clinical used above-mentioned 4 kinds of antibody are all mouse IgG class monoclonal antibody at present, so the nucleotide sequence of mouse IgG antibody Fc fragment general oligonucleotide aglucon is carried out artificial modification, as adding 4 kinds of nucleotide sequence A, B, C, D that do not influence it in conjunction with activity 5 ' terminal people respectively, length 40-80bp promptly becomes 4 kinds of specific markers nucleic acid.With these 4 kinds of different labeling nucleic acids respectively with the corresponding one by one combination of above-mentioned 4 kinds of antibody, fixed by UV-crosslinked, promptly become at 4 kinds of specific nucleic acid marking antibody of synantigen not.
2. by the microarray of present conventional method preparation at above-mentioned A, B, C, D sequence.
3. antigen (as the patients serum) is fixed on the nitrocellulose filter, hatched under 37 mouthfuls 30 minutes jointly with above-mentioned 4 kinds of different nucleic acid marking antibody confining liquid sealing back, behind cleansing solution flushing filter membrane several, filter membrane placed again contain the PCR reactant liquor thin-walled PCR pipe of (comprising primer and Standard PC R reaction system), carry out pcr amplification.The primer is respectively at above-mentioned A, B, C, the artificial sequence of D4 kind (promptly having 8 kinds of primers simultaneously), is marked with fluorescent reagent on the primer simultaneously.With microarray technology the PCR product is carried out qualitative and quantitative analysis, can detect 4 kinds of antigens, thereby primary carcinoma of liver is carried out early diagnosis.
Claims (6)
1. a novel antigens detection method is characterized in that utilizing the special oligonucleotides aglucon of antibody Fc fragment directly to combine with antibody, converts antigen signals to nucleic acid signal, amplifies through pcr amplification then, detects antigen.
2. the described method of claim 1, it is characterized in that said special oligonucleotides aglucon be can with the single stranded DNA of antibody Fc fragment specific bond.
3. the described method of claim 1, it is characterized in that said special oligonucleotides aglucon be can with the single stranded RNA of antibody Fc fragment specific bond.
4. the described method of claim 1 is characterized in that said antibody is monoclonal antibody, polyclonal antibody or phage single chain antibody.
5. the described method of claim 2 to 3, it is characterized in that under the prerequisite that does not change with Fc fragment binding characteristic, aglucon sequence two ends can increase some bases artificially, universal sequence is become carry the diversity aglucon of a large amount of artificial information, to satisfy the needs that detect multiple antigen simultaneously.
6. the detection kit and the device that utilize the described method of claim 1 to make.
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| CN 01144927 CN1428606A (en) | 2001-12-24 | 2001-12-24 | Antigen detection method and detection device made up by using said method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105823878A (en) * | 2016-04-05 | 2016-08-03 | 上海美吉生物医药科技有限公司 | Kit for detecting phenotypes of circulating tumor cells |
| CN106248924A (en) * | 2016-08-02 | 2016-12-21 | 江南大学 | A kind of immune analysis method based on graphene oxide photoactivation horseradish peroxidase |
| CN108414735A (en) * | 2018-02-08 | 2018-08-17 | 斯格特生物 | A kind of large biological molecule immunoassay method mediated based on extension three times-RNA amplification |
-
2001
- 2001-12-24 CN CN 01144927 patent/CN1428606A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105823878A (en) * | 2016-04-05 | 2016-08-03 | 上海美吉生物医药科技有限公司 | Kit for detecting phenotypes of circulating tumor cells |
| CN106248924A (en) * | 2016-08-02 | 2016-12-21 | 江南大学 | A kind of immune analysis method based on graphene oxide photoactivation horseradish peroxidase |
| CN106248924B (en) * | 2016-08-02 | 2018-07-06 | 江南大学 | A kind of immunoassay method based on graphene oxide photoactivation horseradish peroxidase |
| CN108414735A (en) * | 2018-02-08 | 2018-08-17 | 斯格特生物 | A kind of large biological molecule immunoassay method mediated based on extension three times-RNA amplification |
| CN108414735B (en) * | 2018-02-08 | 2020-05-12 | 斯格特生物 | Biomacromolecule immunoassay method based on three-time extension-RNA amplification mediation |
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