CN101382552A - Method for detecting protein content difference - Google Patents
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Abstract
The invention discloses a method for determining the differences of protein contents, which comprises the steps that: a. a plurality of kinds of protein antibodies respectively combined with different oligonucleotide sequences synchronously carry out antigen antibody reaction with one kind of proteantigen to be tested; b. the oligonucleotide sequences of the antigen antibody complex in the step a are coupled together through the coupled reaction by using ligase, and the sequences of a ligation product are led to contain a base sequence representing the source of the protein to be tested; c. the ligation product in the step b is taken as a templet, and a base sequence containing the specificity of the source of the protein to be tested is amplified by adopting the amplification reaction; d. the base sequence representing the source of the protein to be tested in the amplification product sequence is determined by adopting the real-time sequencing technology; and e. the sequence determined in the step d is taken as the source of the protein to be tested, signal intensity of each sequence is compared to obtain the relative contents of the protein to be tested in each source. The method can determine the differences of protein expression content from different sources at tone time with low cost.
Description
Technical field
A kind of method that is used for detecting protein content difference of the present invention belongs to a kind of assay method of protein relative content, is a kind of protein relative content of measuring separate sources by nucleic acid marking proteins react, antigen-antibody reaction, enzyme coupled reaction, nucleic acid amplification reaction and real-time sequencing reaction specifically.
Background technology
The Human Genome Project is fulfiled ahead of schedule (Anal Chem 74 (1): 22A, 2002) because of the development of capillary array electrophoresis sequencing technologies.As capillary array electrophoresis, the two dimensional electrophoresis technology is the development of chromatograph-mass spectrometer coupling technology particularly, has also accelerated the research steps (Lambert, J.P., M.Ethier, et al.Anal Chem 77 (12): 3771-87,2005) of proteomics greatly.But protein molecule than the dna molecular complexity many, set forth the extremely low protein institute's role in physiology course of expression on the molecular level, highly sensitive and high narrow spectrum detection method accordingly just must be arranged.Protein detection is different with DNA detection, concerning DNA detection, can adopt ripe nucleic acid amplification technologies (as PCR) to make the DNA cloning (Saiki of trace, R.K., S.Scharf, et al.Science 230 (4732): 1350-4,1985), can detect the DNA of individual molecule in theory, and specificity is also fine; But concerning protein detection, can not adopt amplification technique that the number of molecules of interest is increased, but adopt direct determination method, so its detection sensitivity is subjected to very big restriction.If can be with a kind of amplification technique amplification protein matter molecule that is similar to PCR, the detection sensitivity of that protein will improve greatly, this variation and detection protein markers relevant with disease development to protein in the analysis list cell has significant meaning, can realize the early diagnosis of disease, major disease such as tumour are played forewarning function.
ULF Landegren seminar has proposed a kind of new aleuroplast outer analysis method, be called ortho position connection method (Proximity Ligation Assay) (Fredriksson, S., M.Gullberg, et al.Nat Biotechnol 20 (5): 473-7,2002).This method is by the single-minded DNA adapter of a kind of height and the affine mechanism of protein, to contain not homotactic two DNA adapters is combined on the cell factor platelet derived growth factor BB to be measured (PDGF-BB) simultaneously, article two, the major part sequence of DNA adapter is identical, afterbody single stranded sequence difference only, be respectively the 3 ' end and the 5 ' end of dna sequence dna, if contain destination protein PDGF-BB in the sample to be tested, adapter is combination with it, when two different adapters with after same PDGF-BB molecule combines, two strands of adapter afterbody are just close mutually, be easy to and complementary catenation sequence hybridization, under the effect of dna ligase, coupled reaction takes place, adopt the amplification of real-time fluorescence PCR method to be connected product, make the detection of protein change detection into, realized the analysis of trace protein DNA with detecting, its detection sensitivity is 1000 times of enzyme linked immunosorbent assay (ELISA), can detect 24000 PDGF-BB molecules.But experimental results show that the probability that connects two kinds of different affinity probe on the same protein molecule simultaneously is not high, only have 1/25; The more important thing is that the DNA adapter kind that can be used in this method at present is limited, limited this The Application of Technology greatly.For this reason, Gullberg etc. use polyclonal antibody (McAb) instead or monoclonal antibody (PcAb) replaces the DNA adapter to measure, successful analysis cell factor (the Gullberg who flies mole level in 11 samples, Gustafsdottir et al.Proc Natl Acad Sci USA 101 (22): 8420-4,2004).But it is bigger to measure background, has limited the further raising of sensitivity.Three ortho position sonde methods of employings such as Schallmeiner descend detection background, can detect the protein (Schallmeiner, E., E.Oksanen, et al.Nat Methods4 (2): 135-7,2007) of 100 molecules.Soderberg etc. also combine this method with the rolling circle amplification mode, be used for the original position analysis of protein, detect the protein distribution situation (Soderberg, O., K.J.Leuchowius, et al.Genet Eng (NY) 28:85-93,2007) in the cell.But it is quantitative that this method depends on real-time fluorescence PCR, need expensive real-time fluorescence PCR instrument, make typical curve, measure loaded down with trivial detailsly, the more important thing is and once only can measure a sample, if be used for the expression difference of certain protein of comparison at three kinds of different tissues or three kinds of different cells, just need measure respectively three times, and then comparative analysis, therefore, not more convenient effective detection method.
Summary of the invention
The objective of the invention is at above-mentioned weak point, adopt nucleic acid marking proteins react, antigen-antibody reaction, enzyme coupled reaction, nucleic acid amplification reaction and real-time sequencing reaction to come quantitative measurement with the expression difference of a kind of protein in a plurality of different tissues or cell, set up a kind of detection technique of not having fluorescently-labeled high sensitivity protein expression difference, be used for the research of comparison protein group.
For convenience's sake, the present invention is called antigen with testing protein, and the protein of combination is called antibody with it.Elder generation's different dna single chain of binding sequence on antibody during mensuration, then with antigen-reactive, after antigen-antibody reacts, the DNA in amplification " Ag-Ab-DNA " compound, feasible detection to protein changes the detection to DNA into.Its main determination step is as follows:
(1) method that combines with antibody specificity of dna single chain and the preparation and the sign of high-purity antibody-DNA combined probe.Protein measuring to be transformed into mensuration, at first DNA must be combined with the antibody protein specificity DNA.DNA and combination of proteins method have a lot, and be wherein more commonly used with the method for streptavidin (STV) reaction based on biotin (biotin).Detecting a kind of antigen needs two kinds of probes at least, and the free-end of the DNA of itself and two kinds of probes is respectively 3 ' and 5 ' end.Because free dna single chain and free antibody can impact the downstream, must antibody purification-DNA compound.
(2) the surperficial and different antibody-DNA combined probe reaction of same antigen molecule.A common antigen has a plurality of determinants, and a kind of antigenic determinant produces a kind of antibody.Therefore same antigen molecule surface can with a plurality of different monoclonal antibodies (each is corresponding to a kind of determinant) combination.Is that example is set forth principle of the present invention with same antigen molecule in conjunction with two different monoclonal antibodies, wherein each monoclonal antibody is combined with not homotactic dna probe, except the sequence difference, free DNA end is also different, be respectively 3 ' end and 5 ' end, when two kinds of antibody-when the DNA combined probe was attached to antigenic surface simultaneously, the end of DNA (3 ' end and 5 ' end) closely linked to each other, local concentration increases, and is easy to take place coupled reaction under complementary series and ligase existence.Also can adopt polyclonal antibody to replace a plurality of different monoclonal antibodies, be about to polyclonal antibody and be divided into several parts (each part is equivalent to a kind of monoclonal antibody), combine with not homotactic dna probe respectively.
(3) antigen by the terminal coupled reaction mark of DNA in Ag-Ab-DNA compound separate sources.In Comparative Proteomics, need in once measuring, analyze the relative expression quantity of a certain specified protein in different tissues or cell.The present invention is by means of coupled reaction, adopts the sequence label method dna marker that one section sequence is different in Ag-Ab-DNA compound.Because it is identical with Tm to connect the length of the dna sequence dna in representative source in the product, so just can not produce any " discrimination " phenomenon during nucleic acid amplification (as PCR), can guarantee the equal proportion amplification, promptly represent the protein DNA of respectively originating behind nucleic acid amplification, to keep relative content constant, can not be subjected to influence as the PCR cycle index.
(4) reduce the background signal that causes because of coupled reaction.In coupled reaction, wish that the terminal coupled reaction of DNA just takes place the antibody-dna probe only be attached to antigenic surface, but terminal coupled reaction also might take place in free in theory antibody-dna probe, so can produce background signal, i.e. false positive signal.The present invention adopts the method for the third antibody that the size of background signal is controlled, the dna single link that is about to be used to connect source specific DNA and antibody-DNA end is incorporated into the third antibody, when all three kinds of antibody combine with determined antigen simultaneously, coupled reaction just takes place, and this can greatly reduce the background signal because of the non-special coupled reaction generation of free antibodies-dna probe end.
(5) amplification contains the separate sources coupled reaction product mixtures of sequence label.After the connection product equal-volume mixing with separate sources, carry out amplified reaction, usually adopt the PCR reaction, promptly adopt a pair of primer to increase, represent the same dna fragmentation of separate sources owing to adopt a pair of universal primer amplification, and the Tm value of amplified production identical (length is identical with the base kind) is as single template, so do not need the special optimization of PCR reaction carrying out.
(6) measure the relative content of representing separate sources antigen in the amplified production with burnt sequencing.Amplified production is a potpourri, and each product is marked with different base sequences, need carry out sequential decoding with the method for order-checking.At present common sequencing reaction is based on the principle of gel electrophoresis, but this method belongs to the qualitative determination sequence, quantitative poor performance, and can not measure the sequence of primer back base.Burnt sequencing is to add each dNTP by circulating one by one successively to carry out sequencing reaction, not only can directly measure the base sequence of primer back, and quantitatively performance is fine, can be by measuring the definite number that repeats base of peak height.When measuring the sequence of source specific DNA amplified production with burnt sequencing, at first use single stranded DNA template and sequencing primer annealing, add one by one again and the corresponding complementary dNTP in antigen source, just can judge the antigen presentation amount difference of separate sources according to peak intensity.
The objective of the invention is to realize by following technical measures:
A kind of method that is used for detecting protein content difference comprises the following steps:
A. the multiple protein that is combined with different oligonucleotide sequences is respectively done antibody while and a kind of testing protein generation antigen-antibody reaction of making antigen;
B. under the effect of ligase, the oligonucleotide sequence in the antigen antibody complex among the step a is linked together, and make to connect and contain one section base sequence of represent testing protein to originate in the product sequence by coupled reaction;
C. be template with the connection product among the step b, adopt one section of amplified reaction amplification to contain the testing protein specific base sequence of originating;
D. adopt the base sequence in the source of the representative testing protein in the amplified production sequence among the real-time sequencing technologies determination step c;
E. with the sequence that records in the steps d source as testing protein, relatively the signal intensity of each sequence just obtains the relative content of testing protein in each source.
Described detecting protein content difference method, wherein antibody is meant with the testing protein to be the monoclonal or the polyclonal antibody protein of antigen preparation.
Described detecting protein content difference method, wherein antigen-antibody reaction is meant the reaction that a kind of antigen takes place with two or more antibody of this antigen simultaneously.
Described detecting protein content difference method, the wherein multiple protein that is combined with different oligonucleotide sequences respectively is meant 5 ' end of not homotactic oligonucleotides (DNA) sequence or method and the protein bound that 3 ' end passes through physics or chemistry, forms DNA-protein combined probe.
Described detecting protein content difference method, wherein one section base sequence of representing testing protein source is meant the section of DNA sequence in the oligonucleotide sequence that is marked on the antibody protein molecule.
On behalf of the base sequence in testing protein source, described detecting protein content difference method wherein be meant the section of DNA sequence that adds in the coupled reaction process for one section.
Described detecting protein content difference method, wherein amplified reaction is meant rolling circle amplification technology (RCA), PCR (PCR), chain substitute amplification (SDA), ligase chain reaction (LCR) and rely on the amplification (NASBA) of nucleotide sequence.
Described detecting protein content difference method, wherein real-time sequencing technologies is meant the burnt sequencing technologies based on bioluminescence reaction.
Described detecting protein content difference method, wherein two or more antibody is meant that 5 ' end of the oligonucleotide sequence of wherein at least a antibody surface combination is free-end, and 3 ' end of the oligonucleotide sequence of at least a antibody surface combination is free-end.
Beneficial effect of the present invention:
The present invention is based on the principle that the ortho position connects, use the protein of the method mark separate sources of sequence label earlier, amplification contains the section of DNA of sequence label again, with real-time sequencing technologies sequence label is decoded at last.Advantage of the present invention is to measure the protein expression amount difference of 2 above separate sources in once analyzing simultaneously, the sample consumption is low, reagent dosage is few, do not need to use fluorescence labeling, need not use laser instrument yet, only need simple bioluminescent detection device as detecting device, can be used for the high-sensitivity detection related sign object, can be used for the functional protein in the analysis of cells; Detect the trace protein mark in the blood; Research and discovery and disease association, the protein molecule relevant with drug effect; Interaction between the research protein molecule; Also can be used for early detection trace viral antigen (as HCV and HIV viral antigen) etc.
Description of drawings
Fig. 1 is the method schematic diagram of detecting protein content difference.
Fig. 2 is Jiao of two kinds of separate sources interleukin 2s measurement result that checks order.
Fig. 3 adds the third antibody to reduce reaction background signal raising mensuration sensitivity of method synoptic diagram.
Fig. 4 is Jiao of three kinds of separate sources interleukin 2s measurement result that checks order.
Embodiment
As shown in Figure 1, the method for a kind of detecting protein content difference of the present invention be divided into five the step carry out:
(1) preparation antibody-DNA combined probe, and carry out purifying.With the testing protein is antigen, prepares its antibody 1 and antibody 2, and combines formation antibody-DNA combined probe 1+3 and 2+4 respectively with dna fragmentation 3 and 4.
(2) will originate among A and the B protein respectively with above-mentioned antibody-DNA combined probe reaction, form antigen-antibody-DNA compound 5 and 6.
(3) add source specific DNA strand 7 and 8 respectively in containing 5 and 6 solution, and add the ortho position respectively and connect dna single chain 9 and 10, under the effect of ligase 11, the specificity of originating ortho position coupled reaction forms respectively and connects product 12 and 13.
(4) get isopyknic source specificity and connect product 12 and 13 mixing, add a pair of primer 14 and 15 and carry out pcr amplification reaction, contain dna fragmentation 16 and 17 in the amplified production.
(5) adopt burnt sequencing reaction to measure the sequence of amplified production 16 and 17 at last, produce the order-checking collection of illustrative plates, wherein peak 18 and 19 is corresponding with source A and B respectively, and peak height is represented the relative expression quantity of the protein of respectively originating.If the antigen in three (or a plurality of) source is arranged, then just can draw three (or a plurality of) peaks by once measuring, relatively the relative height at three (or a plurality of) peaks is just known protein expression situation in each source.
The mensuration of the relative content of 1: two kind of separate sources interleukin 2 of embodiment (IL-2)
1. experiment material: recombination human interleukin-2 (Peprotech, USA); Anti-(R﹠amp more than the biotinylation antihuman interleukin-2; D System, USA); The amine-modified streptavidin of maleimide (Sigma, USA); Magnetic bead (DynabeadsM-280-strptavidin, Dynal AS, Oslo, Norway); Oligonucleotide strand (Invitrogen is synthetic, Shanghai) probe 1:5 '-SH-ttttt ttatg tggtc tatgt cgtcg ttcgc tagta gttcc tgggc tgcac-3 '; Probe 2:5 '-P-tcgaggcgta gaatt ccccc gatgc gcgct gttct tactc atttt t-SH-3 '; Sequence label: 5 '-P-agt tgc cat ctg tcgatc-3 ', 5 '-P-agt tgc cat ctg tca ctg-3 '; Connect strand: 5 '-acgcc tcgac agtga cagat ggcaa ctgtgcagcc cttt-3 ', 5 '-acgcc tcgag atcga cagat ggcaa ctgtg cagcc cttt-3 '; Amplimer: 5 '-Bio-atgtggtcta tgtcg tcgtt cg-3 ', 5 '-tgagt aagaa cagcg cgcat-3 '; Annealing primer: 5 '-ggggg aattc tacgcctcga-3 '.
2. experimental procedure:
(1) preparation of streptavidin-dna probe
At first, the streptavidin that maleimide is amine-modified (0.5nmol) reacted 2 hours respectively with under dna probe 1 and 2 (2nmol) room temperature, and reaction buffer is the PBS solution that 50 μ l contain 5mM EDTA.In order to remove dissociative DNA wherein, in reactant liquor, add isopyknic saturated sulfuric acid amine aqueous solution, placed 2 hours for 65 ℃, centrifugal 30 minutes of room temperature 13,000 * g removes supernatant, and precipitation is dissolved in the PBS solution that contains 5nM EDTA, repeats this step 2 time.In order to remove the free streptavidin in the solution, upwards go on foot the 3M NaAc (pH4.6) that adds 1/10th volumes in the solution, the 10mM Mg (Ac) 2 of 1/10th volumes, 95% cold ethanol of two volumes, room temperature reaction 2 hours, 4 ℃ 13, centrifugal 30 minutes of 000 * g, remove supernatant, precipitation is dissolved in the 10mM Tris-HCl (pH7.4) of 50 μ l, 4 ℃ of preservations.
(2) preparation of ortho position probe
Streptavidin DNA chain (50nM) behind biotinylation antihuman interleukin more than 2 anti-(50nM) and the purifying at room temperature reacted 1 hour in the ratio of 1:1, and reaction buffer is the PBSE (5nM EDTA) that contains 0.1% BSA.4 ℃ of preservations of reacted ortho position probe.
(3) ortho position coupled reaction and burnt order-checking.
With the IL-2 solution of 1 μ l separate sources respectively with 50nM ortho position probe to mixing, 37 ℃ of reactions 1 hour.Reaction system is 10 μ l, and reaction buffer is for containing 1% BSA, 1mM biotin, 16 μ g/ml polyA, the PBS solution of 0.05% polysorbas20.In the IL-2 of separate sources solution system, add respectively and represent the sequence label (0.1mM) in two kinds of sources and connect strand (0.1mM) accordingly, T4 ligase (5U), Tris-HCl (pH7.5) (1mM), KCl (5mM), 2 mercapto ethanol (1mM), EDTA (0.01mM) and glycerine (5%).The reaction cumulative volume is 120 μ l, keeps 15 minutes under 30 ℃ of conditions.
The coupled reaction product equal-volume in two sources is mixed.The connection product 25 μ l that get mixing increase in advance, and amplification system is: amplimer (200nM), and Tris-HCl (pH8.3) is (10mM), KCl (50mM), MgCl2 (1.5mM), dNTP (0.2mM) rTaq enzyme (1.25U), cycling condition is: 95 ℃ of 2min; 95 ℃ of 30sec; 60 ℃ of 1min; 13 cycle indexes.Pre-expansion is increased production thing with 50 times of TE Buffer dilutions, as going on foot the amplification template down.The second step amplification system and amplification cycles condition are all constant, only cycle index are brought up to 35.
Utilize finishing that the magnetic bead of streptavidin is arranged, amplified production made strand, and with annealing primer annealing after, strand is carried out Jiao checks order.
3. experimental result
Fig. 2 is the burnt sequencing result of above-mentioned two source IL2.The relative content of two source IL is represented at peak 20 and 21 respectively among the figure, and the ratio of its peak height or peak area is just represented the relative concentration difference of IL2 in two sources.Peak 22 and 23 is the background signal in the burnt order-checking, can deduct from measurement result.In this example, because the IL2 content in two sources equates that measurement result shows that also IL-2 originates the content ratio near 1:1 two.
Embodiment 2: the mensuration of carrying out protein content difference on the dna molecular that specific sequence directly is marked at antibody protein links to each other of will originating
In the present embodiment, representative source specificity base sequence in the dna single chain 7 and 8 among Fig. 1 is marked at respectively in dna sequence dna 3 or 4, the antigen-antibody that promptly forms after the antigen-antibody reaction-DNA compound 5 is different with the dna sequence dna in 6, contains the source specific sequence.During the specificity of originating ortho position coupled reaction, only need to add and 3 and 4 in the ortho position of sequence complementation be connected dna single chain 9 and carry out coupled reaction.All the other steps are consistent with Fig. 1.
This embodiment need be to distinguishing the different DNA of flag sequence with a kind of antibody, with the protein of difference separate sources.Determination step is with embodiment 1, because the IL2 content in two sources equates that measurement result shows that also IL-2 originates the content ratio near 1:1 two.
Embodiment 3: add the third antibody and reduce reaction background signal raising mensuration sensitivity of method
In the present embodiment, prepare at least 3 kinds of antibody-DNA combined probe, antibody is the antibody with a kind of protein.As shown in Figure 3, remove the antibody 1 among Fig. 1, outside 2, also to increase a kind of antibody 24, and the ortho position that will contain the specific sequence of originating connects dna single chain (9) and antibody 24 connects together, have only when three kinds of antibody all with testing protein generation antigen-antibody reaction after, coupled reaction takes place under the effect of ligase 11.Make the specificity of coupled reaction improve like this, background lowers.
The mensuration of the relative content of 4: three kinds of separate sources interleukin 2s of embodiment (IL-2)
In the present embodiment, utilize embodiment 1 identical method to measure the relative content of three kinds of separate sources interleukin 2s (IL-2), experimental technique is with embodiment 1, difference is exactly to design one group of source specific DNA strand (as 7 among Fig. 1) again to be connected dna single chain (as 7 among Fig. 1) with the ortho position, under the effect of ligase 11, with the specificity ortho position coupled reaction of originating of the antigen protein in the 3rd source.The result just forms respectively and connects three kinds of connections of product product, connects the product mixed in equal amounts with three kinds then, measures sequence according to the method for embodiment 1, and the result as shown in Figure 4.The relative content of three source IL is represented at peak 25,26 and 27 respectively among the figure, and the ratio of its peak height or peak area is just represented the relative concentration difference of IL2 in three sources.
Sequence table
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Claims (9)
1, a kind of method that is used for detecting protein content difference is characterized in that comprising the following steps:
A. with the multiple protein antibody while and a kind of testing protein antigen generation antigen-antibody reaction that is combined with different oligonucleotide sequences respectively;
B. under the effect of ligase, the oligonucleotide sequence in the antigen antibody complex among the step a is linked together, and make to connect and contain one section base sequence of represent testing protein to originate in the product sequence by coupled reaction;
C. be template with the connection product among the step b, adopt one section of amplified reaction amplification to contain the testing protein specific base sequence of originating;
D. adopt the base sequence in the source of the representative testing protein in the amplified production sequence among the real-time sequencing technologies determination step c;
E. with the sequence that records in the steps d source as testing protein, relatively the signal intensity of each sequence just obtains the relative content of testing protein in each source.
2, detecting protein content difference method according to claim 1 is characterized in that it is the monoclonal or the polyclonal antibody protein of antigen preparation that antibody is meant with the testing protein.
3, detecting protein content difference method according to claim 1 is characterized in that antigen-antibody reaction is meant the reaction that a kind of antigen takes place with two or more antibody of this antigen simultaneously.
4, detecting protein content difference method according to claim 1, it is characterized in that the multiple protein that is combined with different oligonucleotide sequences respectively is meant 5 ' end of not homotactic oligonucleotides (DNA) sequence or method and the protein bound that 3 ' end passes through physics or chemistry, forms DNA-protein combined probe.
5, detecting protein content difference method according to claim 1 is characterized in that one section base sequence of representing testing protein to originate is meant the section of DNA sequence in the oligonucleotide sequence that is marked on the antibody protein molecule.
6, detecting protein content difference method according to claim 1 is characterized in that on behalf of the base sequence in testing protein source, one section be meant the section of DNA sequence that adds in the coupled reaction process.
7, detecting protein content difference method according to claim 1, it is characterized in that amplified reaction is meant rolling circle amplification technology (RCA), PCR (PCR), chain substitute amplification (SDA), ligase chain reaction (LCR) and rely on the amplification (NASBA) of nucleotide sequence.
8, detecting protein content difference method according to claim 1 is characterized in that real-time sequencing technologies is meant the burnt sequencing technologies based on bioluminescence reaction.
9, detecting protein content difference method as claimed in claim 3, the antibody that it is characterized in that two or more is meant that 5 ' end of the oligonucleotide sequence of wherein at least a antibody surface combination is free-end, and 3 ' end of the oligonucleotide sequence of at least a antibody surface combination is free-end.
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| CN102460176B (en) * | 2009-05-21 | 2014-09-24 | 李荣秀 | Process for differential polypeptides detection and uses thereof |
| CN102460176A (en) * | 2009-05-21 | 2012-05-16 | 李荣秀 | Process for differential polypeptides detection and uses thereof |
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| EP3041957A4 (en) * | 2013-09-04 | 2017-03-29 | Fluidigm Corporation | Proximity assays for detecting nucleic acids and proteins in a single cell |
| CN103592431B (en) * | 2013-10-15 | 2016-02-24 | 中山大学 | A kind of detection method of amplifying based on nuclease signal |
| CN103592431A (en) * | 2013-10-15 | 2014-02-19 | 中山大学 | Detection method based on nuclease signal amplification |
| CN107267624A (en) * | 2017-07-06 | 2017-10-20 | 中国计量科学研究院 | A kind of protein active concentration mensuration pedestal method based on digital pcr |
| 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 |
| 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 |
| 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 |
| CN113684245A (en) * | 2020-05-19 | 2021-11-23 | 南方医科大学第五附属医院 | Detection kit and detection method for MPT64 protein |
| CN112557666A (en) * | 2020-11-16 | 2021-03-26 | 湖南博奥瑞康生物科技有限公司 | Kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction |
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