CN106932590A - A kind of quantitative determination low-abundance protein and the thereafter method of modified protein - Google Patents
A kind of quantitative determination low-abundance protein and the thereafter method of modified protein Download PDFInfo
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Abstract
The invention belongs to biochemical analysis field, it is related to a kind of quantitative determination low-abundance protein and the thereafter method of modified protein.Enzyme-linked Immunosorbent Assay reaction bonded proximity ligation assay is particularly related to protein and the special quantitative method of rapid sensitive is realized in modification thereafter；The method includes：Antibody protein sandwich complex is fixed on 96 orifice plates；Nucleotide sequence pairing is carried out with reference to proximity ligation assay probe；Connection pairing nucleotide sequence；The signal based on rolling circle amplification is carried out to amplify；Signal detection.The method of the present invention can overcome prior art exist as, test limit is in nanograms per milliliter level, the defect of the albumen of low concentration can not be detected, possess the quick of Enzyme-linked Immunosorbent Assay reaction, accurately, the characteristics of the advantages of being easy to operation and the high specific of proximity ligation assay, high sensitivity, can realize to modified protein rapid sensitive accurate quantitative analysis after low abundance.
Description
Technical field
The invention belongs to biochemical analysis field, it is related to a kind of quantitative determination low-abundance protein and the thereafter side of modified protein
Method.Enzyme-linked Immunosorbent Assay reaction bonded proximity ligation assay is particularly related to protein and rapid sensitive is realized in modification thereafter
Special quantitative method；The method possesses the quick of Enzyme-linked Immunosorbent Assay reaction, accurately, is easy to the advantages of operating and ortho position to connect
The characteristics of high specific of technology, high sensitivity.The method can be realized to modified protein rapid sensitive accurate quantitative analysis after low abundance.
Background technology
Sandwich ELISA reaction (ELISA) is one of quick and precisely quantitative goldstandard of albumen.Capture antibody pair
The enrichment of target protein and washing step make sandwich ELISA react the quantitative objective egg for being capable of quick and precisely low background
In vain.The major defect of the method is exactly its test limit in nanograms per milliliter level, it is impossible to detect the albumen of low concentration.
Proximity ligation assay (PLA) is the important supplement to traditional immunization method, is proposed first just since in 2002
Extensive concern has been obtained due to highly sensitive high specific.The method very various fields are obtained for application, such as tumour mark
The handsome choosing and the excavation of histone modification of will thing.The cardinal principle of proximity ligation assay is exactly that thing to be detected is marked by multiple simultaneously
There is the antibody recognition of nucleotide sequence, after the thing combination to be detected of these antibody and target, the nucleotide sequence of modification is just fixed on
Face position, these nucleotide sequences are connected in the presence of nucleic acid ligase, the nucleotide sequence after connection carries out follow-up as template
Nucleic acid amplification.In brief, proximity ligation assay obtains high specific by the identification of Multiple Antibodies, is obtained by nucleic acid amplification
High sensitivity.
At present, proximity ligation assay is divided three classes：Homogeneous proximity ligation assay, solid phase proximity ligation assay in situ, in vitro
Solid phase proximity ligation assay.Although homogeneous proximity ligation assay step is simple, because all experimental procedures are molten all at one
Lack washing step in liquid, cause homogeneous proximity ligation assay background of a relatively high；Solid phase proximity ligation assay in situ can not be straight
Connect for detecting the fluid sample such as serum sample；In vitro solid phase proximity ligation assay is mainly based upon microballoon and test tube, the method
It is applied to analyze the good effect of all kinds of fluid samples acquirements, reducing background mainly due to enriching step and washing step does
Disturb.But, there is microballoon price costly in practice at present and disturb fluorescence signal, the test tube of modified is with conventional real-time
The defect such as polymerase chain reaction instrument is incompatible.The above shortcoming hinders the application of proximity ligation assay.
Therefore, to need development badly a kind of convenient for this area, quickly, sensitive, and special quantitative approach detects low-abundance protein
And modified protein thereafter.
The content of the invention
It is an object of the invention to provide one kind convenient, quickly, sensitive, special quantitative approach detects low-abundance protein
And modified protein thereafter.Enzyme-linked Immunosorbent Assay reaction bonded proximity ligation assay is particularly related to be modified to protein and thereafter
Realize the special quantitative method of rapid sensitive；The method possesses the quick of Enzyme-linked Immunosorbent Assay reaction, accurately, is easy to operation etc. excellent
The characteristics of high specific of point and proximity ligation assay, high sensitivity.The method can be realized quick to modified protein after low abundance
It is sensitive and accurate quantitative.
To achieve the above object, the technical solution adopted by the present invention is as follows：
1. antibody protein sandwich complex is fixed on 96 orifice plates；
2. nucleotide sequence pairing is carried out with reference to proximity ligation assay probe；
3. nucleotide sequence is matched in connection；
4. the signal based on rolling circle amplification is carried out to amplify；
5. signal detection.
Specifically, a kind of quantitative determination low-abundance protein of the invention and the thereafter method of modified protein, it includes step：
1. 50ng capture antibody adds 1% matter after the carbonate coating buffer 37 degree Celsius 2 hours of the PH 9.6 of 50 μ l
Measure the bovine serum albumin(BSA) 37 degree Celsius of fraction 1 hour；
2. the μ l of solution to be detected 50 are added, after 37 degrees Celsius are incubated 1 hour, (PH in 20mM phosphate buffer solutions is used
7.4) wash three times, every time five minutes；
3. 50ng detection antibodies make 4 degrees Celsius after 12 hours in the bovine serum albumin(BSA) of 1% mass fraction of 50 μ l
Washed with (PH 7.4) in 20mM phosphate buffer solutions three times, every time five minutes；
4. two kinds of ortho position linking probes (the non-primer nucleic acid sequence of the anti-binding of rabbit two, the anti-binding primer nucleic acid sequence of mouse two)
After 1ng/ μ l are dissolved in the incubation 1 hour of 37 degrees Celsius of 1% bovine serum albumin solution, (PH in 20mM phosphate buffer solutions is used
7.4) wash three times, every time five minutes；
5. extension sequence and connection nucleotide sequence are dissolved in T4DNA nucleic acid ligase buffer solutions, final concentration of 100nM.
After 96 orifice plates add 50 μ l, 37 degrees Celsius of solution incubations 40 minutes per hole, after adding 0.3ul T4DNA nucleic acid ligases per hole
37 degrees Celsius are incubated 1 hour, be washed once five minutes using (PH 7.4) in 20mM phosphate buffer solutions；
6. 50 μ l rolling circle amplifications solution (50mM triisopropyls second sulphonyl-hydrochloric acid, 10mM magnesium chlorides, 10mM sulphur are added per hole
Sour ammonium, 0.16mM deoxyribonucleoside triphosphates, 0.25mg/ml bovine serum albumin(BSA)s, 0.05% polysorbas20,0.3ul
Ph29DNA nucleic acid polymerases, pH 7.5) 37 degrees Celsius of incubations are washed for 1.5 hours using (PH 7.4) in 20mM phosphate buffer solutions
Wash one time five minutes；
7. add 37 degrees Celsius of the 100nM HRPO labeling nucleic acid detection probes solution of 50 μ l to be incubated 30 points per hole
Zhong Hou, is washed three times using (PH 7.4) in 20mM phosphate buffer solutions, five minutes every time, detects that optical density is believed using ELIASA
Number.
In the present invention, using commercialized 96 orifice plate, easy Fast Packet quilt can be carried out to capture antibody, capture antibody can be with
Target protein carries out enrichment capture, and ambient interferences can be reduced in complex system；
In the present invention, using ortho position linking probe to double antibodies sandwich in two kinds of primary antibodies simultaneously be identified, can improve
The specificity of the method；
In the present invention, rolling circle amplification is carried out as template using the nucleotide sequence in proximity ligation assay probe, can improved
The sensitivity of the method.
Table 1 is the nucleotide sequence used by this technology
Table 1
This method is used to detect green fluorescent protein, as a result shows that it to be sensitiveer than what conventional Enzyme-linked Immunosorbent Assay reacted
Degree improves two orders of magnitude；Locus specificity phospho-AB and general phospho-AB are wherein used respectively, it is successfully quantitative
The phosphorylation level change of extracellular regulated protein kinase；
Result also shows that this method can be additionally used in the target of quantitative O connections 2-Acetamido-2-deoxy-D-glucose (O-GlcNAc)
Albumen, as a result shows, relative to conventional O-GlcNAc modified proteins quantitative technique, (protein immunization precipitates conjugated protein trace
Method), this method more rapid sensitive.
The present invention can be realized to low-abundance protein and rapid sensitive specific detection is realized in modification thereafter.
Brief description of the drawings
Fig. 1 is the schematic diagram of this method.
Fig. 2 is that ortho position linking probe synthesizes schematic diagram.
The activity (a) of mouse secondary antibody after the linking probe synthesis of Fig. 3 mouse ortho position, the work of rabbit secondary antibody after the synthesis of rabbit ortho position linking probe
Property (b).
The nucleotide sequence connection effect of Fig. 4 secondary antibodies and sulfydryl modification.
Fig. 5 contrasts ELISA and ELISA-PLA and detects effect of the green fluorescent protein (GFP) in different sample adulterations:It is green
Doping (a) of the color fluorescin in serum, doping (b) of the green fluorescent protein in cell extract, green fluorescent protein
Doping (c) in tissue extract, wherein black squares represent ELISA results, and gray squares represent that ELISA-PLA is tied
Really.
Fig. 6 ELISA-PLA detect phosphorylated protein (a), and Western blotting detects phosphorylated protein (b).
Fig. 7 ELISA and ELISA-PLA detect O-GlcNAc modified proteins (a), immune protein precipitation conjugated protein print
Mark detects O-GlcNAc modified proteins (b).
Specific embodiment
Following example is to albumen to utilization Enzyme-linked Immunosorbent Assay reaction bonded proximity ligation assay proposed by the present invention
Further illustrating for the special quantitative new method of rapid sensitive is realized in matter and thereafter modification.
Embodiment 1 synthesizes the active investigation of ortho position linking probe
Ortho position linking probe synthesis is as shown in Figure 2：Respectively take be dissolved in 10 μ l phosphate buffer (PH7.4) 20ug rabbits secondary antibodies and
20ug mouse secondary antibodies, are separately added into 0.5 μ l 8mM sulfo-SMCC, room temperature reaction 2 hours；Use retention surpassing for 3K dalton
Chimney filter ultrafiltration four times, removes excessive sulfo-SMCC；The 100uM sulfydryl modification primers of 4 μ l are added in the mouse secondary antibody of ultrafiltration
Nucleotide sequence, adds the non-primer nucleic acid sequence of 100 μM of sulfydryl modifications of 4 μ l, room temperature reaction 2 hours in the rabbit secondary antibody of ultrafiltration
Afterwards, -20 degrees Celsius of preservations are placed in.
1) activity of mouse secondary antibody after mouse ortho position linking probe synthesizes is investigated
Take the μ l of mouse primary antibody 50 that concentration is 1ng/ μ l to be coated in 96 orifice plates, respectively according to mouse ortho position linking probe after closing
Than HRPO mark mouse secondary antibody ratio for 1,10,50 ratio adds 96 orifice plate room temperatures to combine 1 hour, after washing three times
Developed the color, as a result as shown in Fig. 3 (a), activity is still kept after mouse secondary antibody and nucleic acid reaction；
2) activity of mouse secondary antibody after rabbit ortho position linking probe synthesizes is investigated
After rabbit primary antibody is coated with, above-mentioned experiment is repeated, as a result as shown in Fig. 3 (b), still protected after mouse secondary antibody and nucleic acid reaction
Hold activity；
3) the nucleotide sequence connection effect of mouse secondary antibody and sulfydryl modification is investigated
Mouse ortho position linking probe is taken into 0.5 μ l and is adsorbed onto pvdf membrane, after closing, extension sequence and connection nucleotide sequence are dissolved in
In T4DNA nucleic acid ligase buffer solutions, final concentration of 100nM.200 μ l, 37 degrees Celsius of solution incubations 40 minutes are added on film
Afterwards, per hole add 0.9 μ l T4DNA nucleic acid ligases after 37 degrees Celsius be incubated 1 hour, use (PH in 20mM phosphate buffer solutions
7.4) washed once five minutes.200 μ l rolling circle amplifications solution (50mM triisopropyls second sulphonyl-hydrochloric acid, 10mM chlorine are added on film
Change magnesium, 10mM ammonium sulfate, 0.16mM deoxyribonucleoside triphosphates, 0.25mg/ml bovine serum albumin(BSA)s, 0.05% polysorbas20,
0.3 μ l ph29DNA nucleic acid polymerases, pH 7.5) 37 degrees Celsius be incubated 1.5 hours and use (PH in 20mM phosphate buffer solutions
7.4) washed once five minutes.Add the 100nM HRPO labeling nucleic acid detection probe solution 37 of 50 μ l Celsius per hole
After degree is incubated 30 minutes, washed three times using (PH7.4) in 20mM phosphate buffer solutions, colour developing, as a result as shown in figure 4, mouse two
The anti-nucleotide sequence successful connection with sulfydryl modification；
4) the nucleotide sequence connection effect of rabbit secondary antibody and sulfydryl modification is investigated
Mouse ortho position linking probe is taken after 0.5 μ l are adsorbed onto pvdf membrane, above-mentioned experiment is repeated, as a result as shown in figure 4, rabbit secondary antibody
With the nucleotide sequence successful connection of sulfydryl modification.
Embodiment 2
Contrast ELISA and ELISA-PLA detects effect of the green fluorescent protein (GFP) in different sample adulterations
1) doping of the green fluorescent protein in serum
ELISA experimental procedures：The anti-GFP antibody of 50ng mouse is small in 37 degree Celsius 2 of the carbonate coating buffer of the PH 9.6 of 50 μ l
Shi Hou, adds the bovine serum albumin(BSA) 37 degree Celsius of 1% mass fraction 1 hour.The μ l of solution to be detected 50 are added, 37 degrees Celsius incubate
After educating 1 hour, washed three times, every time five minutes using (PH 7.4) in 20mM phosphate buffer solutions.50ng rabbit-anti GFP antibody exists
4 degrees Celsius after 12 hours in the bovine serum albumin(BSA) of 1% mass fraction of 50 μ l, (PH in 20mM phosphate buffer solutions is used
7.4) wash three times, every time five minutes.Add the 1 of 50 μ l:The rabbit secondary antibody 37 of the HRPO mark of 5000 dilutions is Celsius
Degree 1 hour, develops the color after washing three times, as a result as shown in Fig. 5 (a)；
ELISA-PLA experimental procedures：The carbonate coating buffer 37 of PH 9.6 of the anti-GFP captures antibody of 50ng mouse in 50ul is taken the photograph
After family name's degree 2 hours, the bovine serum albumin(BSA) 37 degree Celsius of 1% mass fraction is added 1 hour.Add solution to be detected 50 μ l, 37
Degree Celsius be incubated 1 hour after, use in 20mM phosphate buffer solutions (PH 7.4) wash three times, every time five minutes.50ng rabbit-antis
GFP detection antibodies are slow using 20mM phosphoric acid 4 degrees Celsius after 12 hours in the bovine serum albumin(BSA) of 1% mass fraction of 50 μ l
(PH 7.4) is rushed in solution to wash three times, every time five minutes.Two kinds of ortho position linking probe (non-primer nucleic acid sequences of the anti-binding of rabbit two
Row, the anti-binding primer nucleic acid sequence of mouse two) 1ng/ μ l are dissolved in after 37 degrees Celsius of 1% bovine serum albumin solution is incubated 1 hour, make
Washed with (PH 7.4) in 20mM phosphate buffer solutions three times, every time five minutes.Extension sequence and connection nucleotide sequence are dissolved in
In T4DNA nucleic acid ligase buffer solutions, final concentration of 100nM.50 μ l, 37 degrees Celsius of incubations of the solution are added per hole in 96 orifice plates
After 40 minutes, 37 degrees Celsius are incubated 1 hour after 0.3 μ l T4DNA nucleic acid ligases of every hole addition, molten using 20mM phosphoric acid buffers
(PH 7.4) washed once five minutes in liquid.50 μ l rolling circle amplifications solution of every hole addition (50mM triisopropyls second sulphonyl-hydrochloric acid,
10mM magnesium chlorides, 10mM ammonium sulfate, 0.16mM deoxyribonucleoside triphosphates, 0.25mg/ml bovine serum albumin(BSA)s, 0.05% tells
Warm 20,0.3 μ l ph29DNA nucleic acid polymerases, pH 7.5) 37 degrees Celsius be incubated 1.5 hours using in 20mM phosphate buffer solutions
(PH 7.4) washed once five minutes.The 100nM HRPO labeling nucleic acid detection probes solution 37 of 50 μ l is added per hole
Degree Celsius be incubated 30 minutes after, use in 20mM phosphate buffer solutions (PH 7.4) wash three times, every time five minutes.Use enzyme mark
Instrument detects optical density signal.As shown in Fig. 5 (a), low two orders of magnitude of test limits of the ELISA-PLA than ELISA；
2) doping of the green fluorescent protein in cell extract
Above-mentioned experiment is repeated, shown in result figure 5 (b)；
3) doping of the green fluorescent protein in tissue extract
Above-mentioned experiment is repeated, shown in result figure 5 (c).
Embodiment 3 detects phosphorylated protein using ELISA-PLA
1) locus specificity phospho-AB experimental group
ELISA-PLA experimental procedures：1：The carbonic acid of PH 9.6 of the anti-ERK1/2 captures antibody of mouse of 1000 dilutions in 50 μ l
After salt coating buffer 37 degree Celsius 2 hours, the bovine serum albumin(BSA) 37 degree Celsius of 1% mass fraction is added 1 hour.1 μ l rat brains
Tissue extract is diluted to 50 μ l, after 37 degrees Celsius are incubated 1 hour, three is washed using (PH 7.4) in 20mM phosphate buffer solutions
It is secondary, five minutes every time.Take the 1 of 50 μ l：After the antibody of rabbit-anti phosphorylated CREB 1/2 of 1000 dilutions adds 96 orifice plates, 4 degree Celsius 12
After hour, washed three times, every time five minutes using (PH 7.4) in 20mM phosphate buffer solutions.Other experimental procedures are ibid
ELISA-PLA, as a result as shown in Fig. 6 (a), the phosphorylation level of the ERK1/2 of chronic pain rat is higher than normal rat；
2) general phospho-AB experimental group
ELISA-PLA experimental procedures：1：The carbonic acid of PH 9.6 of the anti-ERK1/2 captures antibody of mouse of 1000 dilutions in 50ul
After salt coating buffer 37 degree Celsius 2 hours, the bovine serum albumin(BSA) 37 degree Celsius of 1% mass fraction is added 1 hour.1 μ l rat brains
Tissue extract is diluted to 50 μ l, after 37 degrees Celsius are incubated 1 hour, three is washed using (PH 7.4) in 20mM phosphate buffer solutions
It is secondary, five minutes every time.Take the 1 of 50 μ l：After the rabbit-anti phosphotyrosine antibody of 1000 dilutions adds 96 orifice plates, 4 degree Celsius 12
After hour, washed three times, every time five minutes using (PH 7.4) in 20mM phosphate buffer solutions；Other experimental procedures are ibid
ELISA-PLA, as a result as shown in Fig. 6 (a), the phosphorylation level of the ERK1/2 of chronic pain rat is higher than normal rat.Use
General phospho-AB can be obtained and use locus specificity phospho-AB identical effect；
3) Western blotting checking ELISA-PLA results, as a result as shown in Fig. 6 (b), the phosphorus of the ERK1/2 of chronic pain rat
Acidifying level is higher than normal rat, this result verification reliability of ELISA-PLA technologies.
The N acetylglucosamines (O-GlcNAc) that embodiment 4 examines O connections using ELISA-PLA change albumen
1) the AKT albumen of O-GlcNAcization is detected using ELISA-PLA
ELISA-PLA experimental procedures：1：Carbonate bag of the rabbit-anti AKT capture antibody of 1000 dilutions in the PH 9.6 of 50 μ l
After liquid 37 degree Celsius 2 hours, the bovine serum albumin(BSA) 37 degree Celsius of 1% mass fraction is added 1 hour.5 μ l rat cerebral tissues
Extract solution is diluted to 50 μ l, after 37 degrees Celsius are incubated 1 hour, is washed three times using (PH 7.4) in 20mM phosphate buffer solutions,
Five minutes every time.Take the 1 of 50 μ l：After the anti-O-GlcNAcization antibody CTD110.6 of mouse of 1000 dilutions adds 96 orifice plates, 4 degrees Celsius
After 12 hours, washed three times, every time five minutes using (PH 7.4) in 20mM phosphate buffer solutions.Other experimental procedures are ibid
ELISA-PLA, as a result as shown in Fig. 7 (a), ELISA-PLA results show the O-GlcNAc modification levels of the AKT of cerebral ischemia mouse
Normal rat is higher than, and the sensitivity of conventional ELISA method is not enough to distinguish the difference；
2) precipitate conjugated protein trace using protein immunization and verify ELISA-PLA results
Protein immunization precipitates conjugated protein Blot experiment step：Taking 200 μ l brain tissues lysates adds the AKT of 2ul to resist
4 degrees Celsius of body slowly rocks overnight incubation.The protein G-beads for taking 50 μ l are added to 4 degrees Celsius of cell pyrolysis liquid slowly
Rock overnight incubation.After immune precipitation, at 4 DEG C with 3,000g centrifugation 5min, by protein G-beads be centrifuged to
Ttom of pipe；Supernatant is carefully sucked, protein G-beads 1ml lysis buffers are washed 3 times.It is eventually adding the 2 × SDS of 15ul
Sample loading buffer, boiling water boiling 10 minutes.Detect that above-mentioned protein immunization precipitates the AKT albumen of acquisition using protein immunoblotting
O-GlcNAcization level, as a result as shown in Fig. 7 (b), conventional method protein immunization precipitation associated proteins trace shows that cerebral ischemia is small
The O-GlcNAc modification levels of the AKT of mouse are higher than normal rat, demonstrate the reliability of ELISA-PLA, but due to routine
Protein immunization precipitation associated proteins engram technology sensitivity it is relatively low, it is necessary to using more samples, whole experiment process takes
It is longer.
Claims (7)
1. a kind of quantitative determination low-abundance protein and the thereafter method of modified protein, it is characterised in that utilize Enzyme-linked Immunosorbent Assay
Reaction bonded proximity ligation assay (ELISA-PLA) modifies quantitative determination to protein and thereafter, and it includes：
1. antibody protein sandwich complex) is fixed on 96 orifice plates；
2.) nucleotide sequence pairing is carried out with reference to proximity ligation assay probe；
3.) connection pairing nucleotide sequence；
4. the signal based on rolling circle amplification) is carried out to amplify；
5.) signal detection.
2. the method as described in claim 1, it is characterised in that it includes step：
(1) capture antibody adds the cow's serum of 1% mass fraction after the carbonate coating buffer 37 degree Celsius 2 hours of PH 9.6
Albumin 37 degree Celsius 1 hour；
(2) solution to be detected is added, after 37 degrees Celsius are incubated 1 hour, is washed using PH7.4 in 20mM phosphate buffer solutions, often
Secondary five minutes；
(3) detection antibody uses 20mM phosphorus 4 degrees Celsius after 12 hours in the bovine serum albumin(BSA) of 1% mass fraction of 50 μ l
PH 7.4 is washed in acid buffering solution, five minutes every time；
After (4) two kinds of ortho position linking probes are dissolved in the incubation 1 hour of 37 degrees Celsius of 1% bovine serum albumin solution, 20mM phosphorus is used
PH 7.4 is washed in acid buffering solution, five minutes every time；
(5) extension sequence and connection nucleotide sequence are dissolved in T4DNA nucleic acid ligase buffer solutions, final concentration of 100nM；In 96 holes
After plate adds 37 degrees Celsius of the solution to be incubated 40 minutes per hole, added 37 degrees Celsius after 0.3ul T4DNA nucleic acid ligases per hole
It is incubated 1 hour, is washed once five minutes with PH 7.4 in 20mM phosphate buffer solutions；
(6) rolling circle amplification 37 degrees Celsius of incubations of solution are added to be washed using PH 7.4 in 20mM phosphate buffer solutions for 1.5 hours per hole
Wash one time five minutes；
(7) after adding 37 degrees Celsius of 100nM HRPO labeling nucleic acid detection probes solution to be incubated per hole 30 minutes, use
PH 7.4 is washed in 20mM phosphate buffer solutions, five minutes every time, and optical density signal is detected using ELIASA.
3. the method as described in claim 2, it is characterised in that described two its nucleotide sequence of ortho position linking probe are rabbit secondary antibody
With reference to non-primer nucleic acid sequence, the anti-binding primer nucleic acid sequence of mouse two.
4. the method as described in claim 2, it is characterised in that capture antibody is coated on 96 orifice plates.
5. the method as described in claim 2, it is characterised in that determinand is added to 96 orifice plates and is combined with capture antibody.
6. the method as described in claim 2, it is characterised in that detection antibody is added to 96 orifice plates and is combined with determinand.
7. the method as described in claim 2, it is characterised in that by two kinds of ortho position linking probes be added to 96 orifice plates respectively with catch
Obtain antibody and detection antibody is combined.
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| CN108226530A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of proximity ligation assay is for the quantitatively kit of detection Myo contents, method of preparation and use |
| CN116256519A (en) * | 2023-02-17 | 2023-06-13 | 成都诺森医学检验有限公司 | Antigen hypersensitive detection method |
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| VANESSA CASTRO-LÓPEZ，ET AL: "A simple and portable device for the quantification of TNF-α in human plasmaby means of on-chip magnetic bead-based proximity ligation assay", 《BIOSENSORS AND BIOELECTRONICS》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226530A (en) * | 2017-11-27 | 2018-06-29 | 南京天纵易康生物科技股份有限公司 | A kind of proximity ligation assay is for the quantitatively kit of detection Myo contents, method of preparation and use |
| CN116256519A (en) * | 2023-02-17 | 2023-06-13 | 成都诺森医学检验有限公司 | Antigen hypersensitive detection method |
| CN116256519B (en) * | 2023-02-17 | 2024-01-16 | 成都诺森医学检验有限公司 | Antigen hypersensitive detection method |
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