CN100338464C - Protein chip carrier processing method - Google Patents
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- CN100338464C CN100338464C CNB021605041A CN02160504A CN100338464C CN 100338464 C CN100338464 C CN 100338464C CN B021605041 A CNB021605041 A CN B021605041A CN 02160504 A CN02160504 A CN 02160504A CN 100338464 C CN100338464 C CN 100338464C
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- Prior art keywords
- biotin
- solid phase
- avidin
- phase carrier
- protein
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- 108090000623 proteins and genes Proteins 0.000 title abstract description 23
- 102000004169 proteins and genes Human genes 0.000 title abstract description 23
- 238000003672 processing method Methods 0.000 title 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 74
- 229960002685 biotin Drugs 0.000 claims abstract description 66
- 239000011616 biotin Substances 0.000 claims abstract description 66
- 239000007790 solid phase Substances 0.000 claims abstract description 38
- 235000020958 biotin Nutrition 0.000 claims abstract description 37
- 108090001008 Avidin Proteins 0.000 claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000002131 composite material Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000013016 damping Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 239000000020 Nitrocellulose Substances 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 229920001220 nitrocellulos Polymers 0.000 claims description 5
- 239000002985 plastic film Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000002033 PVDF binder Substances 0.000 claims 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims 2
- 238000005070 sampling Methods 0.000 abstract description 12
- 238000001514 detection method Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000009792 diffusion process Methods 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 description 12
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 5
- 238000002493 microarray Methods 0.000 description 5
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- -1 compound compound Chemical class 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003498 protein array Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The present invention provides a solid phase carrier of protein chips. A compound of avidin and biotin is combined on the surface of the solid phase carrier, wherein the amount of biotin in the compound of avidin and biotin is 15 to 40% of the amount of avidin active units. The solid phase carrier of protein chips of the present invention can obviously decrease the diffusion of protein sampling points and increase detection sensitivity. The present invention also provides a method for preparing the solid phase carrier of protein chips.
Description
Technical field
The invention belongs to protein-chip or immunoassays field, relate more specifically to a kind of new protein chip carrier and disposal route thereof.
Background technology
Detect for improving flux and detection efficiency, development in recent years a kind of microarray enzyme linked immunosorbent detection technology of having got up is also referred to as protein biochip technology---and what its utilized is that the specificity that antibody combines with antigen is that immune response comes test sample.
The simplified model that protein-chip makes up is: select a kind of solid phase carrier conjugated protein molecule (antigen or antibody) securely, form the microarray of protein like this, i.e. protein-chip.Make up protein microarray on substrate, each point in the array is being represented an independently biochemical reaction, carries out thereby various reactions is integrated on the very little carrier.
Show that according to data such as Rou-Pan huang, Thomas O.joos the structure flow process of protein-chip is as follows:
(1) protein with purifying is dissolved in the damping fluid,
(2) with the high speed point sample instrument protein spots on film,
(3) point sample instrument has wire dot system and specking formula, point typically have a diameter from 0.2-0.4um, the point sample amount generally is no more than 100nl;
(4) again will be point sample the NC film with the TBS flushing for several times,
(5) seal with bovine serum albumen solution then, stand-by through the room temperature airing.
At first add test substance solution during detection on chip, insulation is washed with abundant reaction, enzyme-added mark reactant liquor, and insulation is with abundant reaction, and flushing adds detection liquid, detects change color or luminous signal with detector.
Protein microarray in order to last method makes up exists following shortcoming:
(1) sensitivity is lower,
(2) background height,
(3) protein array spreads in course of reaction easily, and particularly when density was big, the signal that easily causes difference was owing to distance too closely influences each other for battle array.
Therefore, this area presses for new protein-chip solid phase carrier of exploitation and preparation method thereof, and this solid phase carrier can significantly reduce the diffusion of protein spots sampling point and/or improve detection sensitivity.
Summary of the invention
Purpose of the present invention just provides a kind of new protein-chip solid phase carrier and preparation method thereof, and this solid phase carrier can significantly reduce the diffusion of protein spots sampling point and/or improve detection sensitivity.
In a first aspect of the present invention, a kind of solid phase carrier of protein-chip is provided, in the surface combination of described solid phase carrier the compound of Avidin-biotin is arranged, wherein the quantity of biotin is the 0.15-0.4 of the plain active unit of affinity in Avidin-biotin composite.
In a preference, the quantity of biotin is the 1/5-1/3 that is of the plain active unit of affinity in Avidin-biotin composite.
In another preference, described solid phase carrier is selected from down group: glass sheet, plastic sheet, cellulose nitrate (NC) film, PDVF film.
In another preference, the quantity of the compound of described Avidin-biotin is 5-1000ug/cm
2
In another preference, described Avidin is selected from down group: white of an egg Avidin, Streptavidin, and composition thereof.
In another preference, described biotin is selected from down group: biotin N-maloyl imines ester, long-armed biotin or its potpourri.
In a second aspect of the present invention, the preparation method of the above-mentioned protein-chip solid phase carrier of preparation the present invention is provided, comprise step:
(a) solid phase carrier is soaked in the solution of Avidin-biotin composite, wherein the quantity of biotin is the 0.15-0.4 of the plain active unit of affinity in Avidin-biotin composite;
(b) solid phase carrier of usefulness damping fluid washing step (a);
(c) solid phase carrier of drying steps (b).
In a preference, described solid phase carrier is selected from down group: glass sheet, plastic sheet, nitrocellulose membrane, PDVF film.
In another preference, the concentration of the compound of Avidin-biotin is 5-1000ug/ml in the described solution, preferably is 50-500ug/ml, more preferably is 100-500ug/ml.
In another preference, described Avidin is selected from down group: white of an egg Avidin, Streptavidin, and composition thereof; And described biotin is selected from down group: biotin N-maloyl imines ester, long-armed biotin or its potpourri.
In another preference, soaking conditions is under the room temperature 90-150 minute, or 4 ℃ are spent the night; And drying condition is room temperature airing or freeze drying.
Embodiment
The inventor is extensive studies through going deep into, when finding that surface combination when the solid phase carrier of protein-chip has a kind of compound of specific Avidin-biotin, not only the diffusion of protein spots sampling point can be significantly reduced, and detection sensitivity can be improved.Finished the present invention on this basis.
Protein-chip solid phase carrier of the present invention is made of (a) conventional solid phase carrier and the specific Avidin-biotin composite of the present invention that (b) is incorporated on the solid phase carrier basically.
Being used for the specific Avidin-biotin composite of the present invention is a kind of compound by a certain proportion of Avidin and biotin be combined into.Usually, the Avidin of 1 molecule forms compound compound fully in conjunction with the biotin of 4 molecules.Compound right and wrong of the present invention are compound compound fully, promptly per 1 molecule Avidin with less than the formed compound of the biotin of 4 molecules.Preferable compound be per 1 molecule Avidin with less than the formed compound of the biotin of 0.6-2 molecule, more preferably be per 1 molecule Avidin and less than the formed compound of the biotin of 0.8-1.5 molecule.
The mode of biotin/avidin ratio is to be benchmark with the plain active unit of affinity in another kind of expression Avidin-biotin composite.The plain active unit of affinity be defined as some Avidins energy in conjunction with the quantity of biotin.In compound of the present invention, the quantity of biotin is generally the 0.15-0.4 of the plain active unit of affinity, preferably is 1/5-1/3.
Can be used for Avidin of the present invention is not particularly limited.Representational example comprises (but being not limited to): white of an egg Avidin, Streptavidin or its potpourri etc.
Can be used for biotin of the present invention is not particularly limited.Representational example comprises (but being not limited to): the activation biotin, and the plain N-maloyl of biological example imines ester, long-armed biotin or its potpourri etc.,
As used herein, term " protein-chip solid phase carrier " is exactly a substrate material of putting the protein spots sampling point thereon, and common carrier comprises: glass sheet, plastic sheet, cellulose nitrate (NC) film, PDVF film etc. wherein particularly preferably are the NC film.
The present invention also provides the preparation method of protein-chip solid phase carrier.With the NC film is example, and its disposal route is as follows:
At first, before carrying out the protein point sample NC film is soaked in Avidin-biotin complex solution, wherein the quantity of biotin is the 0.15-0.4 of the plain active unit of affinity in Avidin-biotin composite.
Then, for several times with damping fluid (as the PBS damping fluid) washing.
At last, dried (as room temperature airing or freeze drying).
In Avidin-biotin composite solution, the concentration of Avidin-biotin composite is generally 5-1000ug/ml, preferably is 100ug/ml-500ug/ml.The biotin proportion is the 1/5-1/3 of the plain active unit of affinity in Avidin-biotin composite.
As for soaking temperature and time, be not particularly limited.Usually, the soak time in Avidin-biotin composite solution is under the room temperature 90-150 minute or longer, or 4 ℃ are spent the night.
According to test, handle the NC film by the present invention after, carry out the protein point sample again, through antigen-antibody reaction, the background values of the final sample signal 7-12% that can descend, corresponding sample minimum detectable activity also can reduce more than 8%, thereby detection sensitivity is greatly improved.
In addition, the NC film is handled through affinity element-biotin composite, and the diffusion of protein sampling point also reduces to some extent, and sampling point area ratio is 1.5 times before and after the immune response, and the two untreated area ratio is more than 2.2 times.In other words, point of sample area of the present invention only is about 50% of contrast.Therefore, the NC film of handling through Avidin-biotin, after the luminescence-producing reaction in the same hole between the difference phase mutual interference of light signal reduced greatly, also indicating the integrated level that can further improve protein-chip.
NC film as preparation protein-chip or protein microarray enzyme linked immunoassay carrier, handle and unprocessed comparing through Avidin-biotin composite, therefore, the present invention can be widely used in protein-chip or be the immune response system of substrate with the film.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Present embodiment with the hepatitis B anti-HCV, anti--Core, anti--NS3, anti--NS4, anti--5 kinds of detection of antibodies such as NS5 are that example prepares protein-chip.
(a) preparation of protein-chip solid phase carrier
The aperture 0.4mm water wettability NC film of selecting Whatman company to produce, be soaked in 100ug/ml, biotin under the room temperature and accounted in the affinity element-biotin composite solution of the plain active unit 1/4 of affinity room temperature following 120 minutes, the PBS damping fluid washing of pH 7.8 3 times, the room temperature airing.Used affinity element is a strepto-affinity element in the compound, and biotin is the long-chain biotin.
(b) preparation of protein-chip
With high speed protein point sample instrument with 5 kinds of antigens of correspondence of 5 kinds of antibody of hepatitis C put respectively in above-mentioned through on the NC film that affinity element-biotin is handled with undressed NC film on, the individual sampling point of every array 16 (4 * 4).Use 5% calf serum (BSA) sealing again, the room temperature airing is stand-by.
(c) detect
Serum and above-mentioned two kinds of NC films of the HCV positive are carried out film reaction, and both end reaction results are compared as follows:
Table 1
| Background values | Control treatment background values ratio | Signal value | Difference | Point of sample area before the reaction | Reaction back point of sample area | Reaction back control treatment sampling point area ratio | |
| Contrast | 77.6 | 110/100 | 137.2 | 59.6 | 0.1256 | 0.2889 | 1.53/1.00 |
| Embodiment 1 | 69.0 | 146.8 | 77.8 | 0.1256 | 0.1884 |
Embodiment 2
Repeat the order-checking of embodiment 1, difference only is to have used the Avidin-biotin composite of variable concentrations or different proportion, different solid phase carrier (seeing table 2 for details).
Table 2
| Solid phase carrier | Biotin quantity is the ratio of the plain active unit of affinity | Complex concentration (ug/ml) | Reaction back control treatment sampling point area (experimental point sampling point size/control point sampling point size) |
| NC | 1/3 | 50 | 70.4％ |
| 100 | 69.5％ | ||
| 300 | 67.2％ | ||
| 1/4 | 50 | 68.5％ | |
| 100 | 65.4％ | ||
| 300 | 64.3％ | ||
| PDVF | 1/3 | 50 | 71.4％ |
| 100 | 69.7％ | ||
| 300 | 67.8％ | ||
| 1/4 | 50 | 69.5％ | |
| 100 | 66.1％ | ||
| 300 | 64.9％ |
Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the solid phase carrier of a protein-chip is characterized in that, in the surface combination of described solid phase carrier the compound of Avidin-biotin is arranged, and wherein the quantity of biotin is the 1/5-1/3 of the plain active unit of affinity in Avidin-biotin composite.
2. solid phase carrier as claimed in claim 1 is characterized in that, described solid phase carrier is selected from down group: glass sheet, plastic sheet, nitrocellulose membrane, polyvinylidene fluoride film.
3. solid phase carrier as claimed in claim 1 is characterized in that, the quantity of the compound of described Avidin-biotin is 5-1000ug/cm
2
4. solid phase carrier as claimed in claim 1 is characterized in that, described Avidin is selected from down group: white of an egg Avidin, Streptavidin, and composition thereof.
5. solid phase carrier as claimed in claim 1 is characterized in that, described biotin is selected from down group: biotin N-maloyl imines ester, long-armed biotin or its potpourri.
6. the preparation method of the described protein-chip solid phase carrier of claim 1 is characterized in that, comprises step:
(a) solid phase carrier is soaked in the solution of Avidin-biotin composite, wherein the quantity of biotin is the 1/5-1/3 of the plain active unit of affinity in Avidin-biotin composite;
(b) solid phase carrier of usefulness damping fluid washing step (a);
(c) solid phase carrier of drying steps (b).
7. method as claimed in claim 6 is characterized in that, described solid phase carrier is selected from down group: glass sheet, plastic sheet, nitrocellulose membrane, polyvinylidene fluoride film.
8. method as claimed in claim 5 is characterized in that, the concentration of the compound of Avidin-biotin is 5-1000ug/ml in the described solution.
9. method as claimed in claim 6 is characterized in that, described Avidin is selected from down group: white of an egg Avidin, Streptavidin, and composition thereof; And described biotin is selected from down group: biotin N-maloyl imines ester, long-armed biotin or its potpourri.
10. method as claimed in claim 6 is characterized in that, soaking conditions is under the room temperature 90-150 minute, or 4 ℃ are spent the night; And drying condition is room temperature airing or freeze drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021605041A CN100338464C (en) | 2002-12-27 | 2002-12-27 | Protein chip carrier processing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021605041A CN100338464C (en) | 2002-12-27 | 2002-12-27 | Protein chip carrier processing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1512180A CN1512180A (en) | 2004-07-14 |
| CN100338464C true CN100338464C (en) | 2007-09-19 |
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| CNB021605041A Expired - Fee Related CN100338464C (en) | 2002-12-27 | 2002-12-27 | Protein chip carrier processing method |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539740A (en) * | 2010-12-29 | 2012-07-04 | 河北省健海生物芯片技术有限责任公司 | Protein chip and method for detecting 80 kinds of autoantibodies simultaneously |
| CN102207501A (en) * | 2011-03-22 | 2011-10-05 | 沃克(天津)生物科技有限公司 | Biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and preparation method thereof |
| CN115161277A (en) * | 2022-08-18 | 2022-10-11 | 武汉华美生物工程有限公司 | Method for directly separating immune cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335505A (en) * | 2001-07-11 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip based on labeling streptavidin-biotin technology |
| WO2002031506A1 (en) * | 2000-10-09 | 2002-04-18 | Aviva Biosciences Coropration | Compositions and methods for separation of moieties on chips |
| CN1348993A (en) * | 2001-11-02 | 2002-05-15 | 中国科学院武汉病毒研究所 | Method of proofreading PCR to detect gene mutation on solid phase carrier |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002031506A1 (en) * | 2000-10-09 | 2002-04-18 | Aviva Biosciences Coropration | Compositions and methods for separation of moieties on chips |
| CN1335505A (en) * | 2001-07-11 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip based on labeling streptavidin-biotin technology |
| CN1348993A (en) * | 2001-11-02 | 2002-05-15 | 中国科学院武汉病毒研究所 | Method of proofreading PCR to detect gene mutation on solid phase carrier |
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