CN104198738A - Positive control applied to quantitative detection of protein chip and preparation method - Google Patents
Positive control applied to quantitative detection of protein chip and preparation method Download PDFInfo
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- CN104198738A CN104198738A CN201410471782.7A CN201410471782A CN104198738A CN 104198738 A CN104198738 A CN 104198738A CN 201410471782 A CN201410471782 A CN 201410471782A CN 104198738 A CN104198738 A CN 104198738A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a positive control applied to quantitative detection of a protein chip. 1) a FLAG coated antibody, 2) HA-FLAG polypeptide and 3) an HA biotin labeled antibody are located on a biochip in sequence. The invention further discloses a method for preparing the positive control. The control can characterize whether a whole experiment from antigen and antibody response to development is successful, serves as a reference substance to quantitatively analyze targets in blood serum, and overcomes the defects of poor reliability and failure in standardization in the prior methods.
Description
Technical field
The present invention relates to a kind of positive reference that protein chip quantitatively detects that is applied to.
The invention still further relates to the method for the above-mentioned positive object of reference of preparation.
Background technology
The know-why of protein chip is just proposed in the eighties in last century by Roger Ekin the earliest, it is fixed on a kind of carrier surface by a large amount of protein molecules by the arrangement setting in advance and forms microarray, according to the principle of specific binding between protein molecule, build microfluid biochemical analysis system, detection accurate, quick to biomolecule to realize, large information capacity.Protein chip technology is mainly used in the proteomics research based on chip, is characterized in high flux, at one, only has on the sheet base of tens square millimeters thousands of kinds of protein of mark for detection of other destination proteins.Its ultimate principle be exactly utilize antibody molecule can with the feature of antigen molecule specific binding, free foreign protein is separated with the destination protein that is incorporated into solid phase carrier, and utilize special label to carry out quantitative test to it.The protein chip scope that has a wide range of applications, comprises drug screening, the fields such as medical diagnosis on disease treatment, judicial expertise.
Existing biochip test method comprises luminous detection method and non-luminous detection method two classes, widespread use or luminous detection method, wherein mainly comprise again fluorescence detection, chemiluminescence detecting method and complex light irradiating and detecting method.And the chemiluminescence detecting method that developed recently gets up because of its fast, accurately, favorable reproducibility and the nontoxic feature of reagent safety, demonstrate good application prospect.When object is detected with chemiluminescence detecting method, in order to reduce the impact quantitative on target detection thing of background value and experimental system, a reference point generally to be set, the luminous value of the object recording is compared and obtained relative value with the luminous value of reference point.
Protein chip reference point normally directly carries out the direct coated such as enzyme labeling or biotin labeling in chip surface by certain (a bit) special albumen at present.The effect of this class reference point is to be used to refer to normally carrying out of chemiluminescence reaction, or chip point sample position is determined.Although this class reference point was also once used as the reference point of test substance.But due in conjunction with insecure, easily by washing lotion, rinsed out, affected the accuracy quantitative to object.And this class reference point can only characterize in hybridization reaction and be reacted to chromogenic reaction from biotin and horseradish peroxidase (HRP), cannot characterize the reaction of Ag-Ab, also affected the accuracy quantitative to object.
Summary of the invention
The object of this invention is to provide a kind of positive reference that protein chip quantitatively detects that is applied to.
Another object of the present invention is to provide a kind of method of preparing above-mentioned positive object of reference
For realizing above-mentioned order, the positive reference that is applied to the quantitative detection of protein chip provided by the invention is followed successively by biochip:
1) FLAG coated antibody;
2) HA-FLAG polypeptide;
3) HA biotin labeling antibody.
The method of positive reference in preparation provided by the invention:
A) FLAG coated antibody is fixed on biochip;
B) on biochip, add the standard antigen of the sample that contains polypeptide and each concentration gradient;
C) remove antigenic substance, remove the material of non-specific binding;
D) add biomarker antibody;
E) remove unnecessary antibody.
Wherein, FLAG coated antibody is fixed on biochip with matrix form.
Wherein, for FLAG coated antibody is AFP, CEA, t-PSA and Flag coated antibody.
Wherein, the basic sequence of HA-FLAG polypeptide is: YPYDVPDYA-C-C-C-C-C-C-DYKDDDDK.
Positive reference of the present invention can be monitored immunoreactive each stage of protein chip, synchronizes with pattern detection, effectively reduces manual operation or instrument error and to protein chip, detects the error of bringing.
Embodiment
The positive reference that is applied to the quantitative detection of protein chip provided by the invention, be utilize antibody molecule can with the feature of antigen molecule specific binding, in sample, (particularly serum) sneaks into a kind of artificial synthetic polypeptide, the luminous value that polypeptide is combined with antibody materials is as reference, with the object luminous value recording and its, compare, obtain relative value, with this, carry out the quantitative test of object in sample.
The positive of the present invention is with reference to comprising following components:
1) FLAG coated antibody;
2) HA-FLAG polypeptide, its basic sequence is: YPYDVPDYA-C-C-C-C-C-C-DYKDDDDK
3) HA biotin labeling antibody
By above three kinds of compounds that form " sandwich ".
The present invention further illustrates the present invention for embodiment, but protection domain of the present invention is not limited to following embodiment.
1, detect the concentration of serum tumor mark AFP, CEA, t-PSA
A) AFP, CEA, t-PSA coated antibody and Flag coated antibody are fixed on biochip with matrix form with point sample instrument;
B) on the chip having sealed, add the standard antigen of the blood serum sample that contains artificial synthetic polypeptide and each concentration gradient, reaction 1-1.5 hour;
C) remove antigenic substance, by washing lotion (1 * TBS/1% glycerine/0.1%BSA/0.05% tween), remove the material of non-specific binding;
D) add AFP, CEA, t-PSA antibody and the HA antibody of biomarker, reaction 25-40min;
E) by washing lotion (1 * TBS/1% glycerine/0.1%BSA/0.05% tween), remove unnecessary antibody;
F) add HRP (1/10000), reaction 30min;
G) by washing lotion (1 * TBS/1% glycerine/0.1%BSA/0.05% tween), remove unnecessary HRP;
H) add luminescent solution, colour developing;
I) collect AFP, CEA, t-PSA luminous value and positive reference point luminous value;
J) by AFP, CEA, t-PSA luminous value divided by the positive with reference to luminous value, obtain the relative value of CEA;
K) relative light unit and the theoretical concentration of AFP, CEA, each concentration standard antigen of t-PSA are made to typical curve, then the relative value of AFP, CEA, t-PSA in serum is brought in serum, obtain middle AFP, CEA, the t-PSA concentration in serum.
Feature of the present invention is, after Blast comparison, there is no to have in finder's body protein group the albumen with its homology, be difficult for being degraded by the enzyme in serum, by purity after artificial synthesizing and purifying, can reach more than 95%, price is lower, whether can characterize from antigen and antibody response to the whole Success in Experiment of colour developing, and as reference point, the object in serum is carried out to quantitative test, solved poor reliability that previous methods exists and cannot standardized defect, to realize the successful Application of immuning hybridization technology in diagnosing in vitro.
Correlation data by table 1 and table 2 can be found out, the HA-FLAG of take is better than C-myc with the albumen of biotin from accuracy and repeated two aspects as the measured data of reference point, explanation be take HA-FLAG and is monitored whole immuning hybridization process as reference point, the measured data stability of such reference point is better, and accuracy is higher.
Table 1
Table 2
Claims (8)
1. be applied to the positive reference that protein chip quantitatively detects, on biochip, be followed successively by:
1) FLAG coated antibody;
2) HA-FLAG polypeptide;
3) HA biotin labeling antibody.
2. positive reference according to claim 1, wherein, FLAG coated antibody is fixed on biochip with matrix form.
3. positive reference according to claim 1 and 2, wherein, for FLAG coated antibody is AFP, CEA, t-PSA and Flag coated antibody.
4. positive reference according to claim 1, wherein, the basic sequence of HA-FLAG polypeptide is: YPYDVPDYA-C-C-C-C-C-C-DYKDDDDK.
5. the method for preparing positive reference described in claim 1:
A) FLAG coated antibody is fixed on biochip;
B) on biochip, add the standard antigen of the sample that contains polypeptide and each concentration gradient;
C) remove antigenic substance, remove the material of non-specific binding;
D) add biomarker antibody;
E) remove unnecessary antibody.
6. method according to claim 5, wherein, FLAG coated antibody is fixed on biochip with matrix form.
7. according to the method described in claim 5 or 6, wherein, for FLAG coated antibody is AFP, CEA, t-PSA and Flag coated antibody.
8. method according to claim 5, wherein, the basic sequence of HA-FLAG polypeptide is: YPYDVPDYA-C-C-C-C-C-C-DYKDDDDK.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410471782.7A CN104198738B (en) | 2014-09-16 | 2014-09-16 | A kind of quantitatively positive reference and preparation method of detection of protein chip that be applied to |
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| CN201410471782.7A CN104198738B (en) | 2014-09-16 | 2014-09-16 | A kind of quantitatively positive reference and preparation method of detection of protein chip that be applied to |
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| CN104198738A true CN104198738A (en) | 2014-12-10 |
| CN104198738B CN104198738B (en) | 2016-05-11 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005090546A1 (en) * | 2004-03-19 | 2005-09-29 | Detroit R & D, Inc. | Chip production, hybridization and data interpretation for antibody and protein microarrays |
| US20060014212A1 (en) * | 2002-05-10 | 2006-01-19 | Epitome Biosystems, Inc. | Proteome epitope tags and methods of use thereof in protein modification analysis |
| CN101041697A (en) * | 2006-03-21 | 2007-09-26 | 上海富纯中南生物技术有限公司 | Anti-protein expression label antibody or antibody compound and preparation method and application thereof |
| CN102981002A (en) * | 2012-11-29 | 2013-03-20 | 同昕生物技术（北京）有限公司 | Indirect immunoassay method adopting tag protein humanized chimeric antibody as positive contract and kit |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
-
2014
- 2014-09-16 CN CN201410471782.7A patent/CN104198738B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060014212A1 (en) * | 2002-05-10 | 2006-01-19 | Epitome Biosystems, Inc. | Proteome epitope tags and methods of use thereof in protein modification analysis |
| WO2005090546A1 (en) * | 2004-03-19 | 2005-09-29 | Detroit R & D, Inc. | Chip production, hybridization and data interpretation for antibody and protein microarrays |
| CN101041697A (en) * | 2006-03-21 | 2007-09-26 | 上海富纯中南生物技术有限公司 | Anti-protein expression label antibody or antibody compound and preparation method and application thereof |
| CN102981002A (en) * | 2012-11-29 | 2013-03-20 | 同昕生物技术（北京）有限公司 | Indirect immunoassay method adopting tag protein humanized chimeric antibody as positive contract and kit |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
Non-Patent Citations (1)
| Title |
|---|
| MARTA SANCHEZ-CARBAYO: "Antibody Arrays: Technical Considerations and Clinical Applications in Cancer", 《CLINICAL CHEMISTRY》, vol. 52, 31 December 2006 (2006-12-31), pages 1651 - 1659, XP002572111, DOI: doi:10.1373/clinchem.2005.059592 * |
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Inventor after: Ding Ligong Inventor after: Ma Junlan Inventor after: Cang Baisheng Inventor after: Gong Xiaoyan Inventor before: Ding Ligong Inventor before: Ma Junlan Inventor before: Cang Baisheng Inventor before: Gong Xiaoyan |
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Effective date of registration: 20170407 Address after: 102200 Changping District science and Technology Park, Beijing Chang Hing Road, building No. 1, floor four Patentee after: Beijing Jiuzhou Taikang Biological Technology Co., Ltd. Patentee after: Shandong wotai Biological Technology Co. Ltd. Patentee after: Shandong Taichang Biological Technology Co. Ltd. Address before: 102200 Changping District science and Technology Park, Beijing Chang Hing Road, building No. 1, floor four Patentee before: Beijing Jiuzhou Taikang Biological Technology Co., Ltd. |