CN1584592A - Immune microarray protein chip - Google Patents
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Abstract
An immune microarray protein chip is prepared by printing antibody taken from antibod library or prepared antibody fragment on chip substrate with aldehyde group, fixing antibody or antibody fragment on activated chip substrate by plate printing. The universal signal matter is applied and only one type of signal tag is used for carrying out protin analysis when the chip is used for detection.
Description
Technical field
The present invention relates to protein-chip, particularly a kind of chip, this chip preparation method and detection technique thereof the multiple protein analysis and that can and then be used for proteomics research of being used for based on antibody library.
Background technology
Protein-chip is an another great biotechnology after genetic chip.Genetic chip has been widely used in biological basis research and each field of clinical medicine as the example of success.Yet genetic chip can only detect the variation of the various hereditary information of human body in the DNA/RNA level, and can't detect the variation on protein level.Along with finishing of Human Genome Project examining order, be about to enter the genome times afterwards comprehensively, next step will be studied complicated more protein function, presses for protein biochip technology.
The rudimentary model of protein-chip is based on the ultimate principle similar to genetic chip, and promptly the micro-array by design is in advance fixed a large amount of protein on a certain solid phase carrier.If the protein of being fixed is antibody then claims immune micro-array chip.The combination of antigen-antibody, i.e. immune response is specific, like this antigen (or antibody) is added that various marks can realize the immune detection based on chip.
Any protein can all can produce the specific antibody of oneself by immune response as a kind of antigen.So no matter be that clinical biological sample or the new protein expression sample that has existed can be realized its testing goal by immuno-chip.
Protein-chip of the prior art, as Chinese patent application 01105795.5 and 01113323.6, be with the protein example dot matrix on the microslide of chemical treatment, protein is connected to the immunoassay that is fixed on the microslide and is further used for protein detection or specific antigen or antibody by chemical bond-linking.Such protein-chip is based on protein array, only just can obtain the reaction result of multiple index by serial synchronous reaction once, improved detection speed and efficient greatly, is applicable to any person-portion, detects index the time arbitrarily.But the serious problems that this technology exists have: 1. antigen or antibody with might have a strong impact on original biologically active (a variety of protein be unsuitable for signal mark even can't mark) being connected of semiochemicals; 2. the capture antibody that is adopted in sandwich immunoassays all adopts whole molecule, this under many circumstances, it is non-specific to increase system during as capture antibody and detection antibody homology, reduce signal to noise ratio (S/N ratio), and when the detection of biological sample, increased non-specific jammer meeting, cause the false negative or the false positive of testing result.
Summary of the invention
The present invention is based on the existing defective of above growth requirement and protein-chip, proposed a kind of immune microarray protein biochip technology based on antibody library, this technology comprises design, preparation and the detection technique thereof of protein-chip.Thereby make this protein-chip really become high flux, high efficiency bio-analysis system.
Protein-chip of the present invention, be that antibody or the antibody fragment that utilizes imprint lithography will take from antibody library according to the scheme of design in advance is printed onto on the chip substrate that has aldehyde radical, it is covalently bound to make free amine group on antibody or the antibody fragment and on-chip aldehyde radical form amido link, thereby antibody or its antibody fragment is fixed on the chip substrate of activation; Described antibody is as form on chip the horizontal expansion of capture probe with dot matrix, and the probe dot matrix of accepting all kinds of testing sample then extends longitudinally.
The preparation method of protein-chip of the present invention is as follows:
(1) from the antibody library of specific protein group, chooses antibody;
(2) method of cutting with enzyme prepares antibody fragment;
(3) antibody or antibody fragment are printed onto on the activated chip substrate with the scheme of imprint lithography according to design in advance, make free amine group and on-chip aldehyde radical formation amido link on antibody or the antibody fragment covalently bound;
(4) washing;
(5) remaining reactive group on the sealing chip substrate;
(6) drying.
The method of said chip substrate activation is conventional slide Silicane Method.The method that is used for immune microarray offset printing is that the general point sample instrument of use DNA chip carries out contact sample preparation or spray sample.
Use the detection method that the above-mentioned protein-chip that makes carries out proteomics research and can be immune micro-array chip sandwich assay method, promptly by using the pairing monoclonal antibody to realize sandwich assay to target protein, it is as follows that it detects step:
(1) sample that will need to detect joins on the immune microarray protein-chip.
(2) treat that target protein in the sample and chip antibody or its segment realize immune response and to molecular balance, wherein temperature of reaction: 4～25 ℃; Relative humidity: 70～85%
(3) washing;
(4) add and can detect antibody with the monoclonal or the polyclone of chip capture antibody pairing, formation can directly be carried first semiochemicals of signal, makes two pairing antibody and target protein form double sandwich complex;
(5) washing;
(6) add the secondary signal material
(7) washing;
(8) carrying out imaging after the drying detects.
End in the said chip immune detection also can be introduced affinity element-biotin system, i.e. connector and the plain amplification that realizes detection signal of fluorescein-labeled affinity by using secondary signal material and biotin.The introducing method of this affinity element-biotin system is:
(1) on the protein-chip that forms double sandwich complex, adds biotin and connect the secondary signal material, in temperature of reaction: 4～25 ℃; Relative humidity: under the environment 70～85%), the reaction back forms triple compounds.
(2) washing;
(3) add the affinity element that fluorescein or other luminescent substances connect, and make it to be connected to form the quadruple compound with the chip compound;
(4) washing;
(5) carrying out fluorescence or luminous signal detects.
Above-mentioned secondary signal material can be anti-kind immunoglobulin G (IgG) antibody or Protein A etc.; The method that imaging detects is confocal scanning visualization method or charge-coupled detector(CCD) (CCD) imaging method.
In the above-mentioned immune micro-array chip, the yardstick of each little point is at 100～200 μ m, can prepare the immune microarray more than 2500 in every square centimeter of chip substrate area; So utilize this detection architecture to be expected in the chip range of 2 * 3cm to realize to contain the analyzing and testing of the protein groups of up to ten thousand kinds of components.Thereby realized high throughput testing.
The present invention utilizes the target protein capture probe of antibody as protein-chip, the capture probe of protein-chip is planned as a whole to select and global optimization, comprise necessary molecular modification, for signal system, then introduce the detector probe of non-direct mark, use the universal signal material simultaneously, make entire chip only carry out proteome analysis with a kind of signal tracer, can when realizing that microarray " high flux " detects, also can satisfy the specificity requirement of Protein Detection.Utilize antibody fragment to can further improve the specificity of protein-chip in subsequent reactions, improve signal to noise ratio (S/N ratio), and help introducing universal marker and molecule amplification system as capture probe.The chip detecting system of introducing the signal amplification system of affinity element-biotin system can be increased to detection sensitivity the level of ultramicro-determination.
Description of drawings
Fig. 1 is the immune microarray protein-chip structural representation based on antibody library;
Fig. 2 is a specificity identification experiment imaging results of the present invention;
Fig. 3 is used for the imaging results that the oncoprotein sandwich assay detects for protein-chip of the present invention;
Fig. 4 introduces the imaging results that affinity element-biotin system detects for protein-chip of the present invention is used for oncoprotein.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
As shown in Figure 1, for the present invention is based on the immune microarray protein-chip structural design synoptic diagram of antibody library.All microarray antibody capture probes are all taken from antibody library on its chips, and can be further with the resulting active part of antibody enzymolysis as capture probe.Its design proposal is two-way extension type: antibody or its segment as capture probe increase along laterally pursuing row on chip, until reaching the capacity that chip substrate is allowed; The quantity of testing sample can longitudinally increase line by line, until reaching the capacity that chip substrate allows.
M is the microarray position probe among the figure; A-G reaches and respectively classifies the probe kind thereafter as; The concentration gradient of the various tested target proteins of each behavior of 1-6 (catching coupling with probe) detects band; N behavior target protein detects negative quality control band; P behavior target protein detects the positive quality control band; S1-S4 and each behavior sample detection band later on thereof.Every group of probe (4 identical little points) finally provides an average detection information data by acquisition target albumen.
As shown in Figure 2, be specificity identification experiment imaging results of the present invention.Among the figure:
M classifies antibody fragment microarray azimuth mark as; A classifies the AFP antibody fragment as; B classifies the CEA antibody fragment as; C classifies β-HCG antibody fragment as; D classifies the CA125 antibody fragment as; E classifies the CA19-9 antibody fragment as; F classifies the CA15-3 antibody fragment as.Per four little points obtain average optical data;
S1, S2 and S3 are three kinds of oncoprotein mixed solution sample: S1 (AFP, CEA, CA19-9); S2 (CEA, β-HCG, CA125); S3 (CEA, CA125, CA15-3).
Can draw from imaging results, in detected sample, if contain certain oncoprotein, then it is promptly caught by antibody or antibody fragment and finally provides positive findings, otherwise provide negative findings, thereby reflect that the immune microarray of being made up of different capture probe has high degree of specificity, i.e. selective binding, no cross reaction.
[embodiment 1]-antibody carries out the oncoprotein group with immune microarray protein-chip sandwich assay detection architecture and detects as capture probe, and the step of this chip preparation method and detection oncoprotein group thereof is as follows:
(1) from oncoprotein group antibody library, choose 6 kinds of protein marker-alpha-fetoproteins, carcinomebryonic antigen, human chorionic gonadotrophin, sugar antigen 125 (CA125), sugar antigen 19-9 (CA19-9) and sugar antigen 15-3 (CA15-3)-monoclonal antibody as the target protein capture probe;
(2) these three kinds of antibody are dissolved in respectively in the curing damping fluid, are made into immune microarray with the contact point sample instrument on the chip substrate of aldehyde radicalization, the curing reaction duration is 4-16 hour, 4 ℃, and 75% relative humidity;
(3) wash chip 3 times with cleansing solution, each 1 minute;
(4) chip is immersed in the confining liquid 30-60 minute, room temperature, the chip surface position of sealing all remaining aldehyde groups and having the non-specific adsorption performance;
(5) on chip, add the reactant liquor that contains alpha-fetoprotein, carcinomebryonic antigen, human chorionic gonadotrophin, sugar antigen 125 (CA125), sugar antigen 19-9 (CA19-9) and sugar antigen 15-3 oncoprotein marks such as (CA15-3), under 75% relative humidity, room temperature reaction 3 hours;
(6) wash chip 3 times with cleansing solution, each 1 minute;
(7) the rabbit source polyclonal antibody mixed reaction solution of the above-mentioned oncoprotein of adding, under 75% relative humidity, room temperature reaction 1 hour;
(8) wash chip 3 times with cleansing solution, each 1 minute;
(9) add the fluorescein-labeled goat anti-rabbit igg antibody solution of different sulphur hydracid, under 75% relative humidity, room temperature reaction 1 hour;
(10) wash chip 3 times with cleansing solution, each 1 minute;
(11) carrying out the CCD imaging after the drying detects.
Detect imaging results as shown in Figure 3, wherein:
1.M row and M classify antibody microarray azimuth mark as; The negative Quality Control of N behavior oncoprotein; P behavior oncoprotein positive quality control.
2.A classify AFP antibody as; B classifies CEA antibody as; C classifies β-HCG antibody as; D classifies CA125 antibody as; E classifies CA19-9 antibody as; F classifies CA15-3 antibody as.Per four little points obtain average optical data.
3. detected oncoprotein concentration-AFP:20 μ g/L in the row 1, CEA:20 μ g/L, β-HCG:20 μ g/L, CA125:40KU/L, CA19-9:40KU/L, CA15-3:40KU/L; Detected oncoprotein concentration-AFP:40 μ g/L in the row 2, CEA:40 μ g/L, β-HCG:40 μ g/L, CA125:80KU/L, CA19-9:80KU/L, CA15-3:80KU/L; Detected oncoprotein concentration in the row 3--AFP:80 μ g/L, CEA:80 μ g/L, β-HCG:80 μ g/L, CA125:160KU/L, CA19-9:160KU/L, CA15-3:160KU/L; Detected oncoprotein concentration-AFP:160 μ g/L in the row 4, CEA:160 μ g/L, β-HCG:160 μ g/L, CA125:320KU/L, CA19-9:320KU/L, CA15-3:320KU/L; Detected oncoprotein concentration-AFP:320 μ g/L in the row 5, CEA:320 μ g/L, β-HCG:320 μ g/L, CA125:640KU/L, CA19-9:640KU/L, CA15-3:640KU/L; Detected oncoprotein concentration-AFP:640 μ g/L in the row 6, CEA:640 μ g/L, β-HCG:640 μ tg/L, CA125:1280KU/L, CA19-9:1280KU/L, CA15-3:1280KU/L.
4. can draw from imaging results, 6 kinds of oncoproteins can be detected simultaneously, and the oncoprotein of every kind of variable concentrations finally provides different optical density value (optical density forms graded with the concentration gradient variation).
[embodiment 2]-antibody is as capture probe, and the immune microarray protein-chip detection architecture of introducing affinity element-biotin system carries out the oncoprotein group and detect, this chip preparation method and to detect the step of oncoprotein group as follows:
Wherein step (1) (2) (3) (4) (5) (6) (7) (8) is with embodiment 1;
(9) add biotin labeled goat anti-rabbit igg antibody solution, under 75% relative humidity, room temperature reaction 1 hour;
(10) wash chip 3 times with cleansing solution, each 1 minute;
(11) add the fluorescein-labeled affinity cellulose solution of different sulphur hydracid, under 75% relative humidity, room temperature reaction 1 hour;
(12) wash chip 3 times with cleansing solution, each 1 minute;
(13) carrying out the CCD imaging after the drying detects.
The antibody fragment microarray of using this method is used for imaging results that oncoprotein detects as shown in Figure 4,
Wherein:
1.M row and M classify antibody microarray azimuth mark as; The negative Quality Control of N behavior oncoprotein; P behavior oncoprotein positive quality control.
2.A classify the AFP antibody fragment as; B classifies the CEA antibody fragment as; C classifies β-HCG antibody fragment as; D classifies the CA125 antibody fragment as; E classifies the CA19-9 antibody fragment as; F classifies the CA15-3 antibody fragment as.Per four little points obtain average optical data.
3. detected oncoprotein concentration in the row 1---AFP:20 μ g/L, CEA:20 μ g/L, β-HCG:20 μ g/L, CA125:40KU/L, CA19-9:40KU/L, CA15-3:40KU/L; Detected oncoprotein concentration-AFP:40 μ g/L in the row 2, CEA:40 μ g/L, β-HCG:40 μ g/L, CA125:80KU/L, CA19-9:80KU/L, CA15-3:80KU/L; Detected oncoprotein concentration in the row 3---AFP:80 μ g/L, CEA:80 μ g/L, β-HCG:80 μ g/L, CA125:160KU/L, CA19-9:160KU/L, CA15-3:160KU/L; Detected oncoprotein concentration in the row 4---AFP:160 μ g/L, CEA:160 μ g/L, β-HCG:160 μ g/L, CA125:320KU/L, CA19-9:320KU/L, CA15-3:320KU/L; Detected oncoprotein concentration in the row 5---AFP:320 μ g/L, CEA:320 μ g/L, β-HCG:320 μ g/L, CA125:640KU/L, CA19-9:640KU/L, CA15-3:640KU/L; Detected oncoprotein concentration in the row 6---AFP:640 μ g/L, CEA:640 μ g/L, β-HCG:640 μ g/L, CA125:1280KU/L, CA19-9:1280KU/L, CA15-3:1280KU/L.
4. can draw from imaging results, 6 kinds of oncoproteins can be detected simultaneously, and the oncoprotein of every kind of variable concentrations finally provides different optical density value, and promptly optical density changes with concentration gradient and forms graded; Detection signal is stronger, can provide more excellent signal data.
[embodiment 3]-antibody fragment is not introduced the immune microarray protein-chip detection architecture of affinity element-biotin system and is carried out the detection of oncoprotein group as capture probe, and the step of this chip preparation method and detection oncoprotein group thereof is as follows:
(1) from oncoprotein group antibody library, choose 6 kinds of mark-alpha-fetoproteins, carcinomebryonic antigen, human chorionic gonadotrophin, sugar antigen 125 (CA125), sugar antigen 19-9 (CA19-9) and sugar antigen 15-3 (CA15-3)-monoclonal antibody, carry out according to reported method with pepsin that enzyme is cut and separation and purification obtains antibody fragment F (ab')
2, as capture probe;
(2) with these three kinds of F (ab ')
2Antibody fragment is dissolved in respectively and solidifies in the damping fluid, is made into immune microarray with the contact point sample instrument on the chip substrate of aldehyde radicalization, and the curing reaction duration is 4-16 hour, and 4 ℃, 75% relative humidity;
(3) cleansing solution washing chip is 3 times, each 1 minute;
(4) chip is immersed in the confining liquid 30-60 minute, room temperature, the chip surface position of sealing all remaining aldehyde groups and having the non-specific adsorption performance;
(5) on chip, add the reactant liquor that contains alpha-fetoprotein, carcinomebryonic antigen, human chorionic gonadotrophin, sugar antigen 125 (CA125), sugar antigen 19-9 (CA19-9) and sugar antigen 15-3 oncoprotein marks such as (CA15-3), under 75% relative humidity, room temperature reaction 3 hours;
(6) wash chip 3 times with cleansing solution, each 1 minute;
(7) add the monoclonal antibody mixed reaction solution that contains conduct first semiochemicals that matches with capture probe, under 70% relative humidity, room temperature reaction 1 hour;
(8) wash chip 3 times with cleansing solution, each 1 minute;
(9) add the special sheep anti-mouse igg antibody solution in different sulphur hydracid fluorescein-labeled Fc district, under 70% relative humidity, room temperature reaction 1 hour;
(10) wash chip 3 times with cleansing solution, each 1 minute;
(11) carrying out the CCD imaging after the drying detects.
Testing result shows: 6 kinds of oncoproteins can be detected simultaneously, and the oncoprotein of every kind of variable concentrations finally provides different optical density value, and promptly optical density changes with concentration gradient and forms graded; The result presents better signal to noise ratio (S/N ratio).
[embodiment 4]-antibody fragment is as capture probe, and the immune microarray protein-chip detection architecture of introducing affinity element-biotin system carries out the oncoprotein group and detect, this chip preparation method and to detect the step of oncoprotein group as follows:
Step (1) (2) (3) (4) (5) (6) (7) (8) is with embodiment 3;
(9) add the special sheep anti-mouse igg antibody solution in biotin labeled Fc district, under 75% relative humidity, room temperature reaction 1 hour;
(10) cleansing solution washing chip is 3 times, each 1 minute;
(11) add the fluorescein-labeled affinity cellulose solution of different sulphur hydracid, under 75% relative humidity, room temperature reaction 1 hour;
(12) wash chip 3 times with cleansing solution, each 1 minute;
(13) carrying out the CCD imaging after the drying detects.
Can draw from imaging results, 6 kinds of oncoproteins can be detected simultaneously, and the oncoprotein of every kind of variable concentrations finally provides different optical density value, and promptly optical density changes with concentration gradient and forms graded; The result presents better signal to noise ratio (S/N ratio); Detection signal is stronger, can provide more excellent signal data.
[embodiment 5]-antibody fragment is as capture probe, with Protein A is the secondary signal material, and the immune microarray protein-chip detection architecture of introducing affinity element-biotin system carries out the oncoprotein group and detects, this chip preparation method and to detect the step of oncoprotein group as follows:
Step (1) (2) (3) (4) (5) (6) (7) (8) is with embodiment 3;
(14) add biotin labeled Protein A solution, under 85% relative humidity, room temperature reaction 1 hour;
(15) cleansing solution washing chip is 3 times, each 1 minute;
(16) add the fluorescein-labeled affinity cellulose solution of different sulphur hydracid, under 85% relative humidity, room temperature reaction 1 hour;
(17) wash chip 3 times with cleansing solution, each 1 minute;
(18) carrying out the CCD imaging after the drying detects.
Can draw from imaging results, 6 kinds of oncoproteins can be detected simultaneously, and the oncoprotein of every kind of variable concentrations finally provides different optical density value, and promptly optical density changes with concentration gradient and forms graded; The result presents good signal-to-noise; Detection signal is strong, can provide good signal data.
Above-mentioned capture probe consolidation liquid prescription: 0.5M carbonic acid buffer and substitute thereof, pH 9.1,0.15MNaCl, 40% glycerine, 0.1%NaN
3
Chip cleansing solution prescription: 0.05M phosphate buffer and other substitutes, pH 7.4,0.15M NaCl, 0.2%Tween, 0.1%NaN
3
Chip confining liquid prescription: 0.5M carbonic acid buffer and other substitutes, pH 9.1,0.15M NaCl, 2%BSA, 0.1%NaN
3
Chip reactant liquor prescription: 0.05M phosphate buffer and other substitutes, pH 7.4,0.15M NaCl, 0.5%BSA, 0.1%NaN
3
Can expand the detection method of six kinds of oncoproteins described in the present embodiment and be used for detect suppressing all tumor-marker albumen, more than at least 20 kinds, and and then realize evaluation huge oncoprotein group.
The test findings of the foregoing description has all reached design result, has realized highly sensitive, high special and high-throughout microminiaturized immune detection, with this detection system and self-reacting device logotype, can be applicable to clinical detection and laboratory study.The a plurality of disease markers of synchronous detection will help the clinician disease will be carried out multianalysis; By the healthier and protein groups collection of illustrative plates disease cell, the researchist can better understand and transmission of analysis of cells signal and metabolic pathway, for medicine and pharmacology and diagnostics provide more bases for estimation.
Claims (9)
1. immune microarray protein-chip, it is characterized in that this chip be utilize imprint lithography according in advance the design scheme will take from antibody library antibody or and then the preparation antibody fragment be printed onto on the chip substrate that has aldehyde radical, it is covalently bound to make free amine group on antibody or the antibody fragment and on-chip aldehyde radical form amido link, thereby antibody or its antibody fragment is fixed on the chip substrate of activation; Described antibody is as form on chip the horizontal expansion of capture probe with dot matrix, and the probe dot matrix of accepting all kinds of testing sample then extends longitudinally.
2. the preparation method of protein-chip according to claim 1 is characterized in that:
(1) from the antibody library of specific protein group, chooses antibody;
(2) method of cutting with enzyme prepares antibody fragment;
(3) antibody or antibody fragment are printed onto on the activated chip substrate that has an aldehyde radical according to the scheme of design in advance with imprint lithography, make the free amine group on antibody or the antibody fragment covalently bound with on-chip aldehyde radical formation amido link;
(4) washing;
(5) remaining reactive group on the sealing chip substrate;
(6) drying.
3. preparation method according to claim 2 is characterized in that the method for described chip substrate activation is the slide Silicane Method, and immune microarray offset printing is to adopt general point sample instrument to carry out contact sample preparation or spray sample.
4. use the detection method that protein-chip as claimed in claim 1 or 2 carries out proteome analysis, it is characterized in that the steps include: by using the pairing monoclonal antibody that target protein is carried out sandwich assay
(1) the particular detection sample is joined on the immune microarray protein-chip;
(2) treat that target protein in the sample and chip antibody or its segment are 4～25 ℃ in temperature of reaction, relative humidity is to carry out immune response under 70～85% the condition and to molecular balance;
(3) washing;
(4) add as first semiochemicals can with the detection antibody of chip capture antibody pairing, make two pairing antibody and target protein form double sandwich complex;
(5) washing;
(6) the secondary signal material that is connected with fluorescein or other luminescent substances of adding, and make itself and formed double sandwich complex realize being connected to form triple compounds;
(7) washing;
(8) carrying out imaging after the drying detects.
5. detection method according to claim 4 is characterized in that described secondary signal material is anti-kind immunoglobulin g antibody or Protein A.
6. detection method according to claim 4 is characterized in that the method that described imaging detects is confocal scanning visualization method or charge-coupled detector(CCD) imaging method.
7. detection method according to claim 4, it is characterized in that introducing affinity element-biotin system at the end of said chip immune detection, by the connector and the plain amplification that realizes detection signal of fluorescein-labeled affinity of using secondary signal material and biotin, the introducing method of this affinity element-biotin system is:
(1) on the protein-chip that forms double sandwich complex, adds secondary signal material that biotin connects in temperature of reaction: 4～25 ℃; Relative humidity: react under 70～85% the condition, form triple compounds;
(2) washing;
(3) add the affinity element that fluorescein or other luminescent substances connect, and make it to be connected to form the quadruple compound with the triple compounds of chip;
(4) washing;
(5) carrying out fluorescence or luminous signal detects.
8. detection method according to claim 7 is characterized in that described secondary signal material is anti-kind immunoglobulin g antibody or Protein A.
9. detection method according to claim 7 is characterized in that the method that aforementioned imaging detects is confocal scanning visualization method or charge-coupled detector(CCD) imaging method.
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