CN104655851B - A kind of simultaneous quantitative detects the antibody chip kit of multiple receptors - Google Patents
A kind of simultaneous quantitative detects the antibody chip kit of multiple receptors Download PDFInfo
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- CN104655851B CN104655851B CN201310593767.5A CN201310593767A CN104655851B CN 104655851 B CN104655851 B CN 104655851B CN 201310593767 A CN201310593767 A CN 201310593767A CN 104655851 B CN104655851 B CN 104655851B
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Abstract
The invention discloses the antibody chip kits that a kind of simultaneous quantitative detects multiple receptors.The kit includes：Antibody chip, selection standard Tissue slides are coated with as topical carrier, the normal structure slide using active ammonia group, so that a variety of antigen-specific antibodies is firmly adsorbed on surface of glass slide by untouchable point sample instrument, are formed 16 microarrays；It is used in combination and matches the removable holes 2x8 plastic frame and latex closing frame separates 16 microarrays in non-interfering cell.Each capture antibody has four repetition point samples in each Antibody microarray；Receptor standard items mixture；The detection antibody mixture of biotin labeling；The Streptavidin of Cy3 fluorochrome labels.The antibody chip kit of the present invention can simultaneous quantitative detect multinomial receptor, the dynamic detection range of receptor is wider, the duplicate data for being four times in ELISA can be obtained from single sample experiments, have many advantages, such as highly sensitive, high-throughput, few, cheap, the easy popularization of sample expense.
Description
Technical field
The present invention relates to the antibody chips that field of biomedicine technology more particularly to a kind of simultaneous quantitative detect multiple receptors
Kit.
Technical background
Receptor（receptor）Be it is a kind of can be with extracellular single-minded signaling molecule（Ligand）In conjunction with the egg for causing cell effect
White matter.
Receptor is divided into cell surface receptor and intracellular receptor.Molecular compositing occurs for binding of receptor and ligand, from
And cause cell effect, such as signal transduction, iuntercellular bonding, cell endocytosis cell processes between mediated cell.Receptor is in cell
On film or cell interior energy identification bioactive molecule and in combination, identification and the signal received correctly are amplified and passed
It is delivered to cell interior, to activate or start series of biochemical reactions, finally results in the specific biology effect of the semiochemicals
It answers.There are two functions for usual receptor tool：First, semiochemicals -- the ligand that identification is special, the performance of identification is that the two combines.
Ligand refers to some such semiochemicals, has no other functions in itself other than being combined with receptor, it cannot participate in metabolism and generate
The characteristics of useful products also do not induce any cell activity directly, and there are no enzymes, its unique function is exactly to notify cell in environment
In there are a kind of distinctive signal or stimulus；Second is that identification and the signal received are accurately amplified and are transmitted to cell
Inside starts a series of intracellular biochemical reactions, finally results in specific cell effect, promotes proliferation and the differentiation of target cell, increases
Strong anti-infective and tumor killing cell effect, promotes or inhibits the synthesis of cell factor, promotes inflammatory process, influences cell metabolism
Deng.
Receptor research has very important theoretical and Practical significance, it illustrates hormone, mediator, medicine from molecular level
Object, the mechanism of action of antibody and physiology and pathologic process, it has also become one of scientific and technical forward position.
It is currently used to be included mainly by body detecting method：Enzyme linked immunosorbent assay（ELISA）, radiommunoassay
（Radioimmunoassay, RIA）, Western blot（western blot）, flow cytometer（Flow-Cytometry）Deng.
Wherein, enzyme linked immunosorbent assay is most common method, has the advantages that high sensitivity, specificity is preferable, easy to operate.But it is primary
Experiment can only detect single index, and flux is low, of high cost, and in being detected with the receptor of multi objective characteristic, there are bright for this method
Aobvious deficiency；Radiommunoassay（RIA）It is due to radioactive pollution, seldom using now although high sensitivity；Exempt from
Epidemic disease blotting can measure bulk of molecule, and without non-specific reaction, but cumbersome, and sensitivity is low, and can only detect single
Index is not suitable for the detection for applying to receptor；Flow cytometer can detect the level of receptor on a cellular level, but there are low
The shortcomings of sensitive, small throughput, high cost.
In view of this, it is necessary to a kind of novel a variety of receptor immue quantitative detection reagent boxes are developed, to overcome in the prior art
Urgent problem to be solved realizes high throughput, high sensitivity, high specific and the low cost detection of a variety of receptors.In the present invention,
We select the normal structure slide of no space limitation as carrier, can make the number that receptor is a little detected on same microarray
It can not be limited by space.Since slide has low-down autofluorescence, and fluorescence signal does not have diffusivity, different
Fluorescence signal between point will not be interfering with each other, so can once detect tens receptors simultaneously.The present invention uses antibody sandwich
Method principle carries out multiple ELISA reactions in surface of glass slide, can only once not detect up to tens receptors, and detected
ELISA detection sensitivity of the receptor sensitivity up to single-factor.
Invention content
In view of the deficiencies of the prior art, the purpose of the present invention is to provide the antibody that a kind of simultaneous quantitative detects multiple receptors
Chip agent box, the receptor that high-throughput, highly sensitive, high specific and low cost can be carried out on normal structure slide are quantitatively examined
It surveys.
In order to solve the above-mentioned technical problem, a kind of antibody chip reagent quantitatively detecting multiple receptors of the present invention
Box, including：Antibody chip, including normal structure slide and the specific antibody of fixed 40 kinds of receptors, two in surface of glass slide
Kind positive control；Antibody chip described in four is put in the enterprising rower of chip fixed frame being consistent with conventional 96 hole elisa Plates sizes
The automation mechanized operation of quasi- ELISA systems；Receptor standard items mixture is to be blended in 40 kinds of receptor standard items according to a certain amount
Freeze-drying mixture together；The receptor of biotin labeling detects antibody mixture；It is affine with the strepto- of fluorescent dye Cy3 labels
Element.
The further feature of antibody chip kit according to the present invention, the normal structure slide are with activity
Amino coating processing.
The further feature of antibody chip kit according to the present invention, the normal structure slide are connect with non-
Property point sample instrument is touched by a variety of antigen-specific antibodies o'clock into 2 × 8 identical Antibody microarrays, is used in combination and matches removable 2
× 8 hole frames are segmented in microarray in 16 non-interfering cells；It is clamped by the frame of both sides between slide and frame
It is buckle-shaped at antibody chip.
The further feature of antibody chip kit according to the present invention, the frame are made of three parts：On
The frame folder of layer plastic frame, lower layer's latex closing frame and both sides；Latex closes frame by high transparency double faced adhesive tape in surface point
Have on the slide of microarray, the another side of latex closing frame sticks plastic frame, then is pressed from both sides with frame and clamp their both sides, is formed
The reactor of non-cross pollution between hole, as shown in Figure 1.
The further feature of antibody chip kit according to the present invention, the antibody chip are distinguished with concentration
For 40 kinds of antibody spot samples of 200ug/ml；Each antibody is slow with the specific PBS containing 0.01-10g/100ml bovine albumins
Fliud flushing is prepared；Each antibody has four repetition point samples in each microarray, so that obtaining this receptor from single microarray
It is four times in the experimental data of ELISA.The point sample is to complete printing operation using full-automatic point sample instrument, keeps each specific antibody only
It is vertical to be arranged in the slide.
The further feature of antibody chip kit according to the present invention, the specific antibody are selected from for such as
The antibody of lower receptor：Tumor Necrosis Factor Receptors 4-1BB, leukocyte activation adhesion factor, stimulation molecule B7-1, B cell are ripe
The factor, myeloid cell specificity are rich in leucine glycoprotein, Tumor Necrosis Factor Receptors CD30；Tumor necrosis factor ligand -5,
Carcinomebryonic antigen related cell adhesion molecule 1, death receptor 6, tyrosine kinase, glycoprotein, receptor type tyrosine kinase
ErbB3, E-Selectin, A member of the TNF receptor family 6, people's FMS-like tyrosine kinase 3 ligand, glucocorticoid
Tumor Necrosis Factor Receptors family GAP-associated protein GAP, herpesvirus entry mediator, intercellular adhesion molecule 3, the il-1 of induction
Receptor 4, interleukin-1 receptor 1, Interleukin 2 Receptor γ, interleukin-10 receptor β, interleukin-17 receptor, interleukin-21 by
Body, lysosomal membrane protein53 2, apolipoprotein -2, L-selectin, lymphatic endothelial hyaluronic acid receptor 1, ajor histocompatibility are multiple
It is small to close object I class chain GAP-associated protein GAPs A, major histocompatibility complex I class chain GAP-associated protein GAPs B, 1 receptor 1 of Newland Green albumen, blood
Plate derived growth factor receptor, platelet endothelial cell adhesion molecule 1, mankind's Advanced Glycation Endproducts, cellular immunity ball
Albumen mucin 1, TRAIL mRNA, Trappin-2 albumen, urokinase type plasminogen activation
Agent receptor, vascular cell adhesion molecule, X sex-kinks are ectodermal dysplasia-A2 receptors.Above-mentioned each strain specific antibodies point respectively
Sample forms several independent recognition sites in the slide.One kind of single concentration is respectively fixed on each independent recognition site
Specific antibody.
The further feature of antibody chip kit according to the present invention, the point sample include：By 100-1000pl
The PBS buffer solution of specific antibody protein（Contain 0.01-10g/100ml bovine albumins）Point sample is on the slide, by point
The good slide of sample is put in be stood overnight under room temperature, is saved backup in 2-8 DEG C.
Probe of the present invention using different specific antibodies as quantitatively detection receptor, the property between these antibody are poor
Different big, stability is also poor, and there is the antibody in order to ensure preparing chip higher purity and intact bioactivity, the present invention to adopt
With suitable sampling liquid, the i.e. PBS buffer solution containing 0.01-10g/100ml bovine albumins, carry out lytic antibody.Experiment shows
The sampling liquid can effectively prevent antibody to be denaturalized, to maintain its bioactivity.
Existing detection chip, point sample post-processing generally include hydration, UV crosslinking, elution, closing.Due to this
Invention uses excellent sampling liquid so that the step of prepared by antibody chip has waited until great simplification, without in the prior art
The operating procedure of the active ingredient on point sample rear enclosed slide generally used.
To ensure to develop into high quality, high-throughput antibody chip, in one embodiment of the invention, using the U.S. primary
It is happy（Bio-Rad）Company or platinum Ai Ermo（PerkinElmer）The full automatic point sampling instrument of company's production.Each specific antibody
Dot matrix is arranged in slide, and in specific operating process, the arrangement of each specific antibody can be needed according to experimental design into
Row adjustment controls full-automatic point sample instrument, prepares required intermediate products according to different antibody chip arrangement arrays.
To ensure that antibody chip is easy to operate, sample room not cross contamination, the reaction system that the present invention uses is that had by point
The Tissue slides of antibody chip and contain the removable frame in 16 holes composition.The frame is made of three parts:The upper layer holes 2x8 plastics
The frame folder of frame, lower layer's latex closing frame and both sides.The holes 2x8 plastic frame and latex closing frame will be put on Tissue slides
16 microarrays separate in non-interfering cell, and flexible latex closing frame is located at plastic frame and tissue glass
Among piece so that plastic frame, latex closing frame and slide can be closely affixed by both sides frame folder, so that in each cell
Sample will not interpenetrate and cause cross contamination.The pitch of holes of the size of the removable frame in 16 hole and conventional 96 orifice plates
It is consistent, can be adapted for the automation mechanized operation of standard ELISA system.The reaction system unlike conventional ELISA is in glass
The multiple ELISA of the multiple samples carried out on piece surface is detected, and different from traditional ELISA detection method, antibody chip processing
After the completion, removable frame is removed, slide is subjected to radium-shine scanner scanning, then by Digital Signal Processing software fluorescence
Signal is converted into digital signal, then the concentration of unknown receptor is calculated by special chip processing software.
It is using the present invention detection receptor antibody chip kit, fluorescent assay signal, can simultaneous quantitative detect
40 receptors, and can realize the parallel detection of multisample multi objective, overcome that prior art operation is cumbersome, Testing index
The single, defects such as sensitivity is low, have that cheap, convenient, sensitive, accurate, high-throughput, sample dosage is few, can be in common lab
The advantages that popularization and scale.
Description of the drawings
Fig. 1 present invention detects the chi frame schematic diagram of receptor antibody chip agent box.
Fig. 2 present invention detects the chip point sample schematic diagram of receptor antibody chip agent box.
The standard curve of each recombinant proteins of Fig. 3.
Specific implementation mode
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It should be understood that this
It is a little that examples are only for illustrating the present invention and not for limiting the scope of the present invention.
Embodiment 1：The screening of optimum antibody chip carrier.
Conventional antibody chip is mostly using nitrocellulose filter as carrier, since nitrocellulose filter is multilayered structure, chip
Washing difficulty it is big so that the background of chip is high, as a result fluctuation is big.Nitrocellulose filter is frangible simultaneously, is not easy to large-scale
Operation, the use for larger scale clinical sample be not also universal.Different producers consolidates nitrocellulose filter with different methods
It is scheduled on the surface of slide, wherein there are the SS slides of Whatman, it secures 16 containing nitrocellulose filter on a slide
Cell, in addition Gentel be coated on surface of glass slide with finished cellulose nitrate cellulosic material and produce PATH slides.Except this with
Outside, in order to make antibody be fixed on surface of glass slide, we have screened the carrier of distinct methods activation.With full-automatic point sample instrument Cy3
Then Streptavidin point with Cy5 labels is read on the surface of slide with radium-shine scanner.Experimental result such as following table institute
Show.Nitrocellulose membrane DNA chip has strong signal in the channels Cy3, but their background is also high.With the coated slide of active amino
There is highest signal to background ratio at the channels Cy3.
| Carrier and wavelength of fluorescence | Signal strength | Background signal | Signal to background ratio (S/N) |
| Nitrocellulose filter Cy5 | 12345 | 9177 | 1 |
| Nitrocellulose filter Cy3 | 57206 | 46155 | 1 |
| GentelPATHCy5 | 2086 | 837 | 2 |
| GentelPATHCy3 | 7415 | 959 | 8 |
| WhatmanSSCy5 | 13791 | 5101 | 3 |
| WhatmanSSCy3 | 65328 | 12963 | 5 |
| Active aldehyde radical slide Cy5 | 8063 | 612 | 13 |
| Active aldehyde radical slide Cy3 | 22507 | 722 | 31 |
| Activity hydroxy slide Cy5 | 5123 | 359 | 14 |
| Activity hydroxy slide Cy3 | 58091 | 797 | 73 |
| Active amino slide Cy5 | 8193 | 323 | 25 |
| Active amino slide Cy3 | 43129 | 403 | 107 |
| Blank tissue slide Cy5 | 3869 | 561 | 7 |
| Blank tissue slide Cy3 | 23940 | 1648 | 15 |
Embodiment 2：A kind of simultaneous quantitative detects the preparation of the antibody chip kit of multiple receptors.
Whether there is corresponding receptor in sample to detect, prepare be fixed be directed to following protein specificity it is anti-
The slide of body：Tumor Necrosis Factor Receptors 4-1BB, leukocyte activation adhesion factor, stimulation molecule B7-1, Bcell maturation factor,
Myeloid cell specificity is anti-rich in leucine glycoprotein, Tumor Necrosis Factor Receptors CD30, Tumor necrosis factor ligand -5, cancer embryo
Former related cell adhesion molecule 1, death receptor 6, Axl/Ufo growth factor receptors family tyrosine kinase, glycoprotein,
Receptor type tyrosine kinase ErbB3, E-Selectin, A member of the TNF receptor family 6, people's FMS samples tyrosine kinase 3
Tumor Necrosis Factor Receptors family GAP-associated protein GAP, herpesvirus entry mediator, the cell-cell adhesion of ligand, glucocorticoid inducible
Molecule 3, interleukin-1 receptor 4, interleukin-1 receptor 1, Interleukin 2 Receptor γ, interleukin-10 receptor β, interleukin-17 by
Body, interleukin-21 receptor, lysosomal membrane protein53 2, apolipoprotein -2, L-selectin, lymphatic endothelial hyaluronic acid receptor 1, master
Want histocompatibility complex I class chain GAP-associated protein GAPs A, major histocompatibility complex I class chain GAP-associated protein GAPs B, Newland Green
1 receptor β 1 of albumen, platelet derived growth factor receptor β, platelet endothelial cell adhesion molecule 1, mankind's Advanced Glycation produce eventually
Object receptor, cell immunoglobulin mucin 1, TRAIL mRNA, Trappin-2 albumen, urine
Kinases type plasminogen activator receptor, vascular cell adhesion molecule, X sex-kinks are ectodermal dysplasia-A2 receptors 1.
The preparation of antibody：
Using the specific antibody for listed protein in table 1, source, concentration and its protein being directed to of antibody
Title is described in detail in table 1：
The targeted antigen protein title of 1 specific antibody of table, the purposes of antibody, source, concentration information
2, the preparation and preservation of antibody chip
By the PBS buffer solution containing specific antibody of 100-1000pl（Contain 0.01-10g/100ml bovine albumins）With complete
Auto sample applicator point sample is on slide.Specific chip dot matrix schematic diagram is as shown in Fig. 2, the ox IgG of biotin labeling is used as sun
Property control.There are four repeat point in each array for the positive control of 40 kinds of antibody and two kinds of various concentrations.In the present embodiment
Core chip arrays use arrangement mode as shown in Figure 2, but in fact, in other embodiments, chip array can also be with it
Its arrangement mode is combined, it is not limited to as schemed represented form.There are 16 identical chip battle arrays on every slide
Row.The good slide of point sample is put in and is stood overnight under room temperature, is then evacuated in drier 2 hours dry.After drying
Slide loads onto 16 mating hole frames and one slide is divided into 16 non-interfering cells.After adhesive film closed frame,
Whole chip is encapsulated with air-locked pouch and is then saved backup in 2 DEG C to 8 DEG C.
Wherein, in the present embodiment, full-automatic point sample instrument is the product that Bio Rad Laboratories or platinum Ai Ermo companies produce；
Slide is Corning Incorporated's product.Certainly, in the above-mentioned steps of inventive technique scheme, the use of instrument and material not office
It is limited to enumerating for the present embodiment, but the technical problem of the present invention can be solved, and realize that corresponding technique effect is foundation.
3, the preparation of receptor standard items：
Using for the source of listed recombinant protein in table 2, concentration and its protein title that is directed in table 2
It is described in detail：
Table 2：The title of recombinant protein, source and concentration information in receptor standard items
According to a certain amount after using the phosphate buffer containing 0.1% bovine albumin to dilute above each recombinant protein
It mixes, with freeze-drying drying and in -80 DEG C of preservations after packing.In the present embodiment, for doing standard curve use
Each recombinant protein finally using concentration it is as shown in table 3.But in fact, in other embodiments, for doing standard curve
Recombinant protein concentration can select different sections, it is not limited to the embodiment of table 3.
Embodiment 3：The experiment of receptor is detected with the kit quantification of the present invention.
1, slide chip is completely dried
Slide chip is taken out from box, after equilibrium at room temperature 20-30min, packaging bag is opened, opens sealing
Then chip is placed on vacuum desiccator or drying at room temperature 1-2 hours by item.
2, it presses mode shown in Fig. 3 and gradient dilution is carried out to cytokine standards product gradient
2.1, in the sample diluting liquid to the tubule of cytokine standards mixture of 500 μ l of addition, standard items are re-dissolved.
Before opening tubule, first quickly centrifugation, gently upper and lower lash dissolved powders, and it is Std1 to mark this tubule.
2.2,6 clean centrifuge tubes of label are Std2, Std3 to Std7 respectively, and the sample diluting liquid of 200 μ l of addition arrives
In each tubule.
2.3, the Std1 of 100 μ l of extraction, which is added in Std2, is gently mixed, and 100 μ l are then extracted from Std2 and are added to
In Std3, such gradient dilution to Std7.
2.4, in the sample diluting liquid to another new centrifuge tube for extracting 100 μ l, it is labeled as CNTRL, as negative right
According to.
Note：Because the initial concentration of each cell factor is different, after the gradient dilution of Std1 to Std7, each
The series concentration of cell factor is different, and in the present embodiment, the concentration of gradient recombinant protein dilution is as shown in table 3.
Table 3：The concentration of recombinant cytokine standard items after gradient dilution for making standard curve
3, chip operation flow
3.1, the sample diluting liquid of 100 μ l of each Kong Zhongjia is incubated 30 minutes on room temperature shaker, closes quantitative antibody core
Piece.
3.2, the buffer solution in each hole is pumped, in the titer and sample to hole that add 100 μ l, 4 DEG C of mistakes on shaking table
Night is incubated.
Note：The incubation amount of different samples is different：Blood plasma, serum use sample diluting liquid 1 using preceding：1 dilution；Cell conditioned medium
Liquid can use stoste；The amount of 5-50ug is added in cell or tissue lysate after determination of protein concentration.
3.3, it cleans
The standard items or sample in each hole are pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, per hole
1 × washing lotion I of 150 μ l, every time cleaning will drain washing liquid, dilute 20 × washing lotion I with deionized water.
1 × washing lotion I in each hole is pumped, 1 × washing lotion II is added and cleans 2 times, each 5min room temperature shakers concussion, per hole
1 × washing lotion II of 150 μ l, every time cleaning will drain washing liquid, dilute 20 × washing lotion II with deionized water.
The incubation of 3.4 detection antibody mixtures
Centrifugation detection antibody mixture tubule, then be added 1.4ml sample diluting liquid, after mixing again quickly from
The heart.In the detection antibody to each hole for adding 80 μ l, it is incubated 2 hours on room temperature shaker.
2.3.5 cleaning
The detection antibody in each hole is pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, per 150 μ l of hole
1 × washing lotion I, cleaning every time will drain washing liquid, 1 × washing lotion II is then added and cleans 2 times, each 5min room temperature shakers shake
It swings, 1 × washing lotion II per 150 μ l of hole, cleaning every time will drain washing liquid.
2.3.6Cy3- the incubation of Streptavidin
Centrifuge Cy3- Streptavidin tubules, then be added 1.4ml sample diluting liquid, after mixing again quickly from
The heart.In the Cy3- Streptavidins to each hole for adding 80 μ l, encases slide with aluminium-foil paper and be protected from light incubation, be incubated on room temperature shaker
1 hour.
3.7 cleaning
The Cy3- Streptavidins in each hole are pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, often
1 × washing lotion I of 150 μ l of hole, every time cleaning will drain washing liquid.
3.8 fluoroscopic examination
1）Slide frame is dismantled, not touch the one side of slide printing antibody with hand carefully.
2）Slide is placed in glass slide cleaning pipe, 1 × washing lotion I of about 30ml is added, slide can be entirely covered, in room
15min is shaken on warm shaking table, discards 1 × washing lotion I, 1 × washing lotion II of about 30ml is added, 5min is shaken on room temperature shaker.
3）Remove the residual washing lotion of slide.Slide is placed in glass slide cleaning pipe/drying tube, not lid lid,
1000rpm centrifuges 3min.
4）Using laser scanner such as AxonGenePix scanning signals, using Cy3 or green channel（Stimulating frequency=
532nm）.
2.3.9 the data of chip are extracted and carry out data analysis with analysis software
1）The fluorescent value of biochip is read with GenePix softwares.
2）The numerical value selected after reading is that the median of subtracting point ambient background is read（F532Median-
LocalBackground）.It is as shown in Figure 3 come the standard curve for making each recombinant protein with chip software is specifically quantified.
Embodiment 4：The data analysis of quantitative chip agent box detection receptor.
The fluorescence of detection the Plays product and sample of eight unknown samples of chip pair of the present invention is used in certain experiment
Reading is respectively as shown in table 4 and 5.
Table 4：The standard items fluorescence reading (F532Median-LocalBackground) of three times gradient dilution
Table 5：The fluorescence reading (F532Median-LocalBackground) of unknown sample
It is calculated according to corresponding fluorescence values in the concentration of each standard recombinant protein in table 3 and table 4 unknown in table 5
The concentration of each receptor is as shown in table 6 in sample.
Table 6:The concentration (pg/ml) of cell factor in unknown sample
Embodiment 5：The test of experimental system cross reaction.
Cross reaction test between antibody pair is carried out according to following methods.Different chips first with single antigen concentration
For 100ng/ml not synantigen be incubated, after developing a film again detection antibody response corresponding with each antigen most afterwards through Cy3- chains
Mould Avidin is incubated, and is read after chip scanning.Using the capture antibody of each antigen as horizontal axis, with the antigen of addition and corresponding
Detection antibody is the experimental result that the longitudinal axis can obtain table 7.
Cross reaction test result between 7. antibody pair of table
The experimental results showed that each antibody does not have to can specifically identify the detection antigen of oneself with other antigens
Cross reaction.
Embodiment 6：The detection accuracy of experimental system.
In order to which more traditional ELISA and quantitative chip agent box are to the detection accuracy of same receptor, we are with same 4-
The Ag-Ab of 1BB is to being prepared for the ELISA kit of 4-1BB.Under the same conditions with the 4-1BB ELISA kits
The concentration of 4-1BB in eight identical unknown samples is had detected with quantitative chip agent box.The results are shown in Table 8 for detection.
Table 8. tradition 4-1BB ELISA and 4-1BB in eight identical unknown samples of quantitative chip agent box detection
Concentration
| (pg/ml) | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 |
| 4-1BB ELISA | 7.1 | 44.2 | 16.2 | 21.2 | 32.7 | 21.6 | 10.3 | 148.2 |
| Receptor quantifies chip | 7.9 | 43.8 | 17.7 | 25.7 | 32.6 | 23.5 | 8.8 | 129.5 |
The experimental results showed that having with quantitative chip and the individual event ELISA results detected to 4-1BB very strong related
Property (correlation coefficient r=0.9896).The two not only accuracy having the same, but also have similar detection sensitivity
Embodiment 7：The dielectric film filter rate of experimental system.
The appropriate in different sample medias of the quantitative antibody chip is shown by measuring dielectric film filter rate.
10 times of diluted normal human serum H4522 and 2 times of diluted cell supernatants（CM）In be separately added into various concentration it is each by
Body is detected with the quantitative antibody chip, then calculates dielectric film filter rate=(acceptor density-control sample of intervention sample of sample
Acceptor density)/the acceptor density theoretically intervened.Experiment shows the kit of the invention in human serum and cell supernatant
The rate of recovery reach 80-128%.
The rate of recovery of 9. quantitative antibody chip of table in different medium
In conclusion the invention discloses the antibody chip kits that a kind of simultaneous quantitative detects multiple receptors.The reagent
On the one hand box overcomes Testing index of the conventional ELISA in receptor detection single, takes, the shortcomings of effort, consumptive material, while
Overcome the small throughput in existing multiple-factor detection technique, the weakness such as poor repeatability.The system uses normal structure slide conduct
Topical carrier can complete the reaction of multiple sandwich ELISA in surface of glass slide.The specification of antibody chip complies with standard 96 holes simultaneously
The size of ELISA Plate so that high-throughput sample operation is possibly realized.In addition, we also confirm that the receptor detected with the invention
Concentration can reach the detection sensitivity and accuracy of single ELISA.Finally, by the way that the chip, medium returns in different samples
The chip agent box of the true accurate invention of measurement of yield can apply to serum, the different biological sample such as cell supernatant.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than to the scope of the present invention
Limitation, although being explained in detail to the present invention with reference to preferred embodiment, it will be understood by those of ordinary skill in the art that, can
To be modified or replaced equivalently to technical scheme of the present invention, without departing from the spirit and scope of technical solution of the present invention.
Claims (2)
1. a kind of simultaneous quantitative detects the antibody chip kit of multiple receptors, which is characterized in that the kit includes：
Antibody chip, including fixed 40 kinds of receptors with the processed normal structure slide of active amino packet and in surface of glass slide
Specific antibody, two kinds of positive controls；Antibody chip described in four is put in the core being consistent with conventional 96 hole elisa Plates sizes
The automation mechanized operation of standard ELISA system is carried out on piece fixed frame；The normal structure slide is with untouchable point sample instrument
By a variety of antigen-specific antibodies o'clock at 2 × 8 identical Antibody microarrays, removable 2 × 8 hole frame handle that matches is used in combination
Microarray is segmented in 16 non-interfering cells；Antibody core is formed by the frame clamping buckle of both sides between slide and frame
Piece；The antibody chip is with concentration be respectively 200ug/ml 40 kinds of antibody spot samples；Each antibody is with containing 0.01-
The specific PBS buffer solution of 10g/100ml bovine albumins is prepared；Each antibody has four repetition points in each microarray
Sample, so that obtaining the experimental data that this receptor is four times in ELISA from single microarray；
Receptor standard items mixture is the freeze-drying mixture for mixing 40 kinds of receptor standard items according to a certain amount；
The receptor of biotin labeling detects antibody mixture；With
The Streptavidin of fluorescent dye Cy3 labels；
Wherein, the specific antibody is selected from the antibody for following receptor：Tumor Necrosis Factor Receptors 4-1BB, leucocyte
It is bad rich in leucine glycoprotein, tumour to activate adhesion factor, stimulation molecule B7-1, Bcell maturation factor, myeloid cell specificity
Necrosis factor receptor CD30；Tumor necrosis factor ligand -5, carcinomebryonic antigen related cell adhesion molecule 1, death receptor 6, tyrosine
Kinases, glycoprotein, receptor type tyrosine kinase ErbB3, E-Selectin, A member of the TNF receptor family 6,
People's FMS-like tyrosine kinase 3 ligand, the Tumor Necrosis Factor Receptors family GAP-associated protein GAP of glucocorticoid inducible, herpesviral
Invade mediator, intercellular adhesion molecule 3, interleukin-1 receptor 4, interleukin-1 receptor 1, Interleukin 2 Receptor γ, interleukin-10
Receptor β, interleukin-17 receptor, interleukin-21 receptor, lysosomal membrane protein53 2, apolipoprotein -2, L-selectin, in lymphatic vessel
Skin hyaluronic acid receptor 1, major histocompatibility complex I class chain GAP-associated protein GAPs A, major histocompatibility complex I class chains
GAP-associated protein GAP B, 1 receptor 1 of Newland Green albumen, platelet derived growth factor receptor, platelet endothelial cell adhesion molecule 1,
Mankind's Advanced Glycation Endproducts, cell immunoglobulin mucin 1, TRAIL mRNA,
Trappin-2 albumen, urokinase type plasminogen activator receptor, vascular cell adhesion molecule, the development of X sex-kink ectoderms are not
Good-A2 receptors.
2. antibody chip kit according to claim 1, it is characterised in that：The frame is made of three parts：On
The frame folder of layer plastic frame, lower layer's latex closing frame and both sides；Latex closes frame by high transparency double faced adhesive tape in surface point
Have on the slide of microarray, the another side of latex closing frame sticks plastic frame, then is pressed from both sides with frame and clamp their both sides, is formed
The reactor of non-cross pollution between hole.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106918698B (en) * | 2015-12-25 | 2019-11-22 | 广州瑞博奥生物科技有限公司 | A kind of phospho-AB chip agent box detecting human receptor tyrosine kinase enzyme |
| CN105785032A (en) * | 2016-03-23 | 2016-07-20 | 广州瑞博奥生物科技有限公司 | Antibody chip kit capable of simultaneously and quantitatively detecting plurality of immunoglobulin subtypes |
| CN107091925B (en) * | 2017-06-24 | 2020-06-23 | 浙江玉安康瑞生物科技有限公司 | Kit for detecting periodontitis related protein |
| CN107328941A (en) * | 2017-06-24 | 2017-11-07 | 梧州市兴能农业科技有限公司 | It is a kind of to detect the antibody chip of various kinds of cell adhesion factor simultaneously |
| CN107271683A (en) * | 2017-06-24 | 2017-10-20 | 深圳森阳环保材料科技有限公司 | A kind of kit for screening for detecting pathological myopia |
| CN110967336A (en) * | 2018-09-28 | 2020-04-07 | 陈洁 | Periodontal disease detection kit and use method thereof |
| CN112888946A (en) * | 2018-10-09 | 2021-06-01 | 积水医疗株式会社 | Immunoassay method and assay reagent for leucine-rich alpha 2glycoprotein |
| CN110484974A (en) * | 2019-07-19 | 2019-11-22 | 中国农业科学院郑州果树研究所 | A kind of preparation method of grape antibody library |
| CN111458503A (en) * | 2020-03-12 | 2020-07-28 | 江西业力医疗器械有限公司 | Antibody chip for separating and counting blood cells and preparation method thereof |
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| CN1448719A (en) * | 2003-05-13 | 2003-10-15 | 上海晶泰生物技术有限公司 | Novel biological chip |
| CN1627071A (en) * | 2003-12-10 | 2005-06-15 | 深圳益生堂生物企业有限公司 | Method for carrying out fluorescence quantified measurement for multiple biolotical active substances synchronously |
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|---|---|---|---|---|
| CN1448719A (en) * | 2003-05-13 | 2003-10-15 | 上海晶泰生物技术有限公司 | Novel biological chip |
| CN1627071A (en) * | 2003-12-10 | 2005-06-15 | 深圳益生堂生物企业有限公司 | Method for carrying out fluorescence quantified measurement for multiple biolotical active substances synchronously |
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Address after: No.79, Ruihe Road, Science City, Guangzhou hi tech Industrial Development Zone, Guangdong 510000 Patentee after: Reboo (Guangzhou) Biotechnology Co.,Ltd. Address before: 510663 4th floor, area F, Guangzhou International Business Incubator, 3 Juquan Road, Science City, Luogang District, Guangzhou City, Guangdong Province Patentee before: RAYBIOTECH, Inc. |