CN106918706B - A kind of antibody chip kit detecting periodontosis GAP-associated protein GAP - Google Patents
A kind of antibody chip kit detecting periodontosis GAP-associated protein GAP Download PDFInfo
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Abstract
The present invention relates to technical field of biological, and in particular, to a kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP.The antibody chip of the kit is to contain slide that the hydrophilic reagent of alkyl glycosides is handled as solid phase carrier, and the specific antibody mixture of a variety of periodontosis GAP-associated protein GAPs is under 22~24 DEG C, 30~40% damp condition on point sample to solid phase carrier.Kit of the invention is able to detect multiple clinically used periodontosis GAP-associated protein GAPs; the defects such as prior art operation is cumbersome, Testing index is single, sensitivity is low are overcome, have many advantages, such as that cheap, convenient, sensitive, accurate, high-throughput, sample dosage is few, can be in common lab popularization and scale.
Description
Technical field
The present invention relates to technical field of biological, and in particular, to a kind of antibody core for detecting periodontosis GAP-associated protein GAP
Piece kit.
Background technique
Periodontosis refers to the disease occurred in tooth supporting tissue (periodontium), the gingivitis including only involving gingiva tissue
With the periodontitis two major classes for involving deep layer periodontium (parodontium, alveolar bone, cementum).Periodontal disease is common oral cavity disease
Disease is one of the main reason for causing adult's loss of tooth, and endangers the main oral disease of human teeth and whole body health
Disease.The early symptom of periodontosis is not easy to cause attention, and periodontium long-term chronic is caused to infect, and inflammation recurrent exerbation not only damages
The function of chamber masticatory system is suffered from morning sickness, health can be also seriously affected.
Periodontosis is the disease damaged from simple various focusing depths represented to soft group of group and bony tissue, in the worst case
It will lead to falling off for tooth.Therefore the early diagnosis and prevention of periodontitis are most important.It at present include that oral pocket spy is examined, plaque
It can be used as the index of periodontosis symptom with the clinical detection including X-ray imaging diagnosis, but these detections cannot indicate disease
Development.It is therefore desirable to develop a kind of situation of new detection method reflection disease, effectively prevent and monitor disease
Generation.Level in gingival sulcus fluid (GCF) refers to the liquid penetrated by epithelium in ditch and junctional epithelium from gingival connective tissue in gingival sulcus.
Gums healthy person has minute quantity level in gingival sulcus fluid, penetrates into tissue fluid in gingival sulcus.Because in level in gingival sulcus fluid
Usually there is inflammatory cell, other chemical components and tissue fluid are also not exactly the same；The discharge of level in gingival sulcus fluid and the inflammation at the position
Degree is directly proportional.The liquid component of level in gingival sulcus fluid is mainly derived from serum, and other compositions are then respectively from serum, neighbouring periodontal group
It knits and bacterium.Level in gingival sulcus fluid includes various electrolyte, protein, glucose, enzyme etc., also containing leucocyte, the epithelial cell to fall off
Deng there are also bacterium and other microorganisms.Containing there are many enzyme in level in gingival sulcus fluid, wherein aspartate transaminase, alkaline phosphatase, collagen
Enzyme etc. and the severity and active stage of periodontosis have certain relationship.Gingival crevicular fluid increase be gingivitis early stage main performance it
One, often earlier than the change of Clinical signs.When gingivitis is obvious, level in gingival sulcus fluid be increased significantly.Level in gingival sulcus fluid can pass through non-invasive means
As test strips are acquired detection, therefore the albumen in level in gingival sulcus fluid can be used as detection of the ideal marker for disease.Periodontal
Sick GAP-associated protein GAP includes inflammatory cytokine (such as IL-1 β, IL-6, IL-8, IL-10, IL-12, IFNG, TNFa and CRP), bone
It is metabolized relevant cell factor (for example, OPG, OPN, RANK, RANKL and) and enzyme (such as alkaline phosphatase and aspartic acid turn
Adnosine deaminase).
The common method of detection periodontosis includes that depth is examined in the spy of oral pocket, examination is attached on alveolar bone by measuring
The X-ray photograph of tooth simultaneously examines bleeding with reference to visiting.The above method respectively has advantage and disadvantage, in practical applications, according to respective experiment mesh
And laboratory condition selected.These methods depend critically upon the subjective identification of dentist.It is only pair that depth is examined in spy
The measurement of past attachment loss helps very little in the periodontitis occurred now or its following development.Bleeding energy is examined in spy
Enough show recuperation rather than destructive process.Recently, although the method for also developing many assessment periodontosis activity.So
And do not have a kind of to provide sensitive enough and special test to diagnose periodontitis and its failure mode in the above method.Non- spy
One of anisotropic method will be the result is that the patient that several reality do not suffer from periodontitis will be treated.Clinical observation is for example visited and examines depth not
Enough reliable, because deep oral pocket is not necessarily comprising occurent inflammation, the assessment of X-ray radiophotography x must combine detailed
Clinical observation is to provide correct diagnosis, and only there are pathogens cannot accurately reflect Disease Activity in oral pocket.
And the diagnosis for the Enzymology method based on detection host or bacterial origin protein developed so far is special not enough, because
It is fairly simple for immunological detection, it is rapidly, reproducible, but false positive rate is high, can only detect a GAP-associated protein GAP every time.
In view of this, it is necessary to a kind of novel periodontosis GAP-associated protein GAP detection kit is developed, to overcome the prior art
Middle urgent problem to be solved realizes high throughput, high sensitivity, high specific and the low cost detection of plurality of target albumen.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of antibody for detecting periodontosis GAP-associated protein GAP
Chip agent box, the kit can detect multiple periodontosis GAP-associated protein GAPs simultaneously, overcome prior art operation it is cumbersome, detection refer to
The defects such as single, sensitivity is low are marked, with cheap, convenient, sensitive, accurate, high-throughput, sample dosage is few, can be in routine experimentation
The advantages that room popularization and scale.
To achieve the goals above, the present invention is achieved by the following technical programs:
A kind of antibody chip kit detecting periodontosis GAP-associated protein GAP, including antibody chip, periodontosis GAP-associated protein GAP mark
The periodontosis GAP-associated protein GAP of quasi- product mixture, biotin labeling detects antibody mixture；It is affine with the strepto- of fluorescein Cy3 label
Element；Wherein, the antibody chip is to contain slide that the hydrophilic reagent of alkyl glycosides is handled as solid phase carrier, and a variety of periodontals
The specific antibody mixture of sick GAP-associated protein GAP is under 22~24 DEG C, 30~40% damp condition on point sample to solid phase carrier.
The normal structure slide limited using no space can make a little to detect cell adherence on same microarray as carrier
The number of the factor is not limited by space, in addition, slide has low-down autofluorescence, and fluorescence signal does not have diffusion
Property, the fluorescence signal between difference will not be interfering with each other, so can once detect multiple cell adhesion factor simultaneously.But
Normal structure slide is handled in a variety of ways, will affect background, the detection sensitivity of slide, the present invention is by comparing a variety of
The method for handling slide, discovery is low with the slide background that hydrophilic reagent alkyl glycosides is handled, and detection sensitivity is high, realizes not only
Once it can detecte more than ten of cell adhesion factor, and cell adhesion factor sensitivity detected is up to single-factor
ELISA detection sensitivity.
Preferably, the periodontosis GAP-associated protein GAP detection antibody mixture is c reactive protein, interferon gamma, leucocyte Jie
It is -1 α of element, interleukin-1 ' beta ', interleukin 2, interleukin 4, interleukin-6, interleukin 8, white thin
Born of the same parents' interleukin -10, interleukin 12, Interleukin-17, Macrophage inflammatory protein-α, matrix metalloproteinase 9, base
Matter metalloproteinase 13, osteoprotegerin, osteopontin, bone cerebroysin, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, tumour
The mixture of the specific antibody of necrosin &.
It is highly preferred that the hydrophilic reagent be mass fraction be 0.01~0.2% alkyl glycosides, 0.01~0.1%
Glycerol, the ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method of hydrophilic reagent processing slide is that slide exists
It impregnates 3~5 minutes, dries in hydrophilic reagent.
Most preferably, the hydrophilic reagent be mass fraction be 0.1% alkyl glycosides, 0.05% glycerol, 0.01%
Macrogol 4000 ultra-pure water solution；The method of hydrophilic reagent processing slide is that slide is impregnated 3 points in hydrophilic reagent
Clock dries.
Preferably, periodontosis GAP-associated protein GAP detection antibody mixture point sample under 22 DEG C, 30% damp condition arrives
On solid phase carrier.
Compared with prior art, the invention has the following beneficial effects:
Normal structure slide is handled in a variety of ways, will affect background, the detection sensitivity of slide, the present invention passes through
The method of more a variety of processing slides, discovery is low with the slide background of the hydrophilic reagent processing containing alkyl glycosides, detects sensitive
Degree is high, and realizing not can detecte more than ten of periodontosis GAP-associated protein GAP only once, and periodontosis GAP-associated protein GAP detected
ELISA detection sensitivity of the sensitivity up to single albumen.Antibody chip kit of the invention overcomes prior art operation
The cumbersome, defects such as Testing index is single, sensitivity is low, have that cheap, convenient, sensitive, accurate, high-throughput, sample dosage is few, energy
It is promoted in common lab and the advantages that scale.
Antibody chip kit of the invention is particularly suitable for c reactive protein, interferon gamma, interleukin-1 alpha, white thin
- 1 β of born of the same parents' interleukin, interleukin 2, interleukin 4, interleukin-6, interleukin 8, interleukin 10,
Interleukin 12, Interleukin-17, Macrophage inflammatory protein-α, matrix metalloproteinase 9, matrix metalloprotease
Enzyme 13, osteoprotegerin, osteopontin, bone cerebroysin, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, tumor necrosis factor α,
The combination of the specific antibody of this 20 kinds of periodontosis GAP-associated protein GAPs.Because of the specificity of different periodontosis GAP-associated protein GAPs
Sensitivity of the antibody in chip detecting system of the present invention is different, so, if by the periodontal of some low sensitivitys
The periodontosis GAP-associated protein GAP antibody combination of sick GAP-associated protein GAP antibody and some high sensitivities, will cause the periodontosis phase of low sensitivity
Close the missing inspection of albumen.
Detailed description of the invention
Fig. 1 is antibody chip dot chart.
Fig. 2 is the canonical plotting of periodontosis GAP-associated protein GAP.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
The screening of antibody chip carrier: conventional antibody chip is mostly using nitrocellulose filter as carrier, due to cellulose nitrate
Plain film is multilayered structure, and the washing difficulty of chip is big, so that the background of chip is high, as a result fluctuation is big.Nitrocellulose filter simultaneously
It is frangible, it is not easy to operate on a large scale, the use for larger scale clinical sample is not also universal.And the inexpensive background of traditional slide
Low, this brings breakthrough to diagnosis and being widely used for research field.We have screened does not have to activity methods processing on the market
The also amidized slide point sample effect of slide, either aldehyde radicalization is all unstable.Temperature and slide are obtained by a large amount of screening
The active agent formulation on surface is only the key for determining slide point sample effect.
Use following 5 groups of slide: the 1st group, for amination slide, is purchased from Corning Incorporated, and article No. is
UltraGAPS40019；2nd group is aldehyde radical slide: glutaraldehyde is added in the 1st group of slide and impregnates 40 minutes production aldehyde radical glass
Piece；3rd group is APES slide: common slide being added in the diluted APES of acetone and is impregnated 0.5~1 minute, then is clear with pure acetone
It washes and APES slide is made；4th group is poly-D-lysine slide: the diluted poly-D-lysine of PBS is added in common slide and impregnates 0.5
~1 hour, then cleaned with pure water and poly-D-lysine slide is made；5th group is the slide handled through hydrophilic reagent: by common glass
Piece is impregnated 3~5 minutes with hydrophilic reagent, and room temperature is dried；The hydrophilic reagent is the alkane that mass fraction is 0.01~0.2%
Base glucosides, 0.01~0.1% glycerol, the ultra-pure water solution of 0.01~0.05% Macrogol 4000.Preferably, the parent
Water reagent be mass fraction be 0.1% alkyl glycosides, 0.05% glycerol, 0.01% Macrogol 4000 it is ultrapure water-soluble
Liquid；The method of hydrophilic reagent processing slide is to impregnate slide in hydrophilic reagent 3 minutes, is dried.
The point sample effect of various slides is not quite similar, and the slide point sample effect that hydrophilic coating is added is more apparent, point sample effect
More untreated height.The more other type effects of slide wherein Jing Guo amination and hydrophilic treated are higher.At 22~24 DEG C
Under the conditions of, film/slide point sample effect is more visible, and background value is low；And under the conditions of 27~30 DEG C, no matter which kind of film/slide point
All there are diffusion phenomena in sample effect, and temperature humidity is higher, and diffusion is more obvious.After above-mentioned 5 groups of slide binding antibodies be added secondary antibody and
Substrate, sensitivity the selection result such as the following table 1 at different temperature and damp condition indicate that the index of sensitivity is in table 1
Lowest detection line, unit ng/ml.
Table 1
In summary index, slide of the final choice by the above-mentioned hydrophilic reagent processing for containing alkyl glycosides is as solid phase
Carrier.
Embodiment 2
A kind of simultaneous quantitative detects the preparation of the antibody chip kit of multiple periodontosis GAP-associated protein GAPs: for test sample
In whether there is corresponding receptor, preparation is fixed with the slide of the corresponding specific antibody of 20 kinds of periodontosis GAP-associated protein GAPs, 20 kinds
The corresponding specific antibody of periodontosis GAP-associated protein GAP is ordinary commercial products.
The preparation and preservation of antibody chip: the PBS buffer solution containing specific antibody of 100~1000pl (is contained 0.01
~10g/100ml bovine albumin) with full-automatic point sample instrument point sample on slide.Specific chip dot matrix is with the ox of biotin labeling
IgG is as positive control.There are four repeat point in each array for the positive control of 20 kinds of antibody and two kinds of various concentrations.?
Chip array uses arrangement mode as shown in Figure 1 in the present embodiment, but in fact, in other embodiments, chip array is also
It can be combined with other arrangement modes, it is not limited to form represented by Fig. 1.Have on every slide 16 it is identical
Chip array.The good slide of point sample is put in and is stood overnight under room temperature, is then evacuated in drier 2 hours dry.It is dry
Slide after dry loads onto matched 16 hole frame and one slide is divided into 16 non-interfering cells.U-frame is pressed from both sides from two sides
By 16 hole frames, silicagel pad and standard glass slide sticker tightly together so that standard glass slide is adjacent to the bottom of 16 hole frames of closing
Portion, each of making on 16 hole frames small trellis, 16 hole frame edges mark 1 according to the position in hole respectively at a small reacting hole
Recess number to 16 is recognized with facilitating.After adhesive film closed frame, then the air-locked pouch of whole chip is encapsulated
It is saved backup in 2 DEG C to 8 DEG C.
The preparation of periodontosis GAP-associated protein GAP standard items: the standard items of preparation periodontosis GAP-associated protein GAP.By 20 kinds of periodontosis phases
It is mixed after closing phosphate buffer dilution of the albumen containing 0.1% bovine albumin according to a certain amount, with cold after packing
Freeze seasoning drying and is saved in -80 DEG C.In the present embodiment, for doing every kind of periodontosis GAP-associated protein GAP of standard curve
It is final using concentration it is as shown in table 2.
Table 2 is after gradient dilution for making the concentration of the periodontosis GAP-associated protein GAP standard items of standard curve
| (pg/ml) | Cntrl | Std7 | Std6 | Std5 | Std4 | Std3 | Std2 | Std1 |
| CRP | 0 | 55 | 165 | 494 | 1,481 | 4,444 | 13,333 | 40,000 |
| IFNγ | 0 | 14 | 41 | 123 | 370 | 1,111 | 3,333 | 10,000 |
| IL-1α | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-1β | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-2 | 0 | 5 | 16 | 49 | 148 | 444 | 1,333 | 4,000 |
| IL-4 | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-6 | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-8 | 0 | 1 | 2 | 5 | 15 | 44 | 133 | 400 |
| IL-10 | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-12 | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
| IL-17 | 0 | 14 | 41 | 123 | 370 | 1,111 | 3,333 | 10,000 |
| MIP-1α | 0 | 14 | 41 | 123 | 370 | 1,111 | 3,333 | 10,000 |
| MMP-9 | 0 | 14 | 41 | 123 | 370 | 1,111 | 3,333 | 10,000 |
| MMP-13 | 0 | 27 | 82 | 247 | 741 | 2,222 | 6,667 | 20,000 |
| OPG | 0 | 27 | 82 | 247 | 741 | 2222 | 6,667 | 20,000 |
| OPN | 0 | 137 | 412 | 1,235 | 3,704 | 11,111 | 33,333 | 100,000 |
| Osteoactivin | 0 | 14 | 41 | 123 | 370 | 1,111 | 3,333 | 10,000 |
| RANK | 0 | 137 | 412 | 1,235 | 3,704 | 11,111 | 33,333 | 100,000 |
| TGFβ1 | 0 | 137 | 412 | 1,235 | 3,704 | 11,111 | 33,333 | 100,000 |
| TNFα | 0 | 3 | 8 | 25 | 74 | 222 | 667 | 2,000 |
Embodiment 3
The experimental procedure for detecting a variety of periodontosis GAP-associated protein GAPs with kit quantification of the invention is as follows:
1, slide chip is completely dried: slide chip is taken out from box, after 20~30min of equilibrium at room temperature,
Packaging bag is opened, sealing strip is opened, chip is then placed on vacuum desiccator or drying at room temperature 1~2 hour.
2, gradient dilution is carried out to periodontosis GAP-associated protein GAP by table 2
2.1, the sample diluting liquid of 500 μ l is added into the tubule of periodontosis GAP-associated protein GAP correct mixture, is re-dissolved
Standard items.Before opening tubule, first quickly centrifugation, gently upper and lower lash dissolved powders, and marking this tubule is Std1.
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 for extracting 100 μ l, which is added in Std2, to be gently mixed, and 100 μ l are then extracted from Std2 and are added to
In Std3, such gradient dilution to Std7.
2.4, the sample diluting liquid of 100 μ l is extracted into another new centrifuge tube, is labeled as CNTRL, as negative right
According to.
Note: because the initial concentration of every kind of periodontosis GAP-associated protein GAP is different, the gradient dilution of Std1 to Std7
Afterwards, the series of concentrations of each periodontosis GAP-associated protein GAP is different, in the present embodiment, gradient periodontosis GAP-associated protein GAP dilution
Concentration is as shown in table 2.
3, chip operation process
3.1, the sample diluting liquid of each 100 μ l of Kong Zhongjia is incubated for 30 minutes on room temperature shaker, closes quantitative antibody core
Piece.
3.2, the buffer in each hole is pumped, the titer and sample for adding 100 μ l are into hole, 4 DEG C of mistakes on shaking table
Night is incubated for.
Note: the incubation amount of different samples is different: blood plasma, serum are diluted using preceding with sample diluting liquid 1: 1；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: pumping the standard items or sample in each hole, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker
Concussion, 1 × washing lotion I of every 150 μ l of hole, cleaning will be cleaned washing lotion every time, dilute 20 × washing lotion I with deionized water.It pumps every
1 × washing lotion I in a hole is added 1 × washing lotion II and cleans 2 times, each 5min room temperature shaker concussion, 1 × washing lotion of every 150 μ l of hole
II, cleaning will be cleaned washing lotion every time, dilute 20 × washing lotion II with deionized water.
The incubation of 3.4 detection antibody mixtures: centrifugation detection antibody mixture tubule, the sample that 1.4ml is then added are dilute
Liquid is released, after mixing rapid centrifugation again.The detection antibody of 80 μ l is added into each hole, is incubated for 2 hours on room temperature shaker.
3.5 cleanings: pumping the detection antibody in each hole, and 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion,
1 × washing lotion I of every 150 μ l of hole, cleaning will be cleaned washing lotion every time, and 1 × washing lotion II is then added and cleans 2 times, each room 5min
Warm shaking table concussion, 1 × washing lotion II of every 150 μ l of hole, cleaning will be cleaned washing lotion every time.
The incubation of 3.6Cy3- Streptavidin: centrifugation Cy3- Streptavidin tubule, the sample that 1.4ml is then added are dilute
Liquid is released, after mixing rapid centrifugation again.The Cy3- Streptavidin of 80 μ l is added into each hole, encases glass with aluminium-foil paper
Piece is protected from light incubation, is incubated for 1 hour on room temperature shaker.
3.7 cleanings: pumping the Cy3- Streptavidin in each hole, and 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker
Concussion, 1 × washing lotion I of every 150 μ l of hole, cleaning will be cleaned washing lotion every time.
3.8 fluorescence detection
1) slide frame is dismantled, does not touch the one side of slide printing antibody with hand carefully.
2) slide is placed in glass slide cleaning pipe, adds 1 × washing lotion I of about 30ml, 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) the residual washing lotion of slide is removed.Slide is placed in glass slide cleaning pipe/drying tube, not lid lid,
1000rpm is centrifuged 3min.
4) laser scanner such as AxonGenePix scanning signal is used, using Cy3 or green channel (stimulating frequency
=532nm).
The data of 3.9 chips are extracted and carry out data analysis with analysis software
1) fluorescent value of biochip is read with GenePix software.
2) numerical value selected after reading is that the median of subtracting point ambient background is read
(F532Median-LocalBackground).Make each periodontosis correlation egg with chip software is specifically quantified
White standard curve is as shown in Figure 2.
Embodiment 4
The test of experimental system cross reaction.
The accuracy of antibody chip experiment is limited to the source of selected antigen or antibody to a certain extent, purity with
Specificity, and there is antigenicity, the power of immunogenicity, the rheumatoids of heterologous antibody with application for the biology of protide antibody
The interference of the factor and autoantibody, the high workload screening of rare antibody, because a little screening antibodies are to a large amount of experimental verification of needs.
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 for, after developing a film again detection antibody response corresponding with every kind of antigen most afterwards through Cy3- chain
Mould Avidin is incubated for, and is read after chip scanning.Using the capture antibody of every kind of antigen as horizontal axis, with the antigen of addition and corresponding
Detect the experimental result that antibody is the available table 3 of the longitudinal axis.
Cross reaction test result between 3. antibody pair of table
The experimental results showed that every kind of antibody does not have the detection antigen that can specifically identify oneself with other antigens
Cross reaction.
Embodiment 5
The dielectric film filter rate of experimental system.
The appropriate in different sample medias of the quantitative antibody chip is shown by measurement dielectric film filter rate.?
Each periodontosis phase of various concentration is separately added into 2 times of diluted normal human serums and 2 times of diluted cell supernatants (CM)
Albumen is closed, is detected with the quantitative antibody chip, dielectric film filter rate=(periodontosis correlation egg of intervention sample of sample is then calculated
White concentration-the periodontosis GAP-associated protein GAP concentration of control sample)/the periodontosis GAP-associated protein GAP concentration theoretically intervened.Experiment display
The rate of recovery of the kit of the invention in human serum and cell supernatant is up to 31~126%.
The rate of recovery of 4. quantitative antibody chip of table in different media
| (Pgml) | Spiking | CM | CM+Ag | CM% | Serum | Serum+Ag | Serum% |
| CRP | 20,000 | 0 | 14,411 | 72% | 7,444 | 20,945 | 68% |
| IFNγ | 5,000 | 391 | 4,425 | 81% | 27 | 3,607 | 72% |
| IL-1α | 1,000 | 21 | 746 | 72% | 10 | 654 | 64% |
| IL-1β | 1,000 | 0 | 631 | 63% | 0 | 513 | 51% |
| IL-2 | 2,000 | 432 | 2,536 | 105% | 49 | 2,152 | 105% |
| IL-4 | 1,000 | 14 | 1,244 | 123% | 10 | 1,361 | 135% |
| IL-6 | 1,000 | 1,726 | 2,436 | 71% | 27 | 1,023 | 100% |
| IL-8 | 1,000 | 159 | 1,156 | 100% | 6 | 956 | 95% |
| IL-10 | 1,000 | 9 | 726 | 72% | 1 | 590 | 59% |
| IL-12 | 1,000 | 2 | 1,198 | 120% | 2 | 857 | 85% |
| IL-17 | 5,000 | 0 | 3,461 | 69% | 2 | 3,255 | 65% |
| MIP-1α | 5,000 | 110 | 7,134 | 140% | 0 | 3,934 | 79% |
| MMP-9 | 5,000 | 15 | 2,587 | 51% | 3,368 | 4,067 | 14% |
| MMP-13 | 10,000 | 129 | 9,919 | 98% | 6 | 6,128 | 61% |
| OPG | 10,000 | 34,602 | 39,009 | 44% | 23 | 12,799 | 128% |
| OPN | 50,000 | 958 | 47,043 | 92% | 203 | 28,861 | 57% |
| Osteoactivin | 5,000 | 89 | 4,993 | 98% | 2,493 | 4,538 | 41% |
| RANK | 25,000 | 68 | 19,119 | 76% | 21 | 17,503 | 70% |
| TGFβ1 | 50,000 | 313 | 54,434 | 108% | 0 | 60,182 | 120% |
| TNFα | 1,000 | 42 | 765 | 72% | 16 | 1,301 | 128% |
In conclusion the invention discloses the antibody chip reagents that a kind of simultaneous quantitative detects multiple periodontosis GAP-associated protein GAPs
Box.On the one hand the kit overcomes that Testing index of the conventional ELISA in receptor detection is single, and time-consuming, effort, consumptive material etc. lacks
Point, while the small throughput in existing polyprotein detection technique is also overcomed, the weakness such as poor repeatability.The system uses normal structure
Slide can complete the reaction of multiple sandwich ELISA in surface of glass slide as topical carrier.The specification of antibody chip meets simultaneously
The size of 96 hole elisa Plates of standard, makes it possible high-throughput sample operation.It is detected in addition, we also confirm that with the invention
The concentration of receptor can achieve the detection sensitivity and accuracy of single ELISA.Finally, by the chip in different samples
The chip agent box of the true quasi- invention of the measurement of the middle medium rate of recovery can apply to serum, the different biology such as cell supernatant
Sample.
Claims (3)
1. a kind of antibody chip kit for detecting a variety of periodontosis GAP-associated protein GAPs, including antibody chip, periodontosis GAP-associated protein GAP
The strepto- parent of standard items mixture, the periodontosis GAP-associated protein GAP detection antibody mixture of biotin labeling and fluorescein Cy3 label
And element, which is characterized in that the antibody chip using contain alkyl glycosides hydrophilic reagent handle slide as solid phase carrier, and
The specific antibody mixture of a variety of periodontosis GAP-associated protein GAPs under 22~24 DEG C, 30~40% damp condition point sample to solid phase
On carrier；Wherein, the hydrophilic reagent be mass fraction be 0.01~0.2% alkyl glycosides, 0.01~0.1% glycerol,
The ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method that hydrophilic reagent handles slide is by slide in hydrophilic examination
It impregnates 3~5 minutes, dries in agent；
The periodontosis GAP-associated protein GAP detection antibody mixture is c reactive protein, interferon gamma, interleukin-1 alpha, leucocyte
It is -1 β of interleukin, interleukin 2, interleukin 4, interleukin-6, interleukin 8, interleukin 10, white
Cytokine -12, Interleukin-17, Macrophage inflammatory protein-α, matrix metalloproteinase 9, matrix metalloproteinase
13, the spy of osteoprotegerin, osteopontin, bone cerebroysin, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, tumor necrosis factor α
Heterogenetic antibody mixture.
2. kit according to claim 1, which is characterized in that the periodontosis GAP-associated protein GAP detection antibody mixture exists
22 DEG C, under 30% damp condition on point sample to solid phase carrier.
3. kit according to claim 1, which is characterized in that the hydrophilic reagent is the alkyl that mass fraction is 0.1%
Glucosides, 0.05% glycerol, the ultra-pure water solution of 0.01% Macrogol 4000；The method that hydrophilic reagent handles slide is by glass
Piece impregnates 3 minutes in hydrophilic reagent, dries.
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| CN201510988174.8A CN106918706B (en) | 2015-12-25 | 2015-12-25 | A kind of antibody chip kit detecting periodontosis GAP-associated protein GAP |
| PCT/CN2016/097196 WO2017107540A1 (en) | 2015-12-25 | 2016-08-29 | Antibody-array test kit for detecting proteins related to periodontal disease |
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| CN201510988174.8A CN106918706B (en) | 2015-12-25 | 2015-12-25 | A kind of antibody chip kit detecting periodontosis GAP-associated protein GAP |
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| CN106918706A CN106918706A (en) | 2017-07-04 |
| CN106918706B true CN106918706B (en) | 2019-05-21 |
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| CN108929903B (en) * | 2018-04-23 | 2021-07-16 | 东莞市第五人民医院(东莞市太平人民医院) | Marker and kit for screening, diagnosing and evaluating curative effect of chronic periodontitis |
| CN108593939B (en) * | 2018-07-18 | 2023-11-14 | 瑞博奥(广州)生物科技股份有限公司 | Full-automatic protein chip and application thereof |
| CN110967336A (en) * | 2018-09-28 | 2020-04-07 | 陈洁 | Periodontal disease detection kit and use method thereof |
| CN109459568A (en) * | 2019-01-15 | 2019-03-12 | 西安交通大学 | Paper base periodontitis detection device and periodontitis detection method |
| CN111638370B (en) * | 2020-04-30 | 2023-03-03 | 吉林省格瑞斯特生物技术有限公司 | Gastric function and gastric cancer occurrence risk detection device and preparation method thereof |
| CN111551740B (en) * | 2020-04-30 | 2023-03-03 | 吉林省格瑞斯特生物技术有限公司 | Helicobacter pylori urease IgG and IgM antibody combined detection device and preparation method |
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| CN106918706A (en) | 2017-07-04 |
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