CN106918706A - A kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP - Google Patents
A kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP Download PDFInfo
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Abstract
The present invention relates to technical field of biological, in particular it relates to a kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP.The antibody chip of the kit using the slide of the hydrophilic reagent treatment containing APG as solid phase carrier, and various periodontosis GAP-associated protein GAPs specific antibody mixture under 22~24 DEG C, 30~40% damp condition point sample on solid phase carrier.Kit of the invention can detect multiple clinical conventional periodontosis GAP-associated protein GAPs; overcome that prior art operation is cumbersome, Testing index is single, the low defect of sensitivity, have the advantages that cheap, convenient, sensitive, accurate, high flux, sample consumption are few, can be promoted and scale in common lab.
Description
Technical field
The present invention relates to technical field of biological, in particular it relates to a kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP.
Background technology
Periodontosis refers to that the disease in tooth supporting tissue (periodontium) occurs, including only involves the gingivitis of gingiva tissue and involve the major class of periodontitis two of deep layer periodontium (parodontium, alveolar bone, cementum).Periodontal disease is common mouth disease, is the one of the main reasons for causing adult's loss of tooth, is also the main oral disease for endangering human teeth and whole body health.The early symptom of periodontosis is difficult to draw attention, and causes periodontium long-term chronic to infect, and inflammation recurrent exerbation not only damages the function of oral cavity masticatory system, can also have a strong impact on health.
Periodontosis is the disease damaged to soft group of group and bony tissue from simple various focusing depths represented, and coming off for tooth can be caused in the worst case.Therefore the early diagnosis of periodontitis and prevention are most important.Include that oral pocket is visited at present to examine, bacterial plaque and X-ray imaging diagnosis can be used as the indexs of periodontosis symptom in interior clinical detection, but these detections can not indicate advancing of disease situation.Reflect the situation of disease it is therefore desirable to develop a kind of new detection method, effectively prevention and the generation of monitoring of diseases.Level in gingival sulcus fluid (GCF) refers to penetrate into the liquid in gingival sulcus from gingival connective tissue by epithelium in ditch and junctional epithelium.Gums healthy person has very small amount level in gingival sulcus fluid, tissue fluid is penetrated into gingival sulcus by the osmotic gradient in tissue.Because generally having inflammatory cell in level in gingival sulcus fluid, other chemical compositions are also incomplete same with tissue fluid；The discharge of level in gingival sulcus fluid is directly proportional to the degree of inflammation at the position.The liquid component of level in gingival sulcus fluid is mainly derived from serum, and other compositions are then respectively from serum, neighbouring periodontium and bacterium.Level in gingival sulcus fluid including various electrolyte, protein, glucose, enzyme etc., also containing leucocyte, the epithelial cell for coming off etc., also bacterium and other microorganisms.Contain various enzymes in level in gingival sulcus fluid, wherein aspartate transaminase, alkaline phosphatase, clostridiopetidase A etc. has certain relation with the order of severity and active stage of periodontosis.It is one of main performance of gingivitis early stage that gingival crevicular fluid increases, often earlier than the change of Clinical signs.When gingivitis are obvious, level in gingival sulcus fluid showed increased.Level in gingival sulcus fluid can be acquired detection by non-invasive means such as test strips, therefore albumen in level in gingival sulcus fluid can be used for the detection of disease as preferable mark.Periodontosis GAP-associated protein GAP includes inflammatory cytokine (such as IL-1 β, IL-6, IL-8, IL-10, IL-12, IFNG, TNFa and CRP), the related cell factor of Bone m etabolism is (for example, OPG, OPN, RANK, RANKL and) and enzyme (such as alkaline phosphatase and aspartate transaminase).
Detecting the common method of periodontosis includes that the spy by measuring oral pocket is examined depth, the X-ray photograph of the tooth that examination is attached on alveolar bone and examines bleeding with reference to spy.The above method respectively has advantage and disadvantage, in actual applications, is selected according to respective experiment purpose and laboratory condition.These methods depend critically upon the subjective identification of dentist.It is only the measurement to past attachment loss that depth is examined in spy, and it in the present periodontitis for occurring or helps very little in the development in its future.Spy is examined bleeding and can show that recuperation rather than destructive process.Recently, although the method for also developing many assessment periodontosis activity.However, do not have in the above method it is a kind of there is provided sensitive enough and special experiment to diagnose periodontitis and its failure mode.One result of non-specific method is that several reality are not suffered from the patient of periodontitis and will be treated.Clinical observation for example visits that to examine depth less reliable, because the oral pocket of depth is not necessarily comprising occurent inflammation, the assessment of X-ray radiophotography x must combine detailed clinical observation to provide correct diagnosis, and only there is pathogen in oral pocket can not exactly reflect Disease Activity.It is rapidly, reproducible because immunological detection is fairly simple and the diagnosis of the Enzymology method based on detection host or bacterial origin protein developed so far is special not enough, but false positive rate is high, and a GAP-associated protein GAP can only be detected every time.
In view of this, it is necessary to develop a kind of new periodontosis GAP-associated protein GAP detection kit, to overcome problem demanding prompt solution in the prior art, high flux, high sensitivity, high specific and the low cost detection of plurality of target albumen are realized.
The content of the invention
The invention aims to overcome the deficiencies in the prior art; a kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP is provided; the kit can simultaneously detect multiple periodontosis GAP-associated protein GAPs; overcome that prior art operation is cumbersome, Testing index is single, the low defect of sensitivity, have the advantages that cheap, convenient, sensitive, accurate, high flux, sample consumption are few, can be promoted and scale in common lab..
To achieve these goals, the present invention is achieved by the following technical programs：
A kind of antibody chip kit for detecting periodontosis GAP-associated protein GAP, including antibody chip, periodontosis GAP-associated protein GAP standard items mixture, the periodontosis GAP-associated protein GAP detection antibody mixture of biotin labeling；The Streptavidin marked with fluorescein Cy3；Wherein, the antibody chip is using the slide of the hydrophilic reagent treatment containing APG as solid phase carrier, and various periodontosis GAP-associated protein GAPs specific antibody mixture under 22~24 DEG C, 30~40% damp condition point sample on solid phase carrier.
Using the normal structure slide that is limited without space as carrier, can make a little to detect that the number of CAF is not limited by space on same microarray, in addition, slide has low-down autofluorescence, and fluorescence signal does not have diffusivity, fluorescence signal between difference will not be interfering with each other, so once can simultaneously detect multiple CAFs.But, process normal structure slide in a variety of ways, background, the detection sensitivity of slide can be influenceed, the method that the present invention passes through relatively more various treatment slides, it was found that low with the slide background of hydrophilic reagent APG treatment, detection sensitivity is high, and realizing can only once not detect more than ten CAF, and the CAF sensitivity for being detected is up to the ELISA detection sensitivities of single-factor.
Preferably, the periodontosis GAP-associated protein GAP detection antibody mixture is c reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 ' beta ', interleukin 2, interleukin 4, interleukin-6, interleukin 8, interleukin 10, interleukin 12, Interleukin-17, Macrophage inflammatory protein-α, GELB, mmp-13, osteoprotegerin, osteopontin, bone cytokines, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, the mixture of the specific antibody of tumor necrosis factor α.
It is highly preferred that the hydrophilic reagent is APG, 0.01~0.1% glycerine that mass fraction is 0.01~0.2%, the ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3~5 minutes, is dried.
Most preferably, the hydrophilic reagent is APG, 0.05% glycerine that mass fraction is 0.1%, the ultra-pure water solution of 0.01% Macrogol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3 minutes, is dried.
Preferably, the periodontosis GAP-associated protein GAP detection antibody mixture under 22 DEG C, 30% damp condition point sample on solid phase carrier.
Compared with prior art, the present invention has the advantages that：
Process normal structure slide in a variety of ways, background, the detection sensitivity of slide can be influenceed, the method that the present invention passes through relatively more various treatment slides, it was found that low with the slide background of the hydrophilic reagent treatment containing APG, detection sensitivity is high, realizing can only once not detect more than ten periodontosis GAP-associated protein GAP, and the periodontosis GAP-associated protein GAP for being detected sensitivity up to single albumen ELISA detection sensitivities.Antibody chip kit of the invention overcomes that prior art operation is cumbersome, Testing index is single, the low defect of sensitivity, has the advantages that cheap, convenient, sensitive, accurate, high flux, sample consumption are few, can be promoted and scale in common lab.
Antibody chip kit of the invention is particularly suited for c reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 ' beta ', interleukin 2, interleukin 4, interleukin-6, interleukin 8, interleukin 10, interleukin 12, Interleukin-17, Macrophage inflammatory protein-α, GELB, mmp-13, osteoprotegerin, osteopontin, bone cytokines, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, tumor necrosis factor α, this 20 kinds of combinations of the specific antibody of periodontosis GAP-associated protein GAP.Because, sensitivity of the specific antibody of different periodontosis GAP-associated protein GAPs in chip detecting system of the present invention is different, so, if by the periodontosis GAP-associated protein GAP antibody of some low sensitivitys and the periodontosis GAP-associated protein GAP Antibody Combination of some high sensitivities, the missing inspection of the periodontosis GAP-associated protein GAP of low sensitivity can be caused.
Brief description of the drawings
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 reference to Figure of description and specific embodiment further being elaborated, the embodiment is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is conventional method unless otherwise specified；Material, reagent for being used etc., are the reagent and material for commercially obtaining unless otherwise specified.
Embodiment 1
The screening of antibody chip carrier：Conventional antibody chip is more with nitrocellulose filter as carrier, and because nitrocellulose filter is sandwich construction, the washing difficulty of chip is big, so that the background of chip is high, as a result fluctuates big.Nitrocellulose filter is frangible simultaneously, is not easy to large-scale operation, and the use for larger scale clinical sample is not also universal.And the inexpensive background of traditional slide is low, this brings breakthrough to diagnosis and widely using for research field.We have screened the slide for being processed without activity methods on the market, and either the also amidized slide point sample effect of aldehyde radicalization is all unstable.Show that temperature and the active agent formulation of surface of glass slide are only the key for determining slide point sample effect by substantial amounts of examination.
Using following 5 groups of slide：1st group is amination slide, and purchased from Corning Incorporated, article No. is UltraGAPS40019；2nd group is aldehyde radical slide：Add glutaraldehyde to soak 40 minutes the 1st group of slide and make aldehyde radical slide；3rd group is APES slides：Will common slide add the APES of acetone dilution in soak 0.5~1 minute, then cleaned with pure acetone and be made APES slides；4th group is poly-D-lysine slide：The poly-D-lysine that common slide adds PBS dilutions is soaked into 0.5~1 hour, then is cleaned with pure water and is made poly-D-lysine slide；5th group is the slide processed through hydrophilic reagent：Common slide is soaked 3~5 minutes with hydrophilic reagent, room temperature is dried；The hydrophilic reagent is APG, 0.01~0.1% glycerine that mass fraction is 0.01~0.2%, the ultra-pure water solution of 0.01~0.05% Macrogol 4000.Preferably, the hydrophilic reagent is APG, 0.05% glycerine that mass fraction is 0.1%, the ultra-pure water solution of 0.01% Macrogol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3 minutes, is dried.
The point sample effect of various slides is not quite similar, and adds the slide point sample effect of hydrophilic coating more apparent, the more untreated height of point sample effect.The more other type effects of slide wherein by amination and hydrophilic treated are higher.Under the conditions of 22~24 DEG C, film/slide point sample effect is more visible, and background value is low；And under the conditions of 27~30 DEG C, diffusion phenomena all occurs in the point sample effect of which kind of film/slide, temperature humidity is higher, spreads more obvious.Addition secondary antibody and substrate after above-mentioned 5 groups of slide binding antibodies, the sensitivity the selection result such as table 1 below at different temperature and damp condition, the index that sensitivity is represented in table 1 is lowest detection line, unit ng/ml.
Table 1
In summary index, the slide that final choice is processed by the above-mentioned hydrophilic reagent containing APG is used as solid phase carrier.
Embodiment 2
A kind of preparation of the antibody chip kit of the multiple periodontosis GAP-associated protein GAPs of simultaneous quantitative detection：In order to detect with the presence or absence of corresponding acceptor in sample, preparation is fixed with the slide of the corresponding specific antibody of 20 kinds of periodontosis GAP-associated protein GAPs, and the corresponding specific antibody of 20 kinds of periodontosis GAP-associated protein GAPs is ordinary commercial products.
The preparation of antibody chip and preservation：By the PBS (containing 0.01~10g/100ml BAs) containing specific antibody of 100~1000pl with full-automatic point sample instrument point sample on slide.Specific chip dot matrix is using the ox IgG of biotin labeling as positive control.20 kinds of antibody and two kinds of positive controls of various concentrations have four repetition points in each array.Chip array uses arrangement mode as shown in Figure 1 in the present embodiment, but in fact, in other embodiments, chip array can also be combined with other arrangement modes, it is not limited to the form represented by Fig. 1.There are 16 identical chip arrays on every slide.The good slide of point sample is put under room temperature condition and is stood overnight, be then evacuated in drier and dried 2 hours.Dried slide loads onto 16 supporting hole frameworks and one slide is divided into 16 non-interfering cells.U-frame folder is from both sides by 16 hole frameworks, together with silicagel pad is tight with standard glass slide sticker, so that standard glass slide is adjacent to the bottom of 16 hole frameworks of closing, make each the small trellis on 16 hole frameworks into a small reacting hole, the depression that 16 hole frame edges mark 1 to 16 according to the position in hole respectively is digital to facilitate identification.After with adhesive film closed frame, whole chip is encapsulated with air-locked pouch and then is saved backup in 2 DEG C to 8 DEG C.
The preparation of periodontosis GAP-associated protein GAP standard items：Prepare the standard items of periodontosis GAP-associated protein GAP.Mixed according to a certain amount after 20 kinds of periodontosis GAP-associated protein GAPs are diluted with the phosphate buffer containing 0.1% bovine albumin, dried and in -80 DEG C of preservations with freeze-drying after packing.In the present embodiment, it is as shown in table 2 for doing the final concentration of every kind of periodontosis GAP-associated protein GAP of standard curve.
Table 2 is used for the concentration of the periodontosis GAP-associated protein GAP standard items for making standard curve after gradient dilution
| (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
Detect that the experimental procedure of various periodontosis GAP-associated protein GAPs is as follows with kit of the invention：
1st, slide chip is completely dried：Slide chip is taken out from box, after 20~30min of equilibrium at room temperature, packaging bag is opened, open sealing strip, chip is then placed on vacuum desiccator or drying at room temperature 1~2 hour.
2nd, gradient dilution is carried out to periodontosis GAP-associated protein GAP by table 2
2.1st, the sample diluting liquid of 500 μ l of addition dissolves standard items again in the tubule of periodontosis GAP-associated protein GAP correct mixture.Open tubule before, first quickly centrifugation, gently lash dissolved powders up and down, mark this tubule be Std1.
2.2nd, 6 clean centrifuge tubes of mark are Std2, Std3 to Std7 respectively, and the sample diluting liquid of 200 μ l of addition is in each tubule.
2.3rd, the Std1 of 100 μ l of extraction is gently mixed in being added to Std2,100 μ l is then extracted from Std2 and is added in Std3, such gradient dilution to Std7.
2.4th, the sample diluting liquid of 100 μ l is extracted in another new centrifuge tube, labeled as CNTRL, as negative control.
Note：Because the initial concentration of every kind of periodontosis GAP-associated protein GAP is different, after the gradient dilution of Std1 to Std7, the series concentration of each periodontosis GAP-associated protein GAP is different, and in the present embodiment, the concentration of gradient periodontosis GAP-associated protein GAP dilution is as shown in table 2.
3rd, chip operation flow
3.1st, the sample diluting liquid of each μ l of Kong Zhongjia 100, is incubated 30 minutes on room temperature shaker, closes quantitative antibody chip.
3.2nd, pump the buffer solution in each hole, add the titer and sample of 100 μ l in hole, 4 DEG C of night incubations on shaking table.
Note：The incubation amount of different samples is different：Blood plasma, serum are diluted using preceding use sample diluting liquid 1: 1；Cell supernatant can use stoste；Cell or tissue lysate adds the amount of 5-50ug after determination of protein concentration.
3.3rd, clean：The standard items or sample in each hole are pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, the 1 × washing lotion I per the μ l of hole 150, every time cleaning will drain washing liquid, 20 × washing lotion I is diluted with deionized water.1 × washing lotion the I in each hole is pumped, adds 1 × washing lotion II to clean 2 times, each 5min room temperature shakers concussion, the 1 × washing lotion II per the μ l of hole 150, every time cleaning will drain washing liquid, 20 × washing lotion II is diluted with deionized water.
The incubation of 3.4 detection antibody mixtures：Centrifugation detection antibody mixture tubule, is subsequently adding the sample diluting liquid of 1.4ml, quick centrifugation again after being well mixed.The detection antibody of 80 μ l is added in each hole, is incubated 2 hours on room temperature shaker.
3.5 cleanings：Pump the detection antibody in each hole, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, 1 × washing lotion I per the μ l of hole 150, cleaning every time will drain washing liquid, be subsequently adding 1 × washing lotion II and clean 2 times, each 5min room temperature shakers concussion, 1 × washing lotion II per the μ l of hole 150, every time cleaning will drain washing liquid.
The incubation of 3.6Cy3- Streptavidins：Centrifugation Cy3- Streptavidin tubules, are subsequently adding the sample diluting liquid of 1.4ml, quick centrifugation again after being well mixed.The Cy3- Streptavidins of 80 μ l are added in each hole, slide lucifuge is encased with aluminium-foil paper and is incubated, 1 hour is incubated on room temperature shaker.
3.7 cleanings：The Cy3- Streptavidins in each hole are pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shakers concussion, the 1 × washing lotion I per the μ l of hole 150, every time cleaning will drain washing liquid.
3.8 fluoroscopic examinations
1) slide framework is dismantled, carefully should not touches the one side that slide prints antibody with hand.
2) slide is placed in glass slide cleaning pipe, adds 1 × washing lotion I of about 30ml, can entirely cover slide, 15min is shaken on room temperature shaker, discard 1 × washing lotion I, add 1 × washing lotion II of about 30ml, 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, 3min is centrifuged in 1000rpm.
4) laser scanner such as AxonGenePix scanning signals are 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 softwares.
2) numerical value selected after reading is the median reading of subtracting point ambient background
(F532Median-LocalBackground).It is as shown in Figure 2 to make the standard curve of each periodontosis GAP-associated protein GAP with chip software is specifically quantified.
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 and specificity, and the biological and application of albumen antibody-like has antigenicity, the power of immunogenicity, the interference of the rheumatoid factor and autoantibody of heterologous antibody, the high workload amount screening of rare antibody, because a little screening antibodies are to needing substantial amounts of experimental verification.
Cross reaction test between antibody pair is carried out according to following methods.First with single antigen concentration for the not synantigen of 100ng/ml is incubated, detection antibody reaction corresponding with every kind of antigen again after developing a film most is incubated different chips through Cy3- Streptavidins afterwards, reading after chip scanning.Capture antibody with every kind of antigen can obtain the experimental result of table 3 with the antigen for adding and corresponding detection antibody as transverse axis as the longitudinal axis.
Cross reaction test result between the antibody pair of table 3.
Test result indicate that every kind of antibody does not have cross reaction to can specifically recognize the detection antigen of oneself with other antigens.
Embodiment 5
The dielectric film filter rate of experimental system.
The appropriate in different sample medias of the quantitative antibody chip is shown by determining dielectric film filter rate.Each periodontosis GAP-associated protein GAP of various concentrations is separately added into 2 times of normal human serums for diluting and 2 times of cell supernatants (CM) of dilution, detected with the quantitative antibody chip, then calculate dielectric film filter rate=(the periodontosis GAP-associated protein GAP concentration of the periodontosis GAP-associated protein GAP concentration-control sample of intervention sample)/periodontosis GAP-associated protein GAP concentration for intervening in theory of sample.Experiment shows the rate of recovery of the kit of the invention in human serum and cell supernatant up to 31~126%.
The rate of recovery of the quantitative antibody chip of table 4. in different medium
| (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 sum, the invention discloses a kind of antibody chip kit of the multiple periodontosis GAP-associated protein GAPs of simultaneous quantitative detection.It is single that on the one hand the kit overcomes Testing index of the conventional ELISA in acceptor detection, takes, effort, the shortcomings of consumptive material, while also overcoming the weakness such as the small throughput in existing polyprotein detection technique, poor repeatability.The system uses normal structure slide as topical carrier, and the reaction of multiple sandwich ELISA can be completed in surface of glass slide.The specification of antibody chip meets the size of the hole elisa Plates of standard 96 simultaneously so that high flux sample operation is possibly realized.In addition, we also confirm that the concentration of the acceptor detected with the invention can reach the detection sensitivity and accuracy of single ELISA.Finally, by the chip, the chip agent box of the true standard of measure of the medium rate of recovery invention can apply to the different biological sample such as serum, cell supernatant in different samples.
Claims (5)
1. a kind of antibody chip kit for detecting various periodontosis GAP-associated protein GAPs, including antibody chip, periodontosis GAP-associated protein GAP standard items mixture, the periodontosis GAP-associated protein GAP detection antibody mixture of biotin labeling；The Streptavidin marked with fluorescein Cy3；Characterized in that, the antibody chip is using the slide of the hydrophilic reagent treatment containing APG as solid phase carrier, and various periodontosis GAP-associated protein GAPs specific antibody mixture under 22~24 DEG C, 30~40% damp condition point sample on solid phase carrier.
2. kit according to claim 1, it is characterized in that, the periodontosis GAP-associated protein GAP detection antibody mixture is c reactive protein, interferon gamma, interleukin-1 alpha, interleukin-1 ' beta ', interleukin 2, interleukin 4, interleukin-6, interleukin 8, interleukin 10, interleukin 12, Interleukin-17, Macrophage inflammatory protein-α, GELB, mmp-13, osteoprotegerin, osteopontin, bone cytokines, neoplasm necrosis GAP-associated protein GAP, tumor necrosis factor β 1, the specific antibody mixture of tumor necrosis factor α.
3. kit according to claim 1, it is characterised in that the hydrophilic reagent is APG, 0.01~0.1% glycerine that mass fraction is 0.01~0.2%, the ultra-pure water solution of 0.01~0.05% Macrogol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3~5 minutes, is dried.
4. kit according to claim 1, it is characterised in that the periodontosis GAP-associated protein GAP detection antibody mixture under 22 DEG C, 30% damp condition point sample on solid phase carrier.
5. kit according to claim 3, it is characterised in that the hydrophilic reagent is APG, 0.05% glycerine that mass fraction is 0.1%, the ultra-pure water solution of 0.01% Macrogol 4000；The method of hydrophilic reagent treatment slide is to soak slide in hydrophilic reagent 3 minutes, is dried.
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