CN203909043U - Chip for detecting multiple autoimmune antibodies simultaneously - Google Patents
Chip for detecting multiple autoimmune antibodies simultaneously Download PDFInfo
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- CN203909043U CN203909043U CN201420051743.7U CN201420051743U CN203909043U CN 203909043 U CN203909043 U CN 203909043U CN 201420051743 U CN201420051743 U CN 201420051743U CN 203909043 U CN203909043 U CN 203909043U
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Abstract
The utility model discloses a chip for detecting multiple autoimmune antibodies simultaneously. The chip consists of a glass substrate, seven autoimmune point coatings distributed on the substrate in an array manner, a positive control point coating, a negative control point coating and a blank control point coating, wherein the glass substrate is a glass sheet with a (3-aminopropyl) triethoxysilane modification layer on the surface; the point coatings are round, and 1-2 coating areas are arranged; the point coatings in each coating area are arranged perpendicularly and in parallel into nine rows and five columns and consist of seven groups of five repeated autoimmune point coating detection areas, one group of three repeated positive control point coating areas, one group of three repeated negative control point coating areas and one group of four point coating blank control areas. The chip is high in sensitivity and large in information amount in rheumatic patient serum detection experiments, and compared with an existing detection method, the chip is higher in coincidence rate and has significant clinical application values.
Description
Technical field
The utility model relates to a kind of detection chip, relates in particular to a kind of chip that simultaneously detects various autoimmune antibody, belongs to biochip and diagnostic reagent technical field.
Background technology
Autoimmune disease refers to that body causes autologous tissue to damage caused disease to autoantigen generation immune response.Its incidence of disease in crowd is 3-5%, and especially in the elderly, the incidence of disease is the highest.The diagnostic criteria of general autoimmune disease is except corresponding clinical symptoms in the world at present, and its diagnosis mainly also detects autoantibody in the serum according to patient.Rheumatism is difficult to diagnosis in early days, but in the late period of disease, the multiple tracts of whole body all can be involved, cause huge misery to patient, thus need a kind of highly sensitive and comprehensive diagnostic method, but the detection of autoantibody mainly adopts immunological method at present, conventional have the immune marking, immunofluorescence, an enzyme-linked immunosorbent assay etc., and these methods generally can only detect single antibody of planting, can not detect multiple autoantibodies by massive parallel, and result is difficult to comparison mutually.If need to detect multiple antibody, existing equipment needs relatively a large amount of reagent and clinical samples, has increased the weight of patient's health and financial burden, also makes troubles to clinician.
Biochip refers to by Micrometer-Nanometer Processing Technology and supermolecule self-assembling technique, and the microanalysis unit and the system that build in solid chip list have that information content is large, easy and simple to handle, work efficiency high.In recent years, biochip, as one of representative of new and high technology, has obtained developing rapidly, and technology reaches its maturity, and in biological detection, medical test, medical diagnosis on disease, drug screening and biosome, very important purposes is being brought into play in the aspect such as micro constitutent analysis.
At present, biochip technology increases increasingly for clinical research, the detection of the method that someone adopts chip to 15 kinds of autoantibodies, detection sensitivity and the specificity of finding protein chip are all suitable with ELISA experiment, another researcher detects autoantibody with chip to the dilute serum of 18 kinds of antigens, specificity and susceptibility are also very high, and can reach the detection lower limit of 40fg.But through retrieval, document and the rarely seen report of patent preparing glass-chip and detect rheumatisant's autoantibodies in serum simultaneously by one-time fixing protein, nucleic acid and phosphatide.
Summary of the invention
For the deficiencies in the prior art, problem to be solved in the utility model is to provide a kind of chip that simultaneously detects various autoimmune antibody.
The chip that simultaneously detects various autoimmune antibody described in the utility model, is distributed in seven kinds of autoantigen point coatings on substrate and the some coating of positive control, negative control and blank forms by glass substrate and array;
It is characterized in that:
Described glass substrate is the glass sheet that there is 3-aminopropyl triethoxysilane (APES) decorative layer on surface; Described some coating be shaped as circle, coated areas is established 1～2; The point coating parallel vertical of every coated areas is arranged, horizontal is nine rows, indulging is five row, is made up of the negative control point coated areas of the positive control point coated areas of the autoantigen point coating detection zone of seven groups of 5 repetitions, 1 group of 3 repetition, 1 group of 3 repetition, 1 group 4 some coating blank districts.
Above-mentioned time, detect the chip of various autoimmune antibody, the glass sheet of described 3-aminopropyl triethoxysilane decorative layer refers to one deck 3-aminopropyl triethoxysilane overlayer in the glass sheet die surfaces without any chemical modification.Described glass substrate is rectangle, and area is 73mm～77mm × 25mm～27mm; Thickness is 0.6mm～2.0mm.The described some size that coat side is long-pending, can change according to the variation of the straight footpath ﹑ point number of point and dot spacing.
Wherein, preferred embodiment: the spot diameter of described some coated areas is 0.15mm, dot spacing is 0.5mm, and 1 coated areas is set on a glass substrate.(as shown in Figure 1).
Above-mentioned time, detect the chip of various autoimmune antibody, described seven kinds of autoantigens are respectively ANAs, dsDNA, SSA/Ro-60, SSA/Ro-52, SSB/La, Jo-1, cuorin, positive control is IgG, and negative control is human osteogenic protein-7, and blank is PBST.
Experiment confirms, the present invention adopt seven kinds including protein, phosphatide, nucleic acid, autoantigen can one-time fix on the slide of modifying at APES, obtained detecting the chip of various autoimmune antibody simultaneously, the chip of preparing with this is high in detection rheumatisant Serum experiments medium sensitivity, contain much information, compared with current detection method, coincidence rate higher (seeing Fig. 3,5).In experimentation, also find, conventionally IgG concentration is in the time of 0.5～2.5mg/ml, fluorescent value reaches capacity, and the glass substrate of applying the APES of being coated with layer described in the utility model is in the time that IgG concentration is 0.5mg/ml, fluorescent value just reaches capacity, and IgG concentration is between 0.05～0.5 time, fluorescence intensity and IgG concentration keep good linear relationship (seeing Fig. 4), prove slide, aldehyde radical-H that the more conventional polylysine modification slide of the detection sensitivity of APES slide, aldehyde radical-PBS modify
2the slide that O modifies is high, has clinical value significantly.
Brief description of the drawings
Fig. 1: the audio-visual picture of the chip that simultaneously detects various autoimmune antibody described in the utility model, wherein, 1 is glass substrate; 2 is coated areas.
Fig. 2: on the chip that simultaneously detects various autoimmune antibody described in the utility model, put coating layout viewing, wherein, described some coated areas arrange into:
A1, B1, C1, D1, E1 is autoantigen ANAs point coating detection zone;
A2, B2, C2, D2, E2 is autoantigen dsDNA point coating detection zone;
A3, B3, D3, D3, E3 is autoantigen SSA/Ro-60 point coating detection zone;
A4, B4, D4, D4, E4 is autoantigen SSA/Ro-52 point coating detection zone;
A5, B5, D5, D5, E5 is autoantigen SSB/La point coating detection zone;
A6, B6, D6, D6, E6 is autoantigen Jo-1 point coating detection zone;
A7, B7, D7, D7, E7 is autoantigen cuorin point coating detection zone;
A8, B8, the positive contrast of D8 IgG point coating detection zone;
A9, B9, a coating detection zone, the negative contrast of D9 human osteogenic protein-7;
D8, E8, D9, E9 is blank PBST point coating detection zone.
The slide that Fig. 3 .APES modifies is fixed autoantigen, prepares the point sample distribution plan of microarray
Wherein: 1. positive control; 2. negative control; 3. ANAs; 4. dsDNA; 5. Ro-60/SSA; 6. Ro-52/SSA; 7. La/SSB; 8. Jo-1; 9. cuorin; 10. blank PBST.Each sample has four repetitions.
The linear relationship of the each IgG concentration of Fig. 4 .APES slide and fluorescence intensity.
Fig. 5. prepare the result images of Ag-Ab micro-array chip detection rheumatisant serum with APES slide
Wherein: the clinical current detection method testing result of A: dsDNA+, anti-phospholipid antibody (Acl) IgG+; Micro-array chip testing result: dsDNA+, anti-phospholipid antibody (Acl) IgG+.
The clinical current detection method testing result of B: ANA+, dsDNA+, Acl IgG+, SSA+, SSB+; Micro-array chip testing result: ANA+, dsDNA+, Acl IgG+, SSA+, SSB+.
The clinical current detection method testing result of C: ANA+, dsDNA+, Acl IgG+, SSA+, SSB+; Micro-array chip testing result: ANA+, dsDNA+, Acl IgG+, SSA+, SSB+, Jo-1+.
Embodiment
Embodiment 1
Below in conjunction with the audio-visual picture that detects the chip of various autoimmune antibody described in Fig. 1 simultaneously, and described in Fig. 2, detect on the chip of various autoimmune antibody simultaneously and put coating layout viewing, content of the present utility model is further elaborated, but described example content does not limit the utility model:
The chip that simultaneously detects various autoimmune antibody described in the utility model, is distributed in seven kinds of autoantigen point coatings on substrate and the some coating of positive control, negative control and blank forms by glass substrate and array;
Wherein:
Described glass substrate is the glass sheet that there is 3-aminopropyl triethoxysilane (APES) decorative layer on surface, and shape is rectangle, and area is 25.4mm × 76.2mm, and thickness is 1.2mm; Described some coating be shaped as circle, coated areas is established 1; The point coating parallel vertical of every coated areas is arranged, horizontal is nine rows, indulging is five row, is made up of the negative control point coated areas of the positive control point coated areas of the autoantigen point coating detection zone of seven groups of 5 repetitions, 1 group of 3 repetition, 1 group of 3 repetition, 1 group 4 some coating blank districts; Wherein said seven kinds of autoantigens are respectively ANAs, dsDNA, SSA/Ro-60, SSA/Ro-52, SSB/La, Jo-1, cuorin, and positive control is IgG, and negative control is human osteogenic protein-7, and blank is PBST.On chip, the some coating of coated areas is arranged specifically:
A1, B1, C1, D1, E1 is autoantigen ANAs point coating detection zone;
A2, B2, C2, D2, E2 is autoantigen dsDNA point coating detection zone;
A3, B3, D3, D3, E3 is autoantigen SSA/Ro-60 point coating detection zone;
A4, B4, D4, D4, E4 is autoantigen SSA/Ro-52 point coating detection zone;
A5, B5, D5, D5, E5 is autoantigen SSB/La point coating detection zone;
A6, B6, D6, D6, E6 is autoantigen Jo-1 point coating detection zone;
A7, B7, D7, D7, E7 is autoantigen cuorin point coating detection zone;
A8, B8, the positive contrast of D8 IgG point coating detection zone;
A9, B9, a coating detection zone, the negative contrast of D9 human osteogenic protein-7;
D8, E8, D9, E9 is blank PBST point coating detection zone.
Above-mentioned circular some coating diameter is 0.15mm, and dot spacing is 0.5mm, and the area of some coating microarray is 3.2mm × 6.4mm.
Preparation and the application of embodiment 2 chip that simultaneously detects various autoimmune antibody described in the utility model
(1) slide is embathed with 1N NaOH and the concentrated sulphuric acid respectively, distilled water rinses to slide cleaning, and hydro-extractor dries;
(2) above-mentioned clean slide is put into the APES that volume ratio is the acetone diluted of 1:53, stopped for 25～35 seconds;
(3) take out slide, slightly stop a moment, control dry drop, reenter pure acetone solution and rinse unconjugated APES;
(4) the slide of modifying using above-mentioned APES is as substrate, and fixing protein, nucleic acid and phosphatide preparation detect the Ag-Ab micro-array chip of rheumatisant's serum.
By seven kinds of autoantigens, be ANAs (300 μ g/ml), dsDNA (200 μ g/ml), SSA/Ro-60 (100 μ g/ml), SSA/Ro-52 (200 μ g/ml), SSB/La (300 μ g/ml), Jo-1 (200 μ g/ml), on the slide that cuorin (1000 μ g/ml) is modified at APES with Cartisian point sample instrument point, positive control is the IgG with the 10mg/ml of same sampling liquid dilution, negative control is human osteogenic protein-7 (BMP-7), blank is that PBST (contains NaCl 8.0g in every liter, KCl0.20g, Na2HPO41.44g, KH2PO40.24g, Tween-201ml), on the same slide of modifying at APES with Cartisian point sample instrument point.The point coating in sample coatings district is arranged and is seen Fig. 2.
Above-mentioned micro-array chip temperature in 37 DEG C of hybridizing boxes is incubated to 2h, confining liquid (0.01mol/L PBS, containing 1%BSA, 2.5% sucrose) sealing 30min, PBS washes three times, each 15 seconds, hydro-extractor dries, in each array, add 3 μ L PBS by rheumatisant's serum of 1:2 dilution, in 37 DEG C of hybridizing boxes, temperature is incubated 30min, PBS washes three times, each 15 seconds, hydro-extractor dries, in each array, add the goat anti-human igg of 3 μ L Cy3 marks, 37 DEG C of temperature are incubated 30min, PBS washes three times, each 15 seconds, hydro-extractor dries, scan with ScanArray4000, the parameter of scanner: Laser power and PMT are and are set as 85%, image saves as TIEF file.
Experimental result shows: seven kinds of autoantigens including protein, phosphatide, nucleic acid can be fixed on the slide of APES modification, the micro-array chip of preparing with this slide detects rheumatisant's serum, highly sensitive, contain much information, compared with current detection method, coincidence rate is higher, has clinical value significantly.
Claims (1)
1. detect a chip for various autoimmune antibody simultaneously, be distributed in seven kinds of autoantigen point coatings on substrate and the some coating of positive control, negative control and blank forms by glass substrate and array;
Wherein, described glass substrate is rectangle, and area is 73mm～77mm × 25mm～27mm, and thickness is 0.6mm～2.0mm; Described some coating be shaped as circle, coated areas is established 1～2, the spot diameter of each some coated areas is 0.15mm, dot spacing is 0.5mm;
It is characterized in that:
There is one deck 3-aminopropyl triethoxysilane overlayer on the surface of described glass substrate; Seven kinds of autoantigen point coatings that described array is distributed on substrate are respectively ANAs, dsDNA, SSA/Ro-60, SSA/Ro-52, SSB/La, Jo-1, cuorin, positive control point coating is IgG, negative control point coating is human osteogenic protein-7, and blank point coating is PBST; Point coating parallel vertical in each some coated areas is arranged, horizontal is nine rows, indulging is five row, is made up of the negative control point coated areas of the positive control point coated areas of the autoantigen point coating detection zone of seven groups of 5 repetitions, 1 group of 3 repetition, 1 group of 3 repetition, 1 group of 4 blank point coated areas.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420051743.7U CN203909043U (en) | 2014-01-27 | 2014-01-27 | Chip for detecting multiple autoimmune antibodies simultaneously |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201420051743.7U CN203909043U (en) | 2014-01-27 | 2014-01-27 | Chip for detecting multiple autoimmune antibodies simultaneously |
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| CN203909043U true CN203909043U (en) | 2014-10-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105823873A (en) * | 2015-07-03 | 2016-08-03 | 深圳市亚辉龙生物科技股份有限公司 | Immunoblotting kit for detecting autoimmune nephrosis and preparation method thereof |
| CN113238039A (en) * | 2018-03-07 | 2021-08-10 | 深圳市伯劳特生物制品有限公司 | Detection kit |
-
2014
- 2014-01-27 CN CN201420051743.7U patent/CN203909043U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105823873A (en) * | 2015-07-03 | 2016-08-03 | 深圳市亚辉龙生物科技股份有限公司 | Immunoblotting kit for detecting autoimmune nephrosis and preparation method thereof |
| CN113238039A (en) * | 2018-03-07 | 2021-08-10 | 深圳市伯劳特生物制品有限公司 | Detection kit |
| CN113238039B (en) * | 2018-03-07 | 2023-03-31 | 深圳市伯劳特生物制品有限公司 | Detection kit |
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| C14 | Grant of patent or utility model | ||
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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141029 Termination date: 20200127 |
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| CF01 | Termination of patent right due to non-payment of annual fee |