CN102132159B - Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof - Google Patents
Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof Download PDFInfo
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Abstract
An immunoassay chip for fish lymphocystis disease virus is provided. The chip comprises a chip carrier, an agarose gel layer covering the chip carrier and multiple antibody microarrays immobilized on the agarose gel layer. Chip barriers or line drawn by Super PAP Pen are provided between respective microarrays so as to separate them from each other. Antigen is detected by a sandwich method, which includes immobilizing antibodies specific to the pathogen on a chip substrate, preparing a sample solution to be detected with a target organ infected by virus, incubating the sample solution to be detected and the chip with antibodies specific to the pathogen, then adding specific monoclonal antibody probes with fluorescent labels, and obtaining the result from a CCD scanner. The method is suitable for simultaneously detecting lymphocystis disease virus in multiple samples or different tissues in one sample.
Description
Technical field
The present invention relates to the improvement of marine cultured animal cause of disease detection technique, be specifically related to a kind of for detection of fish lymphocystis disease virus (Lymphocystis disease virus, LCDV) immune detection chip or microarray and preparation method thereof and application, it belongs to immunology, virology and biochip interleaving techniques field.
Background technology
Fish lymphocyst disease is a kind of chronic disease viral disease, and the epidermis of ill fish, fin and afterbody etc. locate to occur the cauliflower-like tumour, affects its commodity value or causes fish dead.The cause of disease of lymphocyst disease is lymphocystis disease virus (LCDV), belongs to Iridoviridae (Iridoviridae).The sick Prevalent district of lymphocyst is wider, is worldwide distribution, and seawater, salt water and the brackishwater fishes of known at least 42 sections more than 125 kinds can be infected by LCDV at present.From lefteye flonder lymphocystic disease in 1997 since China breaks out first on a large scale, this disease all occured in the grouper of the provinces such as China Shandong, Hebei, Guangdong, Zhejiang cultivation, porgy, U.S. snapper, the flat Rockfish of Xu Shi, cobio etc., and the direct and indirect economic loss that causes reaches 10,000,000,000 yuan.The same with other virosis, the fish lymphocyst lacks effective medicine.LCDV infects and to have long characteristics in latent period, makes LCDV very easily along with parent population and fry exchanging and propagate between different regions, therefore, is badly in need of the quick and precisely detection technique of LCDV in the breeding production, early finds the purpose of early preventing in the hope of reaching.
The method that detects fishes virus both at home and abroad roughly comprises electron microscopy, PCR method, dna probe hybridization in situ, immunofluorescence antibody technique, immunohistochemistry technology, enzyme-linked immuno-sorbent assay and western dot blot etc.But these detection technique limitation are large, and how sensitivity, specificity, accuracy can not be taken into account, and can not be used for simultaneously parallel detection of Multi-example.Therefore, it is imperative to set up a kind of detection diagnostic techniques easy, quick, accurate, the Multi-example parallel processing that is applicable to the fish lymphocystis disease virus, and the protein chip technology that new development is got up provides strong technology platform for it, but but the not only reagent of conserve expensive but also the express-analysis of binding kinetics of the reaction of traceization, provide fast means of testing of a cheapness at protein level, had quite wide application prospect at diagnostic field.In addition, the successful development of fish lymphocystis disease virus monoclonal antibody is laid a good foundation for the immuno-chip that utilizes monoclonal antibody to set up fish lymphocyst disease detects diagnostic techniques.
Summary of the invention
For above-mentioned present situation, the purpose of this invention is to provide a kind of immune detection chip that can detect simultaneously lymphocystis disease virus in several samples or the same sample different tissues, be used for quick, sensitive, the accurately detection of marine fish LCDV, and the quarantine of importing and exporting LCDV in the fish.
Another object of the present invention provides the preparation method of above-mentioned fish LCDV immune detection chip.
A further object of the invention provides the application of above-mentioned immune detection chip in cultured fishes LCDV detects.
The objective of the invention is to be realized by following technical scheme:
A kind of fish lymphocystis disease virus (LCDV) immunity detection chip, its structure comprises chip carrier, spread the Ago-Gel layer be overlying on the chip carrier, be fixed with a plurality of Antibody microarrays on the Ago-Gel layer, rules off with chip fence special or Super PAP Pen between each microarray.A liquid: osmotic pressure is less than the hypotonic medium of 360mOsm/kg; B liquid: tween-phosphate buffer (PBST); C liquid: monoclonal antibody (monoclonal antibody) probe of the mouse-anti LCDV of Cy3 mark, the used monoclonal antibody of probe is the anti-LCDV monoclonal antibody of specificity (being called for short: LCDV monoclonal antibody B and monoclonal antibody C) of being secreted respectively by two strain of hybridoma, the preserving number of hybridoma is respectively: CCTCC-C200419 and CCTCC-C200420, preservation date: on Dec 14th, 2004.The antigenic determinant of LCDV and this two strains monoclonal antibody generation specific binding is positioned on the virus envelope.
The present invention is with the actual features of prior art: one, the formation of immunoglobulin (Ig)-IgM is arranged in the Fish, but the mark program of IgM is comparatively complicated in the serum; Its two, be the concept of a colony to the diagnosis of marine cultured animal disease, can reach by the detection to a plurality of individualities the purpose of colony being made diagnosis.Therefore, the present invention is according to these characteristics, adopt the sandwich method detectable antigens, at the fixing antibody of cause of disease of chip slapper base, get to be checked precursor virus target organ and prepare sample liquid to be checked, directly analyte sample fluid and the chip that is fixed with pathogenic autoantibody are hatched, add again fluorescently-labeled specific monoclonal antibody probe, by the ccd scanner reading result.
The preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip comprises the steps:
(1) preparation of antibody and purifying
From the lefteye flounder superficial tumor of suffering from the lymphocyst disease, extract LCDV, the purebred New Zealand white rabbit of conventional method immunity, blood sampling preparation serum obtains the anti-LCDV antibody of rabbit.
Recovery is also cultivated mouse hybridoma cell strain (LCDV monoclonal antibody B and monoclonal antibody C), and the hybridoma of injection some enters mouse peritoneal production ascites, obtains the mouse-anti LCDV monoclonal antibody of a large amount of high concentrations, high specific, high-titer.
Get LCDV monoclonal antibody B and monoclonal antibody C behind the affinitive layer purification, the purebred New Zealand white rabbit of conventional method immunity, blood sampling preparation serum obtains the antibody (antibody of the anti-mouse Ig of rabbit) of the anti-mouse-anti LCDV of rabbit monoclonal antibody.
Use affinity column (HiTrap Protein G SepharoseColumn) the purifying gained antibody of Amersham Phamacia Biotech company.
(2) preparation of the monoclonal antibody probe of Cy3 mark
According to the product description of Amersham Phamacia Biotech company, the mouse-anti LCDV monoclonal antibody after using fluorescein Cy3 to affinitive layer purification carries out mark, and crosses the gel column purifying.
(3) pre-service of microslide
Microslide is embathed with highly basic and the concentrated sulphuric acid respectively, and the distilled water flushing is dried; Microslide after cleaning immersed in the acetic acid solution of 0.4% affine silane, transfer pH to 4.5, room temperature effect 1h, the distilled water flushing is dried.
(4) preparation of Ago-Gel substrate
Preparation 1.2% agarose solution, micro-wave oven boils 3min, and it is dissolved fully, and the 2mL agarose solution is spread on the cleaning slide that the affine silane treatment that overlays on 60 ℃ of preheatings crosses; After agarose solidified, slide was 37 ℃ of lower dried overnight; Use 0.02mol/L NaIO before using
4Activate 30min under the solution room temperature, ultrapure water cleaning down 3 times, and dry up with nitrogen stream, the drying at room temperature place preserves.
(5) antibody is fixing
1. making its concentration with the anti-LCDV antibody of the phosphate buffer that contains 50% glycerine (PBS) of pH7.4 dilution rabbit is 0.5～0.0001mg/mL; It is 0.1mg/mL that the antibody of the anti-mouse Ig of dilution rabbit makes its concentration.
2. with point sample instrument with the zones of different point sample of this antibody diluent in the slide surface of modified, the point sample amount is 50～70nL, spot diameter is 500～600 μ m.Every chip two rows four are listed as totally 84 * 4 inferior arrays, sample that each inferior array is put is consistent, sample that first row is selected is that phosphate glycerine damping fluid is as negative control, second and third row sample of putting is the anti-LCDV antibody of rabbit, and the 4th row sample of putting is that the antibody of the anti-mouse Ig of rabbit reaches fixing contrast as positive control.Separate with chip fence special or Super PAP Pen between each inferior array, form independently reaction member.37 ℃ of saturated humidities are placed 2h, and washing is dried.
3. have the zone of antibody to drip the sealing of 3% bovine serum albumin(BSA) at microslide point, 37 ℃ of saturated humidities are placed 1h, and washing is dried, and 4 ℃ of sealings are preserved, and obtain fish lymphocystis disease virus (LCDV) immunity detection chip.
The application of fish lymphocystis disease virus (LCDV) immunity detection chip of the present invention, described this applying step is as follows:
(1) for detection of Fish Tissue comprise: fish body surface knob, epidermis, spleen, stomach, intestines etc., sampling mixes in the ratio of 1: 10 (W/V) approximately with A liquid, homogenate, multigelation 3～4 times, after the ultrasonication, low-temperature centrifugation, 5000rpm, 15min gets supernatant as test sample, and is to be checked.
(2) difference sample to be checked is added in the different inferior array of said chip, the application of sample amount is 10 μ L/ arrays, and a chip can detect eight samples simultaneously, notes avoiding the liquid between different inferior arrays to mix.37 ℃ of saturated humidities are placed 0.25～2h, wash, and add the C liquid (the mouse-anti LCDV monoclonal antibody of Cy3 mark) of dilution again, 10 μ L/ arrays, and 37 ℃ of saturated humidities are placed 0.25～2h, and washing is dried.
(3) laser scanning
The brilliant core of above-mentioned detection chip
EcoScan
TM-100 ccd scanner scanning imageries, excitation wavelength are 532nm, and the detection wavelength is 585nm, fluorescence signal Lab-chipscanner 2.0 software analysis.
Analysis of test results and judgement: in the inferior array of the image that scanning obtains, the background values size of the signal intensity representative experiment of first row negative control; Second and third lists the positive of existing green fluorescence bright spot, and showing in institute's test sample has LCDV, and redgreen fluorescence bright spot negative shows in institute's test sample without LCDV.Signal is strong and weak relevant with virus concentration in the sample; The 4th row positive control and qualifying point green fluorescence occurs for normal, if there is not green fluorescence, illustrate that detecting operation has problem or antibody protein generation sex change, and then testing result is invalid.
(4) quantitative detection and the lowest detectable limit of virus
LCDV is diluted to 25.6 μ g/mL after the separation and purification, again by 8 antigen concentrations of two doubling dilution gained respectively with microslide on 8 inferior arrays hatch, detect through antibody probe, the result as shown in Figure 3 behind the scanning analysis, change with antigen concentration, fluorescence signal presents the variation of gradient.Take the logarithm of antigen concentration as horizontal ordinate, change take fluorescence signal intensity as the ordinate analyte signal intensity, the result shows that antigen concentration is in 0.8～12.8 μ g/mL scope, preferably linear relationship is arranged between relative signal intensity and the antigen concentration, point out constructed Antibody microarray in certain virus concentration scope, can carry out quantitative test.The minimum antigen concentration that this Antibody microarray can detect is 1.6 μ g/mL.
Described chip carrier has through after the affine silane treatment, and the glass surface of modifying with Ago-Gel, and this surface is three-dimensional porous structure, through NaIO
4After the activation, can make antibody fixed thereon with physisorption and covalently bound dual mode, simultaneously, its hydrophilic environment also helps the activity that remains fixed in top antibody protein.Described antibody is the antibody of the anti-LCDV antibody of rabbit, the anti-mouse Ig of rabbit, but is not limited only to this two kinds of antibody.
Be used for that the anti-LCDV antibody of rabbit optimum concentration is 0.5mg/mL behind the affinitive layer purification of point sample.
After described sample to be checked was added on the chip, 37 ℃ of saturated humidities were placed 30min, add the antibody probe of dilution on the slide after, place 30min under 37 ℃ of saturated humidities.
Individual slide point of described chip has 8 inferior arrays, can increase according to actual needs inferior array quantity, detects when can realize more Multi-example.
The invention has the advantages that the LCDV in a plurality of different tissues that can detect simultaneously a plurality of samples or same individuality, it is simple in structure, with low cost, flux is easy to expand, Parallel testing when realizing Multi-example, increase the comparability between the different specimens data, can detect fast, accurately, easily the LCDV in the multiple cultured fishes body.Simple to sample requirement, a small amount of sample can satisfy the detection needs, and has greatly simplified the sample preparation step of existing detection technique, even the detection of can drawing materials in the situation of not killing cultivated animals.Simultaneously because positive control and the setting of negative control and the repetition point sample of capture antibody increased the reliability of testing result.The immuno-chip reacting phase is to comparatively fast in addition, and operating process is convenient, and general personnel can operate, and can detect cause of disease by relative quantification, is applicable to the fast and accurately detection of virosis in the breeding process.
Description of drawings
Fig. 1. fish lymphocystis disease virus immuno-chip structural representation and point sample distribution plan.Wherein, 1-chip carrier, 2-Ago-Gel layer, 3-Antibody microarray, 4-chip fence special or Super PAP Pen line, 5-label area.Point sample distributes: 1. contain the PBS of 50% glycerine, as negative control; 2., 3. be the anti-LCDV antibody of rabbit; 4. the antibody of the anti-mouse Ig of rabbit reaches fixing contrast as positive control and waits quality control.
Fig. 2. fish lymphocystis disease virus (LCDV) immunity detection chip detects the CCD scintigram (fluorescence intensity of scanner is set as 88%) of LCDV in the separate sources fish sample.A1, A2 show and do not detect LCDV in the sample; A3, B3 show that LCDV concentration is higher in the sample; B2, B4 show and contain a certain amount of LCDV in the sample, but concentration is low than A3, B3; A4, B1 show that LCDV content is lower in the sample.
Fig. 3. the relation of antigen concentration and signal intensity and the detectability of this immuno-chip.The antigen concentration that detects is followed successively by 25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2 μ g/mL.Take the logarithm value of antigen concentration as horizontal ordinate, change take fluorescence signal intensity as the ordinate analyte signal intensity, the result shows that antigen concentration in 0.8～12.8 μ g/mL scope, has preferably linear relationship between relative fluorescence signal intensity and the antigen concentration.
Embodiment
Describe the present invention in detail below in conjunction with accompanying drawing and by specific embodiment.
Instrument of the present invention and reagent are as follows:
Small-sized hand-operated chip point sample system (available from Whatman company); Brilliant core
EcoScan
TM-100 ccd scanners (available from Beijing Bo Ao biotech firm); Cy3 antibody labeling kit (available from GE company); HiTrap Protein G SepharoseColumn (available from GE company); 1640 (available from GIBCO companies); Hyclone (available from HYCLONE company); Bovine serum albumin(BSA) (available from SIGMA company); Tween-20 (available from SIGMA company); Dimethyl sulfoxide (DMSO) (available from SIGMA company).
Embodiment 1:
1. the preparation of antibody and purifying
From the lefteye flounder body surface knob of suffering from lymphocyst, extract LCDV, the purebred New Zealand white rabbit of conventional method immunity, blood sampling preparation serum obtains the anti-LCDV antibody of rabbit.
Recovery is also cultivated mouse hybridoma cell strain monoclonal antibody B and monoclonal antibody C, and the hybridoma of injection some enters mouse peritoneal production ascites, obtains the mouse-anti LCDV monoclonal antibody of a large amount of high concentrations, high specific, high-titer.
The monoclonal antibody B and the monoclonal antibody C that get behind the affinitive layer purification mix, the purebred New Zealand white rabbit of conventional method immunity, and blood sampling preparation serum obtains the antibody of the anti-mouse Ig of rabbit.
Use affinity column (HiTrap Protein G SepharoseColumn) the purifying gained antibody of Amersham Phamacia Biotech company.
2.Cy3 the preparation of the monoclonal antibody probe of mark
According to the Cy3 product description of Amersham Phamacia Biotech company, the mouse-anti LCDV monoclonal antibody after using fluorescein Cy3 to affinitive layer purification carries out mark, and crosses the gel column purifying.
3. the pre-service of microslide
Slide is embathed with highly basic and the concentrated sulphuric acid respectively, and the distilled water flushing is dried; Slide after cleaning immersed in the acetic acid solution of 0.4% affine silane, transfer pH to 4.5, room temperature effect 1h, the distilled water flushing is dried.
4. the preparation of Ago-Gel substrate
Preparation 1.2% agarose solution, micro-wave oven boils 3min and dissolves fully, and the 2mL agarose solution is spread on the cleaning slide that the affine silane treatment that overlays on 60 ℃ of preheatings crosses; After agarose solidified, microslide was 37 ℃ of lower dried overnight; Use 0.02mol/L NaIO before using
4Activate 30min under the solution room temperature, ultrapure water cleaning down 3 times, and dry up with nitrogen stream, the drying at room temperature place preserves.
5. chip structure design and dot matrix distribute
This chip structure as shown in Figure 1, comprise chip carrier (1), spread the Ago-Gel layer (2) that is overlying on the chip carrier, be fixed with two rows, four row totally 84 * 4 Antibody microarray (3) on the Ago-Gel layer, the point sample amount is 50-70nL, and spot diameter is 500～600 μ m.Separate with chip fence special or Super PAP Pen line (4) between each microarray.Described chip carrier is the standard microslide, can reserve label area (5) at slide one end.
6. antibody is fixing
1. with the anti-LCDV antibody of PBS damping fluid dilution rabbit that contains 50% glycerine of pH 7.4, make that its concentration is 0.5,0.1,0.05,0.01,0.005,0.001,0.0005,0.0001mg/mL.
2. with point sample instrument with the zones of different point sample of this variable concentrations antibody diluent in the slide surface of modified, every chip two rows four are listed as totally 84 * 4 inferior arrays, each inferior array is the anti-LCDV antibody of the rabbit of variable concentrations, separate with chip fence special or Super PAP Pen between each inferior array, form independently reaction member.37 ℃ of saturated humidities are placed 2h, and washing is dried.
3. have the zone of antibody to drip 3% bovine serum albumin(BSA) at microslide point and seal, 37 ℃ of saturated humidities are placed 1h, and washing is dried, and 4 ℃ of sealings are preserved, and obtain fish LCDV immune detection chip.
Embodiment 2:
Step 1,2,3,4,5 is with embodiment 1.
6. antibody is fixing
1. with the anti-LCDV antibody of PBS damping fluid dilution rabbit that contains 50% glycerine of pH 7.4, making its concentration is 0.5mg/mL; It is 0.1mg/mL that the antibody of the anti-mouse Ig of dilution rabbit makes its concentration.
2. with point sample instrument with the zones of different point sample of this antibody diluent in the slide surface of modified, dot matrix distributes as shown in Figure 1, every chip two rows four are listed as totally 84 * 4 inferior arrays, sample that each inferior array is put is consistent, wherein, sample that first row is selected is phosphate glycerine damping fluid, as negative control; Second and third row sample of putting is the anti-LCDV antibody of rabbit; The 4th row sample of putting is that the antibody of the anti-mouse Ig of rabbit reaches the qualifying points such as fixing contrast as positive control.Separate with chip fence special Super PAP Pen between each inferior array, form independently reaction member.37 ℃ of saturated humidities are placed 2h, and washing is dried.
3. have the zone of antibody to drip 3% bovine serum albumin(BSA) at microslide point and seal, 37 ℃ of saturated humidities are placed 1h, and washing is dried, and 4 ℃ of sealings are preserved, and obtain fish LCDV immune detection chip.
Embodiment 3:
What detect the employing of fish lymphocystis disease virus is sandwich method.
Step 1,2,3,4,5,6 is with embodiment 2.
7. the detection of cause of disease
1. get Fish Tissue to be checked (epidermis, the gill, stomach or intestines etc.), mix in the ratio of 1: 10 (W/V) approximately with A liquid, homogenate, multigelation 3～4 times, after the ultrasonication, low-temperature centrifugation, 5000rpm * 15min gets supernatant as test sample, and is to be checked;
2. sample to be checked is added in the different inferior arrays of said chip identical carrier, the application of sample amount is 10 μ L/ arrays, and a chip can detect eight samples simultaneously, notes avoiding the liquid between different inferior arrays to mix.37 ℃ of saturated humidities are hatched 15min, 30min, 45min, 60min, 90min, 120min, wash, and add the C liquid of dilution at chip, 10 μ L/ arrays, 37 ℃ of saturated humidities are placed and are hatched 15min, 30min, 45min, 60min, 90min, 120min, and washing is dried;
3. laser scanning
The brilliant core of above-mentioned detection chip
EcoScan
TM-100 ccd scanner scanning imageries, excitation wavelength are 532nm, and the detection wavelength is 585nm, fluorescence signal Lab-chipscanner 2.0 software analysis.
Analysis of test results and judgement: in the inferior array of the image that scanning obtains, the background values size of the signal intensity representative experiment of first row negative control; Second and third lists the positive of existing green fluorescence bright spot, and showing in institute's test sample has LCDV, and redgreen fluorescence bright spot negative shows in institute's test sample without LCDV.Signal is strong and weak relevant with virus concentration in the sample; The 4th row positive control and point of fixity green fluorescence occurs for normal, if there is not green fluorescence, illustrate that detecting operation has problem or antibody protein generation sex change, and testing result is invalid.
Embodiment 4:
The quantitative test of antigen and lowest detectable limit:
Get the fish LCDV immune detection chip for preparing, the LCDV of separation and purification is diluted to 25.6 μ g/mL, again by 8 antigen concentrations of two doubling dilution gained respectively with slide on Antibody microarray hatch, after antibody probe detected, scanning result utilized software processing.The result shows, changes with antigen concentration, and fluorescence signal presents the variation of gradient.Take the logarithm value of antigen concentration as horizontal ordinate, change take fluorescence signal intensity as the ordinate analyte signal intensity, the result shows that antigen concentration is in 0.8～12.8g/mL scope, preferably linear relationship is arranged between relative signal intensity and the antigen concentration, point out constructed Antibody microarray in certain virus concentration scope, can carry out quantitative test.The minimum antigen concentration that this Antibody microarray can detect is 1.6 μ g/mL.
Embodiment 5:
The specific detection of fish LCDV immune detection chip:
Get normal fish tissue (gill, stomach, epidermis etc.), do not infect LCDV through the PCR detection, homogenate after the historrhexis, ultrasonication behind the multigelation, low temperature sedimentation 10min gets supernatant, hatch with the Antibody microarray on the fish lymphocystis disease virus (LCDV) immunity detection chip, scanning after antibody probe detects without fluorescence signal, confirms prepared fish lymphocystis disease virus (LCDV) immunity detection chip and organizes no cross reaction.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, it all is possible making amendment, add and replace for above-described embodiment, and it does not all exceed protection scope of the present invention.
Industrial applicibility
The present invention provides technology platform for the detection diagnosis of all kinds of cause of diseases such as cultured fishes virus, bacterium, be applicable to diseases monitoring in the breeding process and inspection and quarantining for import/export of aquatic livestock etc., have broad application prospects, can effectively avoid cultured fishes transmission of disease and popular.
Claims (3)
1. the preparation method of a fish lymphocystis disease virus (LCDV) immunity detection chip is characterized in that it comprises the steps:
(1) preparation of antibody
The antiangiogenic tumour antiviral antibody of preparation rabbit, the antibody of the anti-mouse Ig of rabbit, mouse-anti lymphocystis disease virus monoclonal antibody, affinity chromatography purifying gained antibody;
(2) preparation of Cy3 labeled monoclonal antibody probe
With the mouse-anti lymphocystis disease virus monoclonal antibody Cy3 mark behind the affinitive layer purification, and cross the gel column purifying;
(3) pre-service of microslide
Microslide is embathed with highly basic and the concentrated sulphuric acid respectively, and the distilled water flushing is dried; Microslide after cleaning immersed in the acetic acid solution of 0.4% affine silane, transfer pH to 4.5, act on 1h under the room temperature, the distilled water flushing is dried;
(4) preparation of Ago-Gel substrate
The agarose solution of preparation 1.2%, micro-wave oven boils 3min, and it is dissolved fully, the 2ml agarose solution is spread overlay on the cleaning microslide that the affine silane treatment of 60 ℃ of preheatings is crossed; After agarose solidifies, with slide 37 ℃ of lower dried overnight; At room temperature use 0.02mol/L NaIO before the use
4Solution activation 30min, ultrapure water flushing 3 times dries up with nitrogen stream, and the drying at room temperature place preserves;
(5) antibody is fixing
1. with the antiangiogenic tumour antiviral antibody of PBS damping fluid dilution rabbit that contains 50% glycerine of pH 7.4, making its concentration is the antibody of 0.5 ~ 0.0001mg/mL, the anti-mouse Ig of dilution rabbit, and making its concentration is 0.1mg/mL;
2. with point sample instrument with the zones of different point sample of this antibody diluent at the Ago-Gel substrate, the point sample amount is 50 ~ 70nL, spot diameter is 500 ~ 600 μ m; Every chip two rows four are listed as totally 84 * 4 inferior arrays, sample that each inferior array is put is consistent, sample that first row is selected is that phosphate glycerine damping fluid is as negative control, second and third row sample of putting is the antiangiogenic tumour antiviral antibody of rabbit, the 4th classifies qualifying point as, and the sample of putting is that the antibody of the anti-mouse Ig of rabbit reaches fixing contrast as positive control; Separate with chip fence special or Super PAP Pen between each inferior array, form independently reaction member; 37 ℃ of saturated humidities are placed 2h, and washing is dried;
3. point has the zone of antibody to drip 3% bovine serum albumin(BSA) to seal on the microslide, and 37 ℃ of saturated humidities are placed 1h, and washing is dried, and 4 ℃ of sealings are preserved, and obtain fish lymphocystis disease virus (LCDV) immunity detection chip.
2. the preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip according to claim 1, it is characterized in that with the mouse-anti lymphocystis disease virus monoclonal antibody behind the Cy3 mark affinitive layer purification as antibody probe, and the antibody that uses the anti-mouse Ig of rabbit is as positive control and fixing contrast.
3. the preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip according to claim 1, it is characterized in that the used monoclonal antibody of Cy3 labelled antibody probe is the antiangiogenic tumour virus envelope of specificity monoclonal antibody B and the monoclonal antibody C that is secreted respectively by two strain of hybridoma, its preserving number is respectively: CCTCC-C200419 and CCTCC-C200420.
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| CN201080002349.9A CN102132159B (en) | 2009-08-07 | 2010-08-03 | Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof |
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| CN200910017833A CN101629955B (en) | 2009-08-07 | 2009-08-07 | Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof |
| CN201080002349.9A CN102132159B (en) | 2009-08-07 | 2010-08-03 | Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof |
| PCT/CN2010/075669 WO2011015131A1 (en) | 2009-08-07 | 2010-08-03 | Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof |
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| CN201080002349.9A Expired - Fee Related CN102132159B (en) | 2009-08-07 | 2010-08-03 | Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof |
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| US20180011120A9 (en) * | 2006-08-03 | 2018-01-11 | AyoxxA Biosystems GmbH | Source controlled decodable microarray system and methods for use |
| CN101629955B (en) * | 2009-08-07 | 2012-09-26 | 中国海洋大学 | Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof |
| CN101893632A (en) * | 2010-07-29 | 2010-11-24 | 中国海洋大学 | Test paper for detecting fish lymphocystis disease virus and preparation method thereof |
| CN101906486B (en) * | 2010-08-03 | 2012-10-17 | 中国水产科学研究院黄海水产研究所 | Gene chip for detecting various fish pathogens and detecting method thereof |
| CN103439514B (en) * | 2013-08-05 | 2015-06-10 | 徐州工程学院 | Pesticide and veterinary drug multi-residue detection method based on microarray detection chip |
| CN112834737B (en) * | 2020-12-30 | 2023-12-22 | 佳维斯(武汉)生物医药有限公司 | Accurate immunofluorescence labeling method for whole tissue sample |
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| WO2011015131A1 (en) | 2011-02-10 |
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