CN101629955B - Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof - Google Patents
Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application thereof Download PDFInfo
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- CN101629955B CN101629955B CN200910017833A CN200910017833A CN101629955B CN 101629955 B CN101629955 B CN 101629955B CN 200910017833 A CN200910017833 A CN 200910017833A CN 200910017833 A CN200910017833 A CN 200910017833A CN 101629955 B CN101629955 B CN 101629955B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/56983—Viruses
Abstract
The invention discloses a fish lymphocystis disease virus (LCDV) immunity detection chip, and the structure of the chip comprises a chip carrier, an agarose gel layer covered on the chip carrier and a plurality of antibody microarrays fixed on the agarose gel layer; wherein, special fences for chips or Super PAP Pen rulings are arranged between the microarrays for separating the microarrays. In the invention, a sandwich method is adopted to detect antigen, pathogenic antibody is fixed on a film support of the chip, the to-be-detected sample solution is prepared by using virus target organ of a sample to be detected, the to-be-detected sample solution and the chip fixed with pathogenic antibody are directly incubated, a specific monoclonal antibody (McAb) probe with a fluorescent mark is added, and then a CCD scanner is used for reading the result. The invention has the advantages of detecting lymphocystis disease virus in various samples or different tissues in the same sample simultaneously, which can be used for rapid, sensitive, and accurate detection on LCDV in marine-culturing fishes and quarantine on LCDV in imported-exported fishes.
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 fish lymphocystis disease virus (Lymphocystis disease virus that is used to detect; 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, influences its commodity value or causes fish dead.The sick cause of disease of lymphocyst is lymphocystis disease virus (LCDV), belongs to Iridoviridae (Iridoviridae).The sick popular area 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.Since 1997 Flounder Lymphocystis first large-scale outbreak of the disease in our country, China's Shandong, Hebei, Guangdong, Zhejiang and other provinces farming grouper, sea bream, American red snapper, rockfish Hsu, cobia etc. This happened disease, caused by the direct and indirect economic losses amounting to ten billion yuan is huge.The same with other virosis, the fish lymphocyst lacks the efficacious therapy 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 detection technique quick and precisely of LCDV in the breeding production, early finds the purpose of early preventing in the hope of reaching.
The method that detects fish virus both at home and abroad roughly comprises electron microscopy, PCR method, dna probe in situ hybridization method, immunofluorescence antibody technique, immunohistochemistry technology, enzyme-linked immuno-sorbent assay and spot immune engram technology etc.But these detection technique limitation are big, and how sensitivity, specificity, accuracy can not be taken into account, and can not be used for the parallel detection simultaneously of various article.Therefore; The detection diagnostic techniques of setting up a kind of easy, quick, accurate, various article parallel processing that is applicable to the fish lymphocystis disease virus is imperative; 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 provides cheapness means of testing fast on protein level, have 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 utilizing monoclonal antibody to set up the sick immuno-chip detection diagnostic techniques of fish lymphocyst.
Summary of the invention
To above-mentioned present situation; The purpose of this invention is to provide a kind of immune detection chip that can detect lymphocystis disease virus in several samples or the same sample different tissues simultaneously; Be used for quick, sensitive, the detection accurately 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 realize by following technical scheme:
A kind of fish lymphocystis disease virus (LCDV) immunity detection chip; Its structure comprises that chip carrier, shop are overlying on Ago-Gel layer on the chip carrier, the Ago-Gel layer and is fixed with a plurality of antibody microarraies, rules off with chip special use fence 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 specific anti LCDV monoclonal antibody of being secreted respectively by two strain of hybridoma (being called for short: LCDV monoclonal antibody B and monoclonal antibody C); The preserving number of hybridoma is respectively: CCTCC-C200419 and CCTCC-C200420, preservation date: on Dec 14th, 2004.LCDV is positioned on the virus envelope with the antigenic determinant that this two strains monoclonal antibody generation specificity combines.
The present invention is with the actual features of prior art: one of which, the formation of immunoglobulin (Ig)-IgM is arranged in the fish body, but the mark program of IgM is comparatively complicated in the serum; Its two, be the notion of a colony to the diagnosis of marine cultured animal disease, can reach the purpose of colony being made diagnosis through detection to a plurality of individualities.Therefore; The present invention is according to these characteristics, adopts sandwich method to detect antigen, the fixing antibody of cause of disease on the 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 fluorescently-labeled specific monoclonal antibody probe again, read the result through ccd scanner.
The preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip comprises the steps:
(1) preparation of antibody and purifying
From suffer from the sick lefteye flounder superficial tumor of lymphocyst, extract LCDV, the purebred NZw of conventional method immunity, blood sampling preparation serum obtains the anti-LCDV antibody of rabbit.
Mouse hybridoma cell strain (LCDV monoclonal antibody B and monoclonal antibody C) is also cultivated in recovery, injects the hybridoma of some and goes into mouse peritoneal production ascites, obtains the mouse-anti LCDV monoclonal antibody that great deal of high concentration, high specific, height are tired.
Get LCDV monoclonal antibody B and monoclonal antibody C behind the affinitive layer purification, the purebred NZw 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 luciferin Cy3 to affinitive layer purification carries out mark, and crosses the gel column purifying.
(3) pre-service of microslide
Microslide is embathed with the 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 2mL agarose solution shop is overlayed on the cleaning slide that the affine silane treatment of 60 ℃ of preheatings crosses; After agarose solidified, slide was 37 ℃ of following dried overnight; Use 0.02mol/L NaIO before using
4Activation 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; The first row sample of selecting 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 special-purpose fence of chip 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 the sealing of 3% bovine serum albumin(BSA) on 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.
The application of fish lymphocystis disease virus (LCDV) immunity detection chip of the present invention, said this applying step is following:
(1) the fish tissue that is used to detect comprises: fish body surface knob, epidermis, spleen, stomach, intestines etc., sampling and A liquid are approximately by the mixed of 1: 10 (W/V), 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 mixing between different inferior arrays.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
Above-mentioned detection chip is with brilliant
EcoScan
TMThe imaging of-100CCD scanner scanning, excitation wavelength is 532nm, and the detection wavelength is 585nm, and fluorescence signal is with Lab-chipscanner 2.0 software analysis.
The testing result Analysis And Evaluation: in the inferior array of the image that scanning obtains, the background values size of the signal intensity representative experiment of the 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, redgreen fluorescence bright spot negative, and showing in institute's test sample does not have LCDV.Signal strong and weak with sample in virus concentration relevant; The 4th row positive control and qualifying point green fluorescence occurs for normal, if there is not green fluorescence, explain that detecting operation has problem or antibody protein generation sex change, and then testing result is invalid.
(4) detection by quantitative and the LDL 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 is as shown in Figure 3 behind the scanning analysis; Change with antigen concentration, fluorescence signal presents the variation of gradient.Logarithm with antigen concentration is a horizontal ordinate; Change for the ordinate analyte signal intensity with the fluorescence signal intensity; The result shows that antigen concentration is in 0.8～12.8 μ g/mL scope; The better linearity relation 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.This antibody microarray institute can detected minimum antigen concentration be 1.6 μ g/mL.
Said chip carrier has through after the affine silane treatment, and the glass surface of modifying with Ago-Gel, and this surface is a 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, 37 ℃ of saturated humidity held 30min.
Individual slide point of said chip has 8 inferior arrays, can increase inferior array quantity according to actual needs, detects when can realize more various article.
The invention has the advantages that the LCDV in a plurality of different tissues that can detect a plurality of samples or same individuality simultaneously; It is simple in structure; With low cost, flux is easy to expand, parallel detection when realizing various article; Increase the comparability between the different specimens data, can detect the LCDV in the multiple cultured fishes body fast, accurately, easily.Simple to sample requirement, small amount of sample can satisfy the detection needs, and has simplified the sample preparation step of existing detection technique greatly, even the detection of can under the situation of not killing cultivated animals, drawing materials.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 accurate detection of virosis in the breeding process.
The present invention also provides technology platform for the detection diagnosis of all kinds of cause of diseases such as cultured fishes virus, bacterium in addition; Be applicable to diseases monitoring and inspection and quarantining for import/export of aquatic livestock in the breeding process etc.; Wide application prospect is arranged, can effectively avoid cultured fishes transmission of disease and popular.
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, special-purpose fence of 4-chip 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 at 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 is detected is followed successively by 25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2 μ g/mL.Logarithm value with antigen concentration is a horizontal ordinate, and for the ordinate analyte signal intensity changes, the result shows antigen concentration in 0.8～12.8 μ g/mL scope with the fluorescence signal intensity, and the better linearity relation is arranged between relative fluorescence signal intensity and the antigen concentration.
Embodiment
Specify the present invention below in conjunction with accompanying drawing and through specific embodiment.
Used instrument of the present invention and reagent are following:
Small-sized hand-operated chip point sample system (available from Whatman company); Brilliant
EcoScan
TM-100CCD scanner (available from Beijing Bo Ao biotech firm); Cy3 antibody labeling kit (available from GE company); HiTrap Protein GSepharose Column (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 NZw of conventional method immunity, blood sampling preparation serum obtains the anti-LCDV antibody of rabbit.
Mouse hybridoma cell strain monoclonal antibody B and monoclonal antibody C are also cultivated in recovery, inject the hybridoma of some and go into mouse peritoneal production ascites, obtain the mouse-anti LCDV monoclonal antibody that great deal of high concentration, high specific, height are tired.
The monoclonal antibody B and the monoclonal antibody C that get behind the affinitive layer purification mix, the purebred NZw 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 luciferin Cy3 to affinitive layer purification carries out mark, and crosses the gel column purifying.
3. the pre-service of microslide
Slide is embathed with the 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 2mL agarose solution shop is overlayed on the cleaning slide that the affine silane treatment of 60 ℃ of preheatings crosses; After agarose solidified, microslide was 37 ℃ of following dried overnight; Use 0.02mol/L NaIO before using
4Activation 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 is as shown in Figure 1; Comprise that chip carrier (1), shop are overlying on the Ago-Gel layer (2) 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 special-purpose fence of chip 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 special-purpose fence of chip 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 microslide, and 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:
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, and sample that each inferior array is put is consistent, wherein; The first row sample of selecting is a 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 qualifying points such as fixing contrast as positive control.Separate with the special-purpose fence Super of chip 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 microslide, and 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.
7. the detection of cause of disease
1. get fish tissue to be checked (epidermis, the gill, stomach or intestines etc.), with the mixed that A liquid is pressed 1: 10 (W/V) approximately, 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 mixing between different inferior arrays.37 ℃ of saturated humidities are hatched 15min, 30min, 45min, 60min, 90min, 120min, wash, and on chip, add the C liquid of dilution; 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
Above-mentioned detection chip is with brilliant
EcoScan
TMThe imaging of-100CCD scanner scanning, excitation wavelength is 532nm, and the detection wavelength is 585nm, and fluorescence signal is with Lab-chipscanner 2.0 software analysis.
The testing result Analysis And Evaluation: in the inferior array of the image that scanning obtains, the background values size of the signal intensity representative experiment of the 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, redgreen fluorescence bright spot negative, and showing in institute's test sample does not have LCDV.Signal strong and weak with sample in virus concentration relevant; The 4th row positive control and point of fixity green fluorescence occurs for normal, if there is not green fluorescence, explain that detecting operation has problem or antibody protein generation sex change, and testing result is invalid.
Embodiment 4:
The quantitative test of antigen and LDL:
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 the 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.Logarithm value with antigen concentration is a horizontal ordinate; Change for the ordinate analyte signal intensity with the fluorescence signal intensity; The result shows that antigen concentration is in 0.8～12.8g/mL scope; The better linearity relation 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.This antibody microarray institute can detected minimum antigen concentration be 1.6 μ g/mL.
Embodiment 5:
The specific detection of fish LCDV immune detection chip:
Get normal fish tissue (gill, stomach, epidermis etc.), detect through PCR and do not infect LCDV, 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, no 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 the foregoing description, and it does not all exceed protection scope of the present invention.
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 (monoclonal antibody), affinity chromatography purifying gained antibody;
(2) preparation of Cy3 labeled monoclonal antibody probe
Mouse-anti lymphocystis disease virus monoclonal antibody behind the affinitive layer purification is used the Cy3 mark, and cross the gel column purifying;
(3) pre-service of microslide
Microslide is embathed with the 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, and 2ml agarose solution shop is overlayed on the cleaning microslide that the affine silane treatment of 60 ℃ of preheatings is crossed; After treating that agarose solidifies, with slide 37 ℃ of following 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; The first row sample of selecting 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, and 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 special-purpose fence of chip 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 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 specific anti lymphocystis disease virus cyst membrane 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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| CN200910017833A CN101629955B (en) | 2009-08-07 | 2009-08-07 | Fish lymphocystis disease virus (LCDV) immunity detection chip and 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 |
| 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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| US20180011120A9 (en) * | 2006-08-03 | 2018-01-11 | AyoxxA Biosystems GmbH | Source controlled decodable microarray system and methods for use |
| 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 |
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| CN102132159B (en) * | 2009-08-07 | 2013-05-29 | 中国海洋大学 | Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof |
Also Published As
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| CN101629955A (en) | 2010-01-20 |
| CN102132159B (en) | 2013-05-29 |
| WO2011015131A1 (en) | 2011-02-10 |
| CN102132159A (en) | 2011-07-20 |
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