CN102132159A

CN102132159A - Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof

Info

Publication number: CN102132159A
Application number: CN2010800023499A
Authority: CN
Inventors: 绳秀珍; 徐晓丽; 战文斌
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2009-08-07
Filing date: 2010-08-03
Publication date: 2011-07-20
Anticipated expiration: 2030-08-03
Also published as: CN101629955A; CN102132159B; WO2011015131A1; CN101629955B

Abstract

The invention discloses a fish lymphocyst virus immune detection chip, which comprises a chip carrier, an agarose gel layer covered on the chip carrier, a plurality of antibody microarrays fixed on the agarose gel layer, each microarray They are separated by chip-specific fences or SuperPAPPen lines. The invention adopts the sandwich method to detect antigens, immobilizes pathogenic antibodies on the chip base, takes virus target organs to prepare sample liquids to be tested, incubates the sample liquids to be tested with chips immobilized with pathogenic antibodies, and then adds fluorescently labeled specificity single Anti-probe, read the result by CCD scanner. The method can simultaneously detect lymphocyst virus in multiple samples or different tissues of the same sample.

Description

Fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application

The present invention relates to the improvement of marine cultured animal Pathogen test technology for specification fish lymphocystis disease virus (LCDV) immunity detection chip and preparation method and application technical field, and in particular to one kind is used to detect fish lymphocystis disease virus（Lymphocyst i s di sease virus, LCDV) immune detection chip or microarray and preparation method and application, it belongs to immunology, virology and biochip interleaving techniques field.Background technology fish Lymphocystis disease is a kind of chronic viral diseases, and cauliflower-like tumour occurs in epidermis, Fin and the afterbody of ill fish etc., influences its commodity value or causes fish dead.The cause of disease of Lymphocystis disease is lymphocystis disease virus（), LCDV Iridoviridae is belonged to（Iridoviridae ).Lymphocystis disease Prevalent district is wider, in worldwide distribution, is currently known seawater, salt water and the brackishwater fishes of more than 125 kinds of at least 42 section and can be infected by LCDV.From lefteye flonder lymphocystic disease in 1997 since China breaks out on a large scale first, China Shandong, Hebei, Guangdong, Zhejiang etc. save grouper, porgy, Scioenops ocellatus, the flat Uranium of Xu Shi, cobio of cultivation etc. and occurred this disease, and the directly or indirectly economic loss caused is up to 10,000,000,000 yuan.As other virosis, fish lymphocyst lacks effective medicine.The characteristics of LCDV infection has incubation period long, makes LCDV easily as parent population is propagated with fry exchanging between different regions, therefore, is badly in need of LCDV quick and precisely detection technique in breeding production, to reach the early purpose for finding early prevention.

The method of detection fishes virus generally comprises electron microscopy, PCR methods, DNA probe hybridization in situ, Immunofluorescent Antibody technology, immunohistochemistry technology, enzyme-linked immuno-sorbent assay and western dot blot etc. both at home and abroad.But these detection technique limitations are big, sensitivity, specificity, it can not take into account more than accuracy, it is impossible to be used in Multi-example parallel detection simultaneously.Therefore, set up a kind of easy, quick, accurate, Multi-example parallel processing Examination and diagnosis suitable for fish lymphocystis disease virus imperative, and the protein chip technology that new development is got up is provided for strong technology platform, the reaction of milligram ammonia can not only save costliness reagent but also can binding kineticses quick analysis, a cheap quickly means of testing is provided on protein level, there is quite wide application prospect in diagnostic field.In addition, the successful development of fish lymphocystis disease virus monoclonal antibody, laid a good foundation for immuno-chip Examination and diagnosis that fish Lymphocystis disease is set up using monoclonal antibody.The content of the invention is directed to above-mentioned present situation, can detect the immune detection chip of lymphocystis disease virus in several samples or same sample different tissues simultaneously it is an object of the invention to provide a kind of, quick, sensitive, the accurate inspection for LCDV in marine fish Survey, and import and export the quarantine of LCDV in fish.

It is a further object to provide the preparation method of above-mentioned fish LCDV immune detection chips.

It is a still further object of the present invention to provide application of the above-mentioned immune detection chip in cultured fishes LCDV detections.The purpose of the present invention is realized by following technical scheme：

A kind of fish lymphocystis disease virus (LCDV) immunity detection chip, its structure includes chip carrier, is coated with being fixed with multiple Antibody microarrays on the Ago-Gel layer on chip carrier, Ago-Gel layer, is ruled off between each microarray with chip fence special or Super PAP Pen.A liquid：Osmotic pressure is less than 360mOsm/kg hypotonic medium；B liquid：Tween-phosphate buffer（PBST ) ；C liquid：The anti-LCDV of mouse of Cy3 marks monoclonal antibody（Monoclonal antibody）Probe, monoclonal antibody used in probe is the specific anti-LCDV monoclonal antibodies secreted respectively by two strain of hybridoma（Referred to as：LCDV monoclonal antibodies B and monoclonal antibody C), the preserving number of hybridoma is respectively：The C200419 standing grain P CCTCC of CCTCC mono--C200420, preservation date：On December 14th, 2004.LCDV is located on virus envelope with the antigenic determinant that two plants of monoclonal antibodies are specifically bound.

The present invention is with the actual features of prior art：First, there is the IgM of immunoglobulin one formation in Fish, but in serum IgM mark program it is complex；Second, being the concept of a colony to the diagnosis of marine cultured animal disease, the purpose of diagnosis can be made to colony by being reached to multiple individual detections.Therefore, the present invention is according to these features, antigen is detected using sandwich method, the antibody of cause of disease is fixed in chip chip base, to be checked precursor virus target organ is taken to prepare measuring samples liquid, directly analyte sample fluid is incubated with being fixed with the chip of pathogenic autoantibody, then adds the specific monoclonal antibody probe of fluorescence labeling, result is read by ccd scanner.

The preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip, comprises the following steps：

(1) preparation and purifying of antibody

LCDV is extracted from the lefteye flounder superficial tumor for suffering from Lymphocystis disease, purebred NZw is immunized in conventional method, and blood sampling prepares serum, obtain rabbit-anti LCDV antibody.

Recover and cultivate mouse hybridoma cell strain（LCDV monoclonal antibodies B and monoclonal antibody C), inject a number of hybridoma and enter mouse peritoneal production ascites, obtain a large amount of high concentrations, high specific, the anti-LCDV monoclonal antibodies of mouse of high-titer.

The LCDV monoclonal antibodies B after affinitive layer purification and monoclonal antibody C is taken, purebred NZw is immunized in conventional method, and blood sampling prepares serum, obtain the antibody of the anti-LCDV monoclonal antibodies of rabbit-anti mouse（Rabbit-anti mouse Ig antibody）.

Use close standing grain mouthful chromatographic column (HiTrap Protein G Sepharose Column) the purifying gained antibody of Amersham Phamacia Biotech companies.

(2) preparation of the monoclonal antibody probes of Cy3 marks

According to the product description of Amersham Phamacia Biotech companies, the anti-LCDV monoclonal antibodies of mouse after affinitive layer purification are marked using fluorescein Cy3, and cross gel column purifying.

(3) pretreatment of slide Slide is embathed with highly basic and the concentrated sulfuric acid respectively, distilled water is rinsed, and is dried；In the acetic acid solution for the affine silane that slide after cleaning is immersed to 0.4 %, pH to 4.5 is adjusted, room temperature effect lh, distilled water is rinsed, dried.

(4) preparation of Ago-Gel substrate

1.2 % agarose solution is prepared, micro-wave oven boils 3min, is completely dissolved it, 2mL agarose solutions are coated with the cleaning slide crossed in 60 °C of affine silane treatments preheated；After agarose solidification, slide is in 37 °C of lower dried overnights；0.02mol/L NaIO are used using preceding₄Solution activates 30min, ultra-pure water cleaning down 3 times at room temperature, and is dried up with nitrogen stream, is preserved at drying at room temperature.

(5) fixation of antibody

1. with the pH7.4 phosphate buffer containing 50 % glycerine（PBS) dilution rabbit-anti LCDV antibody makes its concentration be 0.5 ~ 0.0001 mg/mL;Dilution rabbit-anti mouse Ig antibody makes its concentration be 0.1mg/mL.

2. with point sample instrument by this antibody diluent the slide surface of modified different zones point sample, point sample amount be 50 ~ 70nL, spot diameter be 500 ~ 600 μ ι η.Every row four of chip two arranges the sub- arrays of totally 84 X 4, it is consistent that each Asia array puts sample, first row selects sample and is used as negative control for phosphate-glycerol buffer solution, second and third, which is arranged, puts sample for rabbit-anti LCDV antibody, and the 4th row put antibody of the sample for rabbit-anti mouse Ig as positive control and fixed control.Separated between each sub- array with chip fence special or Super PAP Pen, form independent reaction member.37 °C of saturated humidities place 2h, and washing is dried.

3. the closing of 3 % bovine serum albumin(BSA)s is added dropwise in the region that point has antibody on slide, and 37 °C of saturated humidities place lh, and washing is dried, 4 °C of sealing preserves, obtains fish lymphocystis disease virus (LCDV) immunity detection chip.

The application of the fish lymphocystis disease virus (LCDV) immunity detection chip of the present invention, described applying step is as follows：

(1) Fish Tissue for being used to detect includes：Fish body table knob, epidermis, spleen, stomach, intestines etc., sampling about press 1 with A liquid:10 (W/V) ratio mixing, homogenate, multigelation 3 ~ 4 times, after ultrasonication, low-temperature centrifugation, 5000rpm, 15min take supernatant as detection sample, to be checked.

(2) different measuring samples are added in the sub- array of the difference of said chip, sample-adding amount is the arrays of 10 μ Ι 7, and a chip can detect eight samples simultaneously, note avoiding the liquid between different sub- arrays to mix.37 °C of saturated humidities place 0.25 ~ 2h, washing, then add the C liquid of dilution（The anti-LCDV monoclonal antibodies of mouse of Cy3 marks）, the arrays of 10 μ Ι 7,37 °C of saturated humidities place 0.25 ~ 2h, and washing is dried.

(3) laser scanning

Above-mentioned detection chip crystalline substance core E_C0S_Can- 100 ccd scanner scanning imageries, excitation wavelength is 532nm, and Detection wavelength is 585nm, the fluorescence signal software analysis of Lab-chipscanner 2.0.Analysis of test results is with judging：Scan in the sub- array of obtained image, the signal intensity of first row negative control represents the background values size of experiment；Second and third list existing green fluorescence bright spot for the positive, show there is LCDV in detected sample, redgreen fluorescence bright spot for feminine gender, show in detected sample without LCDV.Signal strength has with virus concentration in sample Close；It is normal that green fluorescence, which occur, in 4th row positive control and qualifying point, if without green fluorescence, illustrating that detection operates problematic or antibody protein to be denatured, then testing result is invalid.

(4) viral quantitative detection and minimum detection limit

Isolate and purify rear LCD V and be diluted to 25.6 g/mL, 8 antigen concentrations as obtained by two doubling dilutions are incubated with 8 sub- arrays on slide respectively again, detected through antibody probe, result is as shown in Figure 3 after scanning analysis, change with antigen concentration, the change of gradient is presented in fluorescence signal.Using the logarithm of antigen concentration as abscissa, change by ordinate analyte signal intensity of fluorescence signal intensity, as a result show antigen concentration in the range of 0.8 ~ 12.8 g/mL, there is preferable linear relationship between relative signal intensity and antigen concentration, point out constructed Antibody microarray to carry out quantitative analysis in the range of certain virus concentration.The minimum antigen concentration that this Antibody microarray can be detected is 1.6 g/mL.

The chip carrier has after affine silane treatment, and the glass surface modified with Ago-Gel, and the surface is three-dimensional porous structure, through NaI0₄After activation, antibody can be made to be fixed thereon with physical absorption and covalent attachment two ways, meanwhile, its hydrophilic environment also helps the activity for remaining fixed in antibody protein above.Described antibody is rabbit-anti LCDV antibody, rabbit-anti mouse Ig antibody, but is not limited only to both antibody.

Rabbit-anti LCDV antibody optimum concentration is 0.5mg/mL after the affinitive layer purification of used point sample.

After described measuring samples are added on chip, 37 °C of saturated humidities are placed on 30min, slide plus after the antibody probe of dilution, and 30min is placed under 37 °C of saturated humidities.

Individual slide point of the chip has 8 sub- arrays, can increase sub- array quantity according to actual needs, be detected while can realizing more Multi-example.

The advantage of the invention is that the LCDV in multiple samples or same individual multiple different tissues can be detected simultaneously, it is simple in construction, it is with low cost, flux is easy to expand, Parallel testing while realizing Multi-example, the comparativity between different specimens data is added, the LCDV in a variety of cultured fishes bodies can quickly, accurately, be easily detected.Simple to sample requirement, a small amount of sample can meet detection needs, and enormously simplify the sample handling procedure of existing detection technique, it might even be possible to detection of being drawn materials in the case where not killing cultivated animals.Simultaneously because the repetition point sample of the setting and capture antibody of positive control and negative control, adds the reliability of testing result.Other immuno-chip reaction is relatively fast, and operating process is convenient, and general staff is operable, and being capable of relative quantification detection cause of disease, it is adaptable to the fast and accurately detection of virosis in breeding process.The fish lymphocystis disease virus immuno-chip structural representation of description of drawings 1. and point sample distribution map.Wherein, 1- chip carriers, 2- Ago-Gels layer, 3- Antibody microarrays, 4- chips fence special or Super PAP Pen line, 5- label areas.Point sample is distributed：1. contain the PBS of 50% glycerine, be used as negative control；2., 3. it is rabbit-anti LCDV antibody；4. rabbit-anti mouse Ig antibody, is used as the quality control such as positive control and fixed control. LCDV CCD scanning figures in Fig. 2 fish lymphocystis disease virus (LCDV) immunity detection chips detection separate sources fish sample（The fluorescence intensity of scanner is set as 88 %).Al, A2 show not detect LCDV in sample;A3, B3 show that LCDV concentration is higher in sample；B2, B4 show to contain a certain amount of LCDV in sample, but concentration is low compared with A3, B3；A4, B1 show that LCDV contents are relatively low in sample.

The test limit of Fig. 3 antigen concentrations and the relation of signal intensity and this immuno-chip.The antigen concentration detected is followed successively by 25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2 g/mL.Using the logarithm value of antigen concentration as abscissa, change by ordinate analyte signal intensity of fluorescence signal intensity, as a result show that antigen concentration in the range of 0.8 ~ 12.8 g/mL, there is preferable linear relationship between relative fluorescence signal intensity and antigen concentration.Embodiment below in conjunction with the accompanying drawings and describes the present invention in detail by specific embodiment.Instrument and reagent of the present invention are as follows：Small-sized hand-operated chip spotting system（Purchased from Whatman companies）；Brilliant core EcoScan^TM- 100 ccd scanners（Purchased from Beijing Bo Ao biotech firms）；Cy3 antibody labeling kits（Purchased from GE companies）；HiTrap Protein G Sepharose Column (are purchased from GE companies）；1640 (are purchased from GIBCO companies）；Hyclone（Purchased from HYCLONE companies）；Bovine serum albumin(BSA)（Purchased from SIGMA companies）；Tween-20 (is purchased from SIGMA companies）；Dimethyl sulfoxide (DMSO)（Purchased from SIGMA companies）.Embodiment 1:

1. the preparation and purifying of antibody

LCDV is extracted from the lefteye flounder body surface knob for suffering from lymphocyst, purebred NZw is immunized in conventional method, and blood sampling prepares serum, obtain rabbit-anti LCDV antibody.

Recover and cultivate mouse hybridoma cell strain monoclonal antibody B and monoclonal antibody C, inject a number of hybridoma and enter mouse peritoneal production ascites, obtain a large amount of high concentrations, high specific, the anti-LCDV monoclonal antibodies of mouse of high-titer.

The monoclonal antibody B after affinitive layer purification and monoclonal antibody C is taken to mix, purebred NZw is immunized in conventional method, and blood sampling prepares serum, obtains rabbit-anti mouse Ig antibody.

Use affinity column (the HiTrap Protein G Sepharose of Amersham Phamacia Biotech companies

Column) purifying gained antibody.

2. the preparation of the monoclonal antibody probes of Cy3 marks

According to the Cy3 product descriptions of Amersham Phamacia Biotech companies, the anti-LCDV monoclonal antibodies of mouse after affinitive layer purification are marked using fluorescein Cy3, and cross gel column purifying.

3. the pretreatment of slide Slide is embathed with highly basic and the concentrated sulfuric acid respectively, distilled water is rinsed, and is dried；In the acetic acid solution for the affine silane that slide after cleaning is immersed to 0.4 %, pH to 4.5 is adjusted, room temperature effect lh, distilled water is rinsed, dried.

4. the preparation of Ago-Gel substrate

1.2 % agarose solution is prepared, micro-wave oven boils 3min and is completely dissolved, 2mL agarose solutions are coated with the cleaning slide crossed in 60 °C of affine silane treatments preheated；After agarose solidification, slide is in 37 °C of lower dried overnights；0.02mol/L NaIO are used using preceding₄Solution activates 30min, ultra-pure water cleaning down 3 times at room temperature, and is dried up with nitrogen stream, is preserved at drying at room temperature.

5. chip structure is designed and dot matrix distribution

The chip structure is as shown in figure 1, including chip carrier（1), it is coated with the Ago-Gel layer on chip carrier（2), it is fixed with the Antibody microarray that two rows four arrange totally 84 X 4 on Ago-Gel layer（3), point sample amount is 50-70nL, and spot diameter is 500 ~ 600 μ ι η.Rule between each microarray with chip fence special or Super PAP Pen（4) separate.Described chip carrier is standard glass slide, and label area can be reserved in slide one end（5 ).

6. the fixation of antibody

1. with the pH 7.4 dilution rabbit-anti LCDV antibody of the PBS containing 50 % glycerine, it is 0.5,0.1,0.05,0.01,0.005,0.001,0.0005,0.0001 mg/mL to make its concentration.

2. with point sample instrument by this various concentrations antibody diluent the slide surface of modified different zones point sample, every row four of chip two arranges the sub- arrays of totally 84 X 4, each Asia array is the rabbit-anti LCDV antibody of various concentrations, separated between each sub- array with chip fence special or Super PAP Pen, form independent reaction member.37 °C of saturated humidities place 2h, and washing is dried.

3. the region that point has antibody on slide is added dropwise 3 % bovine serum albumin(BSA)s and closed, and 37 °C of saturated humidities place lh, and washing is dried, 4 °C of sealing preserves, obtains fish LCDV immune detection chips.Embodiment 2:Step 1,2,3,4,5 be the same as Examples 1.

6. the fixation of antibody

1. the PBS buffer solutions with pH 7.4 containing 50 % glycerine dilute rabbit-anti LCDV antibody, make its concentration be

0.5mg/mL;Dilution rabbit-anti mouse Ig antibody makes its concentration be 0. 1mg/mL.

2. with point sample instrument by this antibody diluent the slide surface of modified different zones point sample, dot matrix distribution is as shown in Figure 1, every row four of chip two arranges the sub- arrays of totally 84 X 4, it is consistent that each Asia array puts sample, wherein, first row selects sample for phosphate-glycerol buffer solution, is used as negative control；Second and third row puts sample for rabbit-anti LCDV antibody；4th row put antibody of the sample for rabbit-anti mouse Ig as the qualifying point such as positive control and fixed control.Separated between each sub- array with chip fence special Super PAP Pen, form independent reaction member.37 °C of saturated humidities are put 2h is put, washs, dries.

3. the region that point has antibody on slide is added dropwise 3 % bovine serum albumin(BSA)s and closed, and 37 °C of saturated humidities place lh, and washing is dried, 4 °C of sealing preserves, obtains fish LCDV immune detection chips.Embodiment 3:Detection fish lymphocystis disease virus uses sandwich method.

Step 1,2,3,4,5,6 be the same as Examples 2.

7. the detection of cause of disease

1. Fish Tissue to be checked is taken（Epidermis, the gill, stomach or intestines etc.）, 1 is about pressed with A liquid:10 (W/V) ratio mixing, homogenate, multigelation 3 ~ 4 times, after ultrasonication, low-temperature centrifugation, 5000rpm X 15min take supernatant as detection sample, to be checked；

2. measuring samples are added in the different sub- arrays of said chip identical carrier, sample-adding amount is the arrays of 10 μ Ι 7, and a chip can detect eight samples simultaneously, note avoiding the liquid between different sub- arrays to mix.37 °C of saturated humidities are incubated 15min, 30min, 45min, 60min, 90min, 120min, washing, add the C liquid of dilution on chip, the arrays of 10 μ Ι 7,37 °C of saturated humidities, which are placed, is incubated 15min, 30min, 45min, 60min, 90min, 120min, washing, dries；3. laser scanning

Above-mentioned detection chip crystalline substance core E_C0S_Can- 100 ccd scanner scanning imageries, excitation wavelength is 532nm, and Detection wavelength is 585nm, the fluorescence signal software analysis of Lab-chipscanner 2.0.Analysis of test results is with judging：Scan in the sub- array of obtained image, the signal intensity of first row negative control represents the background values size of experiment；Second and third list existing green fluorescence bright spot for the positive, show there is LCDV in detected sample, redgreen fluorescence bright spot for feminine gender, show in detected sample without LCDV.Signal strength is relevant with virus concentration in sample；It is normal that green fluorescence, which occur, in 4th row positive control and fixing point, if without green fluorescence, illustrating that detection operates problematic or antibody protein to be denatured, testing result is invalid.Embodiment 4:The quantitative analysis of antigen and minimum detection limit：

Take the fish LCDV immune detection chips prepared, the LCDV isolated and purified is diluted to 25.6 g/mL, 8 antigen concentrations as obtained by two doubling dilutions are incubated with the Antibody microarray on slide respectively again, and after being detected through antibody probe, scanning result utilizes software processing.As a result show, change with antigen concentration, the change of gradient is presented in fluorescence signal.Using the logarithm value of antigen concentration as abscissa, change by ordinate analyte signal intensity of fluorescence signal intensity, as a result show antigen concentration in the range of 0.8 ~ 12.8g/mL, there is preferable linear relationship between relative signal intensity and antigen concentration, point out constructed Antibody microarray to carry out quantitative analysis in the range of certain virus concentration.This Antibody microarray institute energy The minimum antigen concentration detected is 1.6 g/mL.Embodiment 5:The specific detection of fish LCDV immune detection chips：

Take normal fish tissues（The gill, stomach, epidermis etc.）LCDV is uninfected by through PCR detections, it is homogenized after historrhexis, ultrasonication after multigelation, low temperature sedimentation 10min, takes supernatant, it is incubated with the Antibody microarray on fish lymphocystis disease virus (LCDV) immunity detection chip, scanned after being detected through antibody probe, unstressed configuration signal, it was demonstrated that prepared fish lymphocystis disease virus (LCDV) immunity detection chip and tissue no cross reaction.

One of ordinary skill in the art will appreciate that it is all possible within the scope of the present invention, to modify, add and replace for above-described embodiment, and it is all without departing from protection scope of the present invention.The industrial applicibility present invention provides technology platform for the checkout and diagnosis of all kinds of cause of diseases such as cultured fishes virus, bacterium, inspection and quarantining for import/export of diseases monitoring and aquatic livestock suitable for breeding process etc., have broad application prospects, cultured fishes transmission of disease and prevalence can be prevented effectively from.

Claims

1. A fish lymphocyst virus immune detection chip, characterized in that it includes a chip carrier, an agarose gel layer covered on the chip carrier, and a plurality of antibody microarrays are immobilized on the agarose gel layer, each microarray The arrays are separated by chip-specific fences or Super PAP Pens.

2. The fish lymphocyst virus immunoassay chip according to claim 1, characterized in that the chip carrier is a standard glass slide, and the chip carrier has been treated with affinity silane and modified with agarose gel. The glass surface has a three-dimensional porous structure, and after activation by NaI0 ₄ , antibodies can be immobilized on it in two ways: physical adsorption and covalent linkage.

3. The fish lymphocyst virus immunoassay chip according to claim 1 or 2, characterized in that said antibodies are rabbit anti-lymphocyst virus antibody and rabbit anti-mouse Ig antibody.

4. The preparation method of the fish lymphocyst virus immunoassay chip according to claim 1, characterized in that it comprises the following steps:

(1) Antibody preparation

Prepare rabbit anti-lymphocyst virus antibody, rabbit anti-mouse Ig antibody, mouse anti-lymphocyst virus monoclonal antibody (mAb), and purify the obtained antibody by affinity chromatography;

(2) Preparation of Cy3-labeled monoclonal antibody probe

The mouse anti-lymphocyst virus monoclonal antibody purified by affinity chromatography was labeled with Cy3, and purified through a gel column;

( 3 ) Pretreatment of slides

Wash the slides with strong alkali and concentrated sulfuric acid respectively, rinse them with double distilled water, and dry them in the air; immerse the washed slides in 0.4% acetic acid solution of affinity silane, adjust the pH to 4.5, and let them act for 1 hour at room temperature , washed with double distilled water, and dried;

(4) Preparation of agarose gel substrate

Prepare a 1.2% agarose solution, boil it in a microwave oven for 3 minutes to dissolve it completely, spread 2ml of the agarose solution on a clean glass slide treated with affinity silane preheated at 60°C; after the agarose is solidified, place the The slides were dried overnight at 37°C; before use, they were activated with 0.02mol/L NaI0 _solution at room temperature for 30 minutes, rinsed with ultrapure water three times, dried with nitrogen flow, and stored in a dry place at room temperature;

(5) Immobilization of antibodies

① Dilute the rabbit anti-lymphocyst virus antibody with pH 7.4 PBS buffer containing 50% glycerol to a concentration of 0.5-0.0001 mg/mL; dilute the rabbit anti-mouse Ig antibody to a concentration of 0.1 mg/mL;

② Spot the diluted antibody solution on different areas of the agarose gel substrate with a spotting instrument, the spot volume is 50-70nL, and the spot diameter is 500-600μιη. There are eight 4 X 4 subarrays in two rows and four columns per chip. The samples in each subarray are consistent. The samples in the first column are phosphate glycerol buffer as a negative control. The samples in the second and third columns are Rabbit anti-lymphocyst virus antibody, p.

1 The four columns are quality control points, and the samples taken are rabbit anti-mouse Ig antibodies as positive controls and fixed controls. Each subarray is separated by a chip-specific fence or a Super PAP Pen to form an independent reaction unit. Place at 37°C in saturated humidity for 2 hours, wash and dry;

③ Add 3% bovine serum albumin dropwise to the area on the slide where the antibody is spotted for blocking, place in saturated humidity at 37°C for 1h, wash, dry in the air, and seal and store at 4°C to obtain a fish lymphocyst virus immunoassay chip.

5. The preparation method of the fish lymphocyst virus immunoassay chip according to claim 4, characterized in that the mouse anti-lymphocyst virus monoclonal antibody purified by Cy3-labeled affinity chromatography is used as the antibody probe, and the rabbit antibody Mouse Ig antibody was used as positive control and fixed control.

6. The preparation method of the fish lymphocyst virus immunoassay chip according to claim 4, characterized in that the monoclonal antibody used for the Cy3-labeled antibody probe is a specific anti-lymphocyst virus envelope secreted by two hybridoma cells respectively Mab B and Mab O.

7. The application of the fish lymphocystosis virus immunodetection chip according to claim 1 in the detection of cultured fish lymphocystosis virus.

8. The application of the fish lymphocyst virus immunoassay chip according to claim 7, characterized in that it comprises the following steps:

(1) The tissues used for detection include: fish body surface tumors, epidermis, stomach, intestines, etc. After sampling, mix with liquid A at a ratio of 1:10 (W/V), homogenate, and freeze-thaw repeatedly 3~4 times, after sonication, centrifugation at low temperature, 5000rpmX 15min, take the supernatant as a test sample, to be tested;

(2) Add different samples to be tested to different subarrays of the chip above, place at 37°C in saturated humidity for 0.25~2h, wash, then add diluted Cy3-labeled mouse anti-lymphocyst virus monoclonal antibody probe, 37°C Place in C saturated humidity for 0.25~2h, wash and dry;

( 3) Laser scanning

The above slides were scanned and imaged with Crystal® E _CO S _Can ^TM -100 CCD scanner, and the images were analyzed with professional analysis software. According to the strength of the fluorescent signal, the qualitative and quantitative analysis results of the fish lymphocyst virus content in the sample were obtained.

9. The application of the fish lymphocyst virus immune detection chip according to claim 8, characterized in that the sandwich method is used to detect lymphocyst virus in fish, that is, the complex formed by the reaction of the antibody chip and the lymphocyst virus in the sample , is recognized by a highly specific Cy3-labeled antibody probe, and exhibits green fluorescence under a 532nm laser.

10. The application of the fish lymphocyst virus immunoassay chip according to claim 8, characterized in that after the sample to be tested is added to the chip, it is placed in a saturated humidity of 37°C for 30 minutes, and after the diluted fluorescent antibody is added to the slide, 37 °C saturated humidity for 30min.

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