WO2011015131A1

WO2011015131A1 - Immunoassay chip for fish lymphocystis disease virus, preparation method and application thereof

Info

Publication number: WO2011015131A1
Application number: PCT/CN2010/075669
Authority: WO
Inventors: 绳秀珍; 徐晓丽; 战文斌
Original assignee: 中国海洋大学
Priority date: 2009-08-07
Filing date: 2010-08-03
Publication date: 2011-02-10
Also published as: CN101629955B; CN102132159A; CN102132159B; CN101629955A

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 an improvement of a pathogenic detection technique for marine fish culture, and particularly to a method for detecting Lymphocyst is di sease virus (LCDV) The immunodetection chip or microarray and its preparation method and application belong to the field of immunology, virology and biochip crossover technology. BACKGROUND OF THE INVENTION Fish lymphocystosis is a chronic viral disease in which cacao cysts appear on the epidermis, fins, and tails of diseased fish, affecting their commercial value or causing fish to die. The pathogen of lymphocystosis is the lymphocytic virus (LCDV), which belongs to the Iridoviridae family. Lymphocystosis is a widespread area with a worldwide distribution. At least 42 species of seawater, salt water and brackish water fish are known to be infected by LCDV. Since the first large-scale outbreak of gingival lymphocystosis in China in 1997, the grouper, the true carp, the American redfish, the Xu ping uranium, the cobia, etc. have been cultivated in Shandong, Hebei, Guangdong, Zhejiang and other provinces. The direct and indirect economic losses caused by illness have reached tens of billions of yuan. Like other viral diseases, fish lymphocytic virus disease lacks effective therapeutic drugs. LCDV infection has the characteristics of long incubation period, which makes LCDV easy to spread with the exchange of broodstock and fry in different regions. Therefore, the rapid and accurate detection technology of LCDV is urgently needed in aquaculture production, in order to achieve early detection and early prevention.

The methods for detecting fish viruses at home and abroad generally include electron microscopy, PCR, DNA probe in situ hybridization, immunofluorescent antibody technology, immunohistochemistry, enzyme-linked immunosorbent assay and dot immunoblotting. However, these detection techniques have limited limitations, and sensitivity, specificity, and accuracy cannot be balanced. They cannot be used for simultaneous parallel detection of multiple samples. Therefore, it is imperative to establish a simple, rapid, accurate and multi-sample parallel processing detection and diagnosis technology for fish lymphatic cyst virus, and the newly developed protein chip technology provides a powerful technical platform. The micro-quantitative reaction not only saves expensive reagents but also combines rapid analysis of kinetics. It provides a cheap and fast test method at the protein level and has a broad application prospect in the field of diagnosis. In addition, the successful development of monoclonal antibodies against fish lymphocytic virus laid the foundation for the use of monoclonal antibodies to establish immunological chip detection and diagnosis techniques for fish lymphocystosis. SUMMARY OF THE INVENTION In view of the above situation, an object of the present invention is to provide an immunodetection chip capable of simultaneously detecting a plurality of samples or lymphocytic viruses in different tissues of the same sample, for rapid, sensitive and accurate detection of LCDV in marine fish. Measure, and quarantine of LCDV in import and export fish.

Another object of the present invention is to provide a method for preparing the fish LCD immunodetection chip described above.

Still another object of the present invention is to provide an application of the above immunodetection chip for LCDV detection of farmed fish. The object of the invention is achieved by the following technical solutions:

A fish lymphatic cyst virus immunodetection chip, the structure comprising a chip carrier, an agarose gel layer deposited on the chip carrier, and a plurality of antibody microarrays immobilized on the agarose gel layer, used between the microarrays Chip-specific fences or Super PAP Pen lines are separated. Liquid A: hypotonic solution with osmotic pressure less than 360mOsm/kg; liquid B: Tween-phosphate buffer (PBST); liquid C: Cy3-labeled mouse anti-LCDV monoclonal antibody (mAb) probe, probe The monoclonal antibody used is a specific anti-LCDV monoclonal antibody secreted by two hybridoma cells (abbreviation: LCDV monoclonal antibody B and monoclonal antibody C), and the hybridoma cell storage numbers are: CCTCC-C200419 and P CCTCC-C200420, respectively. Date of deposit: December 14, 2004. The antigenic determinant that LCDV specifically binds to the two monoclonal antibodies is located on the viral envelope.

The present invention has the practical characteristics of the prior art: First, the formation of immunoglobulin-IgM in fish, but the labeling procedure of IgM in serum is complicated; Second, the diagnosis of marine animal diseases is a group concept. The diagnosis of the group can be achieved by detecting multiple individuals. Therefore, according to these characteristics, the present invention detects the antigen by a sandwich method, fixes the antibody of the pathogen on the chip substrate, takes the virus target organ to be tested for preparation of the sample liquid to be tested, and directly directly measures the sample liquid to be tested and the chip immobilized with the pathogenic antibody. Incubate, add fluorescently labeled specific monoclonal antibody probes, and read the results by CCD scanner.

The preparation method of the fish lymphocytic virus immunodetection chip comprises the following steps:

(1) Preparation and purification of antibodies

LCDV was extracted from the gingival surface tumor of the lymphocystosis, and the purebred New Zealand white rabbit was immunized by a conventional method, and blood was collected to prepare a rabbit anti-LCDV antibody.

Resuscitation and culture of mouse hybridoma cell lines (LCDV monoclonal antibody B and monoclonal antibody C), injection of a certain number of hybridoma cells into the peritoneal cavity of mice to produce ascites, and obtain a large number of high concentration, high specificity, high titer mouse anti-LCDV single anti.

The affinity-chromatized purified LCDV monoclonal antibody B and monoclonal antibody C were used to immunize pure New Zealand white rabbits by conventional methods, and blood was collected to prepare an antibody against rabbit anti-LCDV monoclonal antibody (rabbit anti-mouse Ig antibody).

The obtained antibody was purified using a Hierrap Protein G Sepharose Column from Amersham Phamacia Biotech.

(2) Preparation of Cy3 labeled monoclonal antibody probe

The mouse anti-LCDV mAb purified by affinity chromatography was labeled with fluorescein Cy3 according to the Amersham Phamacia Biotech product specification and purified by gel column.

(3) Pretreatment of slides The slides were respectively washed with strong alkali and concentrated sulfuric acid, rinsed with double distilled water, and air-dried; the washed slides were immersed in a 0.4% acetic acid solution of affinity silane, adjusted to pH 4.5, and allowed to react at room temperature for 1 h. Rinse with double distilled water and allow to dry.

(4) Preparation of agarose gel substrate

Prepare a 1.2% agarose solution, boil for 3 minutes in a microwave oven, completely dissolve it, and spread 2 mL of agarose solution on a pre-heated affinity silane-treated clean glass slide at 60 °C; after the agarose solidifies, the slide is at 37 Dry overnight at °C; use 0.02mol/L NaIO ₄ solution for 30min at room temperature before use, rinse thoroughly with ultrapure water for 3 times, dry with nitrogen gas, and store at room temperature.

(5) Immobilization of antibodies

1 Dilute the rabbit anti-LCDV antibody to a concentration of 0.5-0.0001 mg/mL with 50% glycerol in phosphate buffer (PBS) at pH 7.4; dilute the rabbit anti-mouse Ig antibody to a concentration of 0.1 mg/mL.

2 Using a spotter, spot the antibody on different areas of the surface of the modified slide. The spotting amount is 50~70nL, and the spot diameter is 500~600μιη. Each chip has 8 4 X 4 subarrays in two rows and four columns. The samples in each subarray are consistent. The samples in the first column are phosphate glycerol buffer as the negative control, and the samples in the second and third columns are The rabbit anti-LCDV antibody, the sample in the fourth column was a rabbit anti-mouse Ig antibody as a positive control and a fixed control. Each sub-array is separated by a chip-specific fence or a Super PAP Pen to form an independent reaction unit. Leave at 37 °C for 2 h, wash and dry.

3 Place the antibody on the slide with 3 % bovine serum albumin, place it at 37 ° C for 1 h, wash, dry, and seal at 4 °C to obtain the fish lymphocytic virus immunoassay chip.

The application of the fish lymphatic cyst virus immunodetection chip of the invention, the application steps are as follows:

(1) Fish tissues used for testing include: fish surface tumors, epidermis, spleen, stomach, intestines, etc. Sampling and mixing with liquid A in a ratio of 1:10 (W/V), homogenizing, repeated After freezing and thawing for 3~4 times, after ultrasonication, centrifuge at low temperature, 5000 rpm, 15 min, take the supernatant as a test sample, and check it.

(2) Add different samples to be tested to different sub-arrays of the above chip, the loading amount is 10μΙ7 array, and one chip can simultaneously detect eight samples, taking care to avoid liquid mixing between different sub-arrays. Place at 37 °C for 0.25~2h, wash, add diluted C solution (Cy3 labeled mouse anti-LCDV monoclonal antibody), 10μΙ7 array, 37 °C saturated humidity for 0.25~2h, wash and dry.

(3) Laser scanning

The above detection chip was scanned with a Crystal Core® E _C0 S _Can TM -100 CCD scanner with an excitation wavelength of 532 nm and a detection wavelength of 585 nm. The fluorescence signal was analyzed by Lab-chipscanner 2.0 software. Analysis and determination of test results: In the image subarray obtained by scanning, the signal intensity of the first column of the negative control represents the background value of the experiment; the second and third columns of the green fluorescent highlights are positive, indicating that there is LCDV in the sample. , No green fluorescent highlights are negative, indicating no LCDV in the sample tested. Signal strength and virus concentration in the sample Guan; Green fluorescence is normal in the fourth column of positive control and quality control points. If there is no green fluorescence, indicating that there is a problem with the detection operation or the antibody protein is denatured, the test result is invalid.

(4) Quantitative detection and minimum detection limit of virus

After separation and purification, LCD V was diluted to 25.6 g/mL, and then the 8 antigen concentrations obtained by double dilution were incubated with 8 subarrays on the slides, and detected by antibody probes. The results of the scan analysis are shown in Fig. 3. It is shown that the fluorescence signal exhibits a gradient change as the antigen concentration changes. Taking the logarithm of the antigen concentration as the abscissa and the fluorescence signal intensity as the ordinate to analyze the signal intensity change, the results show that the antigen concentration is in the range of 0.8~12.8 g/mL, and there is a good linear relationship between the relative signal intensity and the antigen concentration. It is suggested that the constructed antibody microarray can be quantitatively analyzed within a certain range of virus concentration. The lowest antigen concentration detectable by this antibody microarray was 1.6 g/mL.

The chip carrier has a glass surface modified by an affinity silane and modified with an agarose gel. The surface is a three-dimensional porous structure. After activation by NaI0 ₄ , the antibody can be immobilized by physical adsorption and covalent bonding. At the same time, its hydrophilic environment also facilitates the activity of the antibody protein immobilized thereon. The antibody is a rabbit anti-LCDV antibody, a rabbit anti-mouse Ig antibody, but is not limited to these two antibodies.

The optimum concentration of the rabbit anti-LCDV antibody after affinity chromatography for spotting was 0.5 mg/mL.

After the sample to be tested is applied to the chip, it is placed at a saturated humidity of 37 ° C for 30 minutes, and the diluted antibody probe is added to the slide, and then placed at 37 ° C for 30 minutes under saturated humidity.

The chip has a single sub-array of 8 sub-arrays, which can increase the number of sub-arrays according to actual needs, and can realize simultaneous detection of more samples.

The invention has the advantages that the LCDV in a plurality of samples or a plurality of different tissues of the same individual can be simultaneously detected, and the structure is simple, the cost is low, the flux is easy to expand, and simultaneous parallel detection of multiple samples is realized, and data between different specimens is increased. Comparable, fast, accurate and easy to detect LCDV in a variety of farmed fish. The sample requirements are simple, a small number of samples can meet the testing needs, and greatly simplify the sample processing steps of the existing detection technology, and even can be taken without killing the cultured animals. At the same time, the reliability of the test results is increased due to the setting of the positive control and the negative control and the repeated sampling of the captured antibody. In addition, the immune chip reacts relatively quickly, the operation process is convenient, the general personnel can operate, and the pathogen can be detected relatively quantitatively, and is suitable for rapid and accurate detection of viral diseases in the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram of the immunochip of fish lymphatic cyst virus and its spot distribution. Among them, 1-chip carrier, 2-agarose gel layer, 3-antibody microarray, 4-chip dedicated fence or Super PAP Pen line, 5-label area. Spot distribution: 1 PBS containing 50% glycerol as a negative control; 2, 3 for rabbit anti-LCDV antibody; 4 rabbit anti-mouse Ig antibody, as a positive control and fixed control and other quality control. Figure 2. Fish lymphatic cyst virus immunoassay chip detects CCD scans of LCDV in fish samples from different sources (the fluorescence intensity of the scanner is set to 88%). Al and A2 indicate that LCDV is not detected in the sample; A3 and B3 indicate that the LCDV concentration in the sample is higher; B2 and B4 indicate that the sample contains a certain amount of LCDV, but the concentration is lower than A3 and B3; A4 and B1 indicate the LCDV content in the sample. Lower.

Figure 3. Relationship between antigen concentration and signal intensity and detection limits for this immunochip. The concentrations of the antigens detected were 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2 g/mL, respectively. The logarithmic value of the antigen concentration is plotted on the abscissa and the intensity of the signal is analyzed on the ordinate. The results show that the antigen concentration is in the range of 0.8~12.8 g/mL, and there is a good linearity between the relative fluorescence signal intensity and the antigen concentration. relationship. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the accompanying drawings. The present invention is used in instruments and reagents are as follows: small hand-chip spotting system (available from Whatman Corporation); GeeDom ®EcoScan ^TM -100 CCD scanner (available from Beijing Us Biological Company); Antibody Cy3 labeling kit (available from GE Company); HiTrap Protein G Sepharose Column (purchased from GE); 1640 (purchased from GIBCO); fetal bovine serum (purchased from HYCLONE); bovine serum albumin (purchased from SIGMA); Tween-20 (purchased from SIGMA); dimethyl sulfoxide (purchased from SIGMA). Example 1:

1. Preparation and purification of antibodies

LCDV was extracted from the gingival surface tumor of the lymphocytic virus disease, and the purebred New Zealand white rabbit was immunized by a conventional method, and blood was collected to prepare a rabbit anti-LCDV antibody.

The mouse hybridoma cell line monoclonal antibody B and monoclonal antibody C were resuscitated and cultured, and a certain amount of hybridoma cells were injected into the peritoneal cavity of the mouse to produce ascites, and a large amount of high concentration, high specificity and high titer mouse anti-LCDV monoclonal antibody was obtained.

The monoclonal antibody B and the monoclonal antibody C purified by affinity chromatography were mixed, and the purebred New Zealand white rabbits were immunized by a conventional method, and blood was prepared to prepare a rabbit anti-mouse Ig antibody.

Using Amersham Phamacia Biotech's Affinity Chromatography Column ( HiTrap Protein G Sepharose

Column) Purify the resulting antibody.

2. Preparation of Cy3 labeled monoclonal antibody probe

The mouse anti-LCDV monoclonal antibody purified by affinity chromatography was labeled with fluorescein Cy3 according to the Cys product specification of Amersham Phamacia Biotech, and purified by gel column.

3. Pretreatment of slides The slides were respectively washed with strong alkali and concentrated sulfuric acid, rinsed with double distilled water, and air-dried; the washed slides were immersed in 0.4% acetic acid solution of affinity silane, adjusted to pH 4.5, room temperature for lh, double steaming Rinse with water and allow to dry.

4. Preparation of agarose gel substrate

Prepare a 1.2% agarose solution, completely dissolve in a microwave oven for 3 minutes, and spread 2 mL of agarose solution on a pre-heated affinity silane-treated clean glass slide at 60 °C. After the agarose is solidified, the slide is at 37 °C. It was dried overnight; it was incubated with 0.02 mol/L NaIO ₄ solution for 30 min at room temperature before use, and thoroughly rinsed 3 times with ultrapure water, dried with a nitrogen stream, and stored at room temperature.

5. Chip structure design and dot matrix distribution

The structure of the chip is as shown in FIG. 1 , which comprises a chip carrier (1 ), an agarose gel layer (2) deposited on the chip carrier, and two rows and four columns of 8 4 4 4 fixed on the agarose gel layer. The antibody microarray (3) has a spotting amount of 50-70 nL and a spot diameter of 500-600 μm. Each microarray is separated by a chip-specific fence or Super PAP Pen line (4). The chip carrier is a standard slide, and a label area (5) can be left at one end of the slide.

6. Immobilization of antibodies

1 Dilute rabbit anti-LCDV antibody to pH 7.4 in 50% glycerol in PBS buffer to a concentration of 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001 mg/mL.

2 Using a spotter, sample the different concentrations of antibody on different areas of the surface of the modified slide. Each chip has two rows of four columns and four 8 X 4 subarrays. Each subarray has different concentrations. The rabbit anti-LCDV antibody is separated from each sub-array by a chip-specific fence or a Super PAP Pen to form an independent reaction unit. Store at 37 ° C for 2 h, wash and dry.

3 In the area where the antibody was spotted on the slide, 3 % bovine serum albumin was added dropwise, and the mixture was placed at 37 ° C for 1 h, washed, dried, and sealed at 4 ° C to obtain a fish LCD immunodetection chip. Example 2: Steps 1, 2, 3, 4, and 5 are the same as in Example 1.

6. Immobilization of antibodies

1 Dilute rabbit anti-LCDV antibody with 50% glycerol in PBS buffer at pH 7.4 to a concentration of

0.5 mg/mL; diluted rabbit anti-mouse Ig antibody to a concentration of 0.1 mg / mL.

2 Using a spotting device, spot the antibody dilution on different areas of the surface of the modified slide. The dot matrix distribution is shown in Figure 1. Each chip has two rows of four columns and a total of eight 4 X 4 subarrays. The sample points of the sub-arrays are consistent, wherein the sample in the first column is phosphate glycerol buffer as a negative control; the samples in the second and third columns are rabbit anti-LCDV antibodies; The antibody of murine Ig was used as a quality control point such as a positive control and a fixed control. Each sub-array is separated by a chip-specific fence Super PAP Pen to form an independent reaction unit. 37 °C saturated humidity Set for 2h, wash and dry.

3 In the area where the antibody was spotted on the slide, 3 % bovine serum albumin was added dropwise, and the mixture was placed at 37 ° C for 1 h, washed, dried, and sealed at 4 ° C to obtain a fish LCD immunodetection chip. Example 3: Detection of fish lymphocytic virus using a sandwich method.

Steps 1, 2, 3, 4, 5, and 6 are the same as in Embodiment 2.

7. Detection of pathogens

1 Take the fish tissue to be inspected (skin, sputum, stomach or intestine, etc.), mix it with A solution at a ratio of 1:10 (W/V), homogenize, freeze and thaw for 3-4 times, after ultrasonication, Centrifuge at low temperature, 5000 rpm X 15 min, take the supernatant as a test sample, and check it;

2 Add the sample to be tested to the different sub-arrays of the same carrier of the above chip, the loading amount is 10μΙ7 array, and one chip can simultaneously detect eight samples, taking care to avoid liquid mixing between different sub-arrays. Incubate at 37 °C for 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, wash, add diluted C solution on the chip, 10 μΙ7 array, and incubate at 37 °C for 15 min, 30 min, 45 min, 60 min, 90 min, 120 min. , washing, drying; 3 laser scanning

The above detection chip was scanned with a Crystal Core® E _C0 S _Can TM -100 CCD scanner with an excitation wavelength of 532 nm and a detection wavelength of 585 nm. The fluorescence signal was analyzed by Lab-chipscanner 2.0 software. Analysis and determination of test results: In the image subarray obtained by scanning, the signal intensity of the first column of the negative control represents the background value of the experiment; the second and third columns of the green fluorescent highlights are positive, indicating that there is LCDV in the sample. , No green fluorescent highlights are negative, indicating no LCDV in the sample tested. The signal strength is related to the virus concentration in the sample; the green fluorescence in the fourth column of positive control and fixed point is normal. If there is no green fluorescence, it indicates that there is a problem in the detection operation or the antibody protein is denatured, and the detection result is invalid. Example 4: Quantitative analysis of antigens and minimum detection limits:

The prepared fish LCDV immunodetection chip was prepared, and the isolated and purified LCDV was diluted to 25.6 g/mL, and then the 8 antigen concentrations obtained by double dilution were respectively incubated with the antibody microarray on the slide, and detected by the antibody probe. After that, the scan results are processed using computer software. The results show that the fluorescence signal exhibits a gradient change as the antigen concentration changes. The logarithm of the antigen concentration is plotted on the abscissa and the intensity of the fluorescence signal is used as the ordinate. The results show that the antigen concentration is in the range of 0.8~12.8g/mL, and there is a good linear relationship between the relative signal intensity and the antigen concentration. It is suggested that the constructed antibody microarray can be quantitatively analyzed within a certain range of virus concentration. This antibody microarray can The lowest antigen concentration detected was 1.6 g/mL. Example 5: Specific detection of fish LCDV immunodetection chip:

Take normal fish tissue (sputum, stomach, epidermis, etc.), detect uninfected LCDV by PCR, homogenate after tissue disruption, ultrasonically break after repeated freeze-thaw, low temperature sedimentation for 10 min, take supernatant, and fish lymphocytic virus immunodetection chip The antibody microarray was incubated, and after being detected by the antibody probe, there was no fluorescence signal, and it was confirmed that the prepared fish lymphatic cyst virus immunodetection chip did not cross-react with the tissue.

It will be understood by those skilled in the art that modifications, additions and substitutions of the above-described embodiments are possible within the scope of the present invention, and are not beyond the scope of the present invention. Industrial Applicability The present invention provides a technical platform for the detection and diagnosis of various pathogens such as cultured fish viruses and bacteria, and is suitable for disease monitoring in aquaculture processes and entry and exit inspection and quarantine of aquatic animals, and has broad application prospects and can be effective. Avoid the spread and prevalence of farmed fish diseases.

Claims

Claim

1. A fish lymphocytic virus immunodetection chip, which comprises a chip carrier, an agarose gel layer deposited on a chip carrier, and a plurality of antibody microarrays immobilized on the agarose gel layer, each micro The arrays are separated by a chip-specific fence or a Super PAP Pen.

2. The fish lymphatic cyst virus immunodetection chip according to claim 1, wherein the chip carrier is a standard slide, and the chip carrier is treated with an affinity silane and modified with an agarose gel. The surface of the glass is a three-dimensional porous structure. After activation by NaI0 ₄ , the antibody can be immobilized on both the physical adsorption and the covalent linkage.

The fish lymphocytic virus immunodetection chip according to claim 1 or 2, wherein the antibody is a rabbit anti-lymphocytic virus antibody or a rabbit anti-mouse Ig antibody.

The method for preparing a fish lymphatic cyst virus immunodetection chip according to claim 1, characterized in that it comprises the following steps:

(1) Preparation of antibodies

Preparation of rabbit anti-lymphocytic virus antibody, rabbit anti-mouse Ig antibody, mouse anti-lymphocytic virus monoclonal antibody (mAb), affinity chromatography purification of the obtained antibody;

(2) Preparation of Cy3 labeled monoclonal antibody probe

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

(3) Pretreatment of slides

The slides were respectively washed with strong alkali and concentrated sulfuric acid, rinsed with double distilled water, and air-dried; the washed slides were immersed in a 0.4% acetic acid solution of affinity silane, adjusted to pH 4.5, and allowed to act at room temperature for 1 hour. Rinse with double distilled water and dry it;

(4) Preparation of agarose gel substrate

Prepare a 1.2% agarose solution, boil for 3 minutes in a microwave oven, completely dissolve it, and spread 2ml agarose solution on the affinity silane treated clean glass slide preheated at 60 ° C; after the agarose solidifies, The slide was dried overnight at 37 ° C; it was activated with 0.02 mol/L NaI _{4 4} solution for 30 min at room temperature, washed 3 times with ultrapure water, dried with a nitrogen stream, and stored at room temperature in a dry place;

(5) Immobilization of antibodies

The diluted anti-lymphocytic antibody was diluted with 0.5% to 0.0001 mg/mL, and the concentration was 0.5 mg/mL;

2 Using a spotting instrument, the antibody dilution was spotted on different areas of the agarose gel substrate, the spotting amount was 50-70 nL, and the spot diameter was 500-600 μm. Each chip has 8 4 X 4 subarrays in two rows and four columns. The samples in each subarray are consistent. The samples in the first column are phosphate glycerol buffer as the negative control, and the samples in the second and third columns are Rabbit anti-lymphocytic virus antibody, number

1 Four columns were quality control points, and the sample was rabbit anti-mouse Ig antibody as a positive control and a fixed control. Each sub-array is separated by a chip-specific fence or Super PAP Pen to form an independent reaction unit. Store at 37 ° C with saturated humidity for 2 h, wash and dry;

3 The area where the antibody was spotted on the slide was added with 3 % bovine serum albumin for blocking, and the mixture was kept at 37 ° C for 1 h, washed, dried, and sealed at 4 ° C to obtain a fish lymphocytic virus immunodetection chip.

The method for preparing a fish lymphatic cyst virus immunoassay chip according to claim 4, characterized in that the mouse anti-lymphocytic virus monoclonal antibody purified by Cy3 labeling affinity chromatography is used as an antibody probe, and a rabbit anti-resistant antibody is used. The antibody to murine Ig served as a positive control and a fixed control.

The method for preparing a fish lymphatic cyst virus immunodetection chip according to claim 4, wherein the monoclonal antibody used for the Cy3 labeled antibody probe is a specific anti-lymphocytic virus envelope secreted by two hybridoma cells respectively. Monoclonal antibody B and monoclonal antibody.

7. The use of the fish lymphatic cyst virus immunoassay chip according to claim 1 for detecting lymphocytic virus in cultured fish.

The use of the fish lymphatic cyst virus immunodetection chip according to claim 7, characterized in that it comprises the following steps:

(1) The tissues used for testing include: fish surface tumors, epidermis, stomach, intestines, etc., sampled and mixed with liquid A at a ratio of 1: 10 (W/V), homogenized, repeated freeze-thaw 3~4 times, after ultrasonication, centrifuge at low temperature, 5000rpmX 15min, take the supernatant as the test sample, and check it;

(2) Add different samples to be tested to different subarrays of the above chip, place at 37 ° C for 0.25~2 h, wash the paint, and add the diluted monoclonal antibody against Cy3 labeled mouse anti-lymphocytic virus, 37° C saturated humidity is placed for 0.25~2h, washed and dried;

(3) Laser scanning

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

The use of the fish lymphocytic virus immunodetection chip according to claim 8, characterized in that the lymphatic cyst virus in fish is detected by a sandwich method, that is, a complex formed by reacting the antibody chip with a lymphocytic virus in a sample. It is recognized by a highly specific Cy3-labeled antibody probe and exhibits green fluorescence at a laser of 532 nm.

10. The use of the fish lymphocytic virus immunodetection chip according to claim 8, characterized in that after the sample to be tested is applied to the chip, the mixture is placed at 37 ° C for 30 min, and the diluted fluorescent antibody is added to the slide. The humidity was allowed to stand at °C for 30 min.

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