CN102132159B - Fish lymphocyst virus immune detection chip and its preparation method and application - Google Patents

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

Fish lymphocyst virus immune detection chip and its preparation method and application

technical field

The present invention relates to the improvement of seawater culture animal pathogen detection technology, in particular to an immune detection chip or microarray for detecting fish lymphocystis disease virus (LCDV) and its preparation method and application, which belong to immunology, Virology and biochip cross-technical field.

Background technique

Fish lymphocystosis is a chronic viral disease. Cauliflower-like cysts appear on the epidermis, fins and tail of diseased fish, affecting its commercial value or causing fish death. Lymphocystic disease is caused by Lymphocyst virus (LCDV), which belongs to the family Iridoviridae. Lymphocystic disease is endemic in a wide area and is distributed worldwide. It is known that more than 125 species of seawater, brackish water and brackish water fish in at least 42 families can be infected by LCDV. Since the first large-scale outbreak of flounder lymphatic cyst disease in my country in 1997, it has occurred in grouper, red sea bream, American redfish, Xu's flatfish, and cobia in Shandong, Hebei, Guangdong, Zhejiang and other provinces. disease, causing direct and indirect economic losses of tens of billions of dollars. Like other viral diseases, fish lymphocyst virus disease lacks effective drugs. LCDV infection has the characteristics of long incubation period, which makes LCDV very easy to spread with the exchange of broodstock and fry in different regions. Therefore, rapid and accurate detection technology of LCDV is urgently needed in aquaculture production, in order to achieve the purpose of 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, immunohistochemical technology, enzyme-linked immunosorbent assay and dot immunoblotting technology. However, these detection techniques have great limitations, and cannot balance sensitivity, specificity, and accuracy, and cannot be used for simultaneous parallel detection of multiple samples. Therefore, it is imperative to establish a simple, fast, accurate and multi-sample parallel processing detection and diagnosis technology suitable for fish lymphocyst virus, and the newly developed protein chip technology provides a powerful technical platform for it. The microquantified reaction can not only save expensive reagents but also can be combined with rapid analysis of kinetics, providing a cheap and rapid test method at the protein level, and has a very broad application prospect in the field of diagnosis. In addition, the successful development of fish lymphocyst virus monoclonal antibody has laid the foundation for the establishment of immunochip detection and diagnosis technology for fish lymphocyst disease by using monoclonal antibody.

Contents of the invention

In view of the above-mentioned present situation, the object of the present invention is to provide a kind of immunoassay chip that can detect lymphocyst virus in multiple samples or different tissues of the same sample at the same time, which is used for fast, sensitive and accurate detection of LCDV in seawater cultured fish, and Quarantine of LCDV in fish for import and export.

Another object of the present invention is to provide a method for preparing the above-mentioned fish LCDV immune detection chip.

Another object of the present invention is to provide the application of the above-mentioned immune detection chip in the detection of LCDV of cultured fish.

The purpose of the present invention is achieved by the following technical solutions:

An immune detection chip for fish lymphocyst virus, the structure of which includes a chip carrier, an agarose gel layer covered on the chip carrier, and a plurality of antibody microarrays fixed on the agarose gel layer. Chip-specific fences or Super PAP Pens are separated by lines. Solution A: hypotonic solution with osmotic pressure less than 360mOsm/kg; Solution B: Tween-phosphate buffered saline (PBST); Solution C: Cy3-labeled mouse anti-LCDV monoclonal antibody (mAb) probe, probe The monoclonal antibodies used are specific anti-LCDV monoclonal antibodies secreted by two strains of hybridoma cells (abbreviation: LCDV monoclonal antibody B and monoclonal antibody C), and the preservation numbers of the hybridoma cells are respectively: CCTCC-C200419 and CCTCC-C200420. Date: December 14, 2004. The antigenic determinants that specifically bind LCDV to the two monoclonal antibodies are located on the viral envelope.

The present invention is based on the actual characteristics of the prior art: one, there is the formation of immunoglobulin-IgM in the fish body, but the IgM labeling procedure in the serum is relatively complicated; two, the diagnosis of the disease of seawater cultured animals is a concept of a group , the purpose of diagnosing a group can be achieved by detecting multiple individuals. Therefore, according to these characteristics, the present invention adopts the sandwich method to detect the antigen, fixes the antibody of the pathogen on the chip base, takes the target organ of the virus of the individual to be tested to prepare the sample liquid to be tested, and directly combines the sample liquid to be tested with the chip that is fixed with the pathogenic antibody. After incubation, add fluorescently labeled specific monoclonal antibody probes, and read the results by a CCD scanner.

The preparation method of fish lymphocyst virus immune detection chip comprises the following steps:

(1) Preparation and purification of antibodies

LCDV was extracted from the superficial tumor of flounder with lymphocystosis, and purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-LCDV antibodies were obtained.

Resuscitate and cultivate mouse hybridoma cell lines (LCDV monoclonal antibody B and monoclonal antibody C), inject 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 monoclonal antibodies anti.

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

The antibody obtained was purified using an affinity chromatography column (HiTrap Protein G SepharoseColumn) from Amersham Phamacia Biotech.

(2) Preparation of Cy3-labeled monoclonal antibody probes

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

(3) Pretreatment of slides

Wash the glass slides with strong alkali and concentrated sulfuric acid respectively, rinse with double distilled water, and dry them in the air; immerse the cleaned glass 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. Rinse with double distilled water and dry.

(4) Preparation of agarose gel substrate

Prepare a 1.2% agarose solution, boil it in a microwave for 3 minutes to dissolve it completely, spread 2mL of the agarose solution on a clean slide treated with affinity silane preheated at 60°C; after the agarose solidifies, place the slide at 37°C Dry overnight; before use, activate with 0.02mol/L NaIO ₄ solution at room temperature for 30 minutes, rinse thoroughly with ultrapure water three times, blow dry with nitrogen flow, and store in a dry place at room temperature.

(5) Immobilization of antibodies

① Dilute the rabbit anti-LCDV antibody with pH 7.4 phosphate buffered saline (PBS) 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 surface of the modified glass slide with a spotting instrument, the spot volume is 50-70nL, and the spot diameter is 500-600μm. Each chip has two rows and four columns with a total of eight 4×4 subarrays. The samples in each subarray are the same. The samples in the first column are phosphate glycerol buffer as a negative control, and the samples in the second and third columns are Rabbit anti-LCDV antibody, the sample in the fourth column is rabbit anti-mouse Ig antibody as positive control and fixed control. Each subarray is separated by a chip-specific fence or a Super PAP Pen to form an independent reaction unit. Place in saturated humidity at 37°C for 2 hours, wash and dry.

③ Add 3% bovine serum albumin dropwise to the area of the glass slide where the antibody is spotted, place it at 37°C in saturated humidity for 1 hour, wash, dry, and store in a sealed container at 4°C to obtain a fish lymphocyst virus immunoassay chip.

The application of the fish lymphocyst virus immune detection chip of the present invention, described this application step is as follows:

(1) The fish tissues used for detection include: tumors on the surface of fish, epidermis, spleen, stomach, intestines, etc., the samples are mixed with liquid A at a ratio of 1:10 (W/V), homogenized, and repeated Freeze and thaw 3 to 4 times, after ultrasonic crushing, centrifuge at low temperature, 5000rpm, 15min, take the supernatant as the test sample, and wait for the test.

(2) Add different samples to be tested to different subarrays of the above chip, the sample volume is 10 μL/array, one chip can detect eight samples at the same time, pay attention to avoid liquid mixing between different subarrays. Place at 37°C in saturated humidity for 0.25-2h, wash, add diluted solution C (Cy3-labeled mouse anti-LCDV monoclonal antibody), 10 μL/array, place at 37°C in saturated humidity for 0.25-2h, wash, and dry in the air.

(3) Laser scanning

EcoScan^TM-100 CCD扫描仪扫描成像，激发波长为532nm，检测波长为585nm，荧光信号用Lab-chipscanner 2.0软件分析。Crystal core for the above-mentioned detection chip

EcoScan ^TM -100 CCD scanner scans the image, the excitation wavelength is 532nm, the detection wavelength is 585nm, and the fluorescence signal is analyzed by Lab-chipscanner 2.0 software.

Analysis and judgment of test results: In the scanned image subarray, the signal intensity of the negative control in the first column represents the background value of the experiment; the green fluorescent bright spots in the second and third columns are positive, indicating that there is LCDV in the tested sample , no green fluorescent bright spot is negative, indicating that there is no LCDV in the tested sample. The strength of the signal is related to the virus concentration in the sample; it is normal for the positive control and quality control point in the fourth column to have green fluorescence. If there is no green fluorescence, it means that there is a problem with the detection operation or denaturation of the antibody protein, and the test result is invalid.

(4) Quantitative detection and minimum detection limit of virus

After separation and purification, LCDV was diluted to 25.6 μg/mL, and then the 8 antigen concentrations obtained by diluting twice were incubated with 8 subarrays on the glass slide, detected by antibody probes, and the results after scanning analysis are shown in Figure 3 , with the change of antigen concentration, the fluorescent signal presents a gradient change. 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 μg/mL, and there is a good linear relationship between the relative signal intensity and the antigen concentration , suggesting that the constructed antibody microarray can be quantitatively analyzed within a certain range of virus concentration. The lowest antigen concentration that can be detected by this antibody microarray is 1.6μg/mL.

The chip carrier has a glass surface that has been treated with affinity silane and modified with agarose gel. The surface has a three-dimensional porous structure. After activation by NaIO ₄ , antibodies can be immobilized on the surface by physical adsorption and covalent linkage. On it, at the same time, its hydrophilic environment is also conducive to maintaining the activity of the antibody protein immobilized on it. The antibodies are rabbit anti-LCDV antibody and rabbit anti-mouse Ig antibody, but not limited to these two antibodies.

The optimal concentration of rabbit anti-LCDV antibody after affinity chromatography purification used for spotting is 0.5mg/mL.

After the sample to be tested is added to the chip, it is placed at 37° C. in saturated humidity for 30 minutes, and after the diluted antibody probe is added to the glass slide, it is placed at 37° C. in saturated humidity for 30 minutes.

There are 8 sub-arrays on a single slide of the chip, and the number of sub-arrays can be increased according to actual needs, so that simultaneous detection of more samples can be realized.

The advantage of the present invention is that it can detect LCDV in multiple samples or multiple different tissues of the same individual at the same time. It has simple structure, low cost, easy expansion of flux, and realizes parallel detection of multiple samples at the same time, increasing the gap between different sample data. It can quickly, accurately and easily detect LCDV in a variety of cultured fish. The requirements for samples are simple, and a small amount of samples can meet the detection needs, and greatly simplifies the sample processing steps of the existing detection technology, and even can take samples for detection without killing farmed animals. At the same time, due to the setting of positive control and negative control and repeated spotting of capture antibody, the reliability of the detection result is increased. In addition, the immune chip has a relatively fast response and a convenient operation process, which can be operated by ordinary personnel, and can detect pathogens relatively quantitatively, which is suitable for rapid and accurate detection of viral diseases in the breeding process.

Description of drawings

Figure 1. Schematic diagram of fish lymphocyst virus immune chip structure and spot distribution. Among them, 1-chip carrier, 2-agarose gel layer, 3-antibody microarray, 4-chip dedicated fence or Super PAP Pen streak, 5-label area. Sample distribution: ① PBS containing 50% glycerol, as negative control; ②, ③, rabbit anti-LCDV antibody; ④ rabbit anti-mouse Ig antibody, as positive control and fixed control and other quality control.

Fig. 2. CCD scanning images of LCDV in fish samples from different sources detected by the fish lymphocyst virus immunoassay chip (the fluorescence intensity of the scanner is set to 88%). A1 and A2 indicate that no LCDV was detected in the sample; A3 and B3 indicated that the concentration of LCDV in the sample was high; B2 and B4 indicated that there was a certain amount of LCDV in the sample, but the concentration was lower than that of A3 and B3; A4 and B1 indicated that the LCDV content in the sample lower.

Figure 3. The relationship between antigen concentration and signal intensity and the detection limit of this immune chip. The detected antigen concentrations were 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2 μg/mL in turn. The logarithmic value of the antigen concentration is used as the abscissa, and the fluorescence signal intensity is used as the ordinate to analyze the change of the signal intensity. The results show that the antigen concentration is in the range of 0.8-12.8 μg/mL, and there is a good linearity between the relative fluorescence signal intensity and the antigen concentration. relation.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and through specific embodiments.

Instruments and reagents used in the present invention are as follows:

EcoScan^TM-100 CCD扫描仪(购自北京博奥生物公司)；Cy3抗体标记试剂盒(购自GE公司)；HiTrap Protein G SepharoseColumn(购自GE公司)；1640(购自GIBCO公司)；胎牛血清(购自HYCLONE公司)；牛血清白蛋白(购自SIGMA公司)；Tween-20(购自SIGMA公司)；二甲基亚砜(购自SIGMA公司)。Small manual chip pointing system (purchased from Whatman); crystal core

EcoScan ^TM -100 CCD scanner (purchased from Beijing Boao Biological Company); Cy3 Antibody Labeling Kit (purchased from GE Company); HiTrap Protein G SepharoseColumn (purchased from GE Company); 1640 (purchased from GIBCO Company); 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. Antibody preparation and purification

LCDV was extracted from surface tumors of flounder suffering from lymphocyst virus disease, purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-LCDV antibodies were obtained.

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

Monoclonal antibody B and monoclonal antibody C purified by affinity chromatography were mixed, and purebred New Zealand white rabbits were immunized by conventional methods, blood was collected to prepare serum, and rabbit anti-mouse Ig antibodies were obtained.

The antibody obtained was purified using an affinity chromatography column (HiTrap Protein G SepharoseColumn) from Amersham Phamacia Biotech.

2. Preparation of Cy3-labeled monoclonal antibody probes

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

3. Pretreatment of slides

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

4. Preparation of Agarose Gel Substrates

Prepare a 1.2% agarose solution, boil it in a microwave oven for 3 minutes to completely dissolve, spread 2mL of the agarose solution on a clean slide treated with affinity silane preheated at 60°C; after the agarose solidifies, place the slide at 37°C overnight Drying; before use, activate with 0.02mol/L NaIO ₄ solution at room temperature for 30 minutes, rinse thoroughly with ultrapure water three times, blow dry with nitrogen flow, and store in a dry place at room temperature.

5. Chip structure design and lattice distribution

The structure of the chip is shown in Figure 1, including a chip carrier (1), an agarose gel layer (2) covered on the chip carrier, and two rows and four rows of eight 4×4 chips are fixed on the agarose gel layer. Antibody microarray (3) with a sample volume of 50-70 nL and a spot diameter of 500-600 μm. Each microarray is separated by a chip-specific fence or a Super PAP Pen scribe line (4). The chip carrier is a standard glass slide, and a label area (5) can be reserved at one end of the glass slide.

6. Antibody Immobilization

①Dilute the rabbit anti-LCDV antibody with PBS buffer solution containing 50% glycerol at pH 7.4, so that the concentration is 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001mg/mL.

② Spot the antibody dilutions with different concentrations on different areas of the surface of the modified glass slide with a spotting instrument, and each chip has two rows and four columns with a total of 8 4×4 subarrays, and each subarray is a different concentration of antibody. Rabbit anti-LCDV antibody, each subarray is separated by a chip-specific fence or Super PAP Pen to form an independent reaction unit. Place in saturated humidity at 37°C for 2 hours, wash and dry.

③ Add 3% bovine serum albumin dropwise to the area of the glass slide where the antibody is spotted, place it at 37°C in saturated humidity for 1 hour, wash, dry, and store it sealed at 4°C to obtain a fish LCDV immunoassay chip.

Example 2:

Steps 1, 2, 3, 4, 5 are the same as in Example 1.

6. Antibody Immobilization

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

② Spot the diluted antibody solution on different areas of the surface of the modified glass slide with a spotting instrument. The samples in the subarrays are consistent, and 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-LCDV antibodies; the samples in the fourth column are rabbit anti-LCDV antibodies. Mouse Ig antibodies were used as quality control points such as positive control and fixed control. Each sub-array is separated by the chip-specific fence Super PAP Pen to form an independent reaction unit. Place in saturated humidity at 37°C for 2 hours, wash and dry.

③ Add 3% bovine serum albumin dropwise to the area of the glass slide where the antibody is spotted, place it at 37°C in saturated humidity for 1 hour, wash, dry, and store it sealed at 4°C to obtain a fish LCDV immunoassay chip.

Example 3:

The sandwich method was used to detect fish lymphocyst virus.

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

7. Pathogen detection

①Take the fish tissues to be tested (epidermal, gills, stomach or intestines, etc.), mix them with liquid A at a ratio of 1:10 (W/V), homogenate, freeze and thaw repeatedly for 3 to 4 times, and after ultrasonic crushing, Centrifuge at low temperature, 5000rpm×15min, take the supernatant as a test sample, to be tested;

② Add the samples to be tested to different subarrays of the same carrier of the above-mentioned chip. The sample loading volume is 10 μL/array. One chip can detect eight samples at the same time. Pay attention to avoid liquid mixing between different subarrays. Incubate at 37°C with saturated humidity for 15min, 30min, 45min, 60min, 90min, and 120min, wash, add diluted C solution on the chip, 10μL/array, place at 37°C with saturated humidity for 15min, 30min, 45min, 60min, 90min, and 120min, wash, dry;

③Laser scanning

EcoScan^TM-100 CCD扫描仪扫描成像，激发波长为532nm，检测波长为585nm，荧光信号用Lab-chipscanner 2.0软件分析。Crystal core for the above-mentioned detection chip

EcoScan ^TM -100 CCD scanner scans the image, the excitation wavelength is 532nm, the detection wavelength is 585nm, and the fluorescence signal is analyzed by Lab-chipscanner 2.0 software.

Analysis and judgment of test results: In the scanned image subarray, the signal intensity of the negative control in the first column represents the background value of the experiment; the green fluorescent bright spots in the second and third columns are positive, indicating that there is LCDV in the tested sample , no green fluorescent bright spot is negative, indicating that there is no LCDV in the tested sample. The strength of the signal is related to the virus concentration in the sample; green fluorescence is normal for the positive control and fixed point in the fourth column. If there is no green fluorescence, it means that there is a problem with the detection operation or denaturation of the antibody protein, and the detection result is invalid.

Example 4:

Antigen quantitative analysis and minimum detection limit:

Take the prepared fish LCDV immunoassay chip, dilute the isolated and purified LCDV to 25.6 μg/mL, and then incubate with the antibody microarray on the glass slide to the eight antigen concentrations obtained by diluting the purified LCDV to 25.6 μg/mL. Afterwards, the scan results are processed using computer software. The results showed that with the change of the antigen concentration, the fluorescent signal presented a gradient change. The logarithmic value of the antigen concentration is taken as the abscissa, and the fluorescence signal intensity is taken as the ordinate to analyze the change of the signal intensity. 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. , suggesting that the constructed antibody microarray can be quantitatively analyzed within a certain range of virus concentration. The lowest antigen concentration that can be detected by this antibody microarray is 1.6μg/mL.

Example 5:

Specific detection of fish LCDV immunoassay chip:

Take normal fish tissues (gills, stomach, epidermis, etc.), and detect that they are not infected with LCDV by PCR. After the tissue is crushed, it is homogenized. After incubation with the antibody microarray on the antibody probe and scanning after detection by the antibody probe, there was no fluorescent signal, which confirmed that the prepared fish lymphocyst virus immunoassay chip had no cross-reaction with the tissue.

Those skilled in the art will understand that within the protection scope of the present invention, it is possible to modify, add and replace the above-mentioned embodiments, and none of them exceed the protection scope of the present invention.

Industrial Applicability

The invention provides a technical platform for the detection and diagnosis of various pathogens such as viruses and bacteria in cultured fish. It is suitable for disease monitoring in the breeding process and entry-exit inspection and quarantine of aquatic animals. It has broad application prospects and can effectively avoid the The spread and prevalence of similar diseases.

Claims (3)

1. the preparation method of a fish lymphocystis disease virus (LCDV) immunity detection chip is characterized in that it comprises the steps:

(1) preparation of antibody

The antiangiogenic tumour antiviral antibody of preparation rabbit, the antibody of the anti-mouse Ig of rabbit, mouse-anti lymphocystis disease virus monoclonal antibody, affinity chromatography purifying gained antibody;

(2) preparation of Cy3 labeled monoclonal antibody probe

With the mouse-anti lymphocystis disease virus monoclonal antibody Cy3 mark behind the affinitive layer purification, and cross the gel column purifying;

(3) pre-service of microslide

Microslide is embathed with highly basic and the concentrated sulphuric acid respectively, and the distilled water flushing is dried; Microslide after cleaning immersed in the acetic acid solution of 0.4% affine silane, transfer pH to 4.5, act on 1h under the room temperature, the distilled water flushing is dried;

(4) preparation of Ago-Gel substrate

The agarose solution of preparation 1.2%, micro-wave oven boils 3min, and it is dissolved fully, the 2ml agarose solution is spread overlay on the cleaning microslide that the affine silane treatment of 60 ℃ of preheatings is crossed; After agarose solidifies, with slide 37 ℃ of lower dried overnight; At room temperature use 0.02mol/L NaIO before the use ₄Solution activation 30min, ultrapure water flushing 3 times dries up with nitrogen stream, and the drying at room temperature place preserves;

(5) antibody is fixing

1. with the antiangiogenic tumour antiviral antibody of PBS damping fluid dilution rabbit that contains 50% glycerine of pH 7.4, making its concentration is the antibody of 0.5 ~ 0.0001mg/mL, the anti-mouse Ig of dilution rabbit, and making its concentration is 0.1mg/mL;

2. with point sample instrument with the zones of different point sample of this antibody diluent at the Ago-Gel substrate, the point sample amount is 50 ~ 70nL, spot diameter is 500 ~ 600 μ m; Every chip two rows four are listed as totally 84 * 4 inferior arrays, sample that each inferior array is put is consistent, sample that first row is selected is that phosphate glycerine damping fluid is as negative control, second and third row sample of putting is the antiangiogenic tumour antiviral antibody of rabbit, the 4th classifies qualifying point as, and the sample of putting is that the antibody of the anti-mouse Ig of rabbit reaches fixing contrast as positive control; Separate with chip fence special or Super PAP Pen between each inferior array, form independently reaction member; 37 ℃ of saturated humidities are placed 2h, and washing is dried;

3. point has the zone of antibody to drip 3% bovine serum albumin(BSA) to seal on the microslide, and 37 ℃ of saturated humidities are placed 1h, and washing is dried, and 4 ℃ of sealings are preserved, and obtain fish lymphocystis disease virus (LCDV) immunity detection chip.

2. the preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip according to claim 1, it is characterized in that with the mouse-anti lymphocystis disease virus monoclonal antibody behind the Cy3 mark affinitive layer purification as antibody probe, and the antibody that uses the anti-mouse Ig of rabbit is as positive control and fixing contrast.

3. the preparation method of fish lymphocystis disease virus (LCDV) immunity detection chip according to claim 1, it is characterized in that the used monoclonal antibody of Cy3 labelled antibody probe is the antiangiogenic tumour virus envelope of specificity monoclonal antibody B and the monoclonal antibody C that is secreted respectively by two strain of hybridoma, its preserving number is respectively: CCTCC-C200419 and CCTCC-C200420.

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