CN105543176A - Hybridoma cell strain secreting potato-virus-Y-resistant monoclonal antibodies and monoclonal antibody application thereof - Google Patents

Abstract

The invention discloses a hybridoma cell line secreting anti-potato virus Y (PVY) monoclonal antibody and the application of the monoclonal antibody. The PVY virus particles purified by differential centrifugation were used as antigens to immunize BALB/c mice. After cell fusion, screening and cloning, a hybridoma cell line 3B2, which can be stably passaged and secretes anti-PVY monoclonal antibody, was obtained. The preservation number is CGMCC? No. 12001. The indirect ELISA titer of the monoclonal antibody secreted by the cell line reached 10 ^-7 , and the type and subclass of the antibody were IgG1 and kappa light chain. The monoclonal antibody had a specific reaction with the 30kDa PVY coat protein. Using 3B2 monoclonal antibody to establish ACP-ELISA, dot-ELISA and Tissue for detecting PVY on crops? blot-ELISA detection method, in which the sensitivity of ACP-ELISA and dot-ELISA methods to detect diseased leaves reached 1:81920 and 1:10240 times dilution (w/v, g/mL) respectively. The preparation of anti-PVY monoclonal antibody and the establishment of its detection method provide material and technical support for the diagnosis and detection, epidemiological analysis and scientific prevention and control of the potato virus disease.

Description

Hybridoma Cell Line Secreting Potato Virus Y Monoclonal Antibody and Application of Monoclonal Antibody

technical field

The invention relates to the field of biotechnology, in particular to a hybridoma cell line secreting anti-potato virus Y monoclonal antibody and the application of the monoclonal antibody.

Background technique

Potatovirus Y (PVY) is one of the important viruses that harm potato, tobacco and other crops. It spreads widely all over the world and causes serious economic losses. PVY is a representative member of the genus Potyvirus in the family Potyviridae, and was first discovered in potato by Smith in 1931. The genus Potatovirus Y is the largest genus of plant viruses, with about 200 confirmed and possible species. Viruses in this genus can infect many plants such as Solanaceae, Chenopodiaceae, Fabaceae, Cucurbitaceae, etc., and cause significant economic losses. . Since 1953, PVY has been widespread in potato growing areas in Europe. In the 1970s, the virus spread to the Americas. In the early 1990s, the virus began to spread in Asia. Now, the virus is causing harm all over the world. Symptoms caused by PVY vary with different host varieties and virus strains. Typical symptoms include heavy mosaic, necrosis of leaf veins, and necrosis of weeping leaf streaks, which can cause up to 80% loss of potato yield. The study found that the primary source of PVY infection is mainly virus-carrying seed potatoes, and the pathogen can spread long-distance through seed potato transportation. In potato fields, PVY can also spread through disease sap and poison-carrying aphids. PVY particles are curved and linear, without envelope, with a size of 730×11nm. The genome is about 10kb in length. ). The genome contains only one large ORF, encoding a polyprotein of about 360kDa, which can be cleaved into 11 mature functional proteins, including a coat protein of 29.8kDa.

Anti-virus breeding and non-toxic seed potato production are mainly used to control PVY, and rapid, accurate, sensitive and specific virus detection technology is the key to anti-virus breeding and non-toxic seed potato production. Compared with conventional indicator plant detection, electron microscopy detection, molecular biology detection and other methods, serological methods have the advantages of simplicity, economy, easy operation, and large-scale detection. There have been reports about the preparation of PVY antibodies and serological methods, but the detection sensitivity of the reported PVY antibodies and serological methods is not good enough. For this reason, the present invention uses purified PVY virions as antigens to prepare 1 by hybridoma technology. A hybridoma cell line that secretes a specific and sensitive monoclonal antibody against PVY, and establishes a high-throughput serological method and kit for detecting PVY with the secreted monoclonal antibody as the core, so as to contribute to the detection, diagnosis and epidemiology of PVY in my country. Provide material and technical support for the investigation of the virus, the production of non-toxic seed potatoes, the analysis of the function of the virus genome and the establishment of a scientific prevention and control system.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a hybridoma cell line secreting anti-potato virus Y monoclonal antibody and the application of the monoclonal antibody.

Secreting anti-potato virus Y monoclonal antibody hybridoma cell line 3B2, which can secrete anti-potato virus Y monoclonal antibody, the hybridoma cell line 3B2 was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee on January 7, 2016, and the preservation number is It is CGMCC No.12001.

An anti-potato virus Y monoclonal antibody secreted by a hybridoma cell line, the anti-potato virus Y monoclonal antibody ascites indirect ELISA titer reaches 10 ^-7 , the antibody type and subclass are IgG1, kappa light chain, and potato virus Y The coat protein of 30KDa has a specific immune response. Using ACP-ELISA and dot-ELISA methods, it was found that the sensitivity of the monoclonal antibody to detect the diseased leaves infected with potato virus Y reached 1:81920 and 1:10240 times dilution (w/v, g/ mL).

Anti-potato virus Y monoclonal antibody has a specific immune reaction with plant tissues infected with potato virus Y, but not with potato virus A, potato virus X, potato S virus, potato leafroll virus, and healthy tobacco and potato leaf tissues immune response.

The application of anti-potato virus Y monoclonal antibody in the virus detection is various immunological detection methods and immunological kits established with monoclonal antibody as the core.

Compared with the prior art, the present invention has beneficial effects: 1) The hybridoma cell line provided can stably secrete a large amount of anti-PVY specific and sensitive monoclonal antibody, and the ACP-ELISA and dot-ELISA established with the secreted monoclonal antibody as the core Serological methods such as Tissueblot-ELISA and the kits established by these methods can detect the potato virus Y in plants with high specificity, accuracy and sensitivity; Microscope, PCR instrument and other equipment; 3) Utilize the prepared monoclonal antibody of the present invention, can be used effectively in the detection and diagnosis of PVY in the field plant, also can be used for the epidemiological investigation of this virus disease, virus genome function analysis, detoxification Poisonous seed potato production, resistance breeding, scientific prevention and control, etc.

Description of drawings

Figure 1 The specificity analysis of the detection of potato virus Y by dot-ELISA method.

Fig. 2 Sensitivity analysis of PVY detection by dot-ELISA method.

Figure 3 The results of dot-ELISA detection of PVY in field plant samples.

Fig. 4 Results of RT-PCR method for detecting PVY in field plant samples.

Fig. 5 The results of field detection of PVY in plant samples by Tissueblot-ELISA method.

biological deposit

The hybridoma cell line 3B2 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on January 7, 2016, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101, and the preservation number is CGMCCNo.12001 .

detailed description

Secreting anti-potato virus Y monoclonal antibody hybridoma cell line 3B2, which can secrete anti-potato virus Y monoclonal antibody, the hybridoma cell line 3B2 was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee on January 7, 2016, and the preservation number is It is CGMCC No.12001.

An anti-potato virus Y monoclonal antibody secreted by a hybridoma cell line, the anti-potato virus Y monoclonal antibody ascites indirect ELISA titer reaches 10 ^-7 , the antibody type and subclass are IgG1, kappa light chain, and potato virus Y The coat protein of 30KDa has a specific immune response. Using ACP-ELISA and dot-ELISA methods, it was found that the sensitivity of the monoclonal antibody to detect the diseased leaves infected with potato virus Y reached 1:81920 and 1:10240 times dilution (w/v, g/ mL).

Anti-potato virus Y monoclonal antibody has a specific immune reaction with plant tissues infected with potato virus Y, but not with potato virus A, potato virus X, potato S virus, potato leafroll virus, and healthy tobacco and potato leaf tissues immune response.

The application of anti-potato virus Y monoclonal antibody in the virus detection is various immunological detection methods and immunological kits established with monoclonal antibody as the core.

The hybridoma cell strain provided by the invention can secrete a large amount of anti-PVY monoclonal antibody, and the monoclonal antibody secreted by the cell strain has strong specificity, high potency, good stability and high sensitivity. A high-throughput serological method for PVY detection based on this monoclonal antibody can be successfully applied to the detection of PVY in field plants, thus providing a basis for the detection and diagnosis of potato virus Y disease in my country, the production of virus-free seed potatoes, and scientific prevention and control. material and technical support.

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

1. Obtaining hybridoma cells and preparation of monoclonal antibodies

1. Preparation of immunogen and detection antigen

Purify virions using the following procedure:

1) Pre-cool the tissue homogenizer on ice;

2) Weigh 200 g of PVY-infected tobacco tissue, add 200 mL of 0.5M phosphate buffer saline, namely PB buffer (containing 0.01M Na-EDTA and 0.1% mercaptoethanol, pH 7.5) per 100 g of tissue, and homogenize in a tissue homogenizer Slurry for 5-10min to fully homogenize the tobacco tissue, filter the homogenate with double-layer cotton gauze, and centrifuge the filtrate at 6000rpm for 20min to remove plant residues;

3) Add the obtained centrifuged supernatant to a final concentration of 2.5% TritonX-100, 4% PEG (molecular weight: 6000) and 0.1M NaCl while stirring, and then stir at 4°C for more than 4 hours;

4) Centrifuge at 11000rpm for 15min to remove the supernatant;

5) Fully suspend the precipitate with 10 mL of 0.5MPB (containing 0.01MMgCl2 and 0.5M urea) at pH 7.5, centrifuge at 6000rpm for 15min, collect the supernatant in a beaker, place at 4°C, resuspend the precipitate and centrifuge, repeat 3 times;

6) Combine the above centrifuged supernatants and ultracentrifuge at 33000rpm for 100min;

7) Suspend the obtained precipitate with 0.5MPB, centrifuge at 8000rpm for 15min, collect the supernatant, resuspend the precipitate, and centrifuge, repeating 3 times;

8) Add the combined supernatant to the ultracentrifuge tube, absorb 20 mL of 30% sucrose with a syringe needle, add it to the bottom of the ultracentrifuge tube to form a sucrose cushion, and ultracentrifuge at 33000 rpm for 100 min;

9) Suspend the obtained precipitate with an appropriate amount of 0.01MPB (containing 0.01MMgCl2) of pH 7.5, ultracentrifuge at 33000rpm for 100min, and remove the supernatant;

10) The obtained precipitate is suspended with 0.01MPB, and the obtained suspension is the virus purification liquid;

11) After the virus purification solution was negatively stained with 3% phosphotungstic acid (pH6.7), it was placed under a JEOLJEM-1200EX electron microscope and a large number of high-purity PVY particles were found.

2. Immunization of Animals

Immunize 8-week-old BALB/c female mice with purified PVY virus particles: mix 100 μL/mouse of PVY virus particles with an equal volume of Freund’s complete adjuvant, fully emulsify, and inject 0.2 mL of each mouse subcutaneously through the abdomen at multiple points, at intervals After 3 weeks, take the same amount of antigen and the same volume of Freund's incomplete adjuvant as the first vaccine and fully emulsify it, and inject 0.2mL each mouse intraperitoneally for the second time. Splenocytes were fused.

3. Cell Fusion

Take the spleen cells of the above immunized mice and mouse myeloma cells (SP2/0) in the ratio of 5:1 and mix them evenly in serum-free RPMI-1640 (Gibco) medium, centrifuge at 1500rpm for 5min, remove the medium, and contain the cells Add 1 mL of 50% PEG (molecular weight: 1500) fusion agent to the centrifuge tube in a 37°C water bath, and fuse for 2 minutes. Terminate the fusion with serum-free RPMI-1640 medium and centrifuge at 1500 rpm for 5 minutes. Suction the supernatant and suspend the precipitate with HAT medium. Aliquot into 96-well cell plates and culture in a cell culture incubator at 37°C and 5% CO ₂ .

4. Screening of hybridoma cells, positive wells and their cloning

After culturing in the cell incubator for 5 days, replace the medium with HAT medium once, and change the medium with HT medium on the 10th day. When the fused cells cover 5%-20% of the bottom of the well, use the crude extract of potato diseased leaves infected with PVY as the coating The positive wells were screened by conventional indirect ELISA method for antigen, and a total of 298 positive wells were obtained. The specificity and sensitivity of 298 wells were analyzed, and 8 specific and sensitive cell wells were screened out, and cloned by cell limiting dilution method to obtain a hybridoma cell line 3B2 that can secrete specific and sensitive monoclonal antibody against PVY. After more than 4 months of in vitro passage and repeated cryopreservation and recovery, the cell lines can grow well and secrete antibodies stably. After expanded culture, it is used for the preparation of monoclonal antibody ascites and storage in liquid nitrogen.

5. Preparation and purification of monoclonal antibody ascites

8-week-old BALB/c mice were injected intraperitoneally with 0.3mL pristane, and 7-10 days later, 7×10 ⁵ hybridoma cells were injected into the peritoneal cavity. After 7-10 days, the abdomen of the mice was obviously enlarged. Ascites was collected with a needle and centrifuged at 8000rpm After 3 minutes, the supernatant was collected as monoclonal antibody ascites.

Take 1 volume of ascites and 2 volumes of 0.06 MPH4.8 acetate buffer to dilute, add octanoic acid (30 μl/mL ascites) while stirring at room temperature, clarify at 4°C for 1 hour, centrifuge at 12,000 rpm for 20 minutes, collect the supernatant, and then use 50% saturated Precipitate immunoglobulin with ammonium sulfate, place at 4°C for 2h, centrifuge at 3000rpm for 20min, dissolve the precipitate with PBS solution 2 times the volume of ascitic fluid, obtain purified monoclonal antibody after dialysis at 4°C for 24h, and store at -70°C.

6. Subclass identification of monoclonal antibody and ascites titer determination

The purified monoclonal antibody and Sigma's standard anti-BALB/c mouse IgG ₁ , IgG _2a , IgG _2b , IgG ₃ , and IgM antibodies were tested by DAS-ELISA. The test results showed that the type and subclass of 3B2 monoclonal antibody were IgG1, kappa light chain. Using the purified PVY virus particles as antigen, the titer of monoclonal antibody in ascites was detected by conventional indirect ELISA method, and the analysis results showed that the titer of monoclonal antibody in ascites reached 10 ^-7 .

7. Specific detection of monoclonal antibodies

Coat the ELISA plate with crude extracts of diseased leaves infected with potato virus A (PVA), potato virus X (PVX), potato virus S (PVS) and potato leafroll virus (PLRV), and use crude extracts of healthy tobacco and potato leaves As a negative control, the crude extract of tobacco leaves infected with PVY was used as a positive control, and the specificity of monoclonal antibody was analyzed by conventional ACP-ELISA method. The results of specificity analysis showed that the 3B2 monoclonal antibody had a specific immune response to the diseased leaves infected with PVY, but had no reaction with the diseased leaves infected with PVA, PVX, PVS and PLRV, and the crude extracts of healthy tobacco and potato tissues.

2. Establishment of immunological methods and kits for detection of PVY

1. ACP-ELISA method for detection of PVY

1.1 The steps of ACP-ELISA method:

1) Weigh the diseased leaves and healthy leaves respectively, put them into a mortar, add ELISA coating solution at a ratio of 1:20 (w/v, g/mL), grind and homogenate; centrifuge the homogenate at 5000rpm for 3min; 100 μL/well of supernatant was coated with polystyrene plate, and PVY-infected tobacco tissue was used as positive control, and healthy tobacco and potato tissues were used as negative control, at 37°C for 2h or; at 4°C overnight

2) Wash the plate 3 times with PBST, 3 minutes each time, add 250 μL of PBST blocking solution containing 3% skimmed milk powder to each well, and block at 37 ° C for 0.5 h;

3) After washing 3 times with PBST, 100 μL/well of monoclonal antibody ascitic fluid appropriately diluted with blocking solution, at 37°C for 1-2 hours;

4) After washing with PBST for 3 times, add appropriately diluted AP-labeled rabbit anti-mouse IgG secondary antibody (Sigma) 100 μL/well, 37°C for 1-2h;

5) After washing with PBST for 4 times, add PNPP substrate to develop color at room temperature for 30-60 minutes, observe the color change of the substrate with the naked eye, and the sample that develops into yellow-green well is positive, or after 2M sodium hydroxide terminates the reaction, use 680-type enzyme The OD value at 405nm was measured by an immunoassay instrument, and P/N>2.1 was used as a positive judgment standard.

1.2 Establishment of ACP-ELISA detection method:

Use square array test to determine the working concentration of the antibody, that is, use 1:20 times diluted (w/v, g/mL) crude extract of PVY diseased leaves as the coating antigen to coat the polystyrene plate; the ELISA plate is vertically from top to bottom Next, add PVY monoclonal antibody diluted from 1:100 to 1:12800 times with blocking solution, and add AP-labeled goat anti-mouse diluted from 1:1000 to 1:512000 times with blocking solution from left to right. IgG secondary antibody, crude extracts of corresponding dilutions of healthy tobacco or potato plants were used as negative controls, each treatment was repeated three times, and P/N>2.1 was used as a positive judgment standard to determine the optimal antibody in ACP-ELISA working concentration. The results showed that the 3B2 monoclonal antibody was diluted 1:5000 times, and the AP-labeled goat anti-mouse IgG secondary antibody was diluted 1:7000 times as the optimal working concentration. According to the optimal working concentration, the ACP-ELISA method for detecting PVY was established.

1.3 Sensitivity and specificity analysis of ACP-ELISA method

The crude extract of PVY diseased leaves was diluted with coating solution and measured by the established ACP-ELISA method. The results showed that the sensitivity of ACP-ELISA to detect diseased leaves reached 81920 times dilution (w/v, g/mL), indicating that The prepared mAb and the established method have good sensitivity. This method shows a strong positive reaction in detecting the crude extract of PVY diseased leaves, and a negative reaction in detecting diseased leaf tissues infected with PVA, PVX, PVS and PLRV and healthy tobacco and potato tissues, and the difference between negative and positive results is extremely significant, indicating that The specificity of this method and monoclonal antibody is good.

2. The establishment of dot-ELISA method and its application in field testing

The steps of the dot-ELISA method for detecting PVY in plants: Grind the weighed plant tissue in a mortar and add 0.01MPBS (pH7.4) at a ratio of 1:10-30 (w/v, g/mL) to continue Grind and homogenate; centrifuge the homogenate at 5000rpm for 3min; take 2.5μL supernatant and spot on nitrocellulose (NC), and set the crude extracts of healthy and PVY-infected potato leaves as negative and positive controls respectively; dry at room temperature for 10-20min; Immerse the NC membrane in PBST containing 5% skimmed milk powder (0.01MPBS containing 0.05% Tween-20) blocking solution at room temperature for 30min; put the NC membrane into a moderately diluted monoclonal antibody and incubate at room temperature for 30-60min; wash the membrane with PBST for 3 -4 times, 3 min each time; put the NC membrane into moderately diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody, and incubate at room temperature for 30-60 min; wash the membrane 4-5 times with PBST, 3 min each time; 66 μL NBT and 33 μL BCIP substrate ( Promega) was added to 10ml of substrate buffer (0.1MTrisCl, 0.1MNaCl, 0.025MMgCl, pH9.5), mixed evenly, the membrane was put into the substrate solution to develop color, and the results were observed with the naked eye, until the positive control showed purple and was negative When there is no color development in the control, rinse the membrane with tap water to terminate the reaction, and take pictures to record the results.

The optimum working concentration of dot-ELISA monoclonal antibody and enzyme-labeled secondary antibody for detecting potato diseased leaves was determined by conventional square array test. The test showed that the optimum working concentration of 3B2 monoclonal antibody and enzyme-labeled secondary antibody were 1:5000 and 1:5000, respectively. 8000-fold dilution. The dot-ELISA method for detecting PVY was established with the optimum working concentration of the above antibodies. Specificity analysis showed that the method showed a strong positive reaction in the detection of PVY-infected plants, but negative reactions in the detection of PVA, PVX, PVS, PLRV diseased leaves and healthy tobacco and potato leaves (Figure 1). Sensitivity analysis showed that when the crude extract of PVY diseased leaves was diluted to 1:10240 times (w/v, g/mL), the established dot-ELISA still showed purple positive spots, that is, the sensitivity of detecting diseased leaves reached 1:10240 times dilution (Figure 2).

The established dot-ELISA method was used to detect the suspected diseased potato samples collected from Yunnan in 2015. It was found that 20 of the 30 potato samples produced purple positive spots (Figure 3), and the samples were simultaneously tested with RT- Analysis by PCR method showed that PVY-specific gene fragments were amplified in all dot-ELISA positive samples, while no gene fragments were amplified in all dot-ELISA negative samples (Fig. 4). Sequencing indicated that positive samples were indeed infected with PVY. It shows that the dot-ELISA method can be used for the detection of PVY in plants accurately and reliably.

3. Establishment of Tissueblot-ELISA method for detecting PVY and its field sample detection

3.1 Tissueblot-ELISA operation steps:

1) Sample preparation: Take the leaves or stems of the crops, roll the young leaves into a cylindrical shape and cut a cross-section with a blade, and cut the young stems directly into a cross-section;

2) Spotting: press the cross-section on the NC film for 3 sec, and simultaneously use healthy and infected PVY plant tissues as negative and positive controls, respectively, and dry in an oven at 37°C for 5 minutes;

3) Sealing: immerse the spotted NC membrane in PBST (0.01MPBS containing 0.05% Tween-20) blocking solution containing 5% skimmed milk powder and block at room temperature for 30-60min;

4) Primary antibody incubation: NC membrane was incubated in appropriately diluted PVY monoclonal antibody for 1 h at room temperature;

5) Secondary antibody incubation: wash the membrane with PBST for 3 times, then put the NC membrane into appropriately diluted AP enzyme-labeled goat anti-mouse IgG and incubate at room temperature for 1 h;

6) Wash the membrane with PBST 4-5 times, 3 minutes each time;

7) Add substrate for color development: add 66 μl NBT and 33 μl BCIP substrate (Promega) to 10 ml of color development buffer (0.1MTrisCl, 0.1MNaCl, 0.025MMgCl, pH9.5) and mix well, then immerse the membrane in the color development solution for color development React until the positive color development is obvious and the negative control has no background, rinse the membrane in deionized water to terminate the reaction, and record the color development results.

3.2 Determination of optimal working concentration of antibody in Tissueblot-ELISA and detection of field samples

The optimal working concentration of monoclonal antibody and enzyme-labeled secondary antibody in Tissueblot-ELISA was determined by routine array experiments. The combination of monoclonal antibody and enzyme-labeled secondary antibody dilution with the best detection sensitivity and specificity was selected as the optimal working concentration of Tissueblot-ELISA. The results showed that the detection sensitivity and specificity of the Tissueblot-ELISA method were the best when the monoclonal antibody 3B2 and the AP-labeled goat anti-mouse secondary antibody were diluted 1:5000 and 1:8000 times, respectively, and the Tissueblot-ELISA method was established according to the optimal working concentration of the antibody. The established Tissueblot-ELISA method was used to detect the suspected diseased potato samples in the field collected from Yunnan in 2015. It was found that 20 of the 30 potato samples produced purple positive spots (Figure 5), and the samples were simultaneously tested with RT- Analysis by PCR method showed that PVY-specific gene fragments were amplified in all Tissueblot-ELISA positive samples, while no gene fragments were amplified in all Tissueblot-ELISA negative samples (Figure 4), and the PCR product nucleic acid Sequencing indicated that positive samples were indeed infected with PVS. It shows that the Tissueblot-ELISA method can be used for the detection of potato virus Y in potato samples accurately and reliably.

4. Potato virus Y dot-ELISA detection kit

1) Main components of the kit:

The above reagents were stored at 4°C

Nitrocellulose membrane (NC) 10 pieces

2) Operation steps for detecting potato samples:

a. After grinding the weighed plant tissue in a mortar, add 0.01MPBS (pH7.4) at a ratio of 1:10-30 (w/v, g/mL) and continue grinding and homogenizing;

b. The homogenate was centrifuged at 5000rpm for 3min;

c. Take 2.5 μl of the supernatant and spot on the NC membrane, and set healthy and PVY-infected potato tissues as negative and positive controls respectively, and dry at room temperature for 10-20 minutes;

d. Immerse the NC membrane in PBST (0.01 MPBS containing 0.05% Tween-20) blocking solution containing 5% skimmed milk powder and block at room temperature for 30 minutes;

e. NC membrane was incubated in 1:3000 diluted monoclonal antibody for 30-60min at room temperature;

f. Wash the membrane 3-4 times with PBST, 3 minutes each time; put the NC membrane into 1:3000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 30-60 minutes;

g. Wash the membrane with PBST 4-5 times, 3 minutes each time;

h. Add 66 μl of NBT and 33 μl of BCIP substrate into 10 mL of substrate buffer solution (0.1MTrisCl, 0.1MNaCl, 0.025MMgCl, pH9.5), mix well, put the membrane into the substrate solution for color reaction, and observe the results with the naked eye;

i. Rinse the membrane in tap water to terminate the reaction when the positive control has obvious color development (purple), but the negative control has no color development, and take pictures to record the results.

3) Storage and validity period

Store in the dark at 2-8°C, valid for 12 months.

4) Buffer formula:

Phosphate buffer (0.01MPBS, pH7.4):

Add distilled water 950 to dissolve, adjust the pH to 7.4, and set the volume to 1000mL

ELISA washing solution (0.01MPBST): add 0.5mL Tween-20 to 1000mL0.01MPBS

ELISA blocking solution: Add skim milk powder to 0.01MPBST to a final concentration of 5% (w/v, g/mL).

Claims (4)

1. A hybridoma cell line 3B2 secreting anti-potato virus Y monoclonal antibody, characterized in that it can secrete anti-potato virus Y monoclonal antibody, and the hybridoma cell line 3B2 was preserved in Chinese microbial strains on January 7, 2016 General Microorganism Center of the Preservation Management Committee, the deposit number is CGMCCNo.12001. 2. an anti-potato virus Y monoclonal antibody secreted by a hybridoma cell line as claimed in claim 1, characterized in that the monoclonal antibody ascitic fluid indirect ELISA titer reaches 10 ⁻⁷ , and the antibody type and subclass are IgG1, kappa light chain, which has a specific immune reaction with the 30KDa coat protein of potato virus Y. Using ACP-ELISA and dot-ELISA methods, it was found that the sensitivity of monoclonal antibody to detect diseased leaves infected with potato virus Y reached 1:81920 and 1:10240 times dilution, respectively. The unit is g/mL. 3. anti-potato virus monoclonal antibody as claimed in claim 2 is characterized in that this monoclonal antibody has specific immune reaction with the plant tissue that infects potato virus, and with infection potato A virus, potato virus X, potato S virus , potato leafroll virus and healthy tobacco and potato leaf tissues did not produce immune responses. 4. the application of anti-potato virus Y monoclonal antibody as claimed in claim 2 on this virus detection, it is characterized in that take monoclonal antibody as the various immunological detection method and immunological detection kit that core is established.