CN105671001A - Hybridoma cell strain capable of secreting anti-Southern-bean-mosaic-virus monoclonal antibody and application of monoclonal antibody thereof - Google Patents

Abstract

The invention discloses a hybridoma cell strain capable of secreting an anti-Southern-bean-mosaic-virus monoclonal antibody and an application of the monoclonal antibody thereof. Purified Southern bean mosaic virus (SBMV) particles serve as antigen immunization BALB/c mice, and are subjected to cell fusion, cell screening and cell cloning, the hybridoma cell strain 19H9 capable of conducting stable passage and secreting the anti-SBMV monoclonal antibody is obtained, and the collection number is CGMCC No.12004. The ascites indirect ELISA titer of the monoclonal antibody secreted by the hybridoma cell strain is 10<-7>, and the type and the subgenera of the antibody are IgG1 light chains and kappa light chains. The monoclonal antibody and SBMV are subjected to a specific reaction. An ACP-ELISA method for detecting the SBMV in leguminous plants and a dot-ELISA method for detecting the SBMV in leguminous plants are built with the monoclonal antibody 19H9, and the diseased leaf detection sensitivity of the ACP-ELISA method and the dot-ELISA method is 1:81,920 w/v and 1:5,120 g/ml. The hybridoma cell strain 19H9, the obtained monoclonal antibody and the built serological detecting method provide substance supports and technical supports for diagnosing, detecting and scientific preventing and controlling of the virus disease.

Description

Hybridoma Cell Line Secreting Monoclonal Antibody Against Southern Bean Mosaic Virus and Application of Monoclonal Antibody

technical field

The invention relates to the field of biotechnology, in particular to a hybridoma cell strain secreting anti-Southern Bean Mosaic Virus monoclonal antibody and the application of the monoclonal antibody.

Background technique

Southern bean mosaic virus (SBMV) is a typical member of the genus Sobemovirus, mainly including two strains, SBMV-B and SBMV-C. It was first discovered and reported by Zaumeyer and Harter. The virus is mainly distributed in tropical and subtropical regions, and is also distributed in temperate regions. It can infect and harm soybeans, kidney beans, pumpkins and other plants. Bean mosaic virus disease in southern China generally occurs in the field, with many transmission routes and serious damage. Under natural conditions, it can cause mosaic and mottled diseases of kidney bean, cowpea, black mung bean and soybean, resulting in a decrease in the number of flowers, fruits and seeds, and a decrease in yield. Some countries and regions such as Denmark, Australia, Egypt, and East Africa have listed it as a quarantine object. Our country has the conditions to take root, reproduce, and spread. Once it is introduced, there will be endless troubles. At present, it has been included in my country's second-class quarantine pests, and has been quarantined according to law since 1992.

The SBMV virion is an equiaxed and symmetrical icosahedron (T=3), without envelope, with a diameter of about 30 nm. Its genome contains a molecule of linear single-stranded positive-sense RNA with a length of about 4100-4500nt, accounting for about 21% of the weight of the virion. The buoyant density of SBMV in CsCl is 1.36g/cm ³ , and the sedimentation coefficient is 115S. The lethal temperature of the virus is 90-95°C, the dilution limit is 10 ^-5 -10 ^-6 , and the in vitro survival period (18-22°C) is 1.65-20d. There is neither Poly(A) nor tRNA-like structure at the 3' end of the viral RNA. There is a VPg at the 5' end, which may be necessary for virus infection. It contains 4 ORFs. ORF1 encodes a 21kDa P1 protein. The protein is related to the intercellular movement of the virus; the 105kDa P2 protein encoded by ORF2 may be a polymerase; ORF3 is located within ORF2 and encodes an 18kDa protein whose function is still unknown; ORF4 encodes a 30kDa coat protein.

The virus particles mainly exist in the mesophyll cells of the host plant, and are often dispersed or aggregated in the cytoplasm and vacuoles. SBMV-infected plants formed large virus crystals in the cytoplasm, and produced many special vesicle structures containing thin fibrils. Infected cells showed vesicles, and chloroplasts were deformed by extrusion, but no virus particles were found in chloroplasts and mitochondria. The host range of SBMV is very narrow, mostly leguminous plants, and the natural hosts are kidney beans and cowpea. Among them, SBMV-B strain can infect most varieties of kidney bean, while strain SBMV-C can only infect cowpea. Its artificial inoculation hosts are relatively wide, and can infect about 12 genera and 23 species of plants such as soybean, mung bean, red bean, runner bean, cotton bean, pea, broad bean, and sweet-scented clover. The pathogenic symptoms caused by infestation of kidney beans mainly include mottling and mosaic, but the symptoms of different varieties are very different, some are systemic, some are local, some are mild, and some are accompanied by shrinkage and along the veins. Symptoms such as discoloration. SBMV is mainly spread by leaf beetles in the field, and can also be spread mechanically and by seeds.

At present, leguminous plants such as kidney bean and cowpea are planted in a large area and in a wide range in my country, and with the increasing import and export trade and germplasm exchange, the chances of the introduction of this virus are also greatly increased. In order to protect the production of bean crops in my country, To prevent the virus from entering the country from abroad, a set of simple, fast and effective quarantine and detection methods should be established. At present, biological detection, molecular biological detection and electron microscopy detection techniques are generally used for SBMV port detection. These methods are inefficient and not suitable for large-scale sample detection. Serological methods are simple to operate, have good specificity, and can detect large-scale samples at the same time, but they must rely on specific virus monoclonal antibodies. The present invention is mainly aimed at the preparation and detection application of the monoclonal antibody of Southern Bean Mosaic Virus. Using hybridoma technology, a hybridoma cell 19H9 that can secrete anti-SBMV monoclonal antibody is prepared, and the monoclonal antibody secreted by it is the core Serological methods and detection kits for the detection of the virus were established to improve the detection level and speed of the southern bean mosaic virus in my country and prevent the virus from being introduced into China from abroad.

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 monoclonal antibody against southern bean mosaic virus and the application of the monoclonal antibody.

The hybridoma cell line 19H9, which secretes monoclonal antibodies against Southern Bean Mosaic Virus, was deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms on January 7, 2016. The preservation number is CGMCCNo.12004. It can secrete anti-Southern Bean Mosaic Virus Mosaic virus specific monoclonal antibody.

A monoclonal antibody against southern bean mosaic virus secreted by the hybridoma cell line 19H9, the ascites indirect ELISA titer of the monoclonal antibody against southern bean mosaic virus is up to 10 ^-7 , and the antibody type and subclass are IgG1 , kappa light chain. The ACP-ELISA method was used to analyze and found that the sensitivity of the monoclonal antibody to detect diseased leaves reached 1:81920 times dilution (w/v, g/mL).

The monoclonal antibody against Southern bean mosaic virus has specific immune reaction only with the diseased leaf tissue infected with Southern bean mosaic virus, but with common bean mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, cucumber flower Diseased leaf tissues of leaf virus and healthy soybean, edamame, cowpea, pea, broad bean and kidney bean plant tissues did not have any immune response.

The application of anti-Southern bean mosaic virus monoclonal antibody in the detection of anti-Southern bean mosaic virus is a variety of immunological detection methods and immunological kits established with monoclonal antibodies as the core.

Compared with the prior art, the present invention has beneficial effects: 1) The provided hybridoma cell line 19H9 secretes anti-Southern Bean Mosaic Virus-specific monoclonal antibody, and the dot-ELISA and ACP-ELISA established with the monoclonal antibody as the core Immunological methods such as and the test kit that these methods are established can detect southern bean mosaic virus highly specifically, accurately and sensitively; 2) Utilize the prepared monoclonal antibody of the present invention to detect southern bean mosaic virus and do not need expensive electron microscope , PCR instrument and other equipment; 3) the monoclonal antibody prepared by the present invention can be effectively used for the detection of southern bean mosaic virus in ports and field crops.

Description of drawings

Fig. 1 is the sensitivity analysis of detecting SBMV by dot-ELISA method;

Fig. 2 is the specificity analysis that dot-ELISA method detects SBMV;

2 dots above and below column 1 are 2 diseased bean leaves infected with SBMV; 2 dots above and below 2 columns are 2 diseased bean leaves infected with common bean mosaic virus; 2 dots above and below 3 columns are 2 healthy bean leaves; 4 The 2 points above and below the column are 2 healthy soybean leaves; the 2 points above and below the 5 column are 2 healthy soybean leaves; the 2 points above and below the 6 column are diseased leaves infected with cucumber mosaic virus and healthy cowpea leaves respectively; the 2 points above and below 7 columns are 2 The dots are leaves infected with southern cowpea mosaic virus and healthy pea leaves; the upper and lower points of the 8 columns are leaves infected with tobacco mosaic virus and healthy faba bean leaves respectively.

Fig. 3 is the representative result of dot-ELISA method detection SBMV in port leguminous plant sample;

Fig. 4 is a representative result of detection of SBMV in port leguminous plant samples by RT-PCR method.

biological deposit

The hybridoma cell line 19H9 was deposited on January 7, 2016 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101, and the preservation number is CGMCCNo.12004 .

detailed description

The hybridoma cell line 19H9 secreting anti-Southern Bean Mosaic Virus monoclonal antibody was deposited on January 7, 2016 in the General Microbiology Center of the Chinese Microbiological Culture Collection Management Committee, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCCNo.12004. It can Secretes monoclonal antibodies against Southern bean mosaic virus.

A monoclonal antibody against southern bean mosaic virus secreted by the hybridoma cell line 19H9, the ascites indirect ELISA titer of the monoclonal antibody against southern bean mosaic virus is 10 ^-7 , and the antibody type and subclass are IgG1 , kappa light chain. Using ACP-ELISA method analysis, it was found that the sensitivity of the monoclonal antibody to detect diseased leaves reached 1:81,920 times dilution (w/v, g/mL).

The monoclonal antibody against Southern bean mosaic virus has specific immune reaction only with the diseased leaf tissue infected with Southern bean mosaic virus, but with common bean mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, cucumber flower Diseased leaf tissues of leaf virus and healthy soybean, edamame, cowpea, pea, broad bean and kidney bean plant tissues did not have any immune response.

The application of monoclonal antibody against southern bean mosaic virus in the detection of this virus is various immunological detection methods and immunological kits established with monoclonal antibody as the core.

The hybridoma cell strain 19H9 provided by the invention can secrete a large amount of monoclonal antibody against southern bean mosaic virus, and the monoclonal antibody secreted by it has strong specificity, high potency and good stability. The high-throughput serological method for detecting SBMV and its detection kit based on the monoclonal antibody can be successfully applied to the detection of SBMV at ports, so as to improve the detection level of southern bean mosaic virus in my country and prevent the virus from spontaneously developing. Overseas imports domestically to provide material and technical support.

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

1. Obtaining of hybridoma cells and preparation of monoclonal antibodies

1. Preparation of immunogen and detection antigen

Purify virions using the following procedure:

1) Precool the large mortar and tissue stirrer;

2) Weigh 500g of infected SMBV bean diseased leaves, add 200mL of 0.5M phosphate buffer (ie, PB buffer) with pH 7.5 (containing 0.01MNa-EDTA and 0.1% mercaptoethanol) to each 100g of diseased leaves, and homogenize for 2min Finally, filter with double-layer cotton gauze, and centrifuge the filtrate at 6,000 rpm for 20 minutes to remove plant tissue residues;

3) The obtained supernatant was added dropwise while stirring to a final concentration of 2.5% TritonX-100, 4% PEG (molecular weight 6,000) and 0.1M NaCl, and stirred at 4°C for more than 4 hours;

4) Centrifuge at 11,000r/min for 15min to obtain a precipitate;

5) The precipitate was fully suspended in 0.5MPB (containing 0.01MMgCl ₂ and 0.5M urea) with pH 7.5, centrifuged at 6,000rpm for 15min, then the supernatant was sucked out and placed in a centrifuge tube, the precipitate was resuspended, and the centrifugation was repeated 3 times;

6) Combine the supernatants, ultracentrifuge at 33,000rpm for 100min, suspend the obtained pellet with 0.5MPB, centrifuge at 8,000rpm for 15min, collect the supernatant, resuspend the pellet, and repeat centrifugation for 3 times;

7) After combining the supernatants, add them to a centrifuge tube with a 30% sucrose pad at the bottom, and ultracentrifuge at 33,000rpm for 100min;

8) Suspend the obtained precipitate with 0.5MPB (containing 0.01MMgCl ₂ ) at pH 7.5, and the suspension is the virus purification solution;

9) Observing the purified sample with an electron microscope, a large number of high-purity southern bean mosaic virus particles were found. 2. Immunization of Animals

Six-week-old BALB/c female mice weighing 18-20 g were immunized with purified SBMV virus. That is, 100 μL/body of purified SBMV virus particles was mixed with an equal volume of Freund’s complete adjuvant, fully emulsified, and subcutaneously injected with 0.2 mL/body at multiple points on the back and abdomen, with an interval of 3 weeks, and the same amount of antigen and the same volume of Fuchsin as the first vaccine were taken. After the incomplete adjuvant was fully emulsified, the second intraperitoneal injection was performed at 0.2 mL per mouse. After 3 weeks, the double dose of antigen was used for intraperitoneal injection. After 3 days, splenocytes were collected for cell fusion.

3. Cell Fusion

Take the spleen cells of the above immunized mice and mouse myeloma cells (SP2/0) at a ratio of 7:1 and mix them evenly in serum-free RPMI-1640 (Gibco) medium, centrifuge at 1,500rpm for 5min, remove the medium, and place in Add 1 mL of 50% PEG (molecular weight 1,500) to a water bath at 37°C and fuse for 2 minutes, stop the fusion with serum-free RPMI-1640 medium, centrifuge at 1,500 rpm for 5 minutes, suspend the pellet with HAT medium, and distribute to 96-well cells containing feeder cells plate and cultured in a cell culture incubator at 37°C with 5% CO ₂ .

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

After culturing in the cell incubator for 5 days, change the medium once with HAT medium, and change the medium with HT medium on the 10th day. When the fused cells cover 10-30% of the bottom of the well, use the bean leaves infected with SBMV virus and the purified virus as antigens After coating, the positive wells secreting monoclonal antibody were screened by conventional indirect ELISA method, and a total of 137 positive wells were obtained. Five cell wells showing strong positive reactions were selected and cloned by limiting dilution method to obtain a hybridoma cell line 19H9 that can secrete specific monoclonal antibody against SBMV. After more than 6 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 ascitic fluid preparation and liquid nitrogen storage.

5. Preparation and purification of monoclonal antibody ascites

Take 8-week-old BALB/c mice, inject 0.3-0.5mL pristine (Sigma) into the intraperitoneal cavity, and inject ⁸ ×105 hybridoma cells into the abdominal cavity 7-10 days later, and the abdomen of the mice can be seen obviously in 7-10 days after injection. Swell, take the ascites with the injection needle, centrifuge at 8,000 rpm for 3 minutes, and collect the supernatant as the monoclonal antibody ascites. Take 1 volume of ascites and dilute with 2 volumes of 0.06M pH4.8 acetate buffer, add octanoic acid (30uL/mL ascites) while stirring at room temperature, clarify at 4°C for 1 hour, centrifuge at 12,000rpm for 20 minutes, collect the supernatant, and then use 50% saturated Precipitate immunoglobulin with ammonium sulfate, place at 4°C for 2 hours, centrifuge at 3,000 rpm for 20 minutes, dissolve the precipitate with 2 times the volume of PBS solution, and obtain purified ascites antibody after flow dialysis at 4°C for 24 hours, and store at -70°C.

6. Monoclonal antibody type and subclass identification and ascites titer determination

The type and subclass of the monoclonal antibody were identified using the DAS-ELISA kit from Sigma Company for the identification of the monoclonal antibody type. The results showed that the subclass of the 19H9 monoclonal antibody was IgG1 and kappa light chain. The ascites titer of monoclonal antibody was detected by conventional indirect ELISA method, and the result showed that the titer of 19H9 monoclonal antibody ascites reached 10 ^-7 .

7. Specific detection of monoclonal antibodies

Coat ELISA plates with diseased leaf tissues infected with common bean mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, and cucumber mosaic virus, and crude extracts of healthy soybean, edamame, cowpea, pea, and broad bean, The extract of healthy bean leaves was used as a negative control, and the bean juice infected with southern bean mosaic virus was used as a positive control, and the specific reaction of monoclonal antibody was determined by ACP-ELISA method. The ACP-ELISA method is as follows: the diseased leaves infected by the above viruses are added to the ELISA coating liquid at a ratio of 1:30 (w/v, g/mL), and then 100uL/well is coated with an ELISA plate, overnight at 4°C or 2h at 37°C, Make it adsorb to the wells of ELISA polystyrene plates; wash with PBST three times and then block with 3% skimmed milk powder for 30-60min; add appropriately diluted monoclonal antibody 100uL/well, 37°C for 1-2h; after washing with PBST three times, add appropriately diluted Alkaline phosphatase (AP)-labeled goat anti-mouse IgG secondary antibody (Sigma Company) 100uL/well, 37°C for 1-2h; after washing four times with PBST, develop color with PNPP substrate for 30-60min, then use 2M sodium hydroxide The reaction was terminated, and the OD405 value was read with a microplate reader, and the ratio of the negative OD value to the negative OD value was greater than 2.1 as positive. It was found that the 19H9 monoclonal antibody had a specific response to the diseased leaves infected with SBMV, and it had a specific reaction with the diseased leaves infected with common bean mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, cucumber mosaic virus and healthy soybean, edamame, Cowpea, pea, broad bean and bean plant tissues did not show any specific immune response.

2. Establishment of immunological methods and kits for detecting SBMV

1. ACP-ELISA method for detection of SBMV

1.1 Operation steps of ACP-ELISA method:

(1) Diseased leaves were homogenized by adding 0.05M carbonate buffer (pH9.6) at a ratio of 1:30 (w/v, g/mL), centrifuged at 5,000rpm for 3min, and coated with 100uL/well of the centrifuged supernatant on an ELISA plate , taking SBMV diseased leaves as positive control and healthy leaves as negative control, at 37°C for 2h, or at 4°C overnight;

(2) After washing with PBST, seal with 3% skimmed milk powder for 30 minutes;

(3) Add appropriately diluted monoclonal antibody ascites, 100uL/well, 37°C for 1h;

(4) After washing with PBST, add appropriately diluted alkaline phosphatase (AP)-labeled goat anti-mouse IgG secondary antibody (Sigma), 100uL/well, 37°C for 1h;

(5) After washing with PBST, add PNPP nitrophosphate substrate, 100uL/well, room temperature for 30min;

(6) Observe with the naked eye, if the color of the substrate turns yellow-green, it is positive, or after the reaction is terminated with 2M sodium hydroxide, the OD405 is measured with an enzyme-linked immunosorbent assay instrument, and P/N>2.1 is used as the positive judgment standard.

1.2 Determination of detection sensitivity and specificity of ACP-ELISA method

The optimal working concentration of monoclonal antibody and enzyme-labeled secondary antibody in ACP-ELISA detection method was determined by conventional square array test. The test showed that the optimal working concentration of 19H9 monoclonal antibody and enzyme-labeled secondary antibody were 1:5,000 and 1:8,000 times respectively dilution. The ACP-ELISA method for detecting SBMV was established with the optimal working concentration of the antibody, and the crude extract of diseased leaves was diluted 1:20-1:655,360 times (w/v, g/mL), and the healthy The crude leaf extract was used as a negative control, and the above-mentioned ACP-ELISA method was used for detection. The results show that the ACP-ELISA method is still positive to the diseased leaf crude extract of 1:81,920 times dilution (w/v, g/mL), that is, the detection sensitivity of diseased leaves reaches 1:81,920 times dilution (w/v, g/mL), indicating that the monoclonal antibody and the established ACP-ELISA method have high sensitivity and reliability. The method detects that the crude extract of SBMV diseased leaves shows a strong positive reaction, and detects diseased leaves infected with common bean mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, cucumber mosaic virus, and healthy soybeans, soybeans, cowpeas, and peas. , broad bean and kidney bean plant tissues showed negative reactions, and the difference between negative and positive contrast was extremely significant, indicating that the specificity of this method and the monoclonal antibody were very good.

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

2.1 Operation steps of the dot-ELISA method for detecting SBMV in leguminous plants

Weigh the bean leaves and add 0.01MPBS (pH7.4) at a ratio of 1:30 (w/v, g/mL) to homogenate; centrifuge the homogenate at 5,000rpm for 3min; take 3μL of the centrifuged supernatant and put it on nitrocellulose On the (NC) membrane, the crude extracts of healthy and infected SBMV kidney bean leaves were set as negative and positive controls respectively; room temperature drying 10-20min; 0.01MPBS) blocking solution at room temperature for 30min; NC membrane was incubated in moderately diluted monoclonal antibody for 30-60min at room temperature; washed with PBST for 3-4 times, 3min each time; NC membrane was placed in moderately diluted AP enzyme-labeled sheep Incubate in anti-mouse IgG secondary antibody for _30-60min at room temperature; wash the membrane 4-5 times with PBST, 3min each time; pH 9.5) and mix well, put the membrane into the substrate solution to react, and observe the results with naked eyes; when the positive control has obvious color development and the negative has no color development, rinse in tap water to terminate the reaction, and take pictures to record the results.

2.2 Determination of detection sensitivity and specificity of dot-ELISA method

The optimal working concentration of monoclonal antibody and enzyme-labeled secondary antibody in the dot-ELISA detection method was determined by conventional square array test. The test results showed that the optimal working concentration of 19H9 monoclonal antibody and enzyme-labeled secondary antibody were 1:5,000 and 1:5,000, respectively. : 8,000-fold dilution. The dot-ELISA method for detecting SBMV was established with the optimum working concentration of the above antibodies. Sensitivity analysis showed that when the bean diseased leaves were diluted to 1:5, 120 times (w/v, g/mL), the dot-ELISA method based on 19H9 monoclonal antibody still showed purple positive spots in detection, that is, it detected diseased leaves. The sensitivity reached 1:5, 120-fold dilution (w/v, g/mL) (Figure 1). Specificity analysis showed that the method had a strong specific positive reaction in the detection of SBMV-susceptible plants, and the detection of diseased leaves infected with bean common mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus, cucumber mosaic virus and healthy Plant tissues of soybean, edamame, cowpea, pea, broad bean, and kidney bean were all negative (Figure 2).

2.3 Field application of dot-ELISA detection method

The established dot-ELISA method was used to detect the suspected pathogenic leguminous samples provided by the Shanghai Inspection and Quarantine Bureau in 2015. It was found that 23 samples out of 50 tested samples produced purple positive spots (Figure 3), and the samples were further tested at the same time. Using RT-PCR detection, the results showed that SBMV-specific PCR products were amplified in all dot-ELISA positive samples (Figure 4). PCR product nucleic acid sequencing showed that positive samples were indeed infected with SBMV, while dot-ELISA negative samples RT-PCR did not amplify any gene fragments. It shows that the established dot-ELISA method can be used accurately and reliably to detect Southern bean mosaic virus in leguminous samples.

3. Southern Bean Mosaic Virus dot-ELISA Detection Kit

1) Main components of the kit:

All the above reagents were stored at 4°C

Nitrocellulose membrane (NC) 10 sheets 2) Operation steps for detecting legume samples:

a. After weighing the leaves of the leguminous sample, add 0.01MPBS (pH7.4) at a ratio of 1:30 (w/v, g/mL) and then homogenate;

b. Centrifuge the homogenate at 5,000rpm for 3min;

c. Take 3 μL of the supernatant and spot it on the NC membrane, and set the crude extracts of healthy and infected SBMV kidney bean leaves 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 3% skimmed milk powder and block at room temperature for 30 minutes;

e. NC membrane was incubated in 1:2,000 times diluted monoclonal antibody for 30-60min at room temperature;

f. Wash the membrane with PBST 3-4 times, 3 minutes each time; put the NC membrane into 1:3,000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 30-60 minutes;

g. Wash the membrane 4-5 times with PBST, 3min each time; add 66μL NBT and 33μL BCIP substrate to 10mL substrate buffer (0.1MTrisCl, 0.1MNaCl, 0.025MMgCl ₂ , pH9.5) and mix well, and put the membrane into the substrate solution Medium color development 10-20min;

h. Visually observe the results. When the positive control has obvious color development (purple), and the negative control has no color development, rinse with tap water to terminate the reaction, 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 buffered saline (PBS, 0.01M, pH7.4):

Add 950% distilled water to dissolve, adjust the pH to 7.4, and set the volume to 1,000mL

ELISA washing solution (0.01MPBST):

Add 0.5mL Tween-20 to 1,000mL0.01MPBS

ELISA blocking solution:

Skim milk powder was added to 0.01 MPBST to a final concentration of 5% (w/v).

Claims (4)

1. the hybridoma cell strain 19H9 secreting anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS, it is characterized in that secreting the monoclonal antibody specific of anti-bean mosaic virus 4, hybridoma cell strain 19H9 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 7th, 2016, and preserving number is CGMCCNo.12004.

2. the monoclonal antibody of the anti-bean mosaic virus 4 of a hybridoma cell strain 19H9 as claimed in claim 1 secretion, it is characterised in that this monoclonal antibody ascites indirect ELISA titer reaches 10^-7, Antibody types and subclass are IgG1, kappa light chain; Utilizing ACP-ELISA methods analyst to find, the sensitivity of the sick leaf of this monoclonal antibody detection reaches 1:81920 times and dilutes.

3. the monoclonal antibody of anti-bean mosaic virus 4 as claimed in claim 2, it is characterized in that this monoclonal antibody only has a specific immune response with the sick leaf texture infecting bean mosaic virus 4, and with infecting Bean common mosaic virus, southern cowpea mosaic virus, tobacco mosaic virus (TMV), the sick leaf texture of cucumber mosaic virus and the Semen sojae atricolor of health, green soyabeans, Semen vignae sinensis, Semen Pisi sativi, Semen Viciae fabae and bean plant tissue, any immunoreation does not all occur.

4. an anti-MONOCLONAL ANTIBODIES TO SOUTHERN BEAN MOSAIC VIRUS as claimed in claim 2 application on bean mosaic virus 4 detects, it is characterised in that the various immunological detection methods set up with monoclonal antibody for core and immunological detecting kit.