CN111269889B - Hybridoma cell strain secreting monoclonal antibody of tomato mottle mosaic virus and application of monoclonal antibody of hybridoma cell strain - Google Patents
Hybridoma cell strain secreting monoclonal antibody of tomato mottle mosaic virus and application of monoclonal antibody of hybridoma cell strain Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting Tomato mottle mosaic virus (ToMMV) resistant monoclonal antibody and application of the monoclonal antibody. Purified ToMMV virus particles are used as antigen to immunize BALB/c mice, and 1 hybridoma cell strain 2D6 capable of secreting ToMMV-resistant monoclonal antibody is obtained through cell fusion, screening and cloning, and the preservation number is CGMMV NO. 18846. The cell strain secretes monoclonal antibody ascites indirect ELISA titer reaches 10‑8It specifically immunoreacts with ToMMV, but not with Tobacco Mosaic Virus (TMV) and Tomato mosaic virus (ToMV) of the same genus. ACP-ELISA, dot-ELISA and Tissue print-ELISA detection methods for detecting ToMMV in tomato plants are established by using the 2D6 monoclonal antibody, wherein the sensitivity of detecting diseased leaves by the ACP-ELISA and the dot-ELISA methods respectively reaches 1:40,960 and 1:20,480 times of dilution (w/v, g/mL). The creation of a hybridoma cell strain secreting ToMMV high-quality monoclonal antibody and the establishment of a virus serological detection method provide material and technical support for diagnosis and detection of ToMMV, epidemiological investigation and scientific prevention and control.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a hybridoma cell strain secreting a monoclonal antibody of tomato mottle mosaic virus and application of the monoclonal antibody.
Background
Tomato mottle mosaic virus (ToMMV) is a new species of Tobamovirus (Tobamovirus) of the genus Nicotiana of the family Viraviridae, and has a positive-sense single-stranded RNA as its genome. The genome of ToMMV contains four open reading frames, which, like tobacco mosaic virus, expresses four proteins, encoding 1 protein required for replication of 126kD and 1 protein required for replication of 183kD, and also encoding 1 motor protein of 30kD and 1 coat protein of 17.5 kD. In 2013, the new virus is found on tomato in Mexico for the first time, and then the virus is detected on tomatoes in the United states, Israel, Brazil, Spain and the like, and tomatoes in Yunnan province and Hainan province in China are also infected by the virus.
ToMMV can harm a variety of solanaceous crops, and the disease symptoms are mainly manifested as plant dwarfing, chlorotic, mottled and twisted leaves, and serious product loss. The virus has high stability, can survive in the disease residues for a long time, can be attached to the seed surface or mixed in the seed by the disease residues to realize remote transmission, and is easy to infect among plants through mechanical friction. The main solanaceae crops in China, such as tomatoes, hot peppers, tobaccos and the like, are cultivated in a seedling transplanting mode, part of tomatoes (such as cherry tomatoes) are grafted for seedling cultivation, and in addition, the operations of multiple twigs, pruning, shelving, fruit picking and the like are needed in the field management process, so that once the virus is introduced, the virus is easy to spread and spread. The laws of occurrence, transmission pathways, virus detection techniques and viral genome function have been studied only rarely.
In order to investigate the occurrence of the disease of various solanaceae plants in China, strengthen the ToMMV detection and diagnosis technology and scientific guidance and control in China, an economical, effective and high-flux practical detection technology for detecting ToMMV is urgently needed to be established. Compared with the conventional methods such as indicating plant inoculation, electron microscope observation, molecular detection and the like, the serological method has the advantages of simplicity, economy, easy operation, large-scale detection and the like, and is the most practical method for plant virus detection and investigation. The establishment of serological methods, however, relies on specific high quality viral antibodies. The invention takes purified ToMMV virions as antigens to prepare 1 hybridoma cell strain secreting ToMMV specific monoclonal antibodies by a hybridoma technology, and establishes a high-flux serological method and a kit for detecting ToMMV by taking the secreted monoclonal antibodies as a core, thereby providing material and technical support for the detection and diagnosis, epidemiological investigation, virus genome function analysis, tissue culture production, resistance breeding, scientific prevention and control establishment and the like of ToMMV in China.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a hybridoma cell strain secreting monoclonal antibody against tomato mottle mosaic virus and application of the monoclonal antibody.
The hybridoma cell strain 2D6 for secreting tomato mottle mosaic virus monoclonal antibody can specifically secrete anti-tomato mottle mosaic virus monoclonal antibody, and the hybridoma cell strain 2D6 is preserved in China general microbiological culture Collection center (CGMCC) at 11/4 in 2019 with the preservation number of CGMCC NO. 18846.
The tomato mottle mosaic virus monoclonal antibody secreted by the hybridoma cell has the anti-ToMMV virus monoclonal antibody ascites indirect ELISA titer of 10-8The antibody type and subclass is IgG2aAnd kappa light chain has specific immunoreaction with coat protein of 17.5kDa of tomato mottle mosaic virus, and the sensitivity of detecting the crude extract of tomato diseased leaf tissue infected with ToMMV by ACP-ELISA and dot-ELISA methods established by the monoclonal antibody reaches 1:40,960 and 1:20,480 times of dilution (w/v, g/mL) respectively.
The monoclonal antibody resisting the tomato mottle mosaic virus can have specific immunoreaction with the tomato mottle mosaic virus, but does not have immunoreaction with infected tobacco mosaic virus, tomato mosaic virus, infected tomato spotted wilt virus, tomato yellow leaf curl virus, tomato black ring virus, cucumber mosaic virus infected leaves and healthy plant tissues.
The application of the monoclonal antibody for resisting tomato mottle mosaic virus in the virus detection is various immunological detection methods and immunological detection kits established by taking the monoclonal antibody as a core.
Compared with the prior art, the invention has the following beneficial effects: 1) the hybridoma cell strain provided can secrete a large amount of monoclonal antibodies against tomato mottle mosaic virus, and ACP-ELISA, dot-ELISA and Tissue print-ELISA serological detection methods established by taking the monoclonal antibodies as cores and immunological kits thereof can detect the tomato mottle mosaic virus with high specificity, sensitivity and accuracy; 2) the monoclonal antibody prepared by the invention is used for detecting the tomato mottle mosaic virus, has low technical requirements on operators, and does not need expensive equipment such as an electron microscope, a PCR instrument and the like; 3) the monoclonal antibody prepared by the invention can be effectively used for detecting and diagnosing field tomato mottle mosaic virus, and can also be used for epidemiological investigation, virus genome function analysis, resistance breeding, scientific prevention and control and the like of the virus disease.
Drawings
FIG. 1 is a sensitivity analysis of the dot-ELISA method for detection of ToMMV;
FIG. 2 shows the results of detection of ToMMV in tomato samples in field by ACP-ELISA (A), dot-ELISA (B), Tissue print-ELISA (C) and RT-PCR (D). M in the 2D plot represents 1kb DNAmarker.
g2 and h2 are positive control of infected ToMMV diseased leaves and negative control of healthy tomato leaves, respectively, and other samples are field samples.
Biological preservation
The hybridoma cell strain 2D6 secreting tomato mottle mosaic virus monoclonal antibody is preserved in the China general microbiological culture Collection center in 2019, 11 and 4 months, with the address: west road No.1, north chen, chaoyang district, beijing, zip code: 100101 with preservation number of CGMCC NO. 18846.
Detailed Description
The hybridoma cell strain 2D6 secreting tomato mottle mosaic virus monoclonal antibody is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, China institute of microbiology and sciences, in 2019, 11 and 4, with the preservation number of CGMCC NO.18846, and can secrete monoclonal antibody resisting tomato mottle mosaic virus.
The hybridoma cell secretes the tomato mottle mosaic virus monoclonal antibody, and the ascites indirect ELISA titer of the monoclonal antibody reaches 10-8The antibody type and subclass is IgG2aThe kappa light chain has specific immunoreaction with coat protein of 17.5kDa of tomato mottle mosaic virus, and the sensitivity of detecting the crude extract of diseased leaf tissue infected with the tomato mottle mosaic virus by ACP-ELISA and dot-ELISA methods established by the monoclonal antibody reaches 1:40,960 and 1:20,480 times of dilution (w/v, g/mL) respectively.
The monoclonal antibody resisting the tomato mottle mosaic virus can have specific immunoreaction with the tomato mottle mosaic virus, but does not have immunoreaction with infected tobacco mosaic virus, tomato mosaic virus, infected tomato spotted wilt virus, tomato yellow leaf curl virus, tomato black ring virus, cucumber mosaic virus infected leaves and healthy plant tissues.
The application of the monoclonal antibody for resisting tomato mottle mosaic virus in the virus detection is various immunological detection methods and immunological detection kits established by taking the monoclonal antibody as a core.
The hybridoma cell strain provided by the invention can secrete a large amount of monoclonal antibodies for resisting tomato mottle mosaic virus, and the monoclonal antibodies secreted by the cell strain have the advantages of strong specificity, high titer, good stability and high sensitivity. The high-flux serological method for detecting ToMMV established by taking the monoclonal antibody as a core can be successfully applied to detection and diagnosis of ToMMV in fields, thereby providing material and technical support for ToMMV disease detection and diagnosis, tissue culture production and scientific prevention and control in China.
The invention is further illustrated by the following examples and figures.
First, hybridoma cell acquisition and preparation of monoclonal antibody thereof
1. Preparation of immunogen and detection antigen
Viral particles were purified by the following procedure:
1) pre-cooling the tissue homogenizer on ice;
2) weighing 200g of tomato diseased leaf tissue infected with ToMMV, adding 400mL of 0.5M phosphate buffer solution (PB buffer solution (containing 0.01M Na-EDTA and 0.1% mercaptoethanol, pH 7.5), homogenizing in a tissue homogenizer for 5-10min to fully homogenize the plant tissue, filtering the homogenate with a double-layer cotton gauze, and centrifuging the filtrate at 6,000rpm for 20min to remove plant residues;
3) the resulting supernatant was added to a final concentration of 2.5% Triton X-100, 4% PEG (molecular weight 6,000) and 0.1M NaCl with stirring, followed by stirring overnight at 4 ℃;
4) centrifuging at 11,000rpm for 15min, and removing supernatant;
5) with 0.5M PB at pH 7.5 (containing 0.01M MgCl)2And 0.5M urea), the pellet was fully suspended, centrifuged at 6,000rpm for 15min, the supernatant was collected in a beaker and placed at 4 ℃, the pellet was resuspended, centrifuged, and repeated 3 times;
6) mixing the above centrifugated supernatants, and ultracentrifuging at 33,000rpm for 100 min;
7) suspending the obtained precipitate with PB, centrifuging at 8,000rpm for 15min, collecting supernatant, resuspending the precipitate, centrifuging, and repeating for 3 times;
8) adding the combined supernatant into a super-centrifugal tube, sucking 30% of sucrose by using a syringe needle, adding the sucrose to the bottom of the super-centrifugal tube to form a sucrose cushion, and performing ultracentrifugation at 33,000rpm for 100 min;
9) the resulting precipitate was treated with 20mL of 0.01M PB (containing 0.01M MgCl)2pH 7.5), ultracentrifugation at 33,000rpm for 100min, removal of the sucrose cushion;
10) suspending the obtained precipitate with 0.01M PB to obtain suspension as virus purification solution;
11) the virus purified solution is negatively stained by 3 percent phosphotungstic acid (pH 6.7), and then is placed under a JEOL JEM-1200EX electron microscope for observation to discover a large amount of high-purity ToMMV baculovirus particles.
2. Immunizing animals
Immunization of 9-week-old BALB/c female mice with purified ToMMV virions: adding 40 mu L/ToMMV virus particles into 60 mu L/double-antibody normal saline as virus antigen, mixing with equal volume of 100 mu L/Freund complete adjuvant, fully emulsifying, performing intraperitoneal injection on 200 mu L/ToMMV virus particles, spacing for 3 weeks, adding 80 mu L/double-antibody normal saline virus antigen into 20 mu L/ToMMV virus particles for the second time, fully emulsifying with equal volume of Freund incomplete adjuvant, performing intraperitoneal injection on 200 mu L/ToMMV virus particles, performing intraperitoneal injection on the virus antigen with the same volume as that of the first immunization after 3 weeks, and performing fusion on splenocytes 3 days after the third immunization.
3. Cell fusion
Mixing the above immune mouse spleen cell and mouse myeloma cell (SP2/0) at a ratio of 11:1 in serum-free RPMI-1640(Gibco) culture medium, centrifuging at 1,500rpm for 5min, removing the culture medium, adding 1mL of 50% PEG (molecular weight 1,500) fusion agent into centrifuge tube containing cells in 37 deg.C water bath, fusing for 2min, terminating fusion with serum-free RPMI-1640 culture medium, centrifuging at 1,500rpm for 5min, removing supernatant, suspending the precipitate with HAT culture medium, packaging into 96-well cell plate, and culturing at 37 deg.C with 5% CO2Cultured in a cell culture box.
4. Screening of hybridoma cells and positive wells and cell cloning
After 5 days of culture in a cell incubator, the HAT culture medium is used for replacing liquid once, the HT culture medium is used for replacing liquid on the 12 th day, and when the fused cells cover more than 15% of the bottoms of the holes, the tomato disease leaf crude extract infected with ToMMV is used as a coating antigen, and antibody positive holes are screened by an indirect ELISA method, so that 135 positive holes secreting ToMMV are obtained in total. And (2) analyzing the specificity and sensitivity of the 135 positive hole antibody, screening 17 cell holes with better specificity and sensitivity, and cloning by a limiting dilution method to finally obtain 1 hybridoma cell strain 2D6 capable of secreting a ToMMV-resistant highly specific and sensitive monoclonal antibody, namely the hybridoma cell strain with the preservation number of CGMCC NO. 18846. After in vitro passage for more than 3 months and multiple times of cryopreservation recovery, the cell strain can well grow and stably secrete antibodies. After the enlarged culture, the culture medium is used for ascites preparation and liquid nitrogen preservation.
5. Preparation and purification of monoclonal antibody ascites
Taking 12-week-old BALB/c male mouse, injecting 0.4mL pristane into abdominal cavity, injecting 7 × 10 days later into abdominal cavity5And (3) the hybridoma cells are injected into the mouse, the abdomen of the mouse is obviously enlarged 6 to 10 days after the hybridoma cells are injected, ascites is collected by using a needle, the mouse is centrifuged at 8,000rpm for 3min, and the supernatant is collected, namely the monoclonal antibody ascites.
Diluting ascites of 1 volume with 0.85% physiological saline of 2 volumes, adding saturated ammonium sulfate solution (pH 7.0) dropwise under stirring at room temperature, standing overnight at 4 deg.C, centrifuging at 12,000rpm for 20min, suspending and precipitating with 2mL of 0.85% physiological saline, dialyzing in 0.85% physiological saline at 4 deg.C for 24h to obtain purified monoclonal antibody, and storing at-80 deg.C.
6. Subclass identification and ascites titer determination of monoclonal antibodies
The purified monoclonal antibody ascites was mixed with standard anti-BALB/c mouse IgG from Sigma1、IgG2a、IgG2b、IgG3IgM antibody was subjected to a two-way agar diffusion test, and the results analyzed that the subclass of 2D6 monoclonal antibody was IgG2aKappa light chain. The purified virus particles are used as antigen, the indirect ELISA method is used for detecting the titer of the monoclonal antibody ascites, and the analysis result shows that the titer of the monoclonal antibody ascites reaches 10-8。
7. Detection of specificity of monoclonal antibodies
With Tobacco Mosaic Virus (TMV), tomato mosaic virus (ToMV) and Cucumber Mosaic Virus (CMV)) The crude extract of diseased leaves is coated on an ELISA plate, the crude extract of healthy leaves of tomatoes and healthy leaves of tobaccos is used as negative control, the crude extract of diseased leaves of tomatoes infected with ToMMV is used as positive control, and the specificity of the monoclonal antibody is analyzed by an indirect ELISA method. The indirect ELISA method comprises the following steps: grinding the virus-infected diseased leaves and healthy leaves in a mortar respectively, adding an ELISA coating solution according to a ratio of 1:30 (w/v, g/mL) for homogenate, centrifuging at 8,000rpm for 3min, coating an ELISA plate with 100 mu L/hole of supernatant, and standing overnight at 4 ℃ or 2h at 37 ℃; PBST is washed for 3 times and then sealed for 30min by using 3 percent of skimmed milk powder; adding 100 mu L/hole of monoclonal antibody diluted by 1:5,000 times, and incubating for 1h at 37 ℃; PBST was washed 3 times, then 100. mu.L/well of Alkaline Phosphatase (AP) -labeled rabbit anti-mouse IgG secondary antibody (Sigma) diluted 1: 8000 times was added, and incubated at 37 ℃ for 1 hour; PBST is washed for 4 times, is developed for 30-60min by PNPP substrate, and OD is read by a microplate reader after 3mol/L sodium hydroxide stops reaction405The value of (d), with a ratio to the negative OD value of greater than 3.0, is positive. As a result, the 2D6 monoclonal antibody has strong positive immune response to ToMMV, but has no immune response to infected ToMV, TMV and CMV diseased leaves and crude extracts of healthy tobacco and tomato plant tissues.
Second, establishment of serological method for detecting ToMMV
ACP-ELISA detection method for detecting ToMMV
1.1ACP-ELISA method steps:
1) grinding the leaves in a mortar, adding an ELISA coating solution according to a ratio of 1:20(w/v, g/mL), continuing homogenizing for 3min, centrifuging at 5.000rpm for 3min, coating an ELISA plate with supernatant of 100 muL/well, taking tomato leaf tissues infected with ToMMV as positive control, taking healthy tomato leaf tissues as negative control, and coating overnight at 4 ℃ or for 2h at 37 ℃;
2) washing the ELISA plate with PBST for 3 times, each for 3min, adding 250 μ L blocking solution (PBS containing 3% skimmed milk) into each well, and blocking at 37 deg.C for 0.5-1 h;
3) discarding the blocking solution in the ELISA plate, washing with PBST for 3 times, then properly diluting the monoclonal antibody ascites with 100 mu L/well of the blocking solution, and incubating for 1-2h at 37 ℃;
4) removing the single antibody diluent in the ELISA plate, washing with PBST for 3 times, adding a suitably diluted AP-labeled goat anti-mouse IgG secondary antibody (Sigma)100 mu L/well, and reacting at 37 ℃ for 1-2 h;
5) PBST was washed 4 times for 3min each. Adding PNPP substrate, developing at room temperature for 30-60min, observing the hole with naked eye that the substrate color changes into yellow green, or detecting OD with Bio-Rad 680 enzyme-linked immunosorbent assay after 3M sodium hydroxide stops reaction405At a ratio of P/N>3.0 as the positive judgment criterion.
1.2 establishment of method for detecting ToMMV ACP-ELISA
Determining the optimal working concentration of the antibody in the ACP-ELISA method by adopting a matrix test, namely using 1: 30-fold diluted (w/v, g/mL) ToMMV diseased leaf crude extract as an antigen coated ELISA plate; adding 1:1,000-1:512,000-fold diluted ToMMV monoclonal antibody into each transverse column of the ELISA plate for reaction for 1h, adding 1:1,000-1:512,000-fold diluted AP-labeled goat anti-mouse IgG secondary antibody into each longitudinal row of the ELISA plate after PBST washing, taking diluted crude extract of healthy tomato plants as negative control, setting 3 times of repetition for each treatment, and determining the optimum antibody working concentration of the ACP-ELISA. The results show that 2D6 monoclonal antibody is diluted by 1:5,000 times, AP labeled goat anti-mouse IgG secondary antibody is diluted by 1:8,000 times to be the optimal working concentration, and an ACP-ELISA method for detecting ToMMV is established according to the optimal working concentration.
1.3 specificity and sensitivity of the ACP-ELISA method
Respectively taking tomato diseased leaves infected with ToMMV and healthy tomato leaf tissue crude extract as positive control and negative control, taking tomato diseased leaves infected with TMV, tomato diseased leaves infected with ToMV, Tomato Spotted Wilt Virus (TSWV) diseased leaves infected with Tomato Yellow Leaf Curl Virus (TYLCV) diseased leaves infected with Tomato Black Ring Virus (TBRV) diseased leaves infected with CMV and healthy tomato leaves as detection samples, repeating the experiment for three times, and analyzing the specificity of the ToMMV monoclonal antibody and the established ACP-ELISA method. Under the optimal working concentration of the antibody, the method detects that infected leaves infected with ToMMV show strong positive reaction, and detects that crude extract of infected plant leaves such as TMV, ToMV, TSWV, TYLCV, TBRV and CMV shows negative reaction, and the OD value difference is extremely obvious, thereby indicating that the method and the monoclonal antibody have good specificity.
Tobacco diseased leaves infected with ToMMV, healthy tomato leaves and healthy tomato leaves are diluted by PBS buffer solution from 1:20 times to 1:81 times and 920 times (w/v, g/mL), the diluted crude extract is sequentially added to an ELISA plate, the sensitivity of the ToMMV is detected by an ACP-ELISA method, and the experiment is repeated three times. The result shows that the sensitivity of detecting the diseased leaves by the ACP-ELISA reaches 1:40,960 times dilution (w/v, g/mL), which indicates that the prepared monoclonal antibody and the established method have good sensitivity.
Establishment of 2dot-ELISA detection method and specificity and sensitivity analysis thereof
2.1dot-ELISA detection procedure
1) Grinding plant tissues in a mortar, adding 0.01M PBS buffer solution according to the proportion of 1:20(w/v, g/mL) for homogenizing, and centrifuging at 5,000rpm for 3min to obtain supernatant which is plant tissue crude extract;
2) sample application: dropping 2-3 μ L of the crude extractive solution onto Nitrocellulose (NC) membrane, setting crude extractive solutions of healthy and ToMMV-infected diseased leaf blades as negative and positive controls, and oven drying in 37 deg.C incubator for 10 min;
3) soaking NC membrane in PBST (0.01M PBS containing 0.05% Tween-20) containing 5% skimmed milk powder, and sealing at room temperature for 30 min; then placing the NC membrane into the monoclonal antibody diluted by 1:5,000 for incubation for 1h at room temperature;
5) washing the membrane: washing the membrane with PBST for 3 times, each for 3 min;
6) placing the NC membrane into a 1:8,000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody, incubating for 1h at room temperature, washing the membrane for 4-5 times, each time for 3 min;
7) color development: 66 μ L of NBT and 33 μ L of BCIP substrate (Promega) were added to 10ml of substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH9.5) and mixed well, the membrane was blotted with absorbent paper, the membrane was placed in the substrate solution for reaction, and color development was carried out at room temperature in the dark for 15-20 min. The reaction was terminated by visual observation when the positive control showed a purple spot and the negative control did not change color by rinsing the membrane in tap water, photographing and recording the results.
2.2 establishment of method for detecting ToMMV dot-ELISA
And (3) respectively carrying out dilution on the ToMMV monoclonal antibody and the AP-labeled goat anti-mouse IgG secondary antibody in a ratio of 1:1,000 times, and determining the optimal working concentration of the monoclonal antibody and the enzyme-labeled secondary antibody in the dot-ELISA method by using a square matrix experiment. The experimental result shows that the optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody are respectively diluted by 1:5,000 times and 1:8,000 times, and the dot-ELISA method for detecting ToMMV in the plant is established according to the optimal working concentrations of the antibodies.
2.3dot-ELISA method for detecting ToMMV specificity and sensitivity
And respectively taking tomato diseased leaves infected with ToMMV and healthy tomato leaf tissue crude extract as positive control and negative control, and taking infected Tomato Spotted Wilt Virus (TSWV), Tomato Yellow Leaf Curl Virus (TYLCV), Tomato Black Ring Virus (TBRV), TMV, ToMV tomato diseased leaves, cucumber diseased leaves infected with CMV and healthy tomato leaves as detection samples to perform the specificity analysis of the dot-ELISA method. The method detects that tomato diseased leaf crude extract infected with ToMMV shows strong positive reaction, and detects that tomato diseased leaf crude extract infected with TMV, ToMV, TSWV, TYLCV, TBRV and CMV shows negative reaction, which shows that the method and monoclonal antibody have very good specificity.
Tomato diseased leaves infected with ToMMV were ground in a mortar for homogenization, diluted 1:10 to 1:81920 fold in 0.01M PBS (w/v, g/mL), and the same procedure was applied to the crude extract of healthy tomato leaves. The sensitivity of infected ToMMV diseased leaves was measured by dot-ELISA. Sensitivity analysis shows that when the crude extract of ToMMV diseased leaves is diluted to 1:20480 times (w/v, g/mL), the established dot-ELISA still presents purple positive spots, namely, the sensitivity of detecting the diseased leaves reaches 1:20480 times dilution (figure 1).
3. Establishment of Tissue print-ELISA method for detecting ToMMV and field sample detection thereof
3.1Tissue print-ELISA procedure:
1) sample preparation: taking the stems of tomatoes or tobaccos, and cutting a cross section by a blade;
2) sample application: pressing the cross section on NC membrane for 3sec, simultaneously using healthy and infected ToMMV plant tissues as negative and positive control respectively, and oven drying at 37 deg.C for 5 min;
3) and (3) sealing: immersing the NC membrane into PBST (0.01M PBS containing 0.05% Tween-20) sealing solution containing 5% skimmed milk powder, and sealing for 0.5h at room temperature;
4) primary antibody incubation: placing the NC membrane into a ToMMV monoclonal antibody which is diluted properly, and incubating for 1h at room temperature;
5) PBST membrane washing for 3 times, each time for 3 min;
6) and (3) secondary antibody incubation: washing the membrane with PBST for 3 times, putting the NC membrane into the appropriately diluted AP enzyme-labeled goat anti-mouse IgG, and incubating for 1h at room temperature;
7) PBST membrane washing for 3min 4-5 times;
8) adding a substrate for color development: add 66. mu.L NBT and 33. mu.L BCIP substrate (Promega) to 10ml of color buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH9.5) and mix them, immerse the membrane in the color buffer for color reaction until the positive color is obvious and the negative control is not, rinse the membrane in tap water to stop the reaction, record the color result.
3.2 determination of optimum antibody working concentration of Tissue print-ELISA and field sample detection thereof
The optimal working concentration of the monoclonal antibody and the enzyme-labeled secondary antibody in the Tissue print-ELISA is determined by a conventional matrix experiment. And selecting the dilution combination of the primary antibody and the enzyme-labeled secondary antibody with the highest detection sensitivity and specificity as the optimal working concentration of Tissue print-ELISA. The result shows that the detection sensitivity and specificity of the Tissue print-ELISA method are optimal when the monoclonal antibody 2D6 and the AP-labeled goat anti-mouse secondary antibody are diluted by 1:5,000 and 1:8,000 times respectively, and the Tissue print-ELISA method is established according to the optimal working concentration of the antibody.
4. Field sample detection application of serology method
The established ACP-ELISA, dot-ELISA and Tissue print-ELISA methods are used for detecting suspected disease tomato samples in the field collected from Yunnan and Hainan in 2019, and the detection result shows that 16 samples in 38 tomato detection samples generate positive reaction (shown in figures 2A-2C). The samples were simultaneously analyzed by RT-PCR, and the results showed that ToMMV-specific gene fragments were amplified in all serological positive samples, while no ToMMV-specific gene fragments were amplified in all serological negative samples (FIG. 2D), and the nucleic acid sequencing and sequence comparison analysis of PCR products showed that the RT-PCR positive samples indeed infected ToMMV. The ACP-ELISA, dot-ELISA and Tissue print-ELISA methods can be accurately and reliably used for detecting ToMMV in plants.
5. Tomato mottle mosaic virus dot-ELISA detection kit
1) The kit comprises the following main components:
the above reagents were all stored at 4 deg.C
10 nitrocellulose membranes (NC)
Skimmed milk powder 30g
2) The operation steps for detecting the tomato sample comprise:
a. weighing tomato plant tissue, grinding in mortar, adding 0.01M PBS (pH7.4) at a ratio of 1:20-50 (w/v, g/mL), and homogenizing;
b. centrifuging the homogenate at 5,000rpm for 3 min;
c. taking 2.5 μ L of supernatant, sampling on NC membrane, setting healthy and ToMMV-infected tomato tissues as negative and positive controls, and drying at room temperature for 10-20 min;
immersing NC membrane into PBST (0.01M PBS containing 0.05% Tween-20) sealing solution containing 5% skimmed milk powder, and sealing for 30min at room temperature;
e, putting the NC membrane into the monoclonal antibody diluted by 1:5,000 times, and incubating for 30-60min at room temperature;
f. washing membrane with PBST for 3-4 times, each for 3 min; placing the NC membrane into a 1:8,000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody for incubation for 30-60min at room temperature;
PBST washing the membrane for 4-5 times, each time for 3 min;
h.66. mu.L NBT and 33. mu.L BCIP substrate were added to 10mL substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl)2pH9.5), placing the membrane into substrate solution for color development after uniform mixing, observing the result by naked eyes, rinsing the membrane by tap water to stop reaction when positive control shows obvious purple and negative control does not have any color development, and photographing to record the result.
3) Storage and effective period:
storing at 2-8 deg.C in dark place for 12 months.
4) Phosphate buffer (0.01M PBS, pH7.4) formulation:
adding 950mL of distilled water to dissolve, adjusting pH to 7.4, and diluting to 1,000mL
6. Tomato mottle mosaic virus Tissue print-ELISA detection kit
1) The kit comprises the following main components:
the above reagents were all stored at 4 deg.C
10 nitrocellulose membranes (NC)
Skimmed milk powder 30g
2) The operation steps for detecting the tomato sample comprise:
a. sample preparation: taking tomato leaves or stems, rolling young leaves into a cylinder shape, cutting a cross section by a blade, and directly cutting young stems into the cross section;
b. sample application: pressing the cross section on NC membrane for 3sec, simultaneously using healthy and infected ToMMV tissue as negative and positive control respectively, and oven drying at 37 deg.C for 5 min;
immersing the NC membrane into PBST (0.01M PBS containing 0.05 percent Tween-20) sealing liquid containing 5 percent skim milk powder, and sealing for 30min at room temperature;
placing the NC membrane into a monoclonal antibody diluted by 1:5,000 times, and incubating for 30-60min at room temperature;
e. washing membrane with PBST for 3-4 times, each for 3 min; placing the NC membrane into a 1:8,000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody for incubation for 30-60min at room temperature;
PBST washing the membrane for 4-5 times, each time for 3 min;
and g.66 mu L of NBT and 33 mu L of BCIP substrate are added into 10mL of substrate buffer solution (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl and pH9.5), the membrane is placed into the substrate solution to develop color after uniform mixing, the result is observed by naked eyes, the membrane is rinsed by tap water to terminate the reaction when the positive control is obviously purple and the negative control is not developed any color, and the result is recorded by photographing.
3) Storage and effective period:
storing at 2-8 deg.C in dark place for 12 months.
4) Phosphate buffer (0.01M PBS, pH7.4) formulation:
adding 950mL of distilled water to dissolve, adjusting the pH to 7.4, and fixing the volume to 1,000 mL.
Claims (2)
1. The hybridoma cell strain 2D6 secreting tomato mottle mosaic virus monoclonal antibodies is characterized by secreting tomato mottle mosaic virus monoclonal antibodies, and the hybridoma cell strain 2D6 is preserved in the China general microbiological culture Collection center (CGMCC) in 2019, 11 months and 4 days, wherein the preservation number is CGMCM NO. 18846.
2. The tomato mottle mosaic virus monoclonal antibody secreted by hybridoma cell line 2D6 according to claim 1, wherein the ascites indirect ELISA titer of the monoclonal antibody reaches 10-8The antibody type and subclass is IgG2aThe sensitivity of detecting the crude extract of tomato diseased leaf tissue infected with ToMMV by establishing ACP-ELISA and dot-ELISA methods by using the monoclonal antibody reaches 1:40,960 and 1:20,480 times of dilution respectively, wherein the unit is weight/volume and g/mL.
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