LU101592B1 - Hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus, antibody therefrom and antibody preparation method thereof - Google Patents
Hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus, antibody therefrom and antibody preparation method thereof Download PDFInfo
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- LU101592B1 LU101592B1 LU101592A LU101592A LU101592B1 LU 101592 B1 LU101592 B1 LU 101592B1 LU 101592 A LU101592 A LU 101592A LU 101592 A LU101592 A LU 101592A LU 101592 B1 LU101592 B1 LU 101592B1
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- monoclonal antibody
- ringspot virus
- chain variable
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
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Abstract
The present invention provides a hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus as well as antibody therefrom and antibody preparation method thereof, wherein: BALB/c mice are immunized with antigenic peptide CAFGKGVEEIEQTST to obtain a hybridoma cell strain, and the cell strain which can produce monoclonal antibody specifically recognize this antigenic peptide are screened, then monoclonal antibodies specifically recognizing Tomato ringspot virus coat protein AFGKGVEEIEQTST peptide are obtained by ascites preparation method, the lengths of heavy chain variable region and light chain variable region of this monoclonal antibody are respectively 185 and 103 amino acids; the yield of monoclonal antibody is 500-5000 mg/L ascites, the purity is 85%~99.5%. The monoclonal antibody of the present invention has a characteristic of specifically recognizing Tomato ringspot virus coat protein, and it has a broad application prospect in terms of Tomato ringspot virus entry-exit inspection and quarantine.
Description
[0001] The present invention relates to a monoclonal antibody, particularly relates to a hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus as well as antibody therefrom and antibody preparation method thereof.
[0002] Tomato ringspot virus (ToRSV) is a member of virus Comoviridae, its virion is an icosahedron with equal diameter, the diameter is about 30 nm. ToRSV can infect 150 types of crops such as soybean, grape, apple, cherry, tomato and tobacco. At present, international quarantine method for ToRSV comprises SEM observation, biological method, serological test and molecular biology method. Because ToRSV is a spherical virus with a diameter of 28 nm and virus content in the sample is low, it is difficult to be observed and identified under the electron microscope; inoculation and identification of the host require a special isolated greenhouse, and spend a long time, and masking of symptom of the virus tends to affect identification result. The serological method is necessary means for rapid detection of plant virus, and current virus detection at port rely on purchase of imported antiserum, being expensive, but in China there has being no reports on ToRSV serum detection kit for imported plant seeding.
[0003] The present invention screens and obtains a monoclonal-antibody-secreting cell strain, by using Tomato ringspot virus coat protein as the detection target, via selection and synthesis of Tomato ringspot virus specific general antigen peptide, as well as a series of steps such as cross-linked immunity of mouse, cell fusion, antibody activity detection. Sequence of antigen recognition region in antibody gene is obtained by RT- 1
PCR method. Finally, monoclonal antibodies of which the sequence specifically (0101008 recognizing target antigen being known is obtained. a cell strain producing this antibody and corresponding antibody production method. Compared to the method for obtaining monoclonal antibody by immunizing mouse with purified virion, the monoclonal antibody obtained by the present invention has a definite recognition epitope, while the recognition epitope of the monoclonal antibody obtained by virion immunization is not definite. The present invention firstly analyzes a conserved region from the sequence of Tomato ringspot virus coat protein, then selects and designs candidate antigenic peptide from the conserved region, subsequently prepares this antigenic peptide by chemical synthesis method, thus acquisition of antigen is not limited by the sample origin of the carring virus, and does not exceed the permitted scope of imported inspection and quarantine operation. Definite recognition epitope also makes the monoclonal antibody of the present invention has a characteristic of being definite and easy to be recognized compared to the antibodies already on the market and related investigated and reported antibodies.
[0004] The technical problem to be solved by the present invention is to provide a monoclonal antibody against Tomato ringspot virus, the monoclonal antibody has a reaction specificity that recognizes and binds with specific peptide of Tomato ringspot virus coat protein, and lack of binding specificity to other viruses and plant tissue, and lays a foundation for further research and development of Tomato ringspot virus serum detection kit.
[0005] The present invention firstly discloses a monoclonal antibody against Tomato ringspot virus, the sequence of heavy chain variable region of this antibody is as shown in SEQ ID NO:1, the sequence of light chain variable region is as shown in SEQ ID NO:2.
[0006] The lengths of the heavy chain variable region and the light chain variable region in this monoclonal antibody are respectively 185 and 103 amino acids.
[0007] As a preferred embodiment of the present invention, the nucleotide sequences 2encoding said heavy chain and light chain variable region are respectively as shown in SEQ ID NO:3 and SEQ ID NO:4.
[0008] Said monoclonal antibody is prepared by ascites inducing method, its yield is 500~5000 mg/L, and purity is 85%~99.5%.
[0009] The present invention also discloses a hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus, the hybridoma cell strain is collected at China General Microbiological Culture Collection, and the accession number is: CGMCC No.18320.
[0010] The present invention also discloses application of said monoclonal antibody against Tomato ringspot virus in preparation of Tomato ringspot virus detection reagent or kit.
[0011] Said monoclonal antibodies against Tomato ringspot virus are prepared by the steps as follows: (1) Finding out multiple coat protein sequences of Tomato ringspot virus from Genbank (including the protein sequence after gene sequence translation), and selecting conserved fragments by sequence alignment; (2) Selecting for appropriate antigen sequences in accordance with the conserved fragments, and obtaining a preferred antigenic peptide CAFGKGVEEIEQTST, as shown in SEQ ID NO:5; (3) By chemical synthesis method, synthesizing an antigenic peptide CAFGKGVEEIEQTST in which one cysteine residue is added at the N-terminal; (4) Cross-linking said antigenic peptide with KLH; (5) Immunizing mice with the cross-linked antigenic peptide; (6) Preparing hybridoma cells and screening cell strain producing antibody specifically recognizing SEQ ID NO:5 peptide; (7) By RT-PCR method, cloning and determining the sequences of heavy chain and light chain variable regions of the monoclonal antibody in the monoclonal antibody cell strain; 3
(8) By using the ascites inducing method, preparing monoclonal antibodies which can recognize SEQ ID NO:5 peptide and show positive reaction to the sample carrying Tomato ringspot virus; (9) By using ammonium sulfate precipitation and protein G affinity chromatography method, purifying the monoclonal antibodies according to the present invention.
[0012] Preparation process of the monoclonal antibodies by ascites inducing method specifically comprises the steps as follows: (1) 8~15 week old BALB/c mice are injected intraperitoneally with a sterilized paraffin at
0.1~0.5 mL/mouse; (2) After 5~10 days, intraperitoneally inoculated with hybridoma cells suspended in PBS at 5x10°~5%10%mouse; (3) After 5~10 days, the ascites is collected 2~4 times; (4) The ascites is centrifuged at 1000xg for 10 minutes, uppermost adipose tissue is sucked off, cell components and precipitate are removed, and supernatant was collected; (5) 3 mL of ascites supernatant is taken, after centrifugation, precipitation, redissolution, and recentrifugation, it is concentrated to 5 mL; (6) The concentrated solution is passed through a protein G purifying resin, eluted with 10~15 column volumes of pre-elutant, monoclonal antibodies are eluted with 5~10 column volumes of elution buffer, after concentration, to obtain the monoclonal antibodies of the present invention.
[0013] Preferably, the pre-eluant in said step (6) includes 0.1~0.15 M NaCl and 10~40 mM NazHPO4, pH is 7.0; said elution buffer is 0.1-0.3 M glycine, and pH is 3.0.
[0014] The term “monoclonal antibody” as used in the present invention refers to an antibody obtained from a substantially homogenous cell population, namely individual antibodies included in this population are same, except for a few possible naturally occurring mutations. The monoclonal antibody is directed against a single antigen site with high specificity. The monoclonal antibody according to the present invention specifically recognizes the peptide shown in SEQ ID NO:5 which originates from Tomato ringspot virus coat protein, recognizes a unique and definite sequence, and different from the conventional monoclonal antibody by which the recognized sequence 4 |is unknown and obtained by immunizing mouse with Tomato ringspot virus, similarly the monoclonal antibody is definitely different from Tomato ringspot virus polyclonal antibody.
[0015] The present invention has the following beneficial effects: the monoclonal antibody against Tomato ringspot virus according to the present invention underwent antibody titer determination and ELISA, the results indicate that the monoclonal antibody recognizing the peptide as shown in SEQ ID NO:5 has a reaction specificity with Tomato ringspot virus, and can be used as an antibody for Tomato ringspot virus detection, which can lay a good foundation for subsequent development of Tomato ringspot virus serum detection kit, and serve for work needs of entry-exit inspection and quarantine.
[0016] Fig. 1 shows the antigen sequence and position of candidate antigenic peptide 7 on Tomato ringspot virus coat protein.
[0017] The present invention is further explained by the following examples. Example 1
[0018] (1)Sequence of Tomato ringspot virus protein (the sequence accession number is: NP 733973) was retrieved from the Genbank, the length of this sequence is 562 amino acids residues (namely 562 aa); according to the antigenic peptide length requirement of 14 amino acids residue (14aa), the theoretical number of the candidate antigenic peptide segments is 549. By solubility and synthesis difficulty analysis as well as immunogenicity analysis and prediction, 7 candidate antigenic peptides were obtained; the sequences of the candidate antigenic peptides 1-6 is shown as in SEQ ID NO:6-11, the sequence of candidate antigenic peptide 7 is as shown in SEQ ID NO:5, wherein the antigen sequence and position of candidate antigenic peptide 7 on Tomato ringspot virus coat protein is as shown in Fig. 1. For the need of coupling carrier protein-hemocyanin
KLH, after 1 cysteine (C) and 1 cysteine were added at N-terminal or C-terminal, the candidate antigenic peptides are as folows: Candidate antigenic peptide 1: LLRYKEWQRQGFLHC Candidate antigenic peptide 2: GPTEIDLTSTPAPNC Candidate antigenic peptide 3: KLVDRLSVNVILQEC Candidate antigenic peptide 4: GQGAFSLPISTPHAC Candidate antigenic peptide 5: CLLRYKEWQRQGFLH Candidate antigenic peptide 6: CDDKSEVLLRQHPLS Candidate antigenic peptide 7: CAFGKGVEEIEQTST.
[0019] (2) By entrusting a commercial polypeptide synthesis company, seven antigen polypeptides according to step (1) were prepared by solid-state synthesis method, the purities of the polypeptides were more than 98%.
[0020] (3) The candidate antigenic peptide was coupled with KLH by a two-step reaction method (this example is explained by taking “candidate antigenic peptide 7: CAFGKGVEEIEQTST” as an example). Steps of the coupling reaction are as follows: a) 20 mg of SMCC [4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-n- hydroxysuccinimide ester sodium salt] was dissolved in 2 mL of DMF (N,N- dimethylformide).
b) 0.8 ml of KLH (hemocyanin) was added to a 25 ml round-bottom flask, and 1xPBS (pH 7.2) was refilled, to make the final concentration of the protein being 15 mg/ml.
c) the dissolved SMCC solution was slowly dropwise added to 120 mg of KLH protein system, stirred and reacted at room temperature for 1 hour.
d) dialyzed with 1 L of 1xPBS (PH 7.4) solution at 4°C for 6 hours.
e) the dialyzed KLH protein was poured into a 50 mL centrifugal tube, its volume was determined by weighing method (calculated on the basis of density 1 g/cm*), the protein concentration after dialysis was calculated based on the amount of KLH 6 | |protein added before the reaction, then 2.5 mg of KLH-SMCC solution was transferred into a 5 ml centrifugal tube based on its concentration.
f) 3.0 mg of “candidate antigenic peptide 7: CAFGKGVEEIEQTST” was dissolved with 0.6 ml of 1xPBS (pH 7.2) solution.
g) Sulfhydryl group in the polypeptide was detected with Ellman reagent: into a 96-well plate, 100 pl of Ellman reagent stock solution was added, then 10 pl of polypeptide solution was added, its UV absorption value was measured at A=412 nm: if OD value>0.15 then next step was done; if OD value<0.15 and >0.05 then polypeptide was refilled, until meeting the requirement; h) “candidate antigenic peptide 7” solution was dropwise added into a KLH-SMCC tube, mixed uniformly at room temperature with a vertical vortex mixer and reacted for 4 hours.
i) Detecting sulfhydryl group in the polypeptide with Ellman reagent: into 96-well plate 100 pul of Ellman reagent stock solution was added, and 10 ul of coupled polypeptide solution was added, UV absorption valuec was measured at \=412 nm. If OD value<0.03, it shows that cross-linking rate of polypeptide with KLH protein was more than 80%; if OD value>0.03 then KLH protein activated by SMCC was refilled to continue the cross-linking. Obtain coupled antigen of “coupled candidate antigenic peptide 7-KLH”.
[0021] (4) Immunization of mice
[0022] (5) Preparation of hybridma cells
[0023] (6)Cloning of hybridoma cells (by limited dilution method)
[0024] (7)Preparation of monoclonal antibodies by ascites inducing method Example 2
[0025] The steps (1)-(6) of Example 2 are same as Example 1.
[0026] (7)Preparation of monoclonal antibodies by ascites inducing method a) 15 week old BALB/c mice were injected intraperitoneally with sterilized paraffin at 7
0.5 mL/mouse; b) After 10 days, intraperitoneally inoculated with hybridoma cells suspended in PBS at 5x10%/mouse; c) After 10 days, the ascites was collected 4 times; d) The ascites was centrifugationd at 1000xg for 10 minutes, uppermost adipose tissue was sucked off, then cell components and other precipitate were removed, and supernatant was collected; e) 3 mL of ascites supernatant was taken, after centrifugation, precipitation, redissolution, and recentrifugation, it was concentrated to 5 mL; f) the concentrated solution was passed through protein G purifying resin, eluted with column volumes of pre-eluent (00.15 M NaCl, 40 mM Na:HPO4, pH 7.0), monoclonal antibodies were eluted with 10 column volumes of elution buffer (0.3 M glycine, pH 3.0), after being concentrated, to obtain the monoclonal antibodies of the present invention, the yield was 500 mg/L ascites, the purity was 87.2%, the tomato leaves carrying inactivated Tomato ringspot virus were detected, the detection signal was positive. Reaction of this antibody to tomato sample not infected by Tomato ringspot virus was negative, and reactions to Tobacco mosaic virus, Arabis mosaic virus, Bean pod mottle virus etc. were negative.
Example 3
[0027] Steps (1)~(6) of Example 3 are same as Example 1.
[0028] Preparation of monoclonal antibodies by ascites inducing method a) 11.5-week old BALB/c mice were taken and injected intraperitoneally with sterilized paraffin at 0.3 mL/mouse; b) After 7 days, inoculated intraperitoneally with hybridoma cells suspended in PBS at
1.25x10%/ mouse; c) After 7 days, ascites was collected 3 times; d) Ascites was centrifuged at 1000xg for 10 minutes, the uppermost adipose tissue was sucked off, the cell components and other precipitates were removed, and a | 8supernatant was collected; e) 3 mL of ascites supernatant was taken, after centrifugation, precipitation, redissolution, recentrifugation, it was concentrated to 5 mL; f) The concentrated solution was passed through protein G purifying resin, monoclonal antibodies were eluted with 13 column volumes of pre-eluant (0.125 M NaCl, 25 mM Naz:HPO4, pH 7.0), and eluted with 7 column volumes of elution buffer (0.2 M glycine, pH 3.0), after being concentrated, the monoclonal antibodies of the present invention were obtained, the yield was 500~5000 mg/L ascites, the purity was 93.5%, the tomato leaves carrying inactivated Tomato ringspot virus were detected, the detection signal was positive. Reaction of this antibody to tomato sample not infected by Tomato ringspot virus of was negative, and reactions to Tobacco mosaic virus, T7 bacteriophage, Arabis mosaic virus, and Bean pod mottle virus were negative. Example 4
[0029] Steps (1)~(6) of Example 2 are same as Example 1.
[0030] (7)Preparation of monoclonal antibodies by ascites inducing method a) b) 12-week old BALB/c mice were injected intraperitoneally with sterilized paraffin at 0.3 mL/mouse; b) After 7 days, intraperitoneally inoculated with hybridoma cells suspended in PBS at 1x10%/ mouse; c) After 7 days, the ascites was collected 3 times; d) The ascites was centrifuged at 1000xg for 10 minutes, uppermost adipose tissue was sucked off , cell components and other precipitate were removed, and supernatant was collected; .
e) 3 mL of ascites supernatant was taken, after centrifugation, precipitation, redissolution, recentrifugation, it is concentrated to 5 mL; f) The concentrated solution was passed through protein G purifying resin, eluted with column volumes of pre-eluent (0.12 M NaCl, 30 mM Na:HPO4, pH 7.0), the 9monoclonal antibodies were eluted with 7 column volumes of elution buffer (0.15 M glycine, pH 3.0), after being concentrated, to obtain the monoclonal antibodies of the present invention, the yield was 5000 mg/L ascites, the purity was 99.5%, the prepared antibodies were reacted to tomato leaves carrying inactivated Tomato ringspot virus and detected by indirect ELISA method, the detection signal was positive. The reaction of this antibody to tomato sample not infected by Tomato ringspot virus was negative, the reactions to Tobacco mosaic virus, Arabis mosaic virus, Bean pod mottle virus etc. were negative. Example 5
[0031] The implementation steps are same as Example 4, the antigenic peptide is “candidate antigenic peptide 1:LLRYKEWQRQGFLHC”, the monoclonal antibody produced by the hybridoma cell was detected by indirect ELISA method, the reaction to uncoupled “candidate antigenic peptide 1” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody which can be used in natural virion detection was not obtained. Example 6
[0032] The implementation steps are same as Example 4, the antigenic peptide is “candidate antigenic peptide 2:GPTEIDLTSTPAPNC”, the monoclonal antibody produced by the hybridoma cell was detected by indirect ELISA method, the reaction to un-coupled “candidate antigenic peptide 2” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody which can be used in natural virion detection was not obtained. Example 7
[0033] The implementation steps are same as Example 4, the antigenic peptide was “candidate antigenic peptide 3: KLVDRLSVNVILQEC”, the monoclonal antibodyproduced by the hybridoma cell was detect by indirect ELISA method, the reaction to un-coupled “candidate antigenic peptide 3” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody which can be used in natural virion detection was not obtained.
Example 8
[0034] The implementation steps are same as Example 4, the antigenic peptide was “candidate antigenic peptide 4:GQGAFSLPISTPHAC”, the monoclonal antibody produced by the hybridoma cell was detected by indirect ELISA method, reaction to the uncoupled “candidate antigenic peptide 4” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody which can be used in natural virion detection was not obtained. Example 9
[0035] The implementation steps are same as Example 4, the antigenic peptide was “candidate antigenic peptide 5S:CLLRYKEWQRQGFLH”, the monoclonal antibody produced by hybridoma cell was detected by indirect ELISA method, the reaction to the uncoupled “candidate antigenic peptide 5” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody can be used in natural virion detection was not obtained. Example10
[0036] The implementation step are same as Example 4, the antigenic peptide was “candidate antigenic peptide 6:CDDKSEVLLRQHPLS”, the monoclonal antibody produced by hybridoma cell was detected by indirect ELISA method, the reaction to the uncoupled “candidate antigenic peptide 6” was positive, the reaction to tomato leaves carrying inactivated Tomato ringspot virus was negative. The monoclonal antibody which can be used in natural virion detection was not obtained. 11
Claims (7)
1. A monoclonal antibody against Tomato ringspot virus, wherein the sequence of the antibody heavy chain variable region is as shown in SEQ ID NO:1, and the sequence of the light chain variable region is as shown in SEQ ID NO:2.
2. The monoclonal antibody according to claim 1, wherein the lengths of heavy chain variable region and light chain variable region of the monoclonal antibody are 185 and 103 amino acids, respectively.
3. A gene encoding the monoclonal antibody according to claim 1, wherein the gene includes a nucleotide sequence encoding light chain variable region and a nucleotide sequence encoding heavy chain variable region, the nucleotide sequence encoding said heavy chain variable region is as shown in SEQ ID NO:3, the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:4.
4. A hybridoma cell strain secreting monoclonal antibody against Tomato ringspot virus, deposited at China General Microbiological Culture Collection Center, with the accession number of CGMCC No.18320.
5. A preparation method of the monoclonal antibody of claim 1, wherein the method comprises the steps as follows: (1) 8~15 weeks old BALB/c mice are injected intraperitoneally with sterilized paraffin
0.1~0.5 mL/mouse; (2) after 5~10 days, the hybridoma cells according to claim 4 suspended in PBS are inoculated intraperitoneally at 5x105~5x106/mouse; (3) after 5~10 days, ascites is collected 2~4 times; (4) the ascites is centrifugationd at 1000xg for 10 minutes, uppermost adipose tissue is sucked off, cell components and precipitate are removed, and a supernatant is collected; (5) 3 mL of ascites supernatant is taken, after centrifugation, precipitation, redissolution, 12recentrifugation, it is concentrated to 5 mL; (6) the concentrated solution is passed through protein G purifying resin, and eluted with 10-15 column volumes of pre-elutant, the monoclonal antibodies are eluted with 5-10 column volumes of elution buffer, after being concentrated, thereby obtaining the monoclonal antibodies according to the present invention.
6. The preparation method according to claim 5, wherein the pre-eluant in said step (6) includes 0.1~0.15 M NaCl and 10-40 mM Na2HPO4, the pH is 7.0; said elution buffer is
0.1~0.3 M glycine, and the pH is 3.0.
7. Application of the monoclonal antibody against Tomato ringspot virus according to claim 1 in preparation of the detection reagent or kit for Tomato ringspot virus.
13
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