CN1900115A - Method for preparing monoclonal antibody resisting O-type foot and mouth disease virus and antibody and use - Google Patents
Method for preparing monoclonal antibody resisting O-type foot and mouth disease virus and antibody and use Download PDFInfo
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Abstract
本发明公开一种用于检测O型口蹄疫病毒的单克隆抗体的制备方法、以及这种抗体和这种抗体的用途。The invention discloses a preparation method of a monoclonal antibody used for detecting O-type foot-and-mouth disease virus, the antibody and the application of the antibody.
Description
Technical field
The present invention relates to a kind ofly be used to detect the MONOCLONAL ANTIBODIES SPECIFIC FOR method of O type foot and mouth disease virus, this antibody, and the purposes of this antibody.
Background technology
Foot and mouth disease is as great animal epidemic, and countries in the world are all attached great importance to this sick inspection and quarantine, prevention and control.The main method of detection, diagnostics port fever aphthous is to detect morbidity or be with intravital foot and mouth disease virus of malicious animal and antibody horizontal thereof at present, as methods such as isolated viral, ELISA.Directly isolated viral is the reliable diagnostic to foot and mouth disease, but the long cycle can be incured loss through delay the optimum control time to this deadly infectious disease; Need distinguishing though ELISA is simple is natural infection or because the antibody that vaccine immunity produces can not in time be made diagnosis.For the ease of diagnostics port fever aphthous accurately and timely, directly detect foot and mouth disease virus in the animal body, avoid since go out the result slow, do the unfavorable factor that differential diagnosis etc. brings to the anti-system of foot and mouth disease, the foot-and-mouth disease virus resistant monoclonal antibody that the production specificity is high has very strong practical value.
O type foot and mouth disease is a kind of animal epidemic virus of China's pilosity, so O type foot and mouth disease virus is the classical epidemic isolates of China.How early diagnosis finds that the strain of this virus has important meaning to China's livestock industry sound development in practice.But the report that effective, the quick and technical skill that specificity is high of this virus of diagnosis is not arranged so far as yet.
Summary of the invention
Main purpose of the present invention is in order to provide a specific specificity high resisting O-type foot and mouth disease virus MONOCLONAL ANTIBODIES SPECIFIC FOR method.The present invention also provides this antibody that obtains with preceding method, and this antigenic purposes.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus of the present invention is:
(1) with BHK-21 cell proliferation O type foot and mouth disease virus, and collect viral liquid, with the viral liquid freeze thawing several of collecting, make the abundant cracking of cell, allow virus fully discharge, again with the BEI inactivation treatment after cell debris is removed in centrifugation, in gained liquid, add the NaCl that adds PEG6000 and every liter of liquid 40 grams by every liter of liquid 80 grams, the back standing over night stirs, being settled out virus through centrifugal treating again, is 0.1M Tris with content, 0.5M NaCl, 0.05M EDTA, the TNE of pH7.6 suspends and precipitates, and making its volume is 1% of original volume, adds the trieline degrease of 2 times of volumes again, carry out centrifugal treating again, draw supernatant liquor and repeat centrifugal treating, use the resuspended precipitation of the TNE identical again, in resuspended liquid, add the Sodium desoxycholate of 0.2g/L with aforementioned ratio, standing over night behind the mixing, the crude antigen liquid that will spend the night are separated the O type foot-and-mouth disease virus antigen that obtains purifying through sucrose gradient centrifugation.
(2) subcutaneous with injection mouse behind gained O type foot-and-mouth disease virus antigen and the fully emulsified mixing of equivalent adjuvant, injected dose is each 20～80 μ g/ antigens, at interval 2～4 weeks, repeats to inject at least 1 time with method; Immunity for the first time with complete Freund's adjuvant, use Freund's incomplete adjuvant instead for the second time, for the third time without adjuvant, stand-by after all through 2 after the last immunity.
The myeloma cell who gets the X-63, the NS-1 that have obtained in the mouse and SP2/0 clone is stand-by.
(3) cytogamy: with the purifying O type foot-and-mouth disease virus antigen of 20～80 μ g to the mouse peritoneal or the intravenous inoculation immunity of the mode immunization of (2) set by step, behind 3 days after the immunity, get the highest mouse spleen of serum antibody titer and carry out cytogamy, the spleen of getting mouse then obtains splenocyte, add RPMI-1640 in 1: 30 by volume behind the dispersion splenocyte, the more centrifugal supernatant liquor that goes of splenocyte is obtained the splenocyte precipitation.The centrifugal supernatant liquor of cultivating in above-mentioned (2) that goes of bone marrow cells in mice knurl SP2/0 is obtained myeloma cell's precipitation.Precipitate with suspend above-mentioned chrotoplast precipitation and myeloma cell of RPMI-1640, and with splenocyte and myeloma cell by quantity than (5～10): 1 mixes two kinds of cells, the centrifugal then supernatant liquor that goes, fully disperse throw out, in throw out, added concentration in 1: 2 by volume and be 50% polyoxyethylene glycol PEG4000, leave standstill centrifugal removal supernatant liquor after 1～2 minute again, 25～35 times the HAT nutrient solution suspension fused cell that adds the throw out volume again, adding HAT nutrient solution to 140～160 times of throw out volume at last cultivates, get the whiteruss that Balb/c mouse peritoneal injection 0.5mL crosses through autoclaving, press 1-5 * 10 after 7-10 days
6The amount of individual hybridoma/mouse is injected mouse peritoneal, the collection of aseptic technique ascites was carried out after 7-10 days in the injection back, the ascites of gathering discards the lipid layer of the superiors with centrifuging, collect middle layer liquid, be monoclonal antibody, carry out purification process again and obtain highly purified resisting O-type foot and mouth disease virus monoclonal antibody.
In the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus of the present invention, be that the conventional nutrient solution of the monoclonal anti body and function of the resisting O-type foot and mouth disease virus of gained is cultivated, with the mass production monoclonal antibody.
Can adopt big rolling bottle rotary culturing to carry out the mass production of the monoclonal antibody of resisting O-type foot and mouth disease virus in the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus of the present invention.
In the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus of the present invention, with inducing the mass production that method is carried out the monoclonal antibody of resisting O-type foot and mouth disease virus in the homology mouse body,
In the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus of the present invention, adopt ammonium sulfate precipitation and gel chromatography purifying that the monoclonal antibody of resulting resisting O-type foot and mouth disease virus is carried out purifying.
Adopt aforesaid method can obtain the monoclonal antibody of resisting O-type foot and mouth disease virus.
The purposes of the monoclonal antibody of resisting O-type foot and mouth disease virus of the present invention is used it for the ELISA detection kit that preparation detects O type foot and mouth disease virus;
Or use it for the gold-immunochromatographyreagent reagent for assay box that preparation detects O type foot and mouth disease virus.
Discover: resisting O-type foot and mouth disease virus monoclonal antibody of the present invention has the serotype specificity of height, and not with other serotype generation cross reaction, so it has high specificity; Go down to posterity by the Balb/c mouse peritoneal through hybridoma cell strain to secretion resisting O-type foot and mouth disease virus monoclonal antibody, find that the antibody titer in 2～4 generations all is stabilized in 1: 8 * 10
5, antibody titer height of the present invention is described, susceptibility is good, and stable, is fit to the preparation in order to test kit.
Embodiment
Below be a concrete preparation process introduction of the present invention:
(1) the BHK-21 cell proliferation O type foot and mouth disease virus of cultivating with rolling bottle is earlier collected viral liquid.Virus liquid is through freeze thawing 3 times.Adopt 30 ℃ of deactivations of 1%BEI 28 hours, 2% Sulfothiorine stops.The centrifugal 35min of 8000rpm is with after removing cell debris, and the amount that restrains by every liter of liquid 80 adds PEG6000, and the amount that restrains by every liter of liquid 40 adds NaCl, and in 4 ℃ of stirrings 12 hours, 4 ℃ were spent the night; The centrifugal 35min precipitation of 8000rpm/min virus; With TNE (0.1M Tris, 0.5M NaCl, 0.05M EDTA, the pH7.6) precipitation that suspends, making its volume is 1% of original volume.The trieline degrease that adds 2 times of volumes.The centrifugal 20min of 12000rpm.Draw supernatant liquor in new centrifuge tube, the centrifugal 150min of 30000rpm; With the resuspended precipitation of TNE, in resuspended liquid, add the Sodium desoxycholate of 0.2g/L, standing over night behind the mixing.Viral liquid is joined 7.5～45% continuous saccharose gradient, the centrifugal 150min of 140000rpm.Get the component of each gradient, measure protein content with ultraviolet spectrophotometer at 259nm, two-way immune agar diffusion test detects its immunoreactivity.And taking out the albumen of the maximum absorption band part that 259nm determines, this part albumen promptly is the virus antigen albumen of purified high density.To obtain and place-70 ℃ to deposit standby.
(2) preparation immune spleen cell: get aforesaid purifying O type foot-and-mouth disease virus antigen and the abundant mixing of equal-volume adjuvant, inject healthy mice nape portion both sides respectively, pars inguinalis is subcutaneous; The viral dosage of every per injection is 20～80 μ g, after 2～4 weeks, uses the same method and repeats once more to inject at least 1 time at interval; The adjuvant of immune usefulness is a complete Freund's adjuvant for the first time, immune used adjuvant changes Freund's incomplete adjuvant, immunity for the third time into without adjuvant, last immunity 2 weeks of back for the second time, survey the foot-and-mouth disease virus resistant antibody titers in the mice serum, will show the source of the mouse of enough antibody titerss as immune spleen cell.
Measure the method for antibody titers, available radioimmunoassay (RIA) or enzyme linked immunological absorption (ELISA) assay method etc. adopt common ELISA method among the present invention.
(3) preparation myeloma cell: the myeloma cell uses X-63, NS-1 and the SP2/0 clone that has obtained usually from mouse.Before carrying out cytogamy, it was cultivated in conventional nutrient solution 3～4 days, so that when merging, can obtain 2 * 10
7Or more fused cell.The present invention selects SP2/0 myeloma cell usually for use.
(4) cytogamy: to the mouse peritoneal or the intravenous inoculation immunity of the mode immunization of (2) set by step, and the spleen of getting mouse behind 3～4 days after the immunity obtains splenocyte with the purifying O type foot-and-mouth disease virus antigen of the aforementioned gained of 20～80 μ g.Disperse splenocyte, added RPMI-1640 then in 1: 30 by volume, piping and druming obtains the splenocyte precipitation with the centrifugal supernatant liquor that goes of splenocyte for several times more gently.The centrifugal supernatant liquor that goes of bone marrow cells in mice knurl SP2/0 that above-mentioned (3) are cultivated obtains myeloma cell's precipitation.Precipitate with suspend above-mentioned splenocyte precipitation and myeloma cell of RPMI-1640, and with splenocyte and myeloma cell by quantity than (5～10): 1 mixes two kinds of cells, the centrifugal then supernatant liquor that goes, fully disperse throw out, in throw out, added concentration in 1: 2 by volume and be 50% polyoxyethylene glycol PEG, leave standstill the centrifugal supernatant liquor that goes after 1～2 minute again, slowly add 25～35 times HAT nutrient solution of throw out volume in throw out, used HAT solution is: xanthoglobulin 10
-6～10
-3M, aminopterin-induced syndrome 10
-8～10
-7M, thymidine 10
-6～10
-4M, the suspension fused cell is added HAT nutrient solution 140～160 times to the throw out volume at last.Fused cell is added in 96 well culture plates by 100 μ l/ holes, every Kong Zhongzai adds 100 μ l HAT nutrient solution mouse ascites cells again, and used mouse ascites is obtained in the healthy mice body, and (about 1～2 * 10
5Individual/hole) as feeder cell.It is 3～7% CO that above-mentioned culture hole is placed concentration
2In the incubator, be to cultivate 10～14 hours under 35～38 ℃ of conditions,, changed liquid once with every Kong Banliang HAT nutrient solution every 2～3 days since the 3rd day in temperature.
When in observing culture hole, the hybrid cell colony occurring, get culture supernatant earlier, and then add half amount HAT nutrient solution in every hole.Detect resisting O-type foot and mouth disease virus antibody and titre with ELISA again.To show that it is considered as when tangible rising is arranged compared with the control qualified when detection.
Above-mentioned resisting O-type foot and mouth disease virus antibody positive, hybridoma that titre is high transferred in another piece 96 orifice plates clone, obtain hybridoma cell strain.Can make the method that only contains a single hybridoma in each hole through this hybridoma of limiting dilution by a kind of, or a kind of soft agar method of from soft agar medium, separating colony, or a kind of employing micromanipulator, the method of the single hybridoma of picking, or a kind of " sorter clone " method that adopts cell sorter to separate single hybridoma.
(5) preparation monoclonal antibody: cultivate the hybridoma cell strain that obtains in above-mentioned (4) with conventional nutrient solution.The mass production monoclonal antibody can adopt big rolling bottle rotary culturing.Or induce method in the homology mouse body.Wherein big rolling bottle rotary culturing can obtain the foot-and-mouth disease virus resistant monoclonal antibody by cultivating above-mentioned (3) hybridoma purifying.The method that induces in homology mouse (the present invention the uses the Balb/c mouse) body can be by the hybridoma (5 * 10 of generation foot-and-mouth disease virus resistant antibody that above-mentioned (3) are obtained
5～10
6) inject female mice (4～8 age in the week) intraperitoneal of handling through pristane, collect ascites after 2 to 3 weeks, the centrifugal solids component of removing, its supernatant liquor promptly contains the resisting O-type foot and mouth disease virus monoclonal antibody.Be further purified if desired, can adopt ammonium sulfate precipitation and gel chromatography purifying.
Resisting O-type foot and mouth disease virus monoclonal antibody through last cultivation and purification process can perhaps be used to prepare the gold-immunochromatographyreagent reagent for assay box that detects O type foot and mouth disease virus in order to the ELISA detection kit of preparation detection O type foot and mouth disease virus.
The resisting O-type foot and mouth disease virus monoclonal antibody is carried out the indirect ELISA test with bag by Asial, A and C type foot-and-mouth disease virus antigen respectively, the result shows, the resisting O-type foot and mouth disease virus monoclonal antibody that we produce has the serotype specificity of height, not with other serotype generation cross reaction.
Below be relevant detection case:
Table 1 monoclonal antibody ascites and Asial type FMDV antigen-reactive OD
450Value
| 1(2F1) | 2(2F1) | 3(6H4) | 4(6H4) | 5(2G7) | 6(2G7) | 7(+) | 8(-) | 9(0) | |
| A B C D | 0.151 0.078 0.028 0.023 | 0.152 0.075 0.040 0.024 | 0.172 0.107 0.032 0.025 | 0.171 0.106 0.042 0.020 | 0.189 0.084 0.042 0.031 | 0.172 0.102 0.053 0.037 | 0.374 0.313 0.300 0.268 | 0.170 0.146 0.141 0.115 | 0.152 0.102 0.035 0.000 |
The result shows in the table 1, and the OD450 value is between 0.035 ~ 0.152 in the 9th row (0) of blank hole; OD among negative control the 8th row
450Value is 0.141 ~ 0.170; Positive control the 7th row OD
450Value is 0.300 ~ 0.374; The antigenic reacted OD of the monoclonal antibody of all screenings and Asial type
450Value is compared with blank with negative control does not have significant difference, does not react, and has very big difference and compare with the value of positive control, illustrate this monoclonal antibody not with Asial type antigen generation cross reaction.
Table 2 monoclonal antibody ascites and A type FMDV antigen-reactive OD
450Value
| 1(2F1) | 2(2F1) | 3(6H4) | 4(6H4) | 5(2G7) | 6(2G7) | 7(+) | 8(-) | 9(0) | |
| A B C D | 0.012 0.007 0.001 0.000 | 0.016 0.007 0.002 0.002 | 0.012 0.001 0.000 0.000 | 0.009 0.000 0.000 0.000 | 0.004 0.001 0.000 0.000 | 0.013 0.012 0.001 0.000 | 0.253 0.181 0.150 0.061 | 0.105 0.085 0.072 0.030 | 0.000 0.000 0.000 0.000 |
The result shows in the table 2, OD in the 9th row (0) of blank hole
450Value is 0.000; OD among negative control the 8th row
450Value is below 0.105; Positive control the 7th row OD
450Value is between 0.061 ~ 0.253; The antigenic reacted OD of the monoclonal antibody of all screenings and A type
450Value is compared with blank with negative control does not have significant difference, does not react, and has very big difference and compare with the value of positive control, illustrate this monoclonal antibody not with A type antigen generation cross reaction.
Table 3 monoclonal antibody ascites and C type FMDV antigen-reactive OD
450Value
| 1(2F1) | 2(2F1) | 3(6H4) | 4(6H4) | 5(2G7) | 6(2G7) | 7(+) | 8(-) | 9(0) | |
| A B C D | 0.234 0.079 0.055 0.001 | 0.262 0.082 0.058 0.012 | 0.254 0.075 0.049 0.004 | 0.237 0.081 0.049 0.009 | 0.231 0.088 0.022 0.011 | 0.218 0.093 0.033 0.015 | 0.282 0.269 0.216 0.132 | 0.118 0.106 0.085 0.046 | 0.093 0.083 0.020 0.000 |
The result shows in the table 3, in that A is capable when envelope antigen not being diluted, nonspecific reaction is arranged.From the capable raising gradually that begins along with weaker concn of B, nonspecific reaction does not appear.OD in the 9th row (0) of blank hole
450Value is below 0.083; OD among negative control the 8th row
450Value is below 0.106; Positive control the 7th row OD
450Value is between 0.132 ~ 0.269; The antigenic reacted OD of the monoclonal antibody of all screenings and C type
450Value is compared with blank with negative control does not have significant difference, does not react, and has very big difference and compare with the value of positive control, illustrate this monoclonal antibody not with C type antigen generation cross reaction.
Table 4 cell culture fluid supernatant, ascites titration and subgroup identification summary table
| 2F1 | 6H4 | 2G7 | |
| The cell culture fluid supernatant ascites antibody subclass of tiring of tiring | 1∶800 1∶1×10 4 IgG2b | 1∶6400 1∶4×10 5 IgG1 | 1∶1600 1∶1×10 4 IgG1 |
To be accredited as the resisting O-type foot and mouth disease virus monoclonal antibody be IgG to subclass in table 4, and the cell culture fluid supernatant the highest hole 6H4 that tires is 1: 6400, ascites is tired can reach 1: 4 * and 10
5, illustrate that tiring of monoclonal antibody is very high, be fit to carry out the development research of test kit.
Table 5 pair hybridoma stability is identified (tiring of the ascites supernatant after repeatedly going down to posterity)
| 2F1 | 6H4 | 2G7 | |
| The 4th generation of the 3rd generation the 2nd generation the 1st generation | 1∶1×10 4 1∶1×10 4 1∶1×10 4 1∶1×10 4 | 1∶4×10 5 1∶8×10 5 1∶8×10 5 1∶8×10 5 | 1∶1×10 4 1∶1×10 4 1∶1×10 4 1∶1×10 4 |
As seen from Table 5, go down to posterity by the Balb/c mouse peritoneal through hybridoma cell strain to secretion resisting O-type foot and mouth disease virus monoclonal antibody, find that the antibody titer in 2～4 generations all is stabilized in 1: 8 * 10
5, the antibody titer height of tiring not only is described, and resulting hybridoma cell strain is stable, is fit to carry out the development research in downstream.
Show that by above experimental data this resisting O-type foot and mouth disease virus monoclonal antibody of the present invention is the specificity height not only, susceptibility is good, the height of tiring, and also stable, be fit to the preparation detection kit.
Claims (8)
1, the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus is characterized in that:
(1) with BHK-21 cell proliferation O type foot and mouth disease virus, and collect viral liquid, with the viral liquid freeze thawing several of collecting, make the abundant cracking of cell, allow virus fully discharge, again with the BEI inactivation treatment after cell debris is removed in centrifugation, in gained liquid, add the NaCl that adds PEG6000 and every liter of liquid 40 grams by every liter of liquid 80 grams, standing over night after stirring is settled out virus through centrifugal treating again, is 0.1M Tris with content, 0.5M NaCl, the precipitation 0.05M EDTA, the TNE of pH7.6 suspend, making its volume is 1% of original volume, the trieline degrease that adds 2 times of volumes again, carry out centrifugal treating again, draw supernatant liquor and repeat centrifugal treating, use the resuspended precipitation of the TNE identical again with aforementioned ratio, the Sodium desoxycholate that in resuspended liquid, adds 0.2g/L, standing over night behind the mixing, the crude antigen liquid that will spend the night are separated the O type foot-and-mouth disease virus antigen that obtains purifying through sucrose gradient centrifugation
(2) subcutaneous with gained O type foot-and-mouth disease virus antigen and the fully emulsified back injection of equivalent adjuvant mouse, injected dose is each 20～80 μ g/ antigens, at interval 2～4 weeks, repeats to inject at least 1 time with method; Immunity for the first time with complete Freund's adjuvant, use Freund's incomplete adjuvant instead for the second time, for the third time without adjuvant, stand-by after all through 2 after the last immunity,
The myeloma cell who gets the X-63, the NS-1 that have obtained in the mouse and SP2/0 clone is stand-by,
(3) cytogamy: with the purifying O type foot-and-mouth disease virus antigen of 20～80 μ g to the mouse peritoneal or the intravenous inoculation immunity of the mode immunization of (2) set by step, behind 3 days after the immunity, get the highest mouse spleen of serum antibody titer and carry out cytogamy, the spleen of getting mouse then obtains splenocyte, 1: 30 by volume adding RPMI-1640 behind the dispersion splenocyte, again the centrifugal supernatant liquor that goes of splenocyte is obtained the splenocyte precipitation, the centrifugal supernatant liquor of cultivating in above-mentioned (2) that goes of murine myeloma cell SP2/0 is obtained myeloma cell's precipitation.Precipitate with suspend above-mentioned chrotoplast precipitation and myeloma cell of RPMI-1640, and with splenocyte and myeloma cell by quantity than (5～10): 1 mixes two kinds of cells, centrifugal then removal supernatant liquor, fully disperse throw out, in throw out, added concentration in 1: 2 by volume and be 50% polyoxyethylene glycol PEG4000, leave standstill centrifugal removal supernatant liquor after 1～2 minute again, 25～35 times the HAT nutrient solution suspension fused cell that adds the throw out volume again, adding HAT nutrient solution to 140～160 times of throw out volume at last cultivates, get the whiteruss that Balb/c mouse peritoneal injection 0.5mL crosses through autoclaving, press 1-5 * 10 after 7-10 days
6The amount of individual hybridoma/mouse is injected mouse peritoneal, the collection of aseptic technique ascites was carried out after 7-10 days in the injection back, the ascites of gathering discards the lipid layer of the superiors with centrifuging, collect middle layer liquid, be monoclonal antibody, carry out purification process again and obtain highly purified resisting O-type foot and mouth disease virus monoclonal antibody.
2, the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus according to claim 1 is characterized in that the conventional nutrient solution of the monoclonal anti body and function of the resisting O-type foot and mouth disease virus of gained is cultivated, with the mass production monoclonal antibody.
3, the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus according to claim 2 is characterized in that adopting big rolling bottle rotary culturing to carry out the mass production of the monoclonal antibody of resisting O-type foot and mouth disease virus.
4, the MONOCLONAL ANTIBODIES SPECIFIC FOR method of resisting O-type foot and mouth disease virus according to claim 2 is characterized in that adopting in the homology mouse body inducing the mass production that method is carried out the monoclonal antibody of resisting O-type foot and mouth disease virus.
5, according to the MONOCLONAL ANTIBODIES SPECIFIC FOR method of the described arbitrary resisting O-type foot and mouth disease virus of claim 1 to 4, it is characterized in that monoclonal antibody, can adopt ammonium sulfate precipitation and gel chromatography purifying again the resisting O-type foot and mouth disease virus of gained.
6, according to the monoclonal antibody of the resulting resisting O-type foot and mouth disease virus of MONOCLONAL ANTIBODIES SPECIFIC FOR method of the described arbitrary resisting O-type foot and mouth disease virus of claim 1 to 5.
7, the purposes of the monoclonal antibody of resisting O-type foot and mouth disease virus according to claim 6 is characterized in that using it for the ELISA detection kit that preparation detects O type foot and mouth disease virus.
8, the purposes of the monoclonal antibody of resisting O-type foot and mouth disease virus according to claim 6 is characterized in that using it for the gold-immunochromatographyreagent reagent for assay box that preparation detects O type foot and mouth disease virus.
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