TWI276686B - Hybridoma cell line producing monoclonal antibodies against foot-and-mouth disease virus and the monoclonal antibodies therefrom - Google Patents

Abstract

A hybridoma cell line producing monoclonal antibodies against Foot-and-Mouth disease virus, by which the monoclonal antibodies produced are capable of specifically binding to structure protein VP1 (viral protein 1) of Swine adapted Foot-and-Mouth disease virus FMDV O/TWN/97, is provided. The monoclonal antibodies produced by the hybridoma cell line and their preparation, and the method and kit for detecting Foot-and-Mouth disease virus using the monoclonal antibodies are also provided.

Description

1276686 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a single-source antibody against foot-and-mouth disease virus, and a fusion tumor cell line producing the same. [Prior Art] Foot_and_Mouth disease is a highly contagious disease of ancient cloven-hoofed animals. It has a serious impact on livestock, meat production, such as cattle, pigs, sheep, goats, etc., and is listed as the International Organization for Animal Health ( Organization of

Internation Animal Health, the original ΟΙΕ) A table infectious disease (1996), and one of the Chinese cockroach infectious diseases (Peng Xuangui, 1997). Taiwan’s past ten years have been a pest free area for eighteen years (Ryu, E., 1984), and unfortunately broke out in 1997. (Chang, T·C·, et al., 1997) The elimination of cockroaches, the culling, and the death of 180,000 pigs, the direct economic loss is about NT$160 million. The pork channel that is exported to Japan every year and about 1.6 billion US dollars is also completely interrupted, causing serious impact _ pigs and Related industries (Yang, P. C., et' al., 1999). In 1997, the control of the mouth and foot disease epidemic was based on the comprehensive culling of the vaccination site and the comprehensive vaccination strategy of the cloven-hoofed animals. The disease was effectively controlled in May of that year, and the culling policy was revised to be limited to diseased animals. Cooperate with comprehensive vaccination to effectively prevent foot and mouth disease. In addition to the rapid rise in body temperature of the animals infected with foot-and-mouth disease, the most obvious is the presence of blisters in the hoof, inside and outside the mouth, inside the nasal cavity and in the breast of the dam, the degree of which is inconspicuous to extremely severe, and even the death of young animals is extremely different. It is disturbing that foot-and-mouth disease can not rely on g bed symptoms and swine vesicular disease (5 1276686 SVD), vesicular stomatitis (VS), and non-malignant infectious porcine herpes (Vesicular exan) The division of iema of swine (VES) (Leman, A. D., et al., 1992) must be determined by laboratory testing. The laboratory tests are divided into pathogen (antigen) and serological (antibody) determination, that is, the presence or absence of the foot-and-mouth disease virus antigen in the sample, or the specific antibody against the foot-and-mouth disease virus in the serum of the unimmunized animal. Find out whether the diagnosis is a foot-and-mouth disease positive reaction (Kitching, R·P·, 2000). It is used for the diagnosis of B in the outbreak of foot-and-mouth disease and the diagnosis of the foot-and-mouth disease eradication stage. There are four structural proteins of foot-and-mouth disease virus: vpi, VP2, VP3, and VP4, which are classified into seven types of blood by serotype and cross-protection: serotypes: 0, A, C, SAT-1, SAT-2, SAT-3, Asia 1; • In the same way, further subdivided into more than 80 subtypes, almost no cross protection between each serotype, different subtypes of the same serotype Although there is no protection at the same level, the antigenicity of each type is different. Therefore, it is necessary to confirm the type of foot-and-mouth disease virus at the same time as the foot-and-mouth disease virus infection, and benefit from the epidemic prevention and disease removal. jobs. The swine foot-and-mouth disease virus that was raging in Taiwan in 1997 is not a foot-and-mouth disease virus known in the past. It does not infect cattle and sheep ruminants (Chang, TC, et. ale, 'l"7). After the outbreak, the Pirbright was located in the UK. The world's foot-and-mouth disease reference laboratory diagnosis, coupled with animal experiments confirmed by cross-infection between pigs and cattle, was named as the blood/month-type pig-type (SWineadapted) foot-and-mouth disease virus 〇/TWN/97. This type of mouth 1276686 The reason why the foot-and-mouth disease virus only infects pigs and has no infectivity against the reverse animals is related to the change of the nucleic acid sequence of the non-structure protein 3A (Clayton, W·B·, et · al·, 2000). However, there have been no single-source antibodies or detection reagents for detecting swine foot-and-mouth disease virus 0/TWN/97. In order to effectively and effectively prevent swine foot-and-mouth disease in Taiwan, it is imperative to establish a method for effectively diagnosing and/or identifying swine foot-and-mouth disease virus 0/TWN/97. SUMMARY OF THE INVENTION and the present invention provides a single-source antibody against foot-and-mouth disease virus, which is characterized in that it can specifically bind to the structural protein VP1 of porcine foot-and-mouth disease virus 0/TWN/97, and can be used to establish fast and accurate The foot-and-mouth disease virus serological diagnosis technology and diagnostic reagent kits are used to detect foot-and-mouth disease infections and to monitor whether to implement comprehensive immunization. SUMMARY OF THE INVENTION The object of the present invention is to provide a fusion cell strain capable of producing a single-source antibody against S. typhimurium virus. The present invention is characterized in that the single-source antibody produced by the present invention can specifically bind to the VP1 protein antigen of the foot-and-mouth disease virus of porcine type, and does not cross-react with other aquatic diseases viruses and (iv) common viruses in pig farms; The _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 (8) The characteristics of single-source antibodies produced by the Fusion Japonica T5H-12, registered with BCRC _25, deposited at the Food and Jade Development Institute on January 14, 2005. 1276686 A further object of the present invention is to provide a method of preparing the above-described fusion tumor cell line. • Another object of the present invention is to provide a single-antibody against foot-and-mouth disease virus, which is characterized in that the single-source antibody produced by the invention can specifically bind to the VP1 protein of foot-and-mouth disease virus of porcine type, and does not react with other vesicular disease viruses. The common virus in Taiwan pig farms produces cross-fork reactions. A further object of the invention is to provide a process for the preparation of the above described single source antibodies. Further, another object of the present invention is to provide a method for detecting whether or not the foot-and-mouth disease virus is infected, which is characterized in that the single-antibody against foot-and-mouth disease virus of the present invention is used. A further object of the present invention is to provide a kit for detecting foot-and-mouth disease virus, which is characterized in that the single-antibody against foot-and-mouth disease virus of the present invention is used. - Detailed description of the present invention. The present invention provides a fusion tumor cell strain capable of producing a single-source antibody against foot-and-mouth disease virus, as shown in the first figure, which is a standard technique for producing fusion tumors (Harl〇w, E. and

Lane, D·, 1988; Hong, Η·Τ· et·. al·, 1989) Prepared, and repeated the use of porcine foot-and-mouth disease virus 0/TWN/97 as an antigen to repeat BALB/c mice The spleen is immunized to produce a high-valence antibody reaction, and the spleen cells of the mouse are taken out and fused with the myeloma cells, and then screened by immunofluorescence staining to form a fusion tumor having a specific reaction to the swine foot-and-mouth disease virus. The cells are subjected to monoculture and then sputum to the desired single-source fusion tumor cell line, and the mono-antibody produced has a specific binding reaction to the VP1 protein of the pig foot-and-mouth disease virus, and the VP1 protein contains diseased cells and cells. Binding of the 29 amino acid sequence of the key amino acid sequence - RGi motif 1276686 to peptide P29 (as described in Example 5.2). According to a preferred embodiment of the present invention, the invention can be used to produce a fusion tumor cell which can produce a single-source antibody against swine foot-and-mouth disease virus. The $ ’ is named T5H-12 ‘in the past few years! It was deposited on the 14th of the month in the Food Industry Development Research Institute with the registration number BCRC 960225. The invention also provides a method for preparing the above-mentioned fusion tumor cell line, which comprises the following steps: - (1) injecting porcine foot-and-mouth disease virus 0/TWN/97 as an antigen into small white gas, abdominal cavity and spleen, and adding after appropriate time (2) Take out the small spleen cells from the mouse and fuse with the bone-cell strain, and prepare the culture medium of the fusion tumor cell strain with appropriate medium; (3) Take the culture medium of the fusion tumor cell line, and screen out to produce anti-foot-and-mouth disease Disease • Viral VP1 protein fusion tumor cell line. • According to a particular embodiment of the invention, wherein the VP1 protein of step (3) comprises a peptide of 29 amino acid sequences of the RGD motif. The present invention also provides a single chitosan-resistant disease virus, which is heterozygous for the VP1 protein-specific binding of the foot-and-mouth disease virus, and is an important vesicular disease in another pig in Taiwan, and a porcine blister disease (7) ^The ribs are (3) 丨 丨 匕 匕 幻 幻 幻 幻 幻 幻 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无 无/97 Live poison as an antigen. 'The single-source antibody was characterized by a single-source antibody produced by the Fusion Research Cell Line No. 5H-12 deposited with the Food Industry Development Institute on January 14th. 9 1276686 According to a preferred embodiment of the present invention, the single-source anti-system is deposited in the fusion research cell line T5H-12 of BCRC 96〇225, which is deposited in the Food Industry Development Research Institute at the age of 14 years. produce. As shown in the third figure, it is an IgG-type immunoglobulin (IgG2b/c); it can be specifically bound to the 〇/TWN/97 porcine foot-and-mouth disease virus, especially the VP1 protein of the foot-and-mouth disease virus of pig type, which has been confirmed to be compatible with The virus binds to the 29 amino acid sequence peptide pp29, which binds to the key amino acid sequence of the RGD motif, and thus the epitope is located in P29.

The present invention further provides a method for producing the aforementioned single-source antibody, which comprises culturing the fusion cell strain T5H-12 of the present invention in a large amount and harvesting the culture solution. A large amount of culture can be carried out by performing cell culture in vitro or by intraperitoneal injection of ascites in mice. The resulting cell culture supernatant or ascites can be used as it is or after further purification. According to the present invention, a single-source antibody against foot-and-mouth disease virus can be used to detect whether an animal is infected with foot-and-mouth disease virus, and is completed by any conventional immunoretrolysis method and related detection methods, including direct or indirect immunoreactivity analysis methods. One embodiment of the present invention for detecting whether an animal is infected with foot-and-mouth disease virus is detected by a competitive immunological antigen reaction method. The principle is that the animal which is mainly infected by the foot-and-mouth disease will produce antibodies, so the antibody content of the sample is detected and detected by the anti-foot-and-mouth disease virus antibody and the competitive immuno-antibody antibody reaction method of the present invention, and the specific operation method thereof is determined. And the process is as described in Example 5.3 below. The method comprises the following steps: The method for detecting whether an animal is infected by foot-and-mouth disease virus: 10 1276686 (1) Providing a test tank for adsorbing a certain amount of standard foot-and-mouth disease virus protein on the surface of the test tank; (2) providing a proper concentration of the hoof a single-source antibody solution for the virus; (3) taking the sample of the animal to be tested; (4) adding the sample of the subject and the single-source antibody against the foot-and-mouth disease virus of the present invention to the gutter to make it with the test hoof The virulence virus protein produces an immune response; (5) after the sputum is washed, a signal generating tool is added, wherein the signal generating tool can operatively combine with the anti-epidemic virus antibody of the present invention to generate a signal; and (6) The signal determines whether the animal being tested is infected with foot-and-mouth disease virus. The invention also provides a kit for detecting infection of animal shirts by foot-and-mouth disease virus, the pot comprising: ^ (1) a test tank on which a certain amount of standard foot-and-mouth disease virus protein is adsorbed; (2) a suitable concentration of the anti-foot-and-mouth disease virus of the present invention a source antibody solution; (3) a signal generating tool, wherein the signal generating device can be operatively combined with a marker substance on the single-antibody antibody against foot-and-mouth disease virus of the present invention to generate a signal; and a wide range (4) Reagents and cleaning agents. The single test body of the invention can also detect the content of foot-and-mouth disease sputum in the sample by the method of Lin Weiwei. The invention also provides a method for detecting the toxic content of foot-and-mouth disease 1276686 in a liquid sample, comprising the following steps: (1) providing a supporting solid phase, which supports adsorption of a certain amount of the single source of the anti-foot-and-mouth disease virus of the invention on the surface of the solid phase (2) applying a sample of the test animal to the support solid phase to cause an immune reaction with the single-source antibody on the surface of the test cell; Λ (3) adding a signal generating tool, wherein the signal generating tool can operate Sexually combined with the single-source antibody against foot-and-mouth disease virus of the present invention to generate a signal; (4) determining the content of foot-and-mouth disease virus in the sample according to the generated signal. According to the present invention, a kit for detecting a foot-and-mouth disease virus content in a liquid phase sample by an immunoreaction analysis method, comprising: • (1) a supporting solid phase; • (2) a single-source antibody of the foot-and-mouth disease virus of the present invention, attached to The foregoing supports solid phase; (3) a tiger generating tool, wherein the signal generating tool is operably associated with the foot-and-mouth disease virus or its egg-derived segment, and the single-source antibody of the present invention on the aforementioned supporting solid phase Combine to generate a signal. 4 The signal generating means may be any marking substance such as a fluorescent marking substance, a cold 'light marking substance, an enzyme, or a radioactive substance. According to the present invention, preferred embodiments of the signal generating means include catalase, alkaline phosphatase, ?-galactosidase, biotin and the like. The signal generating means can also be a secondary antibody that binds to the single-source antibody of the present invention, and can be combined with the single-antibody antibody of the present invention (sec〇ndary 8111), for example, fluorescent 12 1276686 flag goat anti-mouse secondary antibody. The test cell and the supporting solid phase are composed of a material suitable for protein immobilization, such as a microtiter plate, a beads, a membrane or a strip. The invention is further described in the following examples, which are intended to illustrate the invention and not to be construed as limiting. [Embodiment] 'Example 1: Antigen 劁 by porcine foot-and-mouth disease virus 0/TWN/97 (ie 〇/TW-ChuPei/97, GeneBank accession # AF026168) for the Executive Yuan Agricultural Committee Animal Health Laboratory from '1997 In March, the isolates of the first case of foot-and-mouth disease in Zhubei, Taiwan, were distributed, from the 5 Xuan to the pigs, and in the negative pressure laboratory of the department, the performance was repeated five times in the ΒΗΚ-21 cell line. It is used as an antigen for preparation of immune and antigenic disks. Example 2: Immunization of a tumor cell line for the preparation of a tumor-free vaccination with a porcine foot-and-mouth disease virus 0/TWN/97 in the progeny of the fifth generation (FMDV/K5) as an immunization antigen, containing 1 〇7 immunization anti-protozoal·1 mL was injected into the abdominal cavity of 2-month-old female BALB/c mice, and a second abdominal cavity was performed two weeks later. Two weeks later, blood was collected to detect the anti-foot-and-mouth disease virus towel and the antibody-powered mussels. The mice were selected to be anesthetized with a valence reaction of 256 times. The immunized antigen containing 10 TCIDso was injected into the spleen at multiple points. (H〇ng, τ·, Fu 13 1276686 al·, 1989), two weeks after the interval of blood collection 2 · 2 method to detect anti-foot-and-mouth disease virus and antibody price, select the highest price response, in the first three fusion Days are injected in the same way. The same amount of immunization antigen is injected into the spleen. 1.2 Detection of the reaction of the atmosphere. After solidifying the mouse, 'sterilize the tail with 703⁄4 alcohol dampened cotton, cut the tail tip with a sterile surgical blade, and squeeze the tail from the head to the tail to make the blood flow out of the tail blood vessel. The effluent was taken up with a microcentrifuge tube and the coagulation was placed at 37. 〇 After a small _ hour, move to 4 ° C overnight, centrifuge at 14,000 rpm for 10 minutes every other day, and take the supernatant clarified serum and transfer to another new microcentrifuge tube. The anti-foot-and-mouth disease virus neutralizing antibody is carried out according to the test method provided by OIE Uitching RP, et al., 2000). The procedure is as follows: The serum to be tested is first • The 56°C is not moved. After treatment for 30 minutes, 5 〇 / / L of the test serum was added to each well of a 96-well cell culture plate, and after 2 times serial dilution, an equal amount of i〇〇TCI]) 5 〇 was added. After the standard virus solution of the TWN/97 strain, it was sensed for 60 minutes in an incubator containing 5% (v/v) c〇2 at 37 °C. Thereafter, 1 〇〇 μ of a BHK-21 cell suspension containing 2χ1〇6 cells/mL was added, and the culture was continued in a 5% c〇2, 37c>c incubator. 48 small day, review the occurrence of cytopathic phenomena (cyt〇pathic sputum, cpE) • 隋 亚 以 to inhibit 50% of the cells appear CPE serum maximum dilution or its L 〇 gl. -^ 〇β48) is treated as 0 (negative). ϋ, the production of fusion 澶iE 14 1276686 The production process of the fusion tumor cell line, as shown in the first figure. The spleen was reinforced by the spleen, and the spleen was neutralized by the antibody. The spleen-reinforcing mice were spleened for the last three days before the fusion. The blood was collected from the orbital sinus by the glass capillary ash tube until the animal sacrificed, and then the whole body was sprayed with 7〇% alcohol. , into the aseptic table, cut from the left back (head and tail) and peeling • The cortex separates from the abdominal muscle layer, disinfects the abdominal muscle layer with 70% alcohol, and then cuts the muscle layer in a sterile manner to remove the spleen. RPMI - 丨 64 〇 culture medium in a petri dish. The spleen was transferred into the culture nucleus. The small spleen was firstly acupunctured with 23G, and the RPMI-1640 culture solution was injected into the spleen with a 5 虬 plastic empty needle. The spleen cells were washed out, and the suspension containing the spleen cells was inhaled into a 5 〇虬 centrifuge tube. The above spleen cells were washed with a total of 50 mL of RPMI-1640 medium, and the spleen cells were collected and collected. The spleen was washed twice with 200 xg, 1 minute centrifugation and RPMI_164 清洗 as the cleaning solution. Dirty cells were placed in a 10 mL suspension with RPMM640 and the amount of cells was counted. Myeloma cells (SP2/0-Agl4, ATCC CRL-1581) in RPMI-1640 medium containing 1% fetal calf serum (FCS), Penicillin/Streptomycin (abbreviated as 10% FCS RPMM640) In the medium culture, the culture supernatant (collected as SP2/0 culture supernatant) was collected and stored overnight until the cells proliferated to near the plateau (Plateau). According to the fusion plan, the SP2/0 culture growth for fusion was adjusted, and the plateau was grown in the fusion day; the SP2/0 before the fusion was counted, washed with RPMM640, washed twice, and the suspension was suspended with all the spleen cells. Approximately 1: 3 (SP2/0: spleen fine sputum 1 · 3) The proportions were mixed thoroughly, and the supernatant was removed by centrifugation at 200 xg for 1 minute, and the cell pellet was loosened by tapping the bottom of the tube at 37°. Rotate 15 1276686 tube in C water bath and slowly add 37 mL of pre-heated 1 mL 50% (w/w) PEG-1500 along the tube wall. Control the addition speed to make the whole process for about 45 seconds. Place the spleen with PEG added. The bone tumor cells were mixed at 37. (: The water bath is applied for 75 seconds, then centrifuged at 100 xg for 5 minutes, and slowly added 40 mL 37 along the tube wall. (: Preheat RPMI-1640, centrifuge at 100 xg for 5 minutes, remove the supernatant, tap the centrifuge The cell pellet was loosened at the bottom of the tube, and 4 mL of 20% fetal calf serum (FCS), insulin (Insulin), 20% SP2/0 culture supernatant, Qxaloacetate, Sodium Pyruvate containing Penici 11 in/Streptomycin /Fugizone was added. RPMI-1640 medium (abbreviated as 20% FCS RPMI-1640) was used to suspend the fused cells, and the fused cell suspension was added to the trough cell culture dish in 100 μΙ7 trough, and placed in a 5% C〇2, 37Χ incubator for incubation; overnight And add 20%/^丄, § 2 X HAT (JJypoxanthine ' △minopterin ' Ihymidine, fusion tumor, cell screening) 20% FCSRPMI-1640 medium (abbreviated as HAT medium) to make the final concentration of Η 100 μΜ , A is 〇· 4 μΜ, T is 16 μΜ, and continues to be cultured in a 5% C〇2, 37 ° C incubator. f Example 3: fused to the cell line of the indirect immunofluorescence test ... · · After ten days of cell fusion, the colony can be seen under the microscopic examination (colony) Long • fusion of tumor cells, taking the culture supernatant for indirect immunofluorescence detection (indirect • immimQ-fl_scence観y, referred to as _ for screening of fusion tumor cell lines. IFA use of the antigen (four) for home miscellaneous Bioassay (4) Pressure test 16 1276686 chamber, the process is roughly as follows: · BHK-21 cells are cultured in a 96-well cell culture plate for two days to form a single layer (monolayer), then the culture supernatant is poured out, and 4χ1〇3 TCID5G is added. /100 jL/weu 〇Bali/97 virus dilution, placed in a 5% c〇2, 37 C incubator, after 6 to 8 hours of microscopic examination, when the plaque CPE begins to appear, pour the culture solution , add -2〇〇c 7〇% (v/v) acetone i〇〇/tank, then store in -70 ° C for use, this is the antigen disk. The antigen used for the fusion of tumor cell strains The plate was removed from the Chuanyi and returned to the temperature. After removing the fixative solution, it was washed 3 times with a volume of Phosphate-buffered saline (PBS). The residue was decanted and added to the culture supernatant of the fusion tumor cell. 〇 VL / trough, let stand at room temperature for 1 to 2 hours, pour off the supernatant and wash 3 times with PBS, Net residue, diluted 1-fold fluorescence 〇〇〇 flag goat anti mouse secondary antibody (ICN pharmaceuticals,

Fluorescein-conjugated goat IgG to mouse IgG, IgA, IgM), allowed to stand at room temperature for 1 hour, 'pour off the secondary antibody and wash 3 times with PBS, then add PBS 50 //L/well under a fluorescent microscope Microscopic examination showed whether there was a green fluorescence reaction in the cytoplasm of CPE cells. As shown in the second figure, the cells in the 96-well cell culture plate corresponding to the fusion tumor cells were considered to have secreted antibodies against foot-and-mouth disease virus. The parental hybridoma cells were cultured and the HAT medium was cultured and frozen in liquid nitrogen. 3· 2 single tumor of fusion tumor

After thawing the cryopreserved parental cells, the cells were cultured with HT medium (removed Aminopterin single component in the HAT medium after fusion), and the supernatant was tested by IFA 17 1276686, and the cell scale was determined to be positive. The fusion tumor cells which can be divided into sputum and sputum virus antibodies are successively subjected to single planting. The steps are as follows: the cells are washed up with a micropipette, the cell concentration is calculated by counting chamber slide, and the cell number is adjusted by using ΗΤ culture*. Injecting into a 96-well cell culture plate, each culture well has an average of about one cell. After several days of culture, microscopic examination is performed. The cells in the culture well are determined to have only a single cell population. The supernatant is taken for IFA detection, and the screening reaction is positive. The cells are retained and continue to be cultured; the single planting step is repeated once again, and the resulting single-community cells are monocultured fusion tumor cells. 3·^Incremental plaque culture of the fusion cell branch ^ The proliferation of the obtained single-body fusion tumor cells starts from the 96-well culture plate, and then to the 24-well culture tray, the 6-well culture plate, and the HT culture solution is used. Amplify the passage to the 25T culture flask • stage and gradually replace the HT medium with 10% FCS RPMI-1640 medium. The cells of the ϋ fusion tumor cell line were invited to be deposited. When the cells in the culture flask were proliferated to near the plateau stage, the cells adhering to the bottom of the bottle were washed down with a pipette, and the cell suspension of '2x10 was counted and centrifuged at 1,QQ〇rpiD. After 5 minutes, the cell pellet after removal of the supernatant was suspended in 1 mL of 1% OMSO/FCS (DMSO: FCS ~1:9), transferred to the cell cryotube, and placed in _2 〇χ for 30 minutes. , shift • put into ~80QC for overnight, then move to a liquid nitrogen bucket to save. The present invention screens a fusion tumor cell line which can produce a single-antibody antibody against foot-and-mouth disease virus 0/TWN/97 structural protein, named T5H-12, which was deposited at the Food Industry Development Research Institute on January 14, 2005. The deposit number is BCRC 960225. 18 1276686 Example 4 单 of single-source antibody 4.1 Qualification of single-source antibody The single-source antibody heavy and light chains were identified using the IsoStrip® Mouse Monoclonal Antibody Isotyping Kit (purchased from Roche). The culture supernatant of the fusion tumor cell T5H-12 obtained in Example 3 was operated according to the instructions of the reagent, and the results were as shown in the third figure. After the single-source antibody (T5H-丨2) produced was interviewed, Confirm that the heavy chain is IgG2b, and the light chain is /c chain. 4. 2 Ascites vaginal selection 8 weeks of healthy Balb/c mice, first 1 mL each

Pristane (2' 6,10,14-Tetramethylpentadecane) was injected into the abdominal cavity. After every other week, 3xl〇6 fresh fusion tumor cell T5H-12 was injected into the abdominal cavity of the mouse to observe the change of the abdomen. The abdominal cavity was taken when the abdomen was enlarged. The liquid was centrifuged at 3,000 rpm for 10 minutes, and the supernatant was aspirated, and the mixture was placed in a new tube and frozen in -2 Torr.匸, which contains a high concentration of single-source antibodies.僧 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体Check and observe the green stimuli of the cytoplasm of cpe cells. The highest dilution factor of the fluorescence brightness before the microscopic examination is taken as the IFA price of the ascites. 19 1276686 VP1 and p〇lypeptide The preparation of Ρ29 is as follows: In order to facilitate the subsequent purification and detection of the gene product, the N-terminus and the C-terminus of the VP1 gene are first added with a tag and a His tag. A recombinant VP1 (rVPl) was expressed in the E. coli (疋co//) VP1 gene product in E. coli (Nco//) by the pET expression system (Novagene0 j WI). rVPl is completely expressed in the form of inclusion bodies existing in the bacteria. The cells are obtained by centrifugation, the cell wall and the inclusion bodies are disintegrated, and the sedimentation is completed. The rypi protein is expressed in the rypi protein. Urea urea (urea) binding buffer (binding buffer: 20 Tong Ding 1 ^ -11 (: 1, ? [17.9, 0.5 1% (: 1). Solution containing 1 ^ 1 in the 81 urea environment with metal chelate Purification by metal chelating affinity cloumn -Chelating Sepharose Fast Flow? Amersham Bioscience, followed by refolding of the structure by the SDS assisted rVH protein on the gei fiitration column The refolded rVFI protein sample was then purified by sUperdex 200 column (Amersham Bioscience), and the purified rVH protein was confirmed by SDS-PAGE, and the SDS contained in the purified sample was removed by a scavenging column (Extracti-Gel@, Pierce, IL). ABI peptide synthesizer and Fmoc chemistry, based on 0/TWN/97 VP1 amino acid sequence 13 159 : NGSSKYGDTSTNNVRGDLQVLAQKAERTL, synthetic multi-peptide P29 (Wang, J·H·, et al· 2003) 20 1276686 Take antigen rVP120 pg/mL and P29 20 pg/mL, dissolved in coating buffer (15 mM Na2C〇3, 35 mM Na2HC〇3' pH) 9· 6) 'Enzyme-linked immunosorbent assay (ELISA) to detect the valence of a single-source antibody. The purified, double-coated coat of rVP1 protein or P29 is placed in a 96-well immunoenzyme. In the reaction tray (L0 pg/well), move to a refrigerator at 4 ° C overnight, and wash three times with PBS (named PBST) containing 0.1% Tween-20 for about one minute each time. % skim milk in PBS, incubated in a 37 ° C incubator for one hour, washed three times with one volume of PBST, then add the first antibody (the single-source antibody T5H-12 to be tested, in one volume PBS was diluted in multiples), placed in a 37T incubator for one hour, and washed three times with one volume of PBST. A suitable concentration of biotinylated goat anti-mouse IgG secondary antibody (Vector; 1 : 3000 dilution in PBS; 1 〇〇, g/well) was added to the reaction plate and then allowed to act for one hour at 37QC. The wells were washed 3 times by adding 100 PBS PBST, and then added with a 1:3000 dilution of enzyme streptavidin-peroxidase (Vector; 1 : 3000 dilution in PBS; gL/well), which was allowed to act in 252C for one hour, and the reaction plate was washed three times with PBST. Then, it was added as a substrate for 3', 5, 5'-Tetramethylbenzidine (Sigma, 100 pL/well) for color reaction. It was allowed to stand in the dark for 10 minutes at room temperature, and finally added with The reaction was stopped by the same amount of 1 N H2S〇4, and then the absorbance (abS0rbance) of the sample was measured by spectrophotometer at 450 nm. The absorbance was measured as the vertical axis (χ) value, Τ5Η-12 antibody The different dilution values are plotted on the horizontal axis (γ) value, and the obtained curve takes the middle value of the highest and lowest absorbance values, and the corresponding Υ value is the antibody valence; the antibody titer of the T5IH2 ascites to the rVH protein is 4.5. X 1〇4, the force against more than 29 peptides Then • for 6· 1 X 1〇4 〇 • Single-source antibody particle sensitivity analysis (Enzyme Inhibition Assay, Competition Inhibition Assay • Fix rVP1 protein in 96-well at 4吒Plate for 16 hours, then to exclude non-specific reactions, add 2 5% 5% skim milk powder in PBS for 1 hour per well, wash with PBST, as described in 5.2. • Diluted 4,000 times Single-source antibody T5H-12 ascites in PBS buffer • Eight continuous concentrations of antagonists (rVPl) (1 〇ng, 100 ng, 250 ng, 500 ng, 750 ng, 1, 〇〇〇ng, 1 〇, 〇〇〇ng , just, face ng) preparation, that is, "the competitor is diluted 4, 〇〇〇 times T5H-12 and 稀释νρΐ of different dilution factors, mixed properly. The competitor is placed at 372C for 30 minutes, then 2 Repeat the method to add 50 uL of each different dilution of rVpi competitor to each well of the 96-well plate fixed in ^^1; after 60 minutes at 37%, add 100 uL PBST per well for 3' ELISA detection of biotin-conjugated goat anti-mouse IgG secondary antibody. Steps (described in Section 5.2 above) The number of T5H-12 single-source antibodies still bound to the 96-well plate was detected, and as shown in the fourth panel, the 50% competitive inhibition of rVpl to 5}1-12 single-source antibody binding was approximately 225 ng. 22 1276686 Analysis of the whole-fee fe of single-source antibody (radioactive free sedimentation)

35S BHK-21 lysate Preparation: UMO'BHK—21/75 T-stalks are grown in an incubator containing 5% (v/v) C〇2 at 37°C for 24 hours to grow into m〇n〇layer, after which Serum-containing DMEM medium (GibcoTM 21013-024, without methionine, Hepes 25 mM) was washed twice. After adding 6 mL of serum-free medium for 30 minutes (thirsty), add (10) μα 35Smethi〇nine ( RedivueTM L [S] methionine ' AG1094 ' Amershain Pharmacia Biotech, ❿ Specific activity > lOOOCi/mmol), 120 kL dialysis FCS continued to culture for 20 hours, aspirate supernatant, to ι · 5 mL ripa Dialysis buffer (lysisbuffer) (Talk about RIPA dialysis buffer containing NaC1150mM, NP-40, 1%, Na-deoxycholate 0·5%, SDS 0·1%, pH 7. 5 Tris 50 mM) Dissolve BHK with 35S methionine -21 cells, this is for ΒΗΚ-21 lysate, which is reserved for backup. 35SFMDV lysate Preparation: 3χ107ΒΗΚ-21/150 T yarns were cultured in a 5® % (v/v) C〇2 incubator for 24 hours at 37°C, grown into monolayers, and then serum-free DMEM medium ( Wash once with the above), add 0/TWN/97 5χ108 TCID5〇/6 mL DMEM, ~15 mo· i· (multiplicity infection) • Infected dose, incubate for 30 minutes at 37 ° C with slight shaking. _ (2〇 St-mfeciton, PI, start timing after infection), after sucking unadsorbed virus

Wash three times in DMEM, add 12 mL DMEM thirsty for 30 minutes, then add 240 μΠ 35S methionine (same as above), 240 μM dialyzed FCS 23 1276686 to continue the culture, PI 10 hours ΒΗΚ 100% rounding CPE 50% of the cells floated 'The flask was placed in -80 ° C and frozen, and then taken out at 37 ° C. This step was repeated three times in total, and the cell suspension was aspirated and centrifuged at 1,650 xg for 1 minute. The supernatant was centrifuged, and then 1/100 volume of EI·1 Μ BEI (Mnary-§thyleneimine) was added and stirred at room temperature for 24 hours. The non-dynamic virus solution was filtered through 0.2 μM and then 230 000 x g. After ultra-high speed centrifugation for 8 hours, the settled mass was dissolved in 0·75 mL RIPA, which was 35S FMDV lysate. Radioimmunoassay analysis: using RocheTM Protein g

Immunoprecipitation (referred to as IP) Kit (cat·Ν〇 1719386), according to the instructions for use; the steps are as follows, each]; P reaction using FMDV lysate _ stock solution 12 吣 as antigen, and pr〇tein G at 4. (: 3 hours pre-washing with swaying effect, (Pre-cleaning) After removing the non-specific background, add T5H-12 ascites stock solution 0.2 μί 'lighting at 4 ° C for 1 hour, then add pr〇tein G at 4 ° C sway overnight to adsorb the immune complex of the antigen and the antibody, and then wash the washing buffer 1, 2, 3 (12, 〇〇〇xg after centrifugation for 20 seconds, then go to the supernatant) once. Addition of Protein G sediment to the remaining 10 々L Laemmli sample buffer (this sample buffer contains SDS 2%, Glycerol 10%, - dithiothreitol 100 mM, Bromophenol Blue 0· 〇〇 1%, pH 6 · 8 Tris ' 60 mM) 'Boiled for 3 minutes, centrifuged for 2 sec at i2, 〇〇〇xg for 2 sec., 15% sodium sulphate-polyacrylamide (sds-po 1 yacry 1 am i De ) Colloid (ge 1), protein electrophoresis analysis according to standard method (Maniatis et al., 1982); 24 1276686 After the electrophoresis is completed, the colloid is taken out, and in the Fixation solution (where Isopropanol ·· acetic acid ·· ddw · = 25 : 10 : 65) fixed for 30 minutes, followed by Amplify8 solution (NAMP100 Amersham Pharmacia Biotech TM) was soaked for 15 minutes, then the 3MTM filter paper was lined with the gel, and the cleaning film was applied. The gel was dried in a gel dryer (SlabGel Dryer SE1160, Hoeier Scientific InstrumentsTM) by heating and vacuuming. The dried colloid was dried. Gaiger counter detects the radioactivity of the sample, which is convenient for estimating the X-ray film (Hyperfilm MP, -ersham Bioscience) for autoradiography exposure at -80 °C in the case of intensify screens. The length of time; fully exposed X-ray film temperature _ after washing with Kodak M-35 X-0MAT automatic film processor, as shown in the fifth figure, sample lane, 1 is only 35S BHK-21 lysate, no T5H-12 antibody Participation, sample lane 2 is 35S BHK-21 lysate and T5H-12 antibody, sample lane 3 is only 35S FMDV lysate, no T5H-12 antibody is involved, sample lane 4 is 35S FMDV • lysate and T5H-12 antibody Compared with the 35 § BHK-21 lysate antigen, the T5H-12 antibody and the 35S FMDV lysate IP pattern, due to FMDV replication, assembly of viral eggs The specific mechanism, the IP seizure and sedimentation of the antigen, in addition to the expected VP1 (molecular weight of about 25 1 ^), the fourth visible settling zone in the fifth figure, the 'the darkest color', is also included in the virus The capsid is composed of VP3 which constitutes the primary structure protomer (VP1 with a molecular weight slightly larger than 25kD, the sedimentation concentration is about 1/10 of VP1), VP0 (molecular weight is greater than VP3, about 30kD, sedimentation color concentration 25 1276686) The weakest of the four bands visible, and possible intermediates of the viral protein synthesis process, such as VP0+VP3 (molecular weight greater than 5〇ld), the largest of the visible four bands), etc., when T5H-12 and FMDV lysate in IP When the reaction is combined, the antigen (VP, etc.) is in a complex state, and is extracted by antibodies with strong affinity, and is dissociated separately in subsequent processing and electrophoresis analysis; through sample lane 4 and lane 2 In comparison, it can be confirmed that the antigen corresponding to the T5H-12 antibody is a specific protein of FMDV, especially VP1, and has nothing to do with the host cell BHK. , 5^5_ single-source antibody T5H-12 tombstone analysis Specific indirect immunofluorescence staining (IFA) was used for specific analysis. The Swine vesicular disease (SVD) virus, which has the same blistering disease and has a case in Taiwan, and the antigenic disk prepared by another type of foot-and-mouth disease virus 0/TWN/99 that occurred in Kinmen in 1999. Indirect immunofluorescence staining was performed to confirm whether the T5H-12 single-source antibody cross-reacted with it. In addition, indirect immunofluorescence staining is used to confirm the pathogenic antibodies of T5H-12 and other common viral diseases in Taiwan, such as: Hog cholera, Pseudorabies, Porcine Reproduction and Cross-reaction of viruses such as Porcine reproductive and respiratory syndrome and Porcine circovirus type II, the results are shown below, and there is no cross-reactivity. Table · Single-source antibody T5H-12 and each disease antigen indirect immunofluorescence staining results of the test virus reaction results of foot-and-mouth disease virus 0/TWN/97 + 26 1276686 swine vesicular disease virus - foot and mouth disease virus 0/TWN/99 swine fever Virus————^ ————~M Porcine Pseudorabies Virus Porcine Reproductive and Respiratory Syndrome Virus----- Pig Circovirus Type 2——-_ Review Case 6: Evaluation of Single-source Hanging Body Test Foot-and-Mouth Disease Virus||^ Left-handed immunohistochemical staining method for detection of foot-and-mouth disease turtles using immunohistochemical staining with Vectastin® ABC kit containing Avidin DH and Biotinylated Horseradish Peroxidase®. The detailed steps are as follows. Infected pigs infected with foot-and-mouth disease virus 0/TWN/97, the diseased parts of the skin are fixed with fumarin, embedded in wax, cut into 3 μΜ thick tissue and carried by slide, soaked 3 times at room temperature with Xylene, 3 times each time. After a minute of dewaxing, soak in different concentrations (1%, 1%, 95%, 90%, 80%) of alcohol, each time for 1 minute, then wash with distilled water for 1 minute, then soak in (microwave Heated to about 95 °C in PBS for 3 sec, and then immersed in PBS for 3 minutes at room temperature. Then place the slides carrying the diseased tissue on a sterilized dish (Petri dish) to maintain Humidity of the dyeing process; drip 0.1% trypsin (trypsin 〇. lgm/m 1 〇〇) on the slice and place in a sail oven for 5 minutes' Take out 3 times of washing with room temperature PBS to remove the protease, drip on the slice 3%. 3% hydrogen peroxide (Hydrogen p (10) can, 3 sides 2 2 〇 Feng also ^ 1276686 1980 kL, ready to use before use) placed in a 4 ° c oven for 5 minutes, removed and washed with room temperature PBS 3 times to remove Hydrogen peroxide, diluted 1 〇〇 normal mouse serum drops in PBS. Covered with sections and placed at 40. (: 5 minutes in the oven, gently lick the serum of normal mice, and then drip the 3,000 times diluted T5^H2 ascites in PBS, place in 40 ° C oven for 10 minutes' and then rinse 3 times with room temperature PBS to remove The bound antibody was then diluted 100-fold with a Biotinylated horse anti-mouse IgG (heavy chain and light chain) antibody reagent (Vectastain ABC kit, PK-4002, Vector Laboratories) diluted in a coffee bath and placed in a 40T oven for 5 minutes. The unbound antibody was removed by washing 3 times with PBS at room temperature, and then diluted 1 〇〇 diluted Avidin DH-Bi〇tinylated

Horseradish Peroxidase H (Vectastain® ABC kit), rinsed 3 times at room temperature pbs, dripped on the staining agent chamber (3, 3-diamin〇benzidine 3, .H2〇2 0_5% in PBS, use It is placed in a 4〇〇c oven for 5 minutes, and it is treated with distilled water for 2 minutes. Enhancing solution (H-2200, Vector Laboratories) is allowed to act at room temperature for 15 seconds, then washed with distilled water. 2 'minutes' was finally stained with Hematoxylin (Hematoxylin 1 pi, sodium iodate 0·2 gm , ammonium or potassium alum 50 gm , citric acid 1 gm , chloral hydrate 50 gm , dw · 1 L) for 1 ... minutes. After washing for 2 minutes, the water is dehydrated (the steps are continuous with alcohol 95%, 1%, 1%, v alcohol 100%: xylene : 1 : 1, xylene, xylene treatment) after the film is sealed, the result is like the sixth The map does not. Figure 6A is a 200-fold enlarged porcine lesion skin tissue section epithelial layer 28 i 1276686 (epidermis layer) 'The fading band is positive for foot-and-mouth disease virus antigen, which is T5H-12 single-source antibody and virus VP1 protein Action area. B is a 400-fold magnification section, which is a partial enlargement of the upper epithelial layer of Figure A. The brown granules in the cytoplasm are the areas where the VP1 protein of the foot-and-mouth disease virus antigen is present, and most of the cells with positive cells in the cytoplasm are the spines in the epithelial layer. In the stratum spinosum cells, cells with positive immunohistochemical staining also have pathological hydropic degeneration and the cell boundary becomes inconspicuous. T5H-12 single-source antibody fish disease _ The main virion VP1 protein is located in the cytoplasm and is located in the tissue-specific lesion area, which is consistent with the pathogenic mechanism of foot-and-mouth disease virus. [Simple description of the schema], the first - provides the preparation process of the present invention _ foot-and-mouth disease virus single-source antibody. • $2 is the result of immunofluorescence staining between the single-source antibody (4)-丨2 of the porcine scorpion typhus virus of the present invention. The second figure is the result of Is0 plus i of the single-source antibody Τ5Η-12 heavy bond and the light key of the present invention. Wherein (A) is a heavy chain and (β) is a light chain. The fourth figure shows the results of the enzyme auditory analysis of the single-source antibody Τ5ίΗ2 of the swine foot-and-mouth disease virus of the present invention by rVP1. The fifth figure is the result of radioactive immune deposition analysis of the single-source antibody Τ5Η-12 of the foot-and-mouth disease virus of the present invention. The sixth figure is the result of detecting the foot-and-mouth disease virus by histochemical staining of the single-source antibody Τ5Η_12 of the foot-and-mouth disease virus of the present invention. 29

Claims (1)

1276686 Republic of China Patent Application No. 09410291^^ Chinese application of Zhizhan Yitu, Τ: 士么1 1 + , ......ν—ν/ν,-τ- XI η, patent application scope: ι A fusion tumor that can produce a single-source antibody against foot-and-mouth disease virus, the special purpose of which is that the single-source antibody produced can specifically bind to the reticulum antigen of porcine foot-and-mouth disease virus, and does not react with other water-borne diseases. Taiwan pig farms see the virus cross-reactive; it uses the porcine foot-and-mouth disease virus 0/TW_ live virus as an antigen to immunize Balb/C mice, and the Balb/C mouse spleen cells are fused with myeloma cells into fusion tumor cells. Strain, which was deposited in the Food Industry Development Institute on the date of January _14, 2005, and deposited the fusion cell line T5H-12 with the number BCRC 960225. 2. The fusion tumor cell strain according to the first aspect of the patent application, wherein the single-source antibody produced by the invention can specifically bind to the Vpi protein antigen of the foot-and-mouth disease virus of porcine type, and the VP1 protein contains 29 RGD motifs. The peptide of the amino acid sequence. Φ 3· A method for preparing a fusion tumor cell strain according to claim 1, which comprises the following steps: (1) injecting porcine foot-and-mouth disease virus 0/TWN/97 as an antigen into the abdominal cavity of a mouse and Spleen, and additional immunization at appropriate time; (2) taking out spleen cells of mouse and fusion with myeloma cell line, and preparing fusion cell strain culture medium with appropriate medium; and 1276686 (3) taking the aforementioned fusion tumor cell line The culture solution was screened for a fusion tumor cell line which can produce the VP1 protein of the anti- (porcine) foot-and-mouth disease virus. 4. The method of claim 3, wherein the VP1 protein of step (3) comprises a peptide of the 29 amino acid sequence of moti f (p29). 5. A single-antibody against foot-and-mouth disease virus, which is characterized in that it can specifically bind to the VP1 protein antigen of foot-and-mouth disease virus of pigs, and does not cross-react with other water cancer virus and common virus in Taiwan pig farm; The porcine foot-and-mouth disease virus 0/TWN/97 is used as an antigen. It can be stored in the Food Industry Development Institute and stored in the BCR 96〇225 fusion cell line T5H. 12 single-source antibodies produced. 6. A single-source antibody against foot-and-mouth disease virus, which is characterized in that it can specifically bind to the VP1 protein antigen of foot-and-mouth disease virus of pig type, and does not cross-react with other vesicular disease virus and common virus in Taiwan pig farm; The porcine foot-and-mouth disease virus 0/TWN/97 is used as an antigen, and can be stored in the fusion research cell line T5H-12 of the BCRC 96〇225 deposited with the Food Industry Development Research Institute on January 14, 2005. Single-source antibody competition is combined with foot-and-mouth disease virus. 7. The single-source antibody of claim 5, which binds to 1276686 (P29) of the 29 amino acid sequence of the 阢1) motif of the VP1 protein of porcine foot-and-mouth disease virus. 8. The single-source antibody of claim 6, which binds to the peptide of the 29 amino acid sequence of the RGD motif in the vpi protein of the porcine foot-and-mouth disease virus (P29). 9. A method for preparing a single-source antibody against swine foot-and-mouth disease virus according to claim 5 or 7 of the patent application. The money includes A4 culture. The fusion tumor cell line T5H as described in claim 1 or 2 -12, harvest the culture solution. 10. The method according to claim 9, wherein the large-scale culture is carried out in vitro for cell culture, and the cell culture supernatant is harvested. 11. The method according to claim 9 of the patent scope, wherein the system is alive, then the abdominal cavity of the field produces ascites, and the abdomen and water are harvested. 12. The method of claim 1 or 5, wherein the culture solution obtained can be used directly or after further purification. 13. A method for detecting whether an animal is infected with a foot-and-mouth disease virus, comprising the steps of: adsorbing a certain amount of the standard on the surface of the test tank (〇 providing a test slot for foot-and-mouth disease virus protein; 5, 6, 7 or 8 (2) providing it When the concentration is as specified in the range of 1276686 (' ί the single-antibody solution against foot-and-mouth disease virus; (3) taking the test animal's sample; (4) the test subject and the single-source antibody of step (2) The solution is added to the test tank to cause an immune reaction with the foot-and-mouth disease virus protein on the surface of the gutter; (5) after the test tank is cleared, the signal is generated to generate a Guard, wherein the signal φ generating tool can be operatively combined with the present invention. Anti-foot-and-mouth disease virus single-source antibody combines to generate signals; (6) Determine whether the tested animal is infected by foot-and-mouth disease virus according to the generated signal. 14· A kit for detecting whether an animal is infected with foot-and-mouth disease virus, including: (1) a test tank to which a certain amount of standard foot-and-mouth disease virus protein is adsorbed; (2) an appropriate concentration as described in claim 5, 6, 7 or 8 a single source antibody solution; (3) a 5 tiger generating tool 'where the signal generating tool can operatively combine with the marker substance on the single-antibody antibody against foot-and-mouth disease virus of the present invention to generate a signal (4) related reagents and cleaning agents 1276686 15· A method for detecting the content of foot-and-mouth disease virus in a liquid sample, comprising the following steps: (1) providing a supporting solid phase, which supports adsorption of a certain amount on the surface of the solid phase, as in the scope of patent application 5, 6, a single-source antibody against foot-and-mouth disease virus according to 7 or 8; (2) applying a sample of the test animal to the supporting solid phase to cause an immune reaction with the single-source antibody on the surface of the test cell; 3) adding a signal generating tool, wherein the signal generating tool can operatively combine with the single-source antibody against foot-and-mouth disease virus of the present invention to generate a signal; (4) determining the content of foot-and-mouth disease virus in the sample according to the generated signal. A set of foot-and-mouth disease virus in a liquid phase sample, comprising: (1) a supporting solid phase; (2) a single-source antibody against foot-and-mouth disease virus as described in claim 5, 6, 7 or 8 of the patent application, attached (3) a signal generating tool, wherein the signal generating tool is operably associated with the porcine foot-and-mouth disease virus or a protein fragment thereof, and is conjugated to the single-source antibody attached to the supporting solid phase of the above (2). And the method of claim 13 or claim 15, wherein the method of claim 13 or claim 15 can be carried out by direct or indirect immunization. Γ Ί 276686. The method of claim 15, wherein the support is The micro-reaction disk, the microsphere, the hybridization membrane, or the test paper. 19. The method of claim 13 or 15, wherein the signal generating means is catalase, alkaline phosphatase, β-half Lactose glycan* enzyme, biotin or fluorescent marker. 20. A kit as described in claim 14 or 16, which may be carried out by direct or indirect immunization. $21. The kit of claim 16, wherein the support solid phase is a microreaction disk, a microsphere, a hybrid membrane, or a test strip. - 22. The kit of claim 14 or 16, wherein the signal generating means is catalase, alkaline phosphatase, beta-galactosidase, biotin or fluorescent marker . 6 1276686 十一,图zqq Xu Qiaoqiao (Yangzhi more) 正本„„一一一.一—η Announcement of abdominal cavity and foot and mouth disease virus spleen injection> Balb/c mouse 0/TWN/97 spleen cell myeloma Fine PEG cell fusion HAT screening IFA detection fusion tumor monoculture ψ single-source fusion tumor cell injection ascites cell culture ♦ culture supernatant Foot-and-mouth disease virus 0/TWN/97 single single-source antibody biochemical characteristics First figure 1276686 VP1 competitor (ng) Fourth