TWI249407B - Hybridoma cell line for producing monoclonal antibody against porcine endogenous retrovirus envelope protein and the produced monoclonal antibody and their use - Google Patents

Abstract

The present invention relates to a hybridoma cell line, which produces a monoclonal antibody that specifically binds to porcine endogenous retrovirus (PERV) envelope protein antigen. The present invention also discloses a method of producing the hybridoma, a method of using the monoclonal antibody for detecting PERV and producing a test kit and a pharmaceutical composition comprising the monoclonal antibody.

Description

1249407 A7 B7 V. INSTRUCTION DESCRIPTION (i) FIELD OF THE INVENTION The present invention relates to a fusion tumor cell line which produces an antibody against a porcine endogenous retrovirus outer membrane protein, and the monoclonal antibodies produced thereby and the same use. BACKGROUND OF THE INVENTION Organ transplanting is a very important new technology in human medicine. The difficulty of feeding human organs and the difficulty of feeding other closely related primate (such as orangutan) animals makes organ source a major problem in transplant surgery. Since the organs and functions of pigs are the closest to the size and function of human organs, pigs are receiving increasing attention in the consideration of organ transplantation sources. However, the common infectious diseases of humans and animals, especially viruses, will cause a potential crisis of organ transplantation. The new porcine virus, porcine endogenous retrovirus (PERV), newly discovered by scholars in 1977, has gradually received attention and triggered extensive discussion. There are three types of endogenous retroviruses in pigs, namely type A, type B and type C, the main difference being in their surface antigens, which may affect the tissue affinity of the virus. Almost all vertebrate animals have similar primary viruses, but most are genetically deficient and mostly non-pathogenic. The vast majority of infectious endogenous retroviral pathogens can be prevented by cultivating individual animals with specific pathogens free (S P F). However, naturally occurring in the animal genome or embryonic pathogens, although the original host does not cause any symptoms, but may be activated in xenograft recipients to produce lesions, especially by immunosuppressive drugs. Post-organ transplant recipients, their immune function -4- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1249407 A7 B7 V. INSTRUCTIONS (2) No effective immune protection can be provided. These endogenous retroviruses may be regenerated in immunosuppressed recipients, increasing the risk of xenogeneic (pig) organ transplants. For example, Patience et al. and Wilson et al. successfully released PE HV from pig kidney cell line (PK-15 cell line) and primary peripheral blood mononuclear cells, respectively, and confirmed in vitro. It is infected with human cell lines such as B cells, T cells, and kidney cells (Patience et al., 1997, Nature Med. 3: 282-286; and Wilson et al., 1998, J. Virol. 72(4):3082- 3087). Previous studies have interpreted the PERV genome sequence, so most methods for detecting various types of PERV are performed using nucleic acid detection. Related techniques for such nucleic acid detection are disclosed in WO 97/40167, WO 98/53104, WO 00/00829, WO 00/04191, WO 00/11187, USP 6,100,034 and USP 6,261,806, and the like. However, these methods can only detect those who have been infected with PERV and release the virus in the test sample, and the PERV that may be activated in the recipient of the xenogeneic organ cannot be detected by nucleic acid detection. Therefore, researchers have attempted to perform PERV testing by immunodiagnosis, which involves the preparation of specific antigens, the development of positive control serum and high-valency antibodies. In the preparation of antigens, Matthews et al. first used the PERV whole virus protein released from the infected human kidney epithelial cell line U293 as an antigen, and used Western immunoturbine analysis to detect PERV antibodies (Matthews et al., 1999). Transplantation 67(7): 939-43). Paradis and Galbraith et al. used the recombinant PERV p30-Gag protein as the ____-5^__^ paper scale for the Chinese National Standard (CNS) A4 specification (210X 297 mm) — 1249407 A7 B7 V. Description of invention (3 Antigen, enzyme-linked immunoassay to detect the presence of antibodies in the serum of patients (Paradis et al, 1999, Science 285: 1236-1241; and Galbraith et al, 2000, J. Virol · Methods 90 (2): 115-124). In the study of positive control serum and antibody preparation, early use of other animal retrovirus antibodies to cross-react as a positive control serum, such as SSAV (simian sarcoma-associated virus) p29-Gag ' GALV (gibbon ape leukemia virus p30-Gag 'FeLV (feline leukemia virus) p27-Gag, FeLV gp71-Env, BaEV (baboon endogenous retrovirus) Gag and MuLV (murine leukemia virus) Gag antiserum. The development of multiple antibodies such as PERV p27-Gag, plO-Gag and pl5-Env was subsequently carried out (Krach et al., 2000, Xenotransplantation 7(3): 221-9; Tacke et al., 2000, Virology 268: 87-93 ). However, because the serological detection method of PERV must consider factors such as specific antigens and high-valence antibodies, the difficulties faced are not easily overcome. Therefore, a single antibody with high specificity and sensitivity is still needed for early detection of activated PERV virus. SUMMARY OF THE INVENTION The present invention relates to a fusion tumor cell line which produces a monoclonal antibody that binds to a conserved region of the C-terminus of the outer membrane protein of PERV virus; wherein the monoclonal antibody is deposited with the Food Industry Development Institute, the registration number Binding properties of monoclonal antibodies produced by a fusion cell line of CCRC 9 6 0 1 4 6 . The present invention further relates to a monoclonal antibody produced by the fusion tumor cell of the present invention. The invention further relates to a method for preparing a fusion tumor cell line according to the invention, which mainly comprises the following steps: ________ The paper wave scale is applicable to the Chinese National Standard (CNS) Α4 specification (210 X 297 mm).

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k 1249407 A7 B7 V. Description of the invention (4) a. Preparing a purified PERV outer membrane protein fusion protein; b. injecting the PERV outer membrane protein fusion protein into an animal; c. removing the animal spleen cells and bone marrow: tumor The cell line is fused; d. screening for a fusion cell line that produces a PERV outer membrane protein monoclonal antibody. The invention further relates to a method of detecting the presence of PERV in a sample, comprising: a. contacting the sample with a monoclonal antibody or antigen-binding fragment thereof as in the invention; b. detecting by immunoassay whether a PERV is present in the sample. The invention further relates to a test kit for detecting PERV comprising a monoclonal antibody or antigen-binding fragment thereof according to the invention. The invention further relates to a pharmaceutical composition for the treatment of PERV infection comprising a monoclonal antibody or antigen-binding fragment thereof according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an amino acid sequence alignment analysis of the porcine endogenous retrovirus outer membrane protein gene (PERV ENV). Figure 2 is a flow chart showing the preparation of PERV outer membrane protein monoclonal antibody. Figure 3 is a diagram showing the antibodies secreted by the selected fusion tumor cells (PERV-8E10) by Western blot analysis. Figure 4 is a typing of a monoclonal antibody of the present invention. Fig. 5 shows the results of staining with testin blue after analysis of monoclonal antibodies of the present invention by SDS-PAGE electrophoresis. Fig. 6 is a diagram showing the use of an enzyme-linked immunoassay for detecting the individual antibody of the present invention. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

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1249407 A7 B7 V. Description of invention (5) Price. Fig. 7 is a graph showing the sensitivity of the antibody of the present invention by an enzyme-linked immunoassay. Figure 8 is a graph showing the sensitivity of the monoclonal antibodies of the present invention by Western hybridization assay. Fig. 9 shows the detection of PERV virus by Western hybridization analysis using the monoclonal antibody of the present invention. Fig. 10 shows the detection of PERV virus by cell immunostaining using the monoclonal antibody of the present invention. Fig. 11 is a PERV antigen test of a farm worker using the monoclonal antibody of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention is characterized in that a PERV outer membrane protein fusion protein is prepared for a single antibody fusion tumor cell line and a monoclonal antibody produced. The resulting monoclonal antibodies can be used to establish fast and accurate PERV serological diagnostic techniques for long-term follow-up of xenograft patients and as a powerful tool for detecting and monitoring BERV infection in native pigs. Fusion tumor and preparation thereof The present invention relates to a fusion tumor cell line which can produce a monoclonal antibody which binds to a conserved region of the C-terminus of the outer membrane protein of PERV virus; wherein the monoclonal antibody is deposited in the Food Industry Development Research Institute, The binding characteristics of the monoclonal antibodies produced by the fusion tumor cell line numbered CCRC 960146 were deposited. According to a preferred embodiment of the present invention, the fusion tumor cell line is deposited in the Food Industry Development Research Institute, and the fusion tumor cell number CCRC 960146 is stored - the paper size is applicable to the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) 1249407 A7 B7 V. Inventive Note (6) Strain. The invention further relates to a method for preparing a fusion tumor cell line of the invention, which comprises the following steps: a) preparing a purified PERV outer membrane protein fusion protein; b. injecting the PERV outer membrane protein fusion protein into an animal; c - The spleen cells of the animal are taken out and fused with the myeloma cell line; d. The fusion cell line which produces the PERV outer membrane protein monoclonal antibody is screened. According to the present invention, the construct of the fusion tumor cell line is prepared using standard fusion tumor production techniques; for example, the method described by Harlow et al. and Goding et al. (Harlow et al.: Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1998; And Goding et al.: Monoclonal Antibodies: Principles and Practice). According to the present invention, the PERV sequence used to prepare the fusion tumor has been described in the GenBank database under the registration number AF038601. The PERV sequence was used to design a primer, and the primer was used to select a PERV outer membrane protein gene from pig cells. The present inventors have found that the C-terminal region of all types of PERV outer membrane proteins is extremely conserved. Therefore, the present invention selects a pERV outer membrane protein containing a C-terminal conserved region as an antigen. The PERV outer membrane protein gene containing the C-terminal conserved region is cloned into an appropriate expression vector and transformed into a suitable host to express the PERV outer membrane protein containing the C-terminal conserved region. The outer membrane protein is then isolated and purified as an antigen. According to the present invention, a preferred embodiment of a vector suitable for use in the selection of a PERV outer membrane protein gene having a C-terminal conserved region of the present invention is _ 9 _ This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297). Public ^ " one

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1249407 A7 B7 V. INSTRUCTION DESCRIPTION (7) A vector containing a promoter such as T5, T7, SP6, lac, trp, etc., or a expression vector for a eukaryotic host. Preferably, the vector is a carrier comprising a T7 promoter. Preferred embodiments of the host for expressing the outer membrane protein are colonic bacteria and yeast. Preferably, the host is Escherichia coli. According to the present invention, an antigen, i.e., a PERV outer membrane protein having a C-terminal conserved region, is mixed with an appropriate adjuvant, and then injected into a mouse to produce a plurality of antibodies. Next, the spleen cells of the mouse are taken out and fused with the myeloma cells, and then the fusion tumor cells specific for the PERV outer membrane protein fusion protein are screened by immunoassay, and the desired fusion tumor cell line is obtained by single planting. . Single antibody and preparation thereof The present invention also relates to a monoclonal antibody which is an antibody produced by the fusion tumor cell strain of the present invention. Preferably, the monoclonal resistance system is produced by a fusion cell strain deposited under the Food Industry Development Institute and registered as CCRC 960146. According to the present invention, a monoclonal antibody produced by the fusion tumor cell strain of the present invention can bind to a conserved region of the C-terminus of the outer membrane protein of PERV virus. The monoclonal antibody system of the present invention belongs to the IgG type immunoglobulin, and can specifically bind to the PERV outer membrane protein antigen without cross-reacting to other human retrovirus antigens. According to the present invention, a method for producing a monoclonal antibody of the present invention comprises culturing a large amount of a fusion tumor cell strain (preferably PERV-8E10). The large-scale culture is carried out by performing cell culture outside the living body or by intraperitoneal injection of the animal (preferably in a mouse) to produce ascites in vivo. The obtained cell culture supernatant or ascites can be directly applied to the paper. The Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1249407 A7 B7 5. Inventive Note (8) Use, or further purification After reuse, the purification method includes affinity column chromatography and a dialysis step. Detection Applications and Test Kits The monoclonal antibodies of the present invention have the ability to specifically bind to various types of PERV (i.e., type A, type B, and type C), whereby various PERVs can be widely detected. The invention further relates to a method of detecting the presence of PERV in a sample, comprising: a. contacting the sample with a monoclonal antibody or antigen-binding fragment thereof of the invention; b. detecting by immunoassay whether a PERV is present in the sample. According to the invention, the sample is any sample in which PERV may be present. Preferably, the sample is a blood or cell solution. According to the present invention, the monoclonal antibody is an antibody produced by the fusion tumor cell strain of the present invention. Preferably, the monoclonal antibody is an antibody produced by a fusion cell strain deposited under the number CCRC 960146, deposited in the Food Industry Development Institute. According to the invention, the antigen-binding fragment is any monoclonal antibody fragment of the invention which binds to the PERV outer membrane protein. In accordance with the present invention, the immunoreactivity assay uses standard procedures known in any immunological technique. The immunoreaction assay is based on the specific binding between the monoclonal antibody of the present invention and the outer membrane protein of PERV to form a complex and detect the complex using a suitable device. Preferably, the immune response assay is a fluorescent immunoassay, a radioimmunoassay, and an enzyme immunoassay. For example, the antibody of the present invention is labeled with a fluorescent, radioactive substance or an enzyme, and the labeled antibody and the PE RV outer membrane protein can be specifically combined to form a fluorescent, radiant __- 11 -__ paper scale. Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1249407 A7 B7 V. Description of invention (9) Complex of sexual substance or enzyme label. Next, the presence of PERV can be determined by measuring the fluorescence reaction, radioactivity or enzyme reaction using a suitable device. The invention further relates to a test kit for detecting PER V comprising a monoclonal antibody or antigen-binding fragment thereof according to the invention. In accordance with a preferred embodiment of the present invention, the test kit of the present invention is an immunoassay kit. Preferably, the immunoassay is a fluorescent immunoassay, a radioimmunoassay, and an enzyme immunoassay. The monoclonal antibodies in the test kit can be linked to a supporting solid phase for analysis. The supporting solid phase is any material suitable for protein fixation. For example, a porous carrier material which can provide a capillary phenomenon and adsorb a liquid material, such as a filter paper, a nitrocellulose membrane, a nylon membrane, a glass fiber membrane, etc.; a microreaction disk and a microbead. According to the present invention, the supporting solid phase can be coated with a substance which can be labeled with a fluorescent substance, a radioactive substance or an enzyme (such as catalase, phosphatase, / galactosidase) which can be combined with the antibody of the present invention. For example biotin. According to the present invention, the monoclonal antibody of the test kit is an antibody produced by the fusion tumor cell line of the present invention. Preferably, the monoclonal antibody is an antibody produced by a fusion cell strain deposited under the number CCRC 960146, deposited with the Food Industry Development Institute. According to the invention, the antigen-binding fragment is any monoclonal antibody fragment of the invention which binds to the PERV outer membrane protein. According to the present invention, the monoclonal antibody of the immunoassay may be unlabeled or labeled in combination with fluorescent, radioactive substances or enzymes. Preferably, the anti-system is labeled. More preferably, the anti-system is labeled with a catalase, alkaline phosphatase, point-galactosidase, biotin or camp light. Antibody composition and its application __-12-_._ This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm)

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1249407 A7 B7 V. INSTRUCTION DESCRIPTION (1()) The present invention further relates to a pharmaceutical composition comprising the monoclonal antibody of the present invention and a carrier. According to the present invention, the pharmaceutical composition of the present invention can be formulated by mixing with any of the conventional carriers of the present invention. According to the present invention, the pharmaceutical composition can be used to treat PERV infection. The present invention is described in detail with reference to the accompanying drawings, and the accompanying drawings are not intended to limit the scope of the invention. EXAMPLES Example 1: Anti-buckling, M#舆 purification 1. 1 Gene alignment analysis The present invention was designed with primers in the sequence of PERV (GenBank database registration number AF038601). From pig ST cells, purchased from the National Standard Sterilization Center (ATCC), number ATCC CRL-1746, the πν gene of PERV proviral DNA was selected and reproduced in pBluescript II SK (1) plastid to obtain the env gene. The performance vector was named pST-env (Zhang Yuting et al., 2001, Chinese Veterinary Medicine 27 (2): 94-103). The resulting pST-env plastid DNA was cleaved with restriction enzymes 5am HI and Sac I, and analyzed by colloidal electrophoresis and purified to recover 872 bp DNA fragments, and pET-30a (also cut with 5am HI and Sac I). +) After the vector is conjugated, it is transformed into E. coli NM5 22 competent cells. The plastid DNA of the transgenic strain was isolated, and the direction of the cDNA was selected to be the same as that of the T7 promoter. The sequence was named pST-ENV, and the sequence was determined to confirm that the junction sequence was correct. This is the expression vector containing the sequence encoding the PERV outer membrane protein gene. This plastid has been -13-

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k This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1249407 A7 B7 V. Description of invention (12) Fusion protein. Finally, the bacterial solution was centrifuged at 6,000 rpm for 10 minutes at 4 ° C to precipitate the cells, 150 μl of 50 mM Tris-HCl buffer was added to suspend the cells, and an equal volume of twice the concentration of sodium dodecyl sulfate was added to the stain [Sodium dodecyl sulfate ( SDS) loading dye], vortex and mix, then immerse in a water bath at 100 °C for 5 minutes, then place on ice ice bath, then extract the total protein of the bacteria, with 12.5% sodium lauryl sulfate-polyacrylamide Separation of colloids (SDS-polyacrylamide gel, SDS-PAGE for short) for protein electrophoresis analysis. SDS-PAGE protein electrophoresis analysis was performed according to standard methods known in the art (see Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989). SDS-PAGE was analyzed by coomassie blue and analyzed for ST-env protein size of 36.7 kD. 1.2.2 Soluble analysis of PERV outer membrane protein fusion protein The bacterial solution to be induced was centrifuged at 6,000 rpm for 10 minutes at 4 ° C to remove the supernatant, and then the cells were resuspended in a binding buffer. The cells were disrupted by ultrasonic shattering machine, centrifuged at 10,000 rpm for 10 minutes at 4 ° C, the soluble protein supernatant was aspirated, placed in a new centrifuge tube, and the cells were precipitated with urea-containing binding buffer ( After dissolving, the pellet was centrifuged at 10,000 rpm for 1 C for 1 〇 minutes, and the supernatant containing the insoluble protein was aspirated to another new centrifuge tube. The soluble protein and the insoluble protein solution were each added to an equal volume of twice the concentration of sodium dodecyl sulfate to fill the dye, and the mixture was shaken and mixed, and then placed in an ice bath for 1 minute at 1 ° C for another 5 minutes. #1 was analyzed by protein electrophoresis on a 12.5% SDS-PAGE. As a result, the P E RV outer membrane protein fusion protein was confirmed to be a non-fallable protein. _____-15-_ This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm)

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k 1249407 A7 _B7 V. Inventive Note (13) 1 2.3 Purification of PERV outer membrane protein fusion protein 舆 Confirm that the P ERV outer membrane protein fusion protein expressed by Escherichia coli is an insoluble protein, so use step 1 · 2 · 2 Methods After extracting the insoluble protein, the purified PERV outer membrane protein fusion protein was obtained by a His-Bind affinity chromatography layer (purchased from Novagene), followed by washing, punching and the like. The purified protein was subjected to electrophoresis analysis using a 1 2 · 5 % sodium dodecyl sulfate-polyacrylamide separation gel, and the protein was transferred using a semi-dry protein disruptor (Semi-Dry Transfer Cel) from BIO-RAD. Suppressed with PVDF-Plus hybrid membrane (PVDF-Plus membrane, purchased from MSI), and treated with 5% blocking solution at room temperature for 1 hour, with S-protein assay The antibody (S-Tag Western Blot Kit; Novagen) (S-Tag Western Blot Kit; Novagen) was washed 4 times at room temperature and then washed 4 times with 1 volume of Phosphate-buffered saline (PBS). Finally, a color reaction (S-Tag Western Blot Kit; Novagen) was performed. 1.2.4 The PERV outer membrane protein fusion protein was used to aspirate the purified protein into a dialysis membrane (Dialysis Tubing-3/4 in. Diameter; GIBCOBHL). 1 volume of phosphate buffer at 4t: overnight dialysis, and then concentrated with polyethylene glycol (PEG), take appropriate amount of protein to Bradford staining method (Protein Assay Dye; BIO-RAD) for protein Quantitative analysisReal family 2: argon maid of the epidemic cell line -16 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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k 1249407 A7 B7 V. INSTRUCTIONS (14) 2.1 Immune Vaccination of Rats As shown in Figure 2, healthy BALB/c mice of 6 to 10 weeks were selected for immunization. The first application was performed by mixing 100/z g of the purified PERV outer membrane protein fusion protein antigen with an equal amount of complete adjuvant (Freund's adjuvant complete, purchased from GIBCOBRL) and then injecting it into the abdominal cavity of the mouse. Two weeks later, a second application was performed after mixing with the prepared antigen using Freund's adjuvant incomplete (purchased from Sigma). Two weeks later, the mice were tested for immune response and the antibody titer of the anti-PERV outer membrane protein fusion protein was determined. The well-reacted mice were selected to add three antigens without any adjuvant (100 // g / time) and sacrificed 4-8 days after the last injection to increase the amount of antibody. 2.2 Detection of immune response After fixing the mouse, gently squeeze the mouse tail by hand and find the vein on the back of the tail. At a distance of more than one centimeter from the base of the tail, disinfect with 70% alcohol-soaked cotton, puncture the vein with a 20-gauge needle, and drain the blood into a microcentrifuge tube with a micropipette. After standing at 37 ° C for 1 hour, the blood was transferred to a refrigerator at 4 ° C overnight. Centrifuge at 14,000 rpm for 10 minutes every other day, and take the supernatant into another new microcentrifuge tube, which is serum. The serum was diluted to different folds with one volume of phosphate buffer for Western Blotting and Enzyme-Linked Immunosorbent Assay (ELISA) assays. 2.3 Production of fusion tumor cell line When it is determined that the mouse has been successfully immunized, it is sacrificed by pulling the cervical vertebra. Rats are _-17-_ This paper size applies to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1249407 A7 B7 V. Invention description (15) Left back incision, remove the spleen and place in 10 ml ( Dulbecco's Modified Eagle Medium, referred to as D Μ Ε Μ) in the culture dish. The spleen was transferred to another Petri dish containing 10 ml of DMEM medium, and the spleen was cut with tissue scissors to allow most of the spleen cells to flow out. The cell suspension was aspirated into a 50 ml centrifuge tube, and 10 ml of DMEM medium was added to the dish to break up the spleen cells and repeat. After standing at room temperature for 5 minutes, the supernatant was taken to another 50 ml centrifuge tube, centrifuged at 1,000 rpm for 5 minutes, and after removing the supernatant, 10 ml of DMEM medium was added to suspend the spleen cells and the blood cells were used. The counter counts the cells. On the other hand, myeloma cells (FO cell line, number ATCC CRL-1646) were broken up and cells were counted in a hemocytometer. An appropriate amount of myeloma cells were mixed with spleen cells to have a cell number ratio of 1:3, and after centrifugation at 1,000 rpm for 10 minutes, the supernatant was removed. After allowing to stand in a 37 ° C water bath for 1 minute, 1 ml of 3 7 ° C preheated 50% PEG 1500 was added for cell fusion. Thereafter, 10 ml of DMEM medium was slowly added, and after centrifugation at 600 rpm for 3 minutes, the supernatant was removed. Add 50 ml of HY (HY) containing 20% fetal bovine serum (FBS) and double Penicillin/Streptomycin/Amphotericin B (each concentration is 10,000 U/ml, 10 mg/ml, 0.025 mg/ml) Medium with L-glutamine, bovine Insulin, Oxaloacetate, and sodium pyruvate) The cells were suspended and placed in an incubator at 37 ° C 7% C Ο 2 for four hours. The fused cells were removed and 110 ml of HY medium containing 1% HAT (10 mM sodium hypoxanthin, 40 μΜ aminopeterin, and 1.6 mM thymidine) was added and fully -18- This paper scale was applied to the Chinese National Standard (CNS) A4 specification. (210 x 297 mm)

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1249407 A7 B7 V. INSTRUCTIONS (16) After mixing well, dispense into a 96-well culture dish (200 //1/well) and incubate in a 37 t, 7% C02 incubator for 10 to 15 days. Example 3: Ifc and epileptic cell line 镰逦3.1 Enzyme-linked immunoassay test After one week of cell fusion, a fusion tumor cell of about 30 to 50 cells can be seen, and the culture supernatant is taken as an enzyme. Immunoassay was performed to screen for fusion tumor cell lines. Since the immunizing antigen of the above experiment was prepared by Escherichia coli (BL21(DE3)-RIL), it was necessary to prepare a protein of the host cell coliform as a control group to avoid a false positive reaction. The prepared PERV outer membrane protein fusion protein antigen and E. coli protein were respectively dissolved in a double coating buffer (Coating Buffer; 15 mM Na2C03, 35 mM NaHC03, ρΗ9·6). Then, it was placed in a 96-well immunoenzyme reaction tray (〇·5 pg/well), and moved to a refrigerator at 4 ° C overnight. Wash three times with one volume of PBS for about one minute each time. A 5% skim milk powder solution was added, and the mixture was allowed to stand at 37 ° C for 1 hour, and then washed three times with one volume of P B S.

The culture supernatant of the fusion tumor cells was taken out and injected into a 96-well immunoenzyme reaction disk (50 //1/well) to which the PERV outer membrane protein fusion protein antigen or the large intestinal-like protein was adhered, and the blood was previously separated by cardiac blood collection. The serum was diluted 1,000 times in one volume of PBS as a positive control. After being placed in a 37 ° C incubator for 2 hours, it was washed three times with one volume of PBS. Add a second antibody-catalyst goat anti-mouse IgG peroxidase conjugate (from Chemicon) and place it in a 3 7 °C incubator for 1 hour, then PBS in one volume. Wash three times. Add 100 immune enzyme response receptors (TMB/E _______-19- ___ This paper size applies to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1249407 A7 B7 V. Inventions (17) Solution, 3 3',5,5'-tetramethybenzidine, purchased from Chemicon), was allowed to stand at room temperature for 10 minutes in the dark, and 50 W of 2.5 Μ HC1 was added to terminate the reaction. The absorption spectrum at a wavelength of 450 nm was measured by a spectrophotometer. Comparison of the difference between PERV outer membrane protein fusion protein antigen and Daphnia protein. A number of fusion tumor cells that recognize only the PER V outer membrane protein fusion protein and do not recognize E. coli protein were obtained. 3 23⁄4 paleohybrid assay As the PERV outer membrane protein fusion protein antigen of Example 1.2 is N-terminated with His-Tag protein', it is necessary to prepare a protein with His-Tag E. coli as a control group to avoid pseudo Positive reaction. Add His-Tag protein and PERV outer membrane protein fusion protein to an equal volume of twice the concentration of sodium sulphate (SDS) loading dye, shake and mix, at 1 〇〇 The mixture was placed in an ice bath for 5 minutes, and then placed on ice, and subjected to protein electrophoresis analysis using a S2.5-polyacrylamide gel (SDS-PAGE) of 12.5% sodium dodecyl sulfate-polyacrylamide gel. The membrane was disrupted to a PVDF-Plus hybrid membrane using a semi-dry protein disrupter, and 5% skim milk powder containing 0.1% Tween 20 (dissolved in one volume of PBS) was allowed to stand at room temperature for 1 hour, and then washed with one volume of PBS. Times. The culture supernatant of the selected fusion tumor cells was added to the room temperature for 2 hours, and washed 4 times with one volume of P B S. Add a second antibody-peroxidase argonase goat anti-mouse serum antibody for 1 hour at room temperature, wash 4 times with one volume of PBS, and finally dilute 4CN color solution with one volume of PBS ( 4-〇111〇1*〇-1-naphthol 500 pg/ml, 0.1% H202, 20% methanol) After coloring for 10 minutes at room temperature, it was washed once with PBS of one volume, and the results were interpreted. -20-

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k This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1249407 A7 B7 V. Description of invention (18) As shown in Figure 3, row 1 indicates the protein size marker (ProSieve color protein markers). Top to bottom are 85, 52, 41, 27, 21, 14 and 11 thousand klodalton (kDa); rows 2 and 3 represent the PERV outer membrane protein fusion protein with and without IPTG. Total bacterial protein; line 4 is the total protein of the cell expressing His-Tag protein by IPTG; line 5 is the total protein of Escherichia coli BL21(DE3)-RIL. As a result, it was confirmed that the antibody secreted by the selected fusion tumor cells recognized only the PERV outer membrane protein, which was 36.7 kDa in size and did not react with the His-Tag protein. The single-satellite fusion of the 3·3 fusion tumor was confirmed to be a fusion tumor cell strain capable of secreting PERV outer membrane protein antibody by enzyme-linked immunoassay and western hybridization assay, and the cells in the 96-well culture plate were scattered with a micropipette. Then, the fresh culture solution was diluted 1,000-fold and 100-fold, and then dispensed in another new 96-well culture plate for several days. The above-mentioned enzyme-linked immunoassay test and Western blot analysis test are repeated, and the wells containing only one cell population are screened out, which is a single-merged fusion tumor cell line. 3.4 _ Proliferation culture of the fusion tumor cell line The obtained single-body fusion tumor cells were transferred from a 96-well culture plate to a 24-well culture plate, and transferred to a 6-well culture plate in about three days, and finally transferred to a 25T culture flask. The cells can be stored frozen in about one week. In the subculture process, the culture supernatant was collected first, and then the cells were photographed with 0.05% Trypsin/EDTA, and then fresh DMEM medium containing 20% FBS was added, and centrifuged at 1, rpm for 5 minutes. After removing the supernatant, add fresh DMEM medium containing 20% FBS to make fine ________-21 -_ a paper scale applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1249407 A7 B7 V. Description of invention (19) Cell suspension, after counting the cells by the blood cell counter, take appropriate amount of cells to continue the culture, in which 1/3 volume of the old culture supernatant is added to facilitate cell proliferation, and in the process of subculture, The HY medium containing 20% FBS was replaced with a DMEM medium containing 20% FBS. 3.5 Preservation of the fusion tumor cell The cells in the culture flask were photographed with 0.05% Trypsin/EDTA, and then added to a DMEM medium containing 20% FBS, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed. The cells were suspended by adding fresh DMEM medium containing 20% FBS. After counting by the hemocytometer, fresh DMEM medium containing 20% FBS was added to make the cell concentration 2 X 1 06/500 //1. Add 100/1 of 100% DMSO and 400 //1 DMEM medium in the cell cryotube, mix well, then add the suspended fusion cells (2×106), mix well and seal the bottle. After being placed at -20 ° C for 30 minutes, it was placed at -8 ° C for overnight, and then transferred to a liquid nitrogen drum for storage. The fusion tumor cell line which can produce a monoclonal antibody against PERV outer membrane protein, named PERV-8E10, was deposited in the Food Industry Development Research Institute on February 6, 1991, and the accession number is CCRC 960146. f Example 4: Production and purification of the genus of the stalks 4.1 Typing of the individual antibodies Typing of the monoclonal antibodies was carried out using ImmunoPure® Monoclonal Antibody Isotyping Kit II (purchased from PIERCE). A 50/1/1 coating antibody working solution was added to a 96-well immunoenzyme reaction plate and placed at 4 ° C overnight. After removing the supernatant, add 125 //1 — -22- ^ paper scale for Chinese National Standard (CNS) A4 specification (210X 297 mm) —

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1249407 A7 _____B7_._ V. Description of the invention (2〇) Double the buffering buffer (Mocking buffer) and placed at 37 °C for 1 hour. The supernatant was removed, and after washing four times with 1 2 5 //1 washing buffer, 50 μM of monoclonal antibody (culture supernatant of fusion cell line PERV-8E10) or positive control group (single mouse) was added. IgGl κ chain) was placed at 37 ° C for 1 hour. The supernatant was removed and washed four times with 125 //1 wash buffer, followed by 50 //1 subclass-specific rabbit antimouse immunoglobulins or normal rabbit serum. ), placed at 3 7 ° C for 1 hour. Remove the supernatant, wash it four times with 1 2 5 //1 wash buffer, then add 50 alkaline phosphatase-goat anti-rabbit IgG working solution (alkaline phosphatase conjugated goat anti-rabbit IgG working solution) ) and placed at 37 ° C for 1 hour. After removing the supernatant, wash it four times with 125 //1 wash buffer. Thereafter, a PNPP (/?-nitrophenyl phosphate) solution was added at a concentration of 100 //1 -1, and the color was observed at room temperature for about 30 minutes, and the absorption spectrum at a wavelength of 405 nm was measured by a spectrophotometer. As shown in Fig. 4, the prepared monoclonal antibody (PERV-8E10) was confirmed to be IgG1 after typing (typ), and its light chain was κ chain. 4.2 Ascites preparation Eight weeks old healthy BALB/c mice were selected and injected into the abdominal cavity of mice with an incomplete adjuvant of 500//1. After every other week, 3×106 fresh fusion tumor cells PERV-8E10 were injected into the peritoneal cavity of mice. After 1 day, the ascites was aspirated, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was aspirated into a new tube. The cells were centrifuged at 5 6 ° C for 45 minutes, centrifuged at 3,000 g of 111 for 10 minutes, and the supernatant was aspirated into a new tube and stored at -20 ° C, which contained a high concentration of individual antibodies. -___223-___ This paper size applies to China National Directive (CNS) A4 specification (210X 297 mm)

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k 1249407 A7 B7 V. INSTRUCTION DESCRIPTION (21) 4.3 Purification of single spider body The culture supernatant of the fusion tumor cell line PERV-8E10 was centrifuged at 1,000 rpm for 1 minute at 4 °C. After the supernatant was aspirated and filtered through a 〇·45 #m filter membrane, three times the volume of the starting buffer (Starting buffer, containing 0.05), ^1, -1, HC1, pH 8.6, 0.15 M NaCl, 0.02% NaN3) was added. dilution. The ascites treated as described above was diluted with forty volumes of starting buffer and centrifuged at 10,000 rpm for 20 minutes at 4 QC. The culture supernatant or ascites of the above-mentioned treated fusion tumor cell line PERV-8E10 was passed through a HiTrap Protein-A affinity chromatography layer (purchased from Pharmacia) loaded with hydrazine, washed with a starting buffer, and then with Elute. Buffer (0·05 M glycine-HCl ρΗ2·3, 0.15 M NaCl) was flushed out of IgG antibody, and 1/4 volume (Neutralizing buffer containing 0.5 Μ phosphate buffer, ρΗ7,7) was added to neutralize ρΗ. The purified IgG antibody was obtained, and then placed in a dialysis membrane and dialyzed overnight in a volume of PBS. After taking out, it was dispensed into a centrifuge tube, vacuum-dried, and stored at 4 °C. 4.4 Purity analysis of individual antibodies

The purified IgG antibody was taken and analyzed by protein electrophoresis to determine the antibody purity. The purified IgG antibody was added to an equal volume of twice the concentration of sodium dodecyl sulfate to fill the dye, and the mixture was shaken and mixed at 1 Torr. (: Water bath for 5 minutes. Then place it on ice and analyze it by protein electrophoresis with 12.5% sodium dodecyl sulfate·polyacrylamide separation gel (s D s -PAGE). SDS-PAGE by coomassie (coomassie) Blue) After staining, as shown in Fig. 5, line 1 is the ascites stock solution, and line 2 is the 1 g G antibody purified by the HiTrap Protein-A affinity chromatography column, and its size is heavy chain 50. kDa and light chain 25 kDa. Line 3 indicates protein size mark (ProSieve color __-24-____ This paper size applies to Chinese National Standard (CNS) Α4 size (210 x 297 mm)

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k 1249407 A7 B7 V. Invention description (22) protein markers), from top to bottom are 121, 85, 52, 41, 27, 21, 14 and 1 1 kDa 0 will apply #1 5 : single bee bump效 分 · 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 5.1 The purified PERV outer membrane protein fusion protein was dissolved in double-coating buffer (15 mM Na2C03, 35 mM NaHC03, pH 9.6) and placed in a 96-well immunoenzyme reaction disk (0.5 #g/well). Move to a 4 ° C refrigerator and let it stand overnight. Wash three times with one volume of PBS' for about one minute each time. Add 5% skim milk powder solution, incubate for 1 hour at 37 °C, wash three times with one volume of PBS, then add the first antibody (the monoclonal antibody PERV-8E10 to be tested, in one volume PBS) Diluted differently), placed in a 37 ° C incubator for 1 hour, and washed three times with one volume of PBS. A second antibody-catalase goat anti-mouse serum antibody was added, and after being placed in a 3 7 °c incubator for 1 hour, it was washed three times with one volume of PB S. Then add 100 //1 immunogen reaction receptor (TMB/E solution), let stand at room temperature for 10 minutes in the dark, add 50 //1 2·5 M HC1 to terminate the reaction, and measure by spectrophotometer. Absorption spectrum at a wavelength of 450 nm. As a result, as shown in Fig. 6, the antibody titer of PERV-8E10 ascites was 2χ106, and the purified PERV-8E10IgG antibody was 6.4 χΙΟ4. 5.2 Analysis of the convergence of monoclonal antibodies (enzyme-linked immunoassay _) _ taking different concentrations of antigen y2//g, 2-3//g, 2"4//g, 2_5//g, 2·6 //g), the sensitivity of individual antibodies was detected by enzyme-linked immunoassay. Apply _ -25-_ this paper size to China National Standard (CNS) A4 specification (210 X 297 mm)

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1249407 A7 _B7__ V. Description of the invention (23) Purified PERV outer membrane protein fusion protein antigen is dissolved in double coating buffer (15 mM Na2C03, 35 mM NaHC03, ρΗ9·6), and then different concentrations of antigen are placed in 96-well immunoenzyme. In the reaction tray, move to a 4 ° C refrigerator and let it stand overnight. Wash three times with one volume of P B S for about one minute each time. Thereafter, a 5% skim milk powder solution was added, and the mixture was allowed to act in a 37 ° C incubator for 1 hour, washed three times with one volume of PBS, and then the first antibody (per antibody PERV-8E10 was diluted, and diluted 2,500 in one volume of PBS). After being placed in a 37 ° C incubator for 1 hour, it was washed three times with one volume of PBS. The subsequent steps are the same as the embodiment described in step 5.1. As a result, as shown in Fig. 7, the monoclonal antibody PERV-8E10 was detected by an enzyme-linked immunoassay, and its sensitivity was as high as 2'6 // g of antigen. 5.3 Sensitivity analysis of monoclonal antibodies (Western hybridization assay) Different concentrations of antigen were used to detect the sensitivity of individual antibodies by Western blot analysis. The purified PERV outer membrane protein fusion protein antigen was dissolved in a concentration of sodium dodecyl sulfate loading dye, and different concentrations of antigen were separately taken for protein electrophoresis analysis. The membrane was disrupted to a PVDF-Plus hybrid membrane using a semi-dry protein disrupter, and 5% skim milk powder containing 0.1% Tween 20 (dissolved in one volume of PBS) was allowed to stand at room temperature for 1 hour, and then washed with one volume of PBS. 4 times. The first antibody (per antibody PERV-8E10, diluted 2,500 times in one volume of PBS) was added, and the mixture was allowed to stand at room temperature for 2 hours, and washed 4 times with one volume of PBS. A second antibody is added and the subsequent steps are as described in Example 3.2. As shown in Figure 8, line 1 represents ProSieve color protein markers, 85, 52, 41, 27, 21, 14 and 11 kDa from top to bottom; rows 2, 3, 4, 5, 6 , 7, 8 and 9 respectively, 2·2/^, __-26-___ This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1249407 A7 B7 V. Invention description (24) 2_3 Purified PERV outer membrane protein fusion protein of //g, 2e4//g, 2·5μ, 2_6gg, 2-7yg and 2-8#g. The monoclonal antibody PERV-8E10 was detected by Western blotting analysis and its sensitivity was up to 0.5/5 g of antigen. Example 6: Specificity analysis of monoclonal antibodies In terms of specificity analysis of monoclonal antibodies, since PER V is a type C retrovirus, it is desirable that the prepared PERV monoclonal antibodies are not produced by other human retroviruses. Cross reaction. In this example, a human-linked immunodeficiency virus (Murex HIV-1, 2, 0) and a human tau cell leukemia virus (Murex HTLV I+II) enzyme-linked immunoassay kit (from Murex Biotech) were used. test. Using a 96-well immunoenzyme reaction plate to which ^11乂-1,2,〇 or ^11[]^/-1+11 antigen has been attached, add 50 //1 sample diluent, and then add test samples (single antibody) PERV-8E10) or control group (positive control group is Anti-HIV serum or Anti-HTLV serum; negative control group is normal human serum), placed at 37. (: After 30 minutes, rinse with washing solution five times. Add 50 0/1 conjugate, put it at 3 7 °C for 30 minutes, rinse with washing solution five times. Add 100 //1 receptor The solution was placed at 37 ° C for 30 minutes, and then 50 +/- 1 0.25 M HCl was added to terminate the reaction. Finally, the absorption spectrum at a wavelength of 450 nm was measured by a spectrophotometer. The results are shown in the following table, the monoclonal antibody PERV-8E10 is not It will cross-react with human acquired immunodeficiency virus and human tau cell leukemia virus.

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k antigen antibody OD450 HIV-1, 2 positive control group 0.534 negative control group 0.156 PERV-8E10 ascites 0.169 _____-27- This paper scale applies Chinese national standard (CNS) A4 specification (210X 297 mm) 1249407 A7 B7 V. Invention Description (25 PERV-8E10 IgG 0.160 HTLV-I+II positive control group 1.097 negative control group 0.126 PERV-8E10 ascites 0.128 will be applied to Example 7: susceptibility of the anti-collection PERV cabin capacity 7.1 using Western hybrid analysis PERV virus The antigen used in the above examples is a recombinant fusion protein purified from E. coli. Therefore, whether the selected monoclonal antibodies can recognize the PERV outer membrane protein produced by pig cells, even the infectious PERV virus particles, Further analysis is required. In this experiment, the following cell lines will be cultured, in which the pig kidney cell line PK15 and the human kidney epithelial cell line U293-PK15 which has been infected with PERV have been confirmed by RT-PCR method to release PERV, which The cells of the strain will be used as a positive control group, and other human kidney epithelial cell line U293 will be used as a negative control group. The culture supernatant will be collected in large quantities at an ultra-high speed of 38,000 rpm at 4 °C. Centrifuge for 1 hour to collect virus particles. Add an equal volume of twice the concentration of sodium dodecyl sulfate to fill the dye, mix by shaking, and then bathe at 100 °C for 5 minutes, then place on ice for protein electrophoresis. Analysis. Using a semi-dry protein breaker to break into PVDF-Plus hybrid membrane, 5% defatted milk powder containing 0.1% Tween 20 (dissolved in one volume of PBS) for 1 hour at room temperature, in one volume PBS Wash 4 times. Add the first antibody (the monoclonal antibody of the present invention, diluted 2,500 times with one volume of PBS), apply for 2 hours at room temperature, and wash 4 times with one volume of PBS. Add a second antibody-base. Goat anti-mouse serum antibody antibody, at room temperature -28- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1249407 A7 B7 V. Description of the invention (26 After 1 hour, wash 4 times with 1 volume of PBS, and finally carry out color reaction (2) -

Tag Western Blot Kit ’ from Novagen). As shown in Fig. 9, line 1 indicates ProSieve color protein markers, which are 121, 85, 52, 41, 27, 21, 14 and 11 kDa from top to bottom, respectively; row 2 is a porcine kidney cell strain. The culture supernatant of ρκΐ5; line 3 is the culture supernatant of human kidney epithelial cell line U293; line 4 is the culture supernatant of human kidney epithelial cell line U293-PK15 infected with PERV. To evaluate the ability of monoclonal antibodies to detect p E RV virions from cell lines, we found that the monoclonal antibodies we prepared can bind to the protein of PERV virus released by PK 1 5 and U293-PK15, and the size is 85. kDa and 70 kDa (ENV precursor) and 16 kDa without cross-reaction with U293. 7.2 Detection of PeRV disease by cell immunostaining In the steps of this example, the following cell lines, including porcine kidney cell line PK15 and human renal epithelial cell line U293-PK15 and human kidney epithelial cell line infected with PERV, will be cultured. ^293, cultured in a 6-well culture plate overnight. After removing the culture supernatant, it was washed twice with one volume of PBS. The cells were fixed with 95% alcohol for 1 minute and then washed three times with one volume of pbs for 5 minutes each time. A drop of blocking serum diluted in one volume of p b S was added dropwise for 30 minutes at room temperature, and then washed four times with one volume of PBS. Add the first antibody (single antibody PERV-8E10, diluted 5 times with one volume of PBS; negative control group directly added to one volume of PBS), and doubled in the incubator at 37 °C for 1.5 hours. The volume was washed four times with PBS. Add a second antibody (anti-mouse biotinylated second step -29- -- ---------) This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1249407 A7 B7 V. Inventive Note (27) antibody), after 30 minutes at 37 ° C incubator, washed four times with one volume of PBS. Thereafter, ABC reagent (ABC Reagent, UntiTectTM Mouse ABC kit, purchased from Oncogene) was added, and after washing at 37 ° C for 30 minutes, it was washed four times with one volume of PBS. After adding Novareg substrate (Vector NovaRED substrate kit, available from Vector Laboratories) for 1 minute at room temperature, it was washed twice with deionized water. Finally, HE stain was added and subjected to room temperature for 30 seconds for nuclear staining. After washing twice with deionized water, it was observed with an erect microscope and photographed. As shown in Figure 10, A and B are PK15 cells; C is U293-PK15 cells; D is U293 cells. Among them, A, C and D were hybridized with monoclonal antibody PERV-8 E 10 , and B was a negative control group. The results showed that the prepared monoclonal antibody PERV-8E10 could bind to the PERV virus particles released from PK15 and U293-PK15 without cross-reacting with U293.

Example 8: Applying the ancestral strain of the anti-collision into the field, the PERV Hang JR of the staff of the farm, the PERV antigen was detected by the serum of the farm staff, and the human kidney epithelial cell line U293-PK15 infected with PERV was used. Positive control group. First, extract the serum of the pig farm staff and add an equal volume of 2 times the concentration of sodium dodecyl sulfate to fill the dye. After shaking and mixing, it is placed in a water bath at l ° ° C for 5 minutes, and then placed on an ice bath for protein. Electrophoresis analysis. The PVDF-Plus hybrid membrane was disrupted by a semi-dry protein disruptor, and the procedure was as described in Example 7.1. As shown in Figure 11, line 1 represents the protein size marker, which is 121, 75, 49, 38, 25, 19, and 13 kDa from top to bottom, respectively; line 2 is the person infected by PERV _____-30- _ this Paper scale applies to China National Standards (CNS) A4 specification (210 X 297 mm)

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1249407 A7 B7 V. Description of the invention (28) Culture supernatant of the kidney epithelial cell line U293-PK15. Lines 3 through 10 are the serum of the farm staff. The PERV antigen test of the pig farm staff was carried out using the monoclonal antibody of PERV outer membrane protein. The results showed that there was no PERV antigen expression in the serum test of 8 pig farm staff, while the positive control group U293-PK15 could detect 85. 70 and 16 kD a PERV virus protein. -31 - This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims (1)

——I 109797 Patent Application Chinese Patent Application Scope Replacement (October 94) A8iii; m-i4 B8l , C8\ - D8 6. Patent application scope 1. A fusion tumor cell line which can produce a monoclonal antibody which binds to a conserved region of the C-terminus of the porcine endogenous retrovirus (PERV) outer membrane protein; wherein the monoclonal antibody has a The Institute of Food Industry Development, the binding characteristics of monoclonal antibodies produced by the fusion tumor cell line numbered CCRC 960146. 2. A fusion tumor cell line according to claim 1 of the scope of the patent application, which is deposited in the Food Industry Development Research Institute and has a fusion cell line number CCRC 960146. 3. A monoclonal antibody produced by the fusion tumor cell line of claim 1 of the patent application. 4. The monoclonal antibody according to item 3 of the patent application is produced by a fusion tumor cell line deposited with the Food Industry Development Research Institute under the accession number CCRC 960146. A method for preparing a fusion tumor cell strain according to claim 1, which comprises the following steps: a. preparing a purified PERV outer membrane protein fusion protein; b. injecting the PERV outer membrane protein fusion protein into In vivo; c. The spleen cells of the animal are taken out and fused with the myeloma cell line; d. The fusion tumor cell line producing the antibody of the PERV outer membrane protein is screened. 6. A method for detecting the presence of PERV in a sample, comprising: a. contacting the sample with a monoclonal antibody or antigen-binding fragment thereof as in claim 3; the paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1249407 VI. Patent application scope A BCD b. Borrowing and arbitrarily analyzing and detecting is the existence of pERv in the transmissive towel. 7. The method of claim 6, wherein the sample is a blood or cell solution. 8. According to the method of (4) Fan (4), the monoclonal antibody is deposited in the Food Industry Development Research Institute and deposited with the ccrc reading fusion cell line. 9. The method of claim 6, wherein the immunological analysis is fluorescent immunoassay, radioimmunoassay, and enzyme immunoassay. 10·- A test kit for detecting PERV, which includes a single carcass such as the third item of the patent scope. 11. A kit according to claim 10, wherein the monoclonal antibody is produced by a fusion cell strain deposited with the Food Industry Development Institute under the accession number CCRC 960146. 12. The kit according to item 1 of the scope of the patent application, wherein the kit is an immunoassay kit. 13. The kit according to item 12 of the patent application, wherein the immunoassay is labor light immunoassay, radioimmunoassay and enzyme immunoassay. -2-