1. A preparation method of an anti-African swine fever virus p54 protein monoclonal antibody is characterized by comprising a p54-H22 cell as an immunogen and a p54-293T cell as a detection antigen, wherein an experiment used by the p54-H22 cell is a female Balb/c mouse, the female Balb/c mouse is three-immunity for one week and then is detected by an IPMA method, the serum titer of the female Balb/c mouse can reach 1:25600, the spleen of the female Balb/c mouse with higher titer is taken out through aseptic operation, the spleen is ground into a single cell and then is fused with SP2/0 cell under the action of polyethylene glycol (PEG), the culture medium of the fused cell and the p54-293T cell are used for IPMA detection and screening of positive hybridoma cell strains, and 10 hybridoma cell strains capable of stably secreting the anti-ASFV p54 protein monoclonal antibody are obtained after three times of subcloning, monoclonal antibodies were prepared from p54-H22 female Balb/c mice using common reagents including RPMI-1640 basal medium, RPMI-1640 complete medium, HAT medium, GNK wash and cell culture medium.

2. The method for preparing the monoclonal antibody against the African swine fever virus p54 protein according to claim 1, wherein the monoclonal antibody is prepared from the following components: the preparation method of the common reagent comprises the following steps:

(1) RPMI-1640 basal medium: pouring 10.4g of culture medium powder into 800mL of deionized water, uniformly stirring on a magnetic stirrer, adding deionized water until the final volume is 1L, stirring for 1h, adding 2g of NaHCO3, continuously stirring for 30min, adjusting the pH value to 7.2-7.4, filtering on a super clean bench by using a 0.22 mu m sterile filter head, subpackaging in a sterilization container, and storing at 4 ℃ for later use;

(2) RPMI-1640 complete medium: adding 10mL of inactivated fetal calf serum into 90mL of RPMI-1640 basic culture medium, stirring uniformly, and storing at 4 ℃;

(3) HAT medium: adding 2mL HAT into 98mL RPMI-1640 complete culture medium, stirring, and storing at 4 deg.C;

(4) GNK lotion: weighing 4g of NaCl, 0.2g of KCl, 1g of glucose, 0.005g of Na2HPO4 & 12H2O 1.78, 1.78g of NaH2PO4 & 2H2O 0.39, 0.39g of phenol red by a ten-thousandth balance, adding into 400mL of deionized water, uniformly stirring on a magnetic stirrer, supplementing deionized water to make the final volume be 500mL, adjusting the pH value to 7.2, and storing at 4 ℃ for later use after moist heat sterilization;

(5) cell cryopreservation solution: taking 9mL of serum, adding 1mL of DMSO, and uniformly blowing, wherein the serum is prepared in situ.

3. The method for preparing the monoclonal antibody against the African swine fever virus p54 protein according to claim 2, wherein the method comprises the following steps: the preparation method comprises the following steps:

(1) carrying out an immunity test on the mice;

(2) measuring the serum titer of the mice by an immunoperoxidase monolayer experiment;

(3) culturing myeloma cells;

(4) preparing feeder cells;

(5) preparing splenocytes;

(6) fusing cells;

(7) screening positive holes of hybridoma cells;

(8) subcloning of positive hybridoma cells;

(9) culturing and freezing the positive hybridoma cell strain;

(10) measuring the stability of the positive hybridoma cell strain;

(11) identifying the monoclonal antibody by Western-blot;

(12) and obtaining experimental data.

4. The method for preparing the monoclonal antibody against the African swine fever virus p54 protein according to claim 1, wherein the monoclonal antibody is prepared from the following components: the female Balb/c mouse is immunized by whole cells for 6-8 weeks.

5. The method for preparing the monoclonal antibody against the African swine fever virus p54 protein according to claim 1, wherein the monoclonal antibody is prepared from the following components: the 10 hybridoma cell lines capable of stably secreting ASFV p54 protein monoclonal antibodies are named as 3B3, 4C2, 4C6, 4E3, 5E6, 5G11, 8E6, 10C2, 16B11 and 21A 2.

6. The use of the African swine fever virus (African swine fever) resistant p54 protein monoclonal antibody of claim 1 in preparation of an African swine fever virus detection reagent or kit.

Background

African swine fever is an acute, febrile, highly contagious and virulent disease of pigs caused by African swine fever virus. It features short course of disease, high fatality rate up to 100%, clinical symptoms and pathological changes similar to acute swine fever, easy misdiagnosis, high fever, skin congestion, cyanosis, abortion, edema and organ bleeding. China defines animal diseases and receives high importance from countries all over the world (Sun Huaichang. Chinese preventive veterinary science, 1999, 21 (2): 117-119).

Since 1921, the disease is discovered in kenya, the disease exists in africa countries south of sahara, the disease is transmitted to western europe and ramei successively in 1957, most of the disease is extinguished in time, the indian island in grapevine, southwest spain and italy still has epidemic, and the disease has epidemic tendency in dozens of countries such as africa, europe and america. In 2007, six African swine fever diseases occur continuously in sub-Meinian, and the disease does not exist in China.

African swine fever is assigned to the iridoviridae family in the fourth report of the International Commission on viral classifications, which is listed under the Poxviridae family in the fifth report of the Commission, outside of the vertebrate and entomopoxvirus subfamilies of that family. However, DNA sequence analysis shows that the ASF virus has characteristics between those of poxvirus and iridovirus, and the characteristic of ASFV indicates that it does not belong to any family defined by the International Commission on viral taxonomy, and is a new family, and 9 th International Commission on viral taxonomy in 1995 reports that African swine fever virus is classified as "African swine fever virus-like" which is the only known representative.

At present, no effective vaccine exists, so that the infection condition of pigs is judged by quickly diagnosing the antibody level of the African swine fever, effective control and elimination measures are formulated, and further, the measures such as timely and accurate diagnosis and detection, strict and effective blocking and killing and the like are effective methods for successfully eliminating the African swine fever. The African swine fever antibody diagnosis method comprises agar diffusion test, ELISA, immunoblotting, immunofluorescence and the like. At present, the detection reagent for diagnosing African swine fever in China mainly depends on import, methods and reagents with independent intellectual property rights are few, and in addition, the severe epidemic situation of surrounding countries, the establishment of the detection method and the reagent is urgently needed in China.

Disclosure of Invention

The application aims to provide a preparation method and application of a monoclonal antibody of an anti-African swine fever virus p54 protein.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a preparation method of a P54 protein monoclonal antibody for resisting African swine fever virus comprises the steps of using a p54-H22 cell as an immunogen and using a p54-293T cell as a detection source, using a p54-H22 cell as a female Balb/c mouse, using an IPMA method to determine the female Balb/c mouse after three-week immunization, enabling the serum titer of the female Balb/c mouse to reach 1:25600, taking out the spleen of the female Balb/c mouse with higher titer through aseptic operation, grinding the spleen into a single cell, fusing the single cell with SP2/0 cell under the action of polyethylene glycol (PEG), using a culture medium of the fused cell and the p54-293T cell as IPMA detection to screen positive hybridoma cell strains, obtaining 10 female hybridoma cell strains capable of stably secreting the ASFV p54 protein monoclonal antibody after three times of subcloning, using the Bab/c mouse of the p54-H22 cell to prepare the monoclonal antibody, common reagents include RPMI-1640 basal medium, RPMI-1640 complete medium, HAT medium, GNK lotion, and cell culture medium.

After adopting the structure, the invention has the following advantages:

1. in the experiment, the mice are immunized by using the whole cells, the IPMA titer of the mice reaches a higher level, the specificity of the screened antibody is high, and the screening effect of the hybridoma is obvious;

2. the titer of mouse serum IPMA can reach 1:25600, after cell fusion, 10 monoclonal antibodies are finally screened out by an IPMA method, and experimental data are more accurate and reliable due to a plurality of monoclonal antibodies.

As an improvement, the preparation method of the common reagent comprises the following steps:

(1) RPMI-1640 basal medium: pouring 10.4g of culture medium powder into 800mL of deionized water, uniformly stirring on a magnetic stirrer, adding deionized water until the final volume is 1L, stirring for 1h, adding 2g of NaHCO3, continuously stirring for 30min, adjusting the pH value to 7.2-7.4, filtering on a super clean bench by using a 0.22 mu m sterile filter head, subpackaging in a sterilization container, and storing at 4 ℃ for later use;

(2) RPMI-1640 complete medium: adding 10mL of inactivated fetal calf serum into 90mL of RPMI-1640 basic culture medium, stirring uniformly, and storing at 4 ℃;

(3) HAT medium: adding 2mL HAT into 98mL RPMI-1640 complete culture medium, stirring, and storing at 4 deg.C;

(4) GNK lotion: weighing 4g of NaCl, 0.2g of KCl, 1g of glucose, 0.005g of Na2HPO4 & 12H2O 1.78, 1.78g of NaH2PO4 & 2H2O 0.39, 0.39g of phenol red by a ten-thousandth balance, adding into 400mL of deionized water, uniformly stirring on a magnetic stirrer, supplementing deionized water to make the final volume be 500mL, adjusting the pH value to 7.2, and storing at 4 ℃ for later use after moist heat sterilization;

(5) cell cryopreservation solution: taking 9mL of serum, adding 1mL of DMSO, and uniformly blowing, wherein the serum is prepared in situ.

As an improvement, the preparation method comprises the following steps:

(1) carrying out an immunity test on the mice;

(2) measuring the serum titer of the mice by an immunoperoxidase monolayer experiment;

(3) culturing myeloma cells;

(4) preparing feeder cells;

(5) preparing splenocytes;

(6) fusing cells;

(7) screening positive holes of hybridoma cells;

(8) subcloning of positive hybridoma cells;

(9) culturing and freezing the positive hybridoma cell strain;

(10) measuring the stability of the positive hybridoma cell strain;

(11) and identifying the monoclonal antibody by Western-blot.

As an improvement, the female Balb/c mouse is immunized by whole cells for 6-8 weeks.

As an improvement, the 10 hybridoma cell lines capable of stably secreting anti-ASFV p54 protein monoclonal antibodies are named as 3B3, 4C2, 4C6, 4E3, 5E6, 5G11, 8E6, 10C2, 16B11 and 21A 2.

As an improvement, the African swine fever virus p54 protein-resistant monoclonal antibody is applied to preparation of an African swine fever virus detection reagent or kit.

Drawings

FIG. 1 is a graph showing the result of the IPMA measurement of mouse antiserum titer of the present invention.

FIG. 2 is an IPMA screening graph of the p54 monoclonal antibody of the present invention.

FIG. 3 is a Western-blot identification chart of the monoclonal antibody of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

Combining with attached figures 1-3, p54-H22 cells are used as immunogen and p54-293T cells are used as detection source, the experimental object used by the p54-H22 cells is female Balb/c mice, the female Balb/c mice are three-immunized for one week and then are detected by adopting an IPMA method, the serum titer of the female Balb/c mice can reach 1:25600, spleens of the female Balb/c mice with higher titer are taken out by aseptic technique, single cells are ground and then are fused with SP2/0 cells under the action of polyethylene glycol (PEG), a culture medium of the fused cells and the p54-293T cells are used for IPMA detection and screening of positive hybridoma cell strains, 10 hybridoma cell strains capable of stably secreting anti-ASFV p54 protein monoclonal antibodies are obtained after three times of subcloning, monoclonal antibodies are prepared by the female Balb/c mice of the p54-H22 cells, common reagents include RPMI-1640 basal medium, RPMI-1640 complete medium, HAT medium, GNK lotion, and cell culture medium.

The preparation method of the common reagent comprises the following steps:

(1) RPMI-1640 basal medium: pouring 10.4g of culture medium powder into 800mL of deionized water, uniformly stirring on a magnetic stirrer, adding deionized water until the final volume is 1L, stirring for 1h, adding 2g of NaHCO3, continuously stirring for 30min, adjusting the pH value to 7.2-7.4, filtering on a super clean bench by using a 0.22 mu m sterile filter head, subpackaging in a sterilization container, and storing at 4 ℃ for later use;

(2) RPMI-1640 complete medium: adding 10mL of inactivated fetal calf serum into 90mL of RPMI-1640 basic culture medium, stirring uniformly, and storing at 4 ℃;

(3) HAT medium: adding 2mL HAT into 98mL RPMI-1640 complete culture medium, stirring, and storing at 4 deg.C;

(4) GNK lotion: weighing 4g of NaCl, 0.2g of KCl, 1g of glucose, 0.005g of Na2HPO4 & 12H2O 1.78, 1.78g of NaH2PO4 & 2H2O 0.39, 0.39g of phenol red by a ten-thousandth balance, adding into 400mL of deionized water, uniformly stirring on a magnetic stirrer, supplementing deionized water to make the final volume be 500mL, adjusting the pH value to 7.2, and storing at 4 ℃ for later use after moist heat sterilization;

(5) cell cryopreservation solution: taking 9mL of serum, adding 1mL of DMSO, and uniformly blowing, wherein the serum is prepared in situ.

The preparation method comprises the following steps:

(1) carrying out an immunity test on the mice;

(2) measuring the serum titer of the mice by an immunoperoxidase monolayer experiment;

(3) culturing myeloma cells;

(4) preparing feeder cells;

(5) preparing splenocytes;

(6) fusing cells;

(7) screening positive holes of hybridoma cells;

(8) subcloning of positive hybridoma cells;

(9) culturing and freezing the positive hybridoma cell strain;

(10) measuring the stability of the positive hybridoma cell strain;

(11) identifying the monoclonal antibody by Western-blot;

(12) and obtaining experimental data.

The female Balb/c mouse is immunized by whole cells for 6-8 weeks.

The 10 hybridoma cell lines capable of stably secreting ASFV p54 protein monoclonal antibodies are named as 3B3, 4C2, 4C6, 4E3, 5E6, 5G11, 8E6, 10C2, 16B11 and 21A 2.

The application of the African swine fever virus resisting p54 protein monoclonal antibody in preparation of an African swine fever virus detection reagent or kit.

In the specific implementation of the invention, the implementation case of the preparation method is as follows:

first, to the mouse immunity test

(1) After p54-H22 cells were cultured to a good growth state, the cells were counted, 1X 107 cells were centrifuged at 1000r/min for 10min, the supernatant was discarded, 10mL of sterile PBS was used to wash the cells once, 400. mu.L of sterile PBS buffer was used to resuspend the cells, 2. mu.g of the buffer was addedAdjuvant, mixing gently;

(2) selecting 2 healthy female Balb/c mice with the age of 6-8 weeks, immunizing the mice with the treated cells by adopting a subcutaneous multipoint injection method, wherein each mouse is 200 mu L (5 multiplied by 106 cells);

(3) immunizing the mice once every two weeks for 3 times, wherein the method for immunizing each time is the same;

(4) after one week of the third immunization, tail blood collection is carried out on the mice, and the titer of the mouse multi-antiserum is determined by an IPMA experiment;

(5) selecting mice with higher titer for boosting once, wherein the cell processing method is the same as above in boosting, but adding noAdjuvant, boost immunization by intraperitoneal injection.

Second, the serum titer of the mice is measured by immunoperoxidase monolayer experiment

(1) Culturing pTrip-293T cells in a 96-well plate, removing the culture medium when the cells grow until the confluence reaches about 70%, and fixing the cells with 4% paraformaldehyde for 15min at room temperature;

(2) discarding paraformaldehyde, and permeabilizing the cells at room temperature for 1h with PBS buffer containing 0.3% TritonX-100;

(3) discarding the permeabilization solution, diluting the mouse serum with 1 XPBS, adding the diluted mouse serum into a 96-hole cell culture plate, wherein each hole is 50 mu L, the dilution multiple of the first hole is 1:50, and then diluting each hole in a multiple ratio until reaching the twelfth hole; the negative control is the serum of the non-immune mouse, the dilution is carried out in a multiple proportion way, and then the 96-well plate is placed in an incubator at 37 ℃ for incubation for 1 h;

(4) discarding Mouse serum, washing the plate with PBS for 5 times, diluting HRP-Goat anti Mouse IgG with PBS buffer solution at a ratio of 1:200, adding to the cell well, 100 μ g per well, and incubating at 37 ℃ for 1 h;

(5) discarding HRP-Goat anti Mouse IgG, washing with PBS buffer solution for 5 times, and developing with AEC developing kit, wherein the specific application method comprises: 20 mu L of A solution 20 mu L, B solution 40 mu L, C solution is respectively inoculated into 1mL of deionized water, light and even blowing is carried out, 100 mu L of color development solution is added into each hole, and the cell color development condition is observed by an inverted microscope after the color development is carried out for 10min in a dark place.

Thirdly, culture of myeloma cells

(1) Cell recovery: taking SP2/0 cells frozen in liquid nitrogen, carrying out water bath at 37 ℃ for 1-2min, adding 5mL1640 complete culture medium, centrifuging at 1000r/min for 10min, discarding the supernatant, slightly scattering the cells at the bottom, then carrying out heavy suspension by using 5mL1640 complete culture medium, and putting the cells into a 37 ℃ and 5% CO2 cell incubator for culture;

(2) cell passage: observing the cell density in the culture bottle, removing the original culture medium, adding a new 1640 complete culture medium, blowing and beating the culture medium along the wall of the bottle by using an electric pipette, uniformly dividing the cells into two cell culture bottles, and culturing the cells in a 5% CO2 cell culture box at 37 ℃;

(3) on the day of fusion, the cell state was observed, GNK was used to blow out the cells, centrifugation was carried out at 1000r/min for 10min, the supernatant was discarded, 20mL of GNK was added and blown uniformly, and the cells were counted.

Fourth, preparation of feeder cells

(1) 24h before cell fusion, taking a young Kunming female mouse with good health condition, killing the female mouse by using a cervical dislocation method, and putting the female mouse into 75% alcohol for surface disinfection for 5 min;

(2) fixing a mouse on a sterilized dissection plate, placing the mouse in a super clean workbench, and sucking 100mL of HAT culture medium precooled at 4 ℃ for standby based on a disposable sterile culture dish;

(3) the abdominal epidermis of the mouse is cut (without cutting the peritoneum) by a set of sterile scissors and tweezers, the peritoneum is exposed, and the abdominal cavity of the mouse is lightly wiped by an alcohol cotton ball;

(4) taking a 20mL syringe, sucking about 10mL of cold HAT culture medium, gently clamping the peritoneum of the mouse by using forceps, puncturing the peritoneum by using a needle (avoiding a fat layer inside the peritoneum and preventing the needle from being blocked), and slowly injecting the culture medium into the abdominal cavity of the mouse;

(5) gently kneading the abdomen of the mouse with alcohol cotton, sucking out the culture medium in the abdominal cavity, injecting the culture medium into a disposable plate filled with HAT culture medium in advance, and mixing uniformly;

(6) the cell suspension was uniformly added to a 96-well cell culture plate at 100. mu.L per well, and cultured in a cell incubator.

Preparation of spleen cells

(1) After 3 days of boosting immunity of the mice, picking up eyeballs, dropping blood into a centrifugal tube, standing at 37 ℃ for 2h, centrifuging at 3000r/min for 10min, slightly sucking upper serum without sucking blood cells, subpackaging the serum, storing at-20 ℃ for later use, and soaking and sterilizing the mice in 75% alcohol for 5 min;

(2) mice were fixed on the dissecting plate and placed in a clean bench. Wetting a 120-mesh nylon gauze on the beaker with GNK lotion;

(3) the skin of the abdomen of the mouse was lifted with sterilized forceps and cut with scissors without cutting the peritoneum. Then, a set of sterilized scissors forceps is changed, the peritoneum is cut open and pulled open, the spleen of the mouse is taken out, fat around the spleen is cut off, and then the spleen is placed on a nylon net soaked by GNK lotion;

(4) slowly grinding the spleen of the mouse by using scissors and tweezers, adding GNK lotion dropwise while grinding the edge, filtering the ground spleen cells by using nylon gauze, then pouring the spleen into a beaker, and keeping the spleen moist;

(5) discarding nylon gauze, transferring the spleen cell suspension in the beaker into a 50mL sterile centrifuge tube, and centrifuging for 10min at 1000 r/min;

(6) discarding the supernatant, slightly scattering the cell precipitate with fingers, adding 20mL of GNK washing solution into a centrifuge tube to resuspend the cell precipitate, taking a small amount of cell suspension for counting, and then placing the cell suspension in a cell culture box for later use.

Sixthly, cell fusion

(1) Taking myeloma cells in a logarithmic growth phase, discarding a culture medium, sucking 5mL of RPMI 1640 complete culture medium preheated to 37 ℃ by using an electric pipette, slightly blowing down adherent myeloma cells, transferring a cell suspension into a sterile centrifuge tube, centrifuging at 1000r/min for 10min, removing a supernatant, adding 20mL of GNK washing liquid to resuspend the cells, and taking a small amount of cell suspension for counting;

(2) taking tumor cells and splenocytes with the cell number ratio of 1:8, adding into a centrifuge tube, gently blowing uniformly, centrifuging at 1000r/min for 10min, removing supernatant, and gently scattering cell precipitate;

(3) placing the cell sediment in a 37 ℃ water bath under a sterile environment, sucking 1mL of PEG 1500, slowly adding the PEG 1500 into the cells, shaking the centrifuge tube while adding the PEG 1500, adding the PEG 1500 within 90s, and slightly shaking the centrifuge tube for about 1min after adding the PEG 1500, so that myeloma cells and spleen cells are fully fused;

(4) slowly dripping 15mL of preheated GNK lotion into the fused cells in a water bath at 37 ℃, standing for 5min, adding 25mL of GNK lotion, uniformly mixing, centrifuging for 10min at 1000r/min, removing supernatant, slightly scattering cell precipitates, and adding 10mL of HAT culture medium to resuspend the cell precipitates;

(5) adding 90mL of HAT culture medium at 37 ℃ into a sterile disposable plate, sucking 10mL of cell suspension in a centrifuge tube, uniformly mixing with the HAT culture medium, paving the mixture into a 96-well cell culture plate, wherein each well is 100 mu L, and putting the cells into a cell culture box for culture.

Seventh, screening of hybridoma cell positive wells

On day 5 after cell fusion, fresh HAT medium was added to the cell culture plates at 50. mu.L/well; about 8 days after cell fusion, the cells were fused into larger cell masses in the wells, and the positive wells were screened by the IPMA method using the cell culture supernatant.

Eight, subcloning of Positive hybridoma cells

One day before subcloning after screening positive hybridoma wells by the IPMA method, feeder cells were prepared in the same manner as 3.2.3, except that HAT medium was replaced with HT medium. The cells in the IPMA positive holes are blown evenly, the living cells are counted accurately, the cell suspension volume is calculated according to the result after counting, the cell suspension volume is added into the needed HT culture medium, the cell suspension volume is paved in a 96-hole cell culture plate after evenly mixing, each hole is 100 mu L and is divided into 3 cell density gradients (the cell density of the front four rows is 30 cells/mL, the cell density of the middle four rows is 15 cells/mL, the cell density of the rear four rows is 7.5 cells/mL), and the subcloned cell culture plate is placed in a 37 ℃ cell culture box with 5% CO2 for culture. Observing the cell growth state under a microscope after one week, accurately recording the number of cell masses in each hole, collecting the culture supernatant of the subcloned cell hole when the cell mass in the hole is larger, and screening positive clones by an IPMA method, wherein the method is the same as 3.2.2. And carrying out expanded culture on the obtained hybridoma cells in the positive hole to a 24-hole plate and carrying out subcloning for 2-3 times to ensure that the finally obtained hybridoma cell strain can stably secrete the monoclonal antibody and is monoclonal.

Nine, culture and cryopreservation of positive hybridoma cell strain

(1) And (3) expanding and culturing cells: the screened positive monoclonal hybridoma cells were gently pipetted down from a 96-well plate into a 24-well plate and supplemented with medium to 500. mu.L. Standing and culturing for 2-3 days, and transferring the cells to a 25cm2 cell culture bottle for culture when the confluence degree of the cells reaches 80% and the cell state is good;

(2) freezing and storing cells: blowing down the cells at the bottom of a 25cm2 cell culture bottle by using an electric pipette, centrifuging the cell suspension for 10min at 1000r/min, removing the supernatant, dispersing and precipitating, adding 1mL of cryopreservation solution, uniformly mixing, adding into a cryopreservation tube, and slowly cooling in a cryopreservation box at-80 ℃ for storage.

Ten, culture and cryopreservation of positive hybridoma cell strain

(1) Resuscitating the hybridoma cells;

(2) culturing the recovered cells, collecting cell culture supernatant when the cells grow to be full of a 25cm2 cell culture bottle, measuring the titer of the antibody in the culture supernatant by using an IPMA method, specifically referring to 3.2.2, repeatedly freezing and thawing the screened positive hybridoma cells for 3 times, and if the titer of the supernatant does not change greatly, indicating that the cell strain can stably secrete the monoclonal antibody.

Eleven, Western-blot identification of monoclonal antibody

Culturing p54-H22 cells and pLVX-H22 cells in a 25cm2 cell culture flask, when the cell growth state is good, using RIPA lysate (strong) to lyse the cells, collecting supernatant, using p54-H22 cells to lyse the supernatant as antigen, using the prepared monoclonal antibody supernatant as primary antibody, and carrying out Western-blot experiment.

Twelve, experimental results

Mouse antiserum one week after the three-immunization is separated, the titer of the mouse antiserum is measured by an IPMA method, and the result is shown in figure 1, and the titer of the mouse multi-antiserum IPMA reaches 1: 25600.

Screening positive clones by an IPMA method after cell fusion, obtaining 10 hybridoma cell strains resisting p54 protein after three times of subcloning, and as shown in figure 2, the hybridoma cell strains are named as 3B3, 4C2, 4C6, 4E3, 5E6, 5G11, 8E6, 10C2, 16B11 and 21A2, NC is negative control, and mouse serum before immunization; PC: positive control, pre-fusion mouse serum.

Western-blot As shown in FIG. 3, monoclonal antibodies 4C2, 4C6, 4E3, 5E6, 5G11, 10C2, 16B11 and 21A2 reacted strongly with the p54-H22 cell lysis supernatant but not with the pLVX-H22 cell lysis supernatant, indicating that these monoclonal antibodies recognize linear epitopes of the p54 protein. While the remaining 2 monoclonal antibodies likely bound to conformational epitopes of the p54 protein. These 2 Wb negative monoclonal antibodies were not studied further.

The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.