Method for preparing bovine mastitis enterococcus faecalis ESP monoclonal antibody

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing a bovine mastitis enterococcus faecalis ESP monoclonal antibody. The enterococcus faecalis surface protein ESP is a surface protein with an adhesion effect, and the expression of an esp gene can promote the adhesion of bacteria to an inorganic surface and the adhesion among bacteria in a biofilm, so that the biofilm is more stable and enhanced, and the enterococcus faecalis surface protein ESP is considered to be a unique characteristic of pathogenic enterococcus faecalis. Antigenicity analysis and bioinformatics analysis are carried out, and an experimental foundation is laid for development of corresponding genetic engineering vaccines and preparation of rapid antigen detection kits. The invention provides a bovine mastitis enterococcus faecalis ESP monoclonal antibody which is secreted by a hybridoma cell strain 1A7 and / or 6F6 and can be used for detecting bovine mastitis enterococcus faecalis, preparing a detection reagent, preparing colloidal gold rapid detection test paper and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for preparing ESP monoclonal antibody against bovine mastitis Enterococcus faecalis. Background Art

[0002] There are more than 150 types of pathogenic microorganisms that cause dairy cow mastitis, and most of them are pathogenic bacteria. In northern my country, dairy cow mastitis was previously caused by Streptococcus agalactiae, Staphylococcus aureus, and Escherichia coli. However, follow-up screening since 2016 has found that more than 70% of dairy cow mastitis in eastern northern my country is caused by Enterococcus faecalis.

[0003] Enterococcus faecalis, also known as fecal streptococcus, belongs to group D according to Lancefield serological classification. It is a newly discovered nosocomial infection and bovine milk-derived pathogen. Its infected population is mainly critically ill patients, people with serious underlying diseases, the elderly, young children, and people with low immune function. The above-mentioned population is also the group with high milk consumption. It can cause sepsis, endocarditis, pelvic infection, abdominal infection, urinary tract infection, radiculitis, arteritis, neonatal sepsis, etc. Enterococcus faecalis is one of the resident flora in the intestines of humans and animals. It is widely present in the soil of the living environment of humans and animals, human and animal excrement, the intestines of the body, the upper respiratory tract, the oral cavity, and the urogenital tract. The esp gene is directly related to the adsorption, infection and formation of biofilms of the pathogen. The formation of biofilms will significantly increase the drug resistance and resistance of bacteria. The surface protein ESP of Enterococcus faecalis is a surface protein with adhesion, which plays an important role in the formation of biofilms. The formation of Enterococcus faecalis biofilm is regulated by multiple factors. The expression of the esp gene can promote the adhesion of bacteria to inorganic surfaces and the adhesion between bacteria in the biofilm, thereby making the biofilm more stable and enhanced. Therefore, the esp gene is considered to be a unique feature of pathogenic Enterococcus faecalis.

[0004] In this study, the esp gene of Enterococcus faecalis from bovine milk in northern my country was cloned, and the recombinant plasmid was constructed. After transformation into DE3, the expression was induced and purified. Antigenicity analysis and bioinformatics analysis were also performed, laying an experimental foundation for the development of corresponding genetic engineering vaccines and the preparation of antigen rapid detection kits. Summary of the invention

[0005] The main purpose of the present invention is to solve the above existing problems and provide a method for preparing ESP monoclonal antibody against Enterococcus faecalis of bovine mastitis.

[0006] The above purpose is achieved by the following technical solutions: A hybridoma cell line 1A7 secreting a monoclonal antibody against Enterococcus faecalis ESP of bovine mastitis is prepared by the following method: 1) BALB / c mice were immunized three times with the purified recombinant ESP protein, and spleen cells were isolated from the mice; 2) Take mouse spleen cells, fuse them with myeloma cells, and screen them.

[0007] A hybridoma cell line 6F6 secreting a monoclonal antibody against Enterococcus faecalis ESP of bovine mastitis is prepared by the following method: 1) BALB / c mice were immunized three times with the purified recombinant ESP protein, and spleen cells were isolated from the mice; 2) Take mouse spleen cells, fuse them with myeloma cells, and screen them.

[0008] A monoclonal antibody against ESP of Enterococcus faecalis causing bovine mastitis, which is secreted by hybridoma cell lines 1A7 and / or 6F6.

[0009] A method for preparing bovine mastitis Enterococcus faecalis ESP monoclonal antibody, characterized in that: The preparation method of the ESP monoclonal antibody comprises the following steps: 1) Immunization of mice: Purified recombinant ESP protein was used to immunize BALB / c mice multiple times. Blood was collected from the tail vein of the mice one week after immunization, and the serum titer of the immunized mice was detected by indirect ELISA method using ESP protein as the coating antigen; 2) Cell fusion: Isolate the spleen cells from step 1) and fuse them with mouse myeloma cells; 3) Hybridoma cell screening: Take the supernatant and use the ESP protein-coated ELISA plate to screen the hybridoma cells by the indirect ELISA method; 4) Cloning of hybridoma cells: Cloning of hybridoma cells is done by limiting dilution method; 5) Mouse ascites: hybridoma cells were inoculated into the mouse peritoneal cavity; 6) Monoclonal antibody purification: Collect and purify ascites to obtain monoclonal antibodies.

[0010] In step 5 of the monoclonal antibody preparation method, the amount of hybridoma cells inoculated is 5×10 5 / 0.2ml.

[0011] In step 2 of the monoclonal antibody preparation method, the concentration of spleen cells is 2.1×10 8 Pieces / ml.

[0012] In step 2 of the monoclonal antibody preparation method, the concentration of myeloma cells is 2×10 7 Pieces / ml.

[0013] The use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in detecting bovine mastitis Enterococcus faecalis.

[0014] Use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in preparing a reagent for detecting bovine mastitis Enterococcus faecalis.

[0015] The use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in preparing a colloidal gold rapid test paper for detecting bovine mastitis Enterococcus faecalis.

[0016] The beneficial effects of the present invention are: Enterococcus faecalis is a conditional pathogen. When the body's homeostasis is destroyed, its own system becomes disordered, and the bacteria immediately transform into pathogenic bacteria, producing pathogenic factors that are harmful to the body, causing diseases in humans and animals. The formation of Enterococcus faecalis biofilm is regulated by many factors. ESP can promote the adhesion of bacteria to inorganic surfaces and the adhesion between bacteria in the biofilm, thereby making the biofilm more stable and enhanced. The cloned and expressed surface protein ESP has relatively stable properties and antigen accessibility. Animal immunization and Western blot analysis show that the protein is antigenic, which lays the foundation for the development of corresponding genetic engineering vaccines and is of great significance to the study of the pathogenic mechanism of Enterococcus faecalis and the development of detection antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The attached pictures are as follows: Figure 1 is a diagram showing the cloning results of the hybridoma cells 1A7 and 6F6 involved in the present invention; Figure 2 It is a diagram showing the purification results of the ESP protein monoclonal antibodies 1A7 and 6F6 involved in the present invention; Figure 3 This is a result diagram of the application of Western blotting to detect the reactivity of 1A7 and 6F6 with ESP protein involved in the present invention. DETAILED DESCRIPTION

[0018] A method for preparing monoclonal antibody against Enterococcus faecalis ESP of bovine mastitis.

[0019] Reference: Cloning, expression and antigenicity analysis of the surface protein esp gene of pathogenic Enterococcus faecalis in bovine mastitis in Inner Mongolia, Shi Jingnan, Xilingaowa, Wu Jinhua, et al., Chinese Journal of Pathogenic Biology, 2023, 18(01): 23-29.

[0020] The recombinant ESP protein described in the present application can be derived from the above references.

[0021] 1. Methods

[0022] 1. Immunize mice.

[0023] The purified recombinant ESP protein was used to immunize BALB / c mice multiple times. The concentration of the purified ESP protein was adjusted with phosphate buffer (0.01 mol / L, pH 7.4). After the protein concentration was adjusted to 1 mg / mL, it was filtered and sterilized for use. The first immunization of mice was 150 μg / mouse, and an equal volume of Freund's complete adjuvant was added to emulsify it completely. The mouse epidermis was pinched and the BALB / c mice were injected subcutaneously at multiple points on the back of the neck. After the first immunization, the second and third immunizations were carried out 2 weeks apart, and an equal volume of Freund's incomplete adjuvant was added to emulsify it completely. The immunization dose was 150 μg / mouse, and the route was the same as the first immunization.

[0024] 2. Detect the immune titer of mouse serum.

[0025] One week after the third immunization, the tail vein of the mice was collected, and the indirect ELISA method with ESP protein as the coating antigen was used to detect the titer of the immunized mouse serum. The ELISA plate was coated with purified ESP protein (2ug / ml), coated overnight at 4°C, and blocked with blocking solution at 37°C for 1h. To detect the titer of mouse serum, the serum was diluted 2 times from 1:1000, and the sample was added after dilution, incubated at 37°C for 30 min, washed 3 times with washing solution, and then the secondary antibody was added, incubated at 37°C for 30min, washed 3 times with washing solution, and color was developed. The color development time was 10 min, and the temperature was 37°C. 50 μl 2M H2SO4 was added to each well to stop and measure the absorbance value. The blank control was 0.1mol PBS, and the negative control was negative serum diluted 1:1000 times. One mouse with a higher antibody titer was selected for impact immunization at 100 μg / mouse.

[0026] 3. Cell fusion.

[0027] 3.1 Preparation of myeloma cells.

[0028] 36 hours before fusion, expand the myeloma cells in a 100 ml cell culture flask and prepare 2-3 flasks. On the day of fusion, use an elbow dropper to gently blow the cells off the flask wall and collect them in a 50 ml centrifuge tube. Centrifuge at 1000 rpm for 5-10 minutes and discard the supernatant. Add 30 ml of incomplete culture medium and centrifuge and wash once in the same way. Then resuspend the cells in 10 ml of incomplete culture medium and mix well.

[0029] 3.2 Preparation of splenocytes.

[0030] Take BALB / c mice that have been immunized for 3 days, remove the eyeballs to collect blood, and separate the serum as the positive control serum for antibody detection. At the same time, kill the mice by cervical dislocation, soak in 75% alcohol for 5 minutes, fix on the dissection table, lift the skin on the left abdomen, and you can see the spleen. Change the ophthalmic scissors and forceps, cut the peritoneum with sterile surgical scissors in the clean bench, remove the spleen, place it in a plate containing 10 ml of incomplete culture medium, wash it gently, and carefully peel off the surrounding connective tissue. Move the spleen to another plate containing 10 ml of incomplete culture medium, gently squeeze the spleen with elbow forceps, so that the spleen cells enter the incomplete culture medium in the plate. Use a pipette to blow several times to make a single cell suspension. Centrifuge at 1000rpm for 10 minutes, harvest the spleen cell suspension, centrifuge and wash twice with incomplete culture medium, then resuspend the cells in 10ml of incomplete culture medium and mix well. Take the above suspension, add trypan blue dye, count the cells, and get 2.1×10 8 splenocytes / ml.

[0031] 3.3 Preparation of feeder cells According to the method for collecting mouse spleen cells, the mouse was killed, the body surface was disinfected and fixed, and the abdominal skin was lifted from the posterior abdomen with sterilized scissors and forceps to expose the peritoneum. The peritoneum was wiped and disinfected with an alcohol cotton ball. 10 ml of incomplete culture medium was injected into the abdominal cavity with a syringe, and care was taken to avoid penetration of the intestine. The syringe was fixed with the right hand so that the needle remained in the abdominal cavity. The left hand held the alcohol cotton ball and gently massaged the abdomen for 1 min, and then the injected culture medium was aspirated. Centrifuge at 1000 rpm for 10 min and discard the supernatant. First, suspend the precipitated cells with 5 ml of HAT culture medium. According to the cell counting results, add HAT culture medium to make the cell concentration 2×10 5 / ml, and store in a cell culture incubator for later use. Add the above cell suspension to a 96-well plate, 2 drops per well, i.e. 0.1ml, and then culture in a cell culture incubator at 37°C and 6% CO2.

[0032] 3.4 Cell fusion and selective culture of hybridoma cells.

[0033] 2.1×10 8 splenocytes and 2×10 7Myeloma cells SP2 / 0 were mixed in a 50 ml centrifuge tube, and incomplete culture medium was added to 30 ml, and the mixture was thoroughly mixed. Centrifuge at 1000 rpm for 10 min, and the supernatant was aspirated as much as possible. Tap the bottom of the fusion tube lightly on the palm of your hand to loosen and evenly disperse the precipitated cells, and preheat in a 40°C water bath. Use a 1 ml pipette to add 1 ml of 50% PEG (PH 8.0) preheated to 40°C within 1 min, and stir gently while adding. Use a 10 ml pipette to add 25 ml of incomplete culture medium preheated to 37°C within 90 seconds, let stand at 37°C for 10 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 5 ml of HAT culture medium, gently blow and aspirate the precipitated cells to suspend and mix, and then add HAT culture medium containing peritoneal macrophages to 100 ml. Dispense into 96-well cell culture plates, 0.10-0.15 ml per well; then place the cell culture plates in a 37°C, 6% CO2 incubator for culture. After 5 days, replace 1 / 2 of the culture medium with HAT culture medium, after 10 days, replace HAT culture medium with HT culture medium, and after 14 days, use ordinary complete culture medium. Observe the growth of hybridoma cells frequently, and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0034] 4. Hybridoma cell screening.

[0035] Take 100 μl of supernatant and use 2 μg / ml ESP protein coated ELISA plate to screen hybridoma cells by indirect ELISA method. Add cell culture supernatant, SP2 / 0 culture supernatant as negative control, 1×PBS as blank control, positive serum diluted 1:1000 times as positive control, incubate at 37°C for 15 min, wash 3 times with washing solution, add secondary antibody, incubate at 37°C for 30 min, wash 3 times with washing solution, add color development solution, incubate at 37°C for 10 min, add 50 μl stop solution to each well, and measure the absorbance value after termination.

[0036] 5. Cloning of hybridoma cells.

[0037] The hybridoma cells were cloned by limiting dilution method. Feeder cells were prepared, and the hybridoma cell suspension to be cloned was prepared. The counted hybridoma cells were accurately serially diluted until there were 10 cells per ml. 0.1 ml of cell suspension was inoculated into each well so that each well contained 1 cell. The cells were cultured in a cell culture incubator at 37°C and 6% CO2 for 10 days, and observed under an inverted microscope. The wells where only a single clone grew were marked, and the supernatant was taken for antibody detection. After two subclones, cells with stable genotypes and stable secretory phenotypes were obtained. The cells in the antibody-positive wells were expanded and cultured, and then frozen.

[0038] 6. Cryopreservation and recovery of hybridoma cells.

[0039] 6.1 Cryopreservation of hybridoma cells.

[0040] The hybridoma cells were centrifuged at 2000 rpm for 5 min and resuspended in pre-cooled cryopreservation buffer at a concentration of 10 7 / ml, divided into 1ml bottles, sealed and placed on ice, and marked with cell name and freezing date. Placed in a cell freezing box pre-cooled at 4℃, stored in a minus 80 refrigerator overnight, and then transferred to liquid nitrogen for long-term storage.

[0041] 6.2 Recovery of hybridoma cells.

[0042] Take out the ampoule from liquid nitrogen and immediately melt it in a 37°C water bath. When the last bit of ice is about to melt, take it out of the water bath and place it on an ice bath. Dilute with 5 ml HT medium, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend in an appropriate amount of HT medium, transfer to a culture bottle, and culture in a 37°C, 6% CO2 cell culture incubator.

[0043] 7. Preparation of Mouse Ascites.

[0044] Mice were sensitized by intraperitoneal inoculation of liquid paraffin, 0.3 ml per mouse. Ten days later, hybridoma cells diluted in serum-free medium were intraperitoneally inoculated, 5 × 10 cells per mouse. 5 / 0.2ml. After an interval of 5 days, observe the ascites production of mice every day. If the abdomen is obviously swollen and the skin feels tense when touched, the ascites can be collected with a 16-gauge needle. Generally, it can be collected 2 to 3 times in a row. Usually, 5 to 10ml of ascites can be collected from each mouse. Centrifuge the ascites at 2000 rpm for 5 minutes to remove cell components and other precipitates, collect the supernatant, determine the antibody titer, and freeze at -80°C for later use.

[0045] 8. Purification of Monoclonal Antibodies.

[0046] The ascites was purified by the method of octanoic acid-ammonium sulfate, that is, the ascites was filtered with double-layer filter paper to preliminarily remove impurities, fat and cell debris. Centrifuge at 4°C, 12000 rpm for 15 min, collect the supernatant and discard the precipitate. Accurately quantify the volume of ascites, add 3 volumes of acetate buffer to one volume of ascites, mix with magnetic stirring, and add 2mol / L HCL to adjust the pH to 4.5. Slowly add octanoic acid, 33μL / mL ascites under magnetic stirring, stir at room temperature for half an hour after addition, and then stand at 4°C for 2h. After standing, centrifuge at 4°C, 12000 rpm for 5min, collect the supernatant, filter with double-layer filter paper, and collect the filtrate. Accurately measure the volume of the filtrate, add 1 / 10 volume of 0.1M pH 7.4 PBS, adjust the pH to 7.4 with 2mol / L NaOH, and record the volume of NaOH added. Add ammonium sulfate solid to 0.277g / mL, stirring while adding, and add it within 30 minutes, and place it at 4°C overnight. Centrifuge at 12000 rpm for 15 minutes, and discard the supernatant. Dissolve the precipitate with a certain volume of 0.01 mol / L PBS, and then dialyze it with 0.01 mol / L PBS overnight. Collect the dialysate, centrifuge at 12000 rpm for 15 minutes, take the supernatant, store it at -20°C for later use, and then analyze the purity by SDS-PAGE electrophoresis.

[0047] 9. Identification of monoclonal antibodies by Western blotting.

[0048] The purified recombinant protein ESP and bacterial protein containing pET-30a empty plasmid were subjected to SDS-PAGE electrophoresis and electrotransferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk at 37℃ for 1 h, then immersed in TBST solution containing 0.1 μg / mL monoclonal antibody and incubated at 37℃ for 1 h. The membrane was washed 3 times with TBST Buffer; the membrane was then immersed in TBST solution containing 0.1 μg / mL HRP-labeled goat anti-monoclonal mouse IgG and incubated at 37℃ for 1 h. After washing 3 times with TBST Buffer, Western blotting was performed for identification.

[0049] 2. Results

[0050] 1. Detect the mouse serum immune titer results.

[0051] After three immunizations, the serum titers of the three mice detected by indirect ELISA were all high. See the table below for details.

[0052] .

[0053] 2. Hybridoma cell screening results.

[0054] In the initial screening of hybridoma cells, 12 positive monoclonal cell lines were screened out. The 12 cell lines were subcultured and finally two hybridoma cell lines that could stably secrete anti-ESP protein were detected, namely 1A7 and 6F6. (In the following tables: the black number boxes are the positive strains screened, and the gray parts are blank control, negative control, and positive control respectively).

[0055] Fusion cell plate screening (plate 1) see the table below: .

[0056] Fusion cell plate screening (plate 2) see the table below: .

[0057] Fusion cell plate screening (plate three) see the table below: .

[0058] Fusion cell plate screening (plate 4) is shown in the table below: .

[0059] Fusion cell plate screening (plate 5) see the table below: .

[0060] Fusion cell plate screening (plate six) see the table below: .

[0061] 3. Cloning results of hybridoma cells.

[0062] The limited dilution method was used to clone hybridoma cells. Two positive hybridoma cell lines 1A7 and 6F6 were cloned in two 96-well cell culture plates, and the wells where only a single clone grew were circled. The supernatant was taken for antibody detection. After two subclones, the positive rate of indirect ELISA detection was 99%, that is, cells with stable genotype and stable secretory phenotype were obtained. The cells in the antibody-positive wells were taken for expansion culture and cryopreservation. See the attached for details. Figure 1 .

[0063] 4. Purification of ESP protein monoclonal antibodies 1A7, 6F6.

[0064] The ascites was purified by the caprylic acid-ammonium sulfate method, and then dialyzed against phosphate buffer (0.01 mol / L, pH 7.4) overnight and then tested by SDS-PAGE. The results showed that the monoclonal antibody was cleaved into a heavy chain of about 55 kDa and a light chain of about 25 kDa. Figure 2 .

[0065] 5. Western blotting identification.

[0066] Western blotting was used to detect the reactivity of 1A7, 6F6 and ESP proteins. The results showed that they could specifically recognize ESP protein, with a specific band appearing at about 37 kDa, and did not react with bacterial proteins expressed by pET-30a empty plasmid. Figure 3 .

[0067] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A hybridoma cell line 1A7 secreting monoclonal antibody against Enterococcus faecalis, characterized in that: The hybridoma cell line was prepared by the following method: 1) BALB / c mice were immunized three times with purified recombinant ESP protein, and spleen cells were isolated from the mice; 2) Take mouse spleen cells, fuse them with myeloma cells, and screen them.

2. A hybridoma cell line 6F6 secreting monoclonal antibody against Enterococcus faecalis, characterized in that: The hybridoma cell line was prepared by the following method: 1) BALB / c mice were immunized three times with purified recombinant ESP protein, and spleen cells were isolated from the mice; 2) Take mouse spleen cells, fuse them with myeloma cells, and screen them.

3. A bovine mastitis Enterococcus faecalis ESP monoclonal antibody, characterized in that: It is secreted by the hybridoma cell line 1A7 and / or 6F6 according to claim 1 or 2.

4. A method for preparing the bovine mastitis Enterococcus faecalis ESP monoclonal antibody as claimed in claim 3, characterized in that: The preparation method of the ESP monoclonal antibody comprises the following steps: 1) Immunization of mice: BALB / c mice were immunized multiple times with purified recombinant ESP protein. Blood was collected from the tail vein of the mice one week after immunization, and the serum titer of the immunized mice was detected by indirect ELISA method using ESP protein as the coating antigen; 2) Cell fusion: Isolate the spleen cells from step 1) and fuse them with mouse myeloma cells; 3) Hybridoma cell screening: Take the supernatant and use the ESP protein-coated ELISA plate to screen the hybridoma cells by the indirect ELISA method; 4) Cloning of hybridoma cells: Cloning of hybridoma cells is done by limiting dilution method; 5) Mouse ascites: hybridoma cells were inoculated into the mouse peritoneal cavity; 6) Monoclonal antibody purification: Collect and purify ascites to obtain monoclonal antibodies.

5. The method for preparing ESP monoclonal antibody against Enterococcus faecalis of bovine mastitis according to claim 4, characterized in that: The amount of hybridoma cells inoculated in step 5) is 5×10 per mouse. 5 / 0.2ml.

6. The method for preparing ESP monoclonal antibody against Enterococcus faecalis of bovine mastitis according to claim 4, characterized in that: The concentration of splenocytes in step 2) was 2.1×10 8 Pieces / ml.

7. The method for preparing ESP monoclonal antibody against Enterococcus faecalis of bovine mastitis according to claim 4, characterized in that: The concentration of myeloma cells in step 2) is 2×10 7 Pieces / ml.

8. The bovine mastitis Enterococcus faecalis ESP monoclonal antibody prepared by the method for preparing the bovine mastitis Enterococcus faecalis ESP monoclonal antibody according to claim 4, characterized in that: The use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in detecting bovine mastitis Enterococcus faecalis.

9. The bovine mastitis Enterococcus faecalis ESP monoclonal antibody prepared by the method for preparing the bovine mastitis Enterococcus faecalis ESP monoclonal antibody according to claim 4, characterized in that: The use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in preparing a reagent for detecting bovine mastitis Enterococcus faecalis.

10. The bovine mastitis Enterococcus faecalis ESP monoclonal antibody prepared by the method for preparing the bovine mastitis Enterococcus faecalis ESP monoclonal antibody according to claim 4, characterized in that: The use of the bovine mastitis Enterococcus faecalis ESP monoclonal antibody in preparing a colloidal gold rapid test paper for detecting bovine mastitis Enterococcus faecalis.

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