CN108048408B - Bovine monocyte chemotactic protein-1 hybridoma cell strain, monoclonal antibody secreted by same and application - Google Patents

Abstract

The invention provides a hybridoma cell strain 4G9 secreting monoclonal antibody against bovine monocyte chemotactic protein 1(MCP-1) and a monoclonal antibody MAb4G9 secreted by the hybridoma cell strain. The monoclonal antibody MAb4G9 has the advantages of high titer, good specificity and strong affinity with natural bovine MCP-1 antigen, and Western-Blot detection kit, direct immunofluorescence (DFA) detection kit and Flow Cytometry (FCM) detection kit established based on the monoclonal antibody MAb4G9 have good sensitivity and specificity, and can be used for non-diagnosis purpose researches such as detection of recombinant bovine MCP-1, detection of isolated tissues, epitope identification research, import and export inspection and quarantine, epidemiological research and the like.

Description

Bovine monocyte chemoattractant protein-1 hybridoma cell line and its secreted monoclonal antibody and application

technical field

The present invention relates to a hybridoma cell line, in particular to a hybridoma cell line that secretes anti-bovine monocyte chemotactic protein 1 (MCP-1) monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell line and its application.

Background technique

Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC subfamily of chemokines. MCP-1 mainly chemoattracts monocytes, and makes various inflammatory cells, especially monocytes, aggregate to the lesion site, thereby playing an important role in body defense, chronic inflammation and anti-tumor. Under the action of specific chemokines, monocytes in blood migrate and aggregate in different diseased tissues to play important biological effects, such as phagocytosis and killing of pathogenic microorganisms, presenting antigens and secreting a variety of biologically active substances. Studies have shown that the changes in the expression level of MCP-1 are related to the development of infectious diseases, abnormal immune function, inflammatory diseases and tumors.

The immunological detection of MCP-1 is an experimental method established on the basis of specific anti-MCP-1 monoclonal antibody (MAb) for qualitative and quantitative analysis of MCP-1 in samples. Using different monoclonal antibody markers and detection techniques, a variety of detection methods can be established, such as enzyme immunoassay (Enzyme Immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, referred to as ELISA), flow cytometry Flow Cytometry (FCM) analysis method, etc. The FCM of MCP-1 is highly sensitive and widely used. As one of the important indicators of cellular immune status, the FCM detection method of MCP-1 is widely used in the research of basic and clinical medicine, the evaluation of vaccine immune effect, organ transplantation, allergic reactions and the diagnosis of various pathogenic microorganism infections.

However, the anti-bovine MCP-1 (BoMCP-1) antibody prepared in the prior art often encounters great problems when applied to the above-mentioned detection method, especially when it is used to detect natural bovine MCP-1, the effect is not good. Therefore, a bovine MCP-1 monoclonal antibody with high titer, good specificity, and antigenic affinity with natural bovine MCP-1 that can be used for bovine clinical detection is obtained, which is related to the evaluation of bovine immune status and the diagnosis of infectious diseases. research is of great significance.

SUMMARY OF THE INVENTION

In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a hybridoma cell line that secretes bovine MCP-1 monoclonal antibody, and the hybridoma cell line and the monoclonal antibody secreted by the hybridoma cell line are used in bovine immune detection. application in the field.

The anti-bovine MCP-1 monoclonal antibody secreted by the hybridoma cell line has a high titer, and it can specifically bind to the natural bovine MCP-1 with high affinity. and specificity.

The first aspect of the present invention provides a hybridoma cell line that secretes bovine MCP-1 monoclonal antibody, wherein the hybridoma cell line is a hybridoma cell line 4G9 or a passaged cell line thereof, and the hybridoma cell line 4G9 The deposit number is CCTCCNO: C2017281.

The second aspect of the present invention provides a bovine MCP-1 monoclonal antibody 4G9, which is secreted and produced by the above hybridoma cell line or its passaged cell line.

The third aspect of the present invention provides a Western-Blot detection kit for bovine MCP-1, the Western-Blot detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the detection antibody is horseradish peroxidase The above bovine MCP-1 monoclonal antibody 4G9 (HRP-4G9) labeled with oxidase.

The fourth aspect of the present invention provides a direct immunofluorescence DFA detection kit for bovine MCP-1, the direct immunofluorescence DFA detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the detection antibody is an The above bovine MCP-1 monoclonal antibody 4G9 (FITC-4G9) labeled with fluorescein thiocyanate.

A fifth aspect of the present invention provides a FCM detection kit for bovine MCP-1, the FCM detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the detection antibody is a fluorescein isothiocyanate label The aforementioned bovine MCP-1 monoclonal antibody 4G9 (FITC-4G9).

The sixth aspect of the present invention provides the application of the above-mentioned hybridoma cell line secreting bovine MCP-1 monoclonal antibody or the above-mentioned bovine MCP-1 monoclonal antibody MAb 4G9 in the preparation of detection reagents or diagnostic reagents for bovine immune status.

The seventh aspect of the present invention provides the application of the Western-Blot detection kit, the direct immunofluorescence DFA detection kit or the FCM detection kit in the detection of bovine MCP-1 for non-diagnostic purposes, the non-diagnostic detection includes: Detection of recombinantly expressed bovine MCP-1, detection of isolated tissues, epitope identification research, import and export inspection and quarantine, and epidemiological research.

Technical effect of the present invention:

The monoclonal antibody secreted by the hybridoma cell line of the present invention has the advantages of high titer, good specificity and strong binding affinity with natural antigen, and can be used for detecting natural bovine MCP-1.

The Western-Blot detection kit established based on this can effectively detect the bovine MCP-1 protein expressed by the E. coli expression system and the natural bovine MCP-1 protein secreted by bovine peripheral blood mononuclear cells (PBMC), with good sensitivity and specificity. sex.

Based on this, the established direct immunofluorescence (DFA) detection kit can effectively detect bovine MCP-1 expressed and secreted by eukaryotic plasmids and bovine MCP-1 secreted by bovine peripheral blood mononuclear cells, with good sensitivity and specificity.

At the same time, the FCM detection kit for bovine MCP-1 established based on this can detect a higher level of bovine MCP-1-secreting monocytes when detecting bovine peripheral blood mononuclear cell samples, with good sensitivity and specificity.

Description of drawings

Fig. 1 is a diagram showing the detection result of recombinant bovine MCP-1 protein by the Western-Blot method of the present invention: A. detection result of recombinant His-BoMCP-1 protein, B. detection result of recombinant GST-BoMCP-1 protein.

Figure 2 is a diagram showing the detection results of bovine peripheral blood mononuclear cells by the direct immunofluorescence (DFA) method of the present invention: A. The detection results of unstimulated bovine peripheral blood mononuclear cells; B. The results of LPS stimulated bovine peripheral blood mononuclear cells.

Figure 3 is a diagram showing the detection results of bovine peripheral blood mononuclear cells in the FCM kit of the present invention: A. The detection results of unstimulated bovine peripheral blood mononuclear cells, and B. the results of LPS-stimulated bovine peripheral blood mononuclear cells.

Detailed ways

Embodiments of the present invention will be described below.

The purpose of the present invention is to provide a hybridoma cell line that secretes bovine MCP-1 monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell line, and the application of the hybridoma cell line and the monoclonal antibody secreted by the hybridoma cell line in the field of immunodetection application.

One aspect of the present invention provides a hybridoma cell line that secretes bovine MCP-1 monoclonal antibody, wherein the hybridoma cell line is hybridoma cell line 4G9 or a passaged cell line thereof. The hybridoma cell line 4G9 has the deposit number CCTCCNO: C2017281; it is deposited in the China Center for Type Culture Collection; the deposit address is China. Wuhan. Wuhan University, and the deposit date is December 7, 2017.

The second aspect of the present invention provides a bovine MCP-1 monoclonal antibody 4G9 secreted by the hybridoma cell line 4G9 of the present invention or its passaged cell line.

The third aspect of the present invention provides a Western-Blot detection kit for bovine MCP-1, the Western-Blot detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the detection antibody is horseradish peroxidase The above bovine MCP-1 monoclonal antibody 4G9 (HRP-4G9) labeled with oxidase.

Preferably, the Western-Blot detection kit also includes one or more of the following reagents:

1) Blocking solution;

2) Substrate solution; and

3) Washing solution.

The Western-Blot detection kit of the present invention can be used to detect recombinant bovine MCP-1 protein.

The method for detecting recombinant bovine MCP-1 protein using the Western-Blot detection kit of the present invention comprises the following steps:

1. SDS-PAGE electrophoresis,

The purified proteins rHis-BoMCP-1 and rGST-BoMCP-1 were added to protein loading buffer respectively, boiled at 100 °C for 10 min, and subjected to SDS-PAGE electrophoresis;

2. Transfer,

After electrophoresis, soak the gel in the transfer buffer for 5 minutes, and soak the NC membrane and filter paper at the same time. From top to bottom, the order is 2 pieces of filter paper - NC membrane - gel - 2 pieces of filter paper. Transfer to a semi-dry transfer machine, taking care to remove air bubbles. Transfer 2h at 0.8mA/ ^cm2 in Bio-Rad semi-Dry transfer Cell system;

3. Detection,

①Block with PBS containing 5% skimmed milk, and block overnight with shaking at room temperature;

② Wash the membrane with PBST, wash 4 times for 10 minutes each time, and dry the residual washing solution on the membrane as much as possible;

③ Add the detection antibody (HRP-4G9) diluted 1:1000 with PBS, and shake at room temperature for 2 hours;

④ Wash the membrane with PBST for 4 times, 10 min each time, and dry the residual washing solution on the membrane as much as possible;

⑤ Color development, tap water to terminate the reaction, and a scanner to take pictures of the reaction results.

The results show that the Western-Blot kit of the present invention can effectively detect the bovine MCP-1 protein expressed by the E. coli expression system, and has good sensitivity and specificity.

The Western-Blot detection kit for bovine MCP-1 of the present invention can be used to detect bovine peripheral blood mononuclear cell samples.

The method for detecting bovine peripheral blood mononuclear cell sample using the Western-Blot kit of bovine MCP-1 of the present invention comprises the following steps:

1. Preparation of bovine peripheral blood mononuclear cells,

① Aseptically take 5 mL of bovine blood to be tested and add it to a blood collection tube containing heparin sodium, invert and mix to obtain anticoagulation after blood collection;

② After diluting anticoagulant and sterile PBS 1:1, slowly add the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation medium at a ratio of 1:1 to form a clear interface, and centrifuge at 2000rmp at room temperature 20-30min;

③ It can be seen that the peripheral blood mononuclear cells exist in the cloudy layer. Use a sterile dropper to suck the peripheral blood mononuclear cell layer into a clean centrifuge tube, add sterile PBS, mix the cells, and centrifuge at 2000rmp at 4°C 10min, repeated twice to obtain precipitated cells;

④ Discard the supernatant medium, add complete 1640 medium to resuspend the pelleted cells, take 10 μL of cell suspension, add 10 μL of phenol blue, mix well, add to a hemocytometer, count under a microscope, and use complete 1640 medium to suspend the cell suspension. Dilute to ¹ x 107 cells/mL.

2. Cell incubation,

Add the following reagents to a 24-well cell culture plate: 500 μL of culture medium to each control well, 500 μL of 10 μg/mL LPS to each positive well. Add 500 μL of the diluted cell suspension to each well, incubate at 37°C, 5% _CO2 incubator for 24-48 hours, and collect cells as test samples;

3. SDS-PAGE electrophoresis,

Cells were lysed with Western lysis buffer, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken. The cell lysis supernatant was added to the protein loading buffer, boiled at 100 °C for 10 min, and subjected to SDS-PAGE electrophoresis;

4. Transfer,

After electrophoresis, soak the gel in the transfer buffer for 5 minutes, and soak the NC membrane and filter paper at the same time. In the order from top to bottom, 2 pieces of filter paper-NC membrane-gel-2 pieces of filter paper are laid and then transferred. Transfer to a semi-dry transfer machine, taking care to remove air bubbles. Transfer in Bio-Rad semi-Dry transfer Cell system at 0.8mA/cm2 for 2h;

5. Detection,

①Block with PBS containing 5% skimmed milk, and block overnight with shaking at room temperature;

② Wash the membrane with PBST, wash 4 times for 10 minutes each time, and dry the residual washing solution on the membrane as much as possible;

③ Add the detection antibody (HRP-4G9) diluted 1:1000 with PBS, and shake at room temperature for 2 hours;

④ Wash the membrane with PBST for 4 times, 10 min each time, and dry the residual washing solution on the membrane as much as possible;

⑤ Color development, tap water to terminate the reaction, and a scanner to take pictures of the reaction results.

The results show that the Western-Blot detection kit of the present invention can effectively detect the natural bovine MCP-1 protein secreted by bovine peripheral blood mononuclear cells, and has good sensitivity and specificity.

The fourth aspect of the present invention provides a direct immunofluorescence (DFA) detection kit for bovine MCP-1, the direct immunofluorescence (DFA) detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the The detection antibody was the above-mentioned bovine MCP-1 monoclonal antibody 4G9 (FITC-4G9) labeled with fluorescein isothiocyanate.

Preferably, the direct immunofluorescence method further includes the following reagents:

1) a fixative; and

2) Washing solution.

The direct immunofluorescence (DFA) detection method of the present invention can be used to analyze recombinant bovine MCP-1 protein.

The detection method of utilizing the direct immunofluorescence (DFA) detection method of the present invention to detect recombinant bovine MCP-1 protein comprises the following steps:

1. Transfection,

①24h before transfection, digest and inoculate HEK293T cells in a 24-well plate, 2×10 ⁵ cells/well;

1.5μL至含Opti-MEM的1.5mL指形管中至总体积50μL；②Join

1.5 μL into a 1.5 mL finger tube with Opti-MEM to a total volume of 50 μL;

③ Add 1 μL each of recombinant plasmid pVAX1-GFP-BoMCP-1 and empty vector plasmid pVAX1, and 2 μL of P3000 ^TM to a 1.5 mL finger tube containing Opti-MEM to a total volume of 50 μL;

Opti-MEM 50μL，静置5min，分别加入细胞板中，置培养箱继续培养；④Add the containing

Opti-MEM 50μL, let stand for 5 minutes, add them to the cell plate respectively, and continue to culture in the incubator;

2. Cytokine detection,

①Aspirate the culture supernatant of HEK293T(pVAX1-GFP-BoMCP-1) and HEK293T(pVAX1) cells, wash twice with 500μL PBS, add 500μL -20℃ pre-cooled ice methanol and fix at -20℃ for 10min;

② Wash three times with 500 μL PBS, add FITC-4G9 diluted 1:1000, and incubate in a 37°C water bath for 2 hours;

③ Rinse three times with 500 μL PBS and observe with a fluorescence inverted microscope.

The results showed that the direct immunofluorescence (DFA) method could effectively detect bovine MCP-1 protein in eukaryotic expression system with good sensitivity and specificity.

The direct immunofluorescence (DFA) detection method of the present invention can be used to analyze bovine peripheral blood mononuclear cell samples.

The detection method for detecting bovine peripheral blood mononuclear cell samples using the direct immunofluorescence (DFA) detection method of the present invention comprises the following steps:

1. Preparation of bovine peripheral blood mononuclear cells,

① Aseptically take 5 mL of bovine blood to be tested and add it to a blood collection tube containing heparin sodium, invert and mix to obtain anticoagulation after blood collection;

② After diluting the anticoagulant and sterile PBS 1:1, slowly add the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation medium at a ratio of 1:1 to form a clear interface, at room temperature 2000rmp Centrifuge for 20-30min;

③ It can be seen that the peripheral blood mononuclear cells exist in the cloudy layer. Use a sterile dropper to suck the peripheral blood mononuclear cell layer into a clean centrifuge tube, add sterile PBS, mix the cells, and centrifuge at 2000rmp at 4°C 10min, repeated twice to obtain precipitated cells;

④ Discard the supernatant medium, add complete 1640 medium to resuspend the pelleted cells, take 10 μL of cell suspension, add 10 μL of phenol blue, mix well, add to a hemocytometer, count under a microscope, and use complete 1640 medium to suspend the cell suspension. Dilute to ¹ x 107 cells/mL.

2. Cell incubation,

Add the following reagents to a 24-well cell culture plate: 500 μL of culture medium to each control well, 500 μL of 10 μg/mL LPS to each positive well. Add 500 μL of diluted cell suspension to each well, place at 37°C, 5% CO ₂ incubator for 24-48 hours, and collect cells; 3. Cytokine detection,

①Aspirate the culture supernatant of the cells, wash twice with 500μL PBS, add 500μL -20℃ pre-cooled ice methanol and fix at -20℃ for 10min;

② Wash three times with 500 μL PBS, add FITC-4G9 diluted 1:1000, and incubate in a 37°C water bath for 2 hours;

③ Wash three times with 500 μL of PBS and observe with a fluorescent inverted microscope.

The results show that when detecting bovine peripheral blood mononuclear cell samples, the positive sample well (B) has obvious green fluorescence, while the control sample well (A) has no green fluorescence, which indicates that the kit can effectively detect LPS-stimulated bovine peripheral blood. Cells that secrete bovine MCP-1 have better sensitivity and specificity.

A fifth aspect of the present invention provides a FCM detection kit for bovine MCP-1, the FCM detection kit includes a detection antibody and a bacterial lipopolysaccharide LPS stimulator, wherein the detection antibody is the above-mentioned fluorescein isothiocyanate-labeled Bovine MCP-1 monoclonal antibody 4G9.

Preferably, the FCM detection kit may also include one or more of the following reagents:

1) The blocking agent Brefeldin (Brefeldin, BFA);

2) Fixatives, membrane-breaking agents; and

3) Washing solution.

The above reagents are all common reagents in FCM detection and are not limited by specific detection items, so they can be selectively added according to needs, and can also be configured by the operator or purchased separately. For the convenience of the author, the best choice is to include the blocking agent BFA, fixative, membrane breaking agent and washing solution in the kit.

The washing solution can be a washing solution commonly used in FCM detection kits, such as a phosphate buffered solution containing 1% BSA and the like. Concentrated or unconcentrated washes can be selected as required.

As an embodiment of the present invention, the kit may also selectively include other general reagents required for FCM detection, such as cell culture medium, phosphate buffer solution, and the like.

Usually, in the kit of the present invention, each reagent is stored separately.

The present invention is further based on the above-mentioned FCM detection kit for bovine MCP-1, and establishes a FCM detection method for bovine MCP-1 with better specificity and sensitivity, which is used to detect monocytes secreting bovine MCP-1, so as to detect the bovine MCP-1. Research on immune status evaluation and disease diagnosis.

The FCM detection kit of the present invention can be used for analyzing bovine peripheral blood mononuclear cell samples.

The detection method that utilizes the FCM detection kit of the present invention to detect bovine peripheral blood mononuclear cell samples comprises the following steps:

1) Preparation of bovine peripheral blood mononuclear cell suspension;

2) Cell incubation:

①Add the following reagents to a 96-well cell culture plate: 50 μL of culture medium to each control well, 50 μL of 10 μg/mL LPS to each positive well. Add 50 μL of bovine peripheral blood mononuclear cell suspension to each well, and place the 96-well cell culture plate in a 37°C, 5% CO ₂ incubator for 4-6 hours;

② Add the cytokine secretion blocking agent BFA, and culture in a 37°C, 5% CO ₂ incubator for 16 hours.

3) Cytokine detection;

① Collect the cultured cells the next day, wash the cells with PBS containing 1% BSA, centrifuge at 2000 rpm at 4°C for 10 min, and discard the supernatant;

②Add fixative and let stand at room temperature for 15min; wash cells with PBS containing 1% BSA; centrifuge at 4°C at 2000rpm for 10min, discard the supernatant, repeat twice;

③ Dilute the FITC-labeled monoclonal antibody 4G9 specific for bovine MCP-1 to 0.5 μg/mL with a working concentration of membrane-breaking agent, let it stand at room temperature for 15 min, and wash the cells with PBS containing 1% BSA; Discard the supernatant and repeat twice;

④ Resuspend the cells in 200 μL PBS, and perform FACS to detect the proportion of cells secreting bovine MCP-1.

The above method was used to separate bovine peripheral blood mononuclear cells by density gradient centrifugation. The LPS-stimulated bovine peripheral blood mononuclear cells were used as positive samples, the unstimulated bovine peripheral blood mononuclear cells were used as control samples, and FITC-4G9 was used for detection. Antibody, FACS results showed that this method can effectively detect the proportion of cells secreting bovine MCP-1.

The sixth aspect of the present invention relates to the use of the hybridoma cell line secreting bovine MCP-1 monoclonal antibody or the bovine MCP-1 monoclonal antibody MAb 4G9 of the present invention in the preparation of detection reagents or diagnostic reagents for bovine immune status application.

The seventh aspect of the present invention provides the application of the Western-Blot detection kit, the direct immunofluorescence DFA detection kit or the FCM detection kit of the present invention in the detection of bovine MCP-1 for non-diagnostic purposes. The detection includes the detection of recombinantly expressed bovine MCP-1, the detection of isolated tissues, epitope identification research, import and export inspection and quarantine, and epidemiological research.

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the spirit of the present invention.

Unless otherwise specified, the test methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the art. conventional techniques. These techniques are well described in the existing literature, see Sambrook et al. MOLECULARCLONING: A LABORATARY MANUAL, Second edition, 2001; Ausubel et al., CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; theseries METHODS IN ENZYMOLOGY , Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE ANG FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, ed.), AcademicPress, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999 et al.

Example 1: Acquisition of Hybridoma Cell Lines

The acquisition of the hybridoma cell line 4G9 with the deposit number of CCTCC NO: C2017281.

1. Animal Immunization

The specific immunization procedure is as follows: for the first immunization, 100 μg of bovine MCP-1 purified protein fully emulsified in Freund’s complete adjuvant was injected subcutaneously in the abdomen at multiple points, and 2 weeks later, 100 μg of purified protein fully emulsified in incomplete Freund’s adjuvant was injected subcutaneously in the abdomen at multiple points. The protein was immunized for the second time, and 100 μg of purified protein without adjuvant was injected intraperitoneally after 2 weeks for the third immunization. After 7 days, blood was collected to measure the serum antibody titer, and mice with higher titers were selected for intraperitoneal booster immunization of 100 μg without adjuvant. purified protein.

2. Cell fusion

The specific steps are as follows: 3 days after boosting immunization by tail vein injection, a small amount of blood is collected, serum is separated, and frozen at -20°C as a positive clone control during screening. The immunized mice were killed according to the biosafety method, immersed in 75% alcohol for 5 minutes, and the splenocytes were aseptically collected and fused with the myeloma cells SP2/0 in the logarithmic growth phase under the action of polyethylene glycol PEG (MW4000), and the mice were treated with ICR. Peritoneal macrophages were used as feeder cells, and the fused cells and feeder cells were suspended in HAT medium, divided into 96-well plates, and cultured in a 37°C, 5% CO ₂ incubator. After 5 days, fresh HAT medium was added, and after 10 days, HT medium was used for culture, and regular observation, medium change and detection were performed.

3. Establishment of an indirect ELISA detection method

Positive clones were screened by indirect ELISA. The phalanx assay determines the coating concentration of the detection antigen.

The detection antigen was serially diluted with coating buffer, and 100 μL per well was used to coat the ELISA plate, overnight at 4°C; PBST was washed 3 times, and 200 μL of blocking solution was added to each well, overnight at 4°C; the immunized mouse serum was longitudinally diluted, 100 μL per well. , normal mouse serum was diluted at the same fold as a negative control, incubated at 37°C for 2h; washed 3 times with PBST, added the enzyme-labeled secondary antibody at the working concentration, 100 μL per well, incubated at 37°C for 1h; after washing with PBST, add 3,3 The ',5,5'-tetramethylbenzidine (TMB) color was developed, and the OD ₄₅₀ value was determined by an enzyme-linked detector to determine the optimal coating concentration for detecting the antigen.

According to the coating concentration of the detection antigen determined by the square array test, 100 μL/well of the diluted detection antigen was added to the ELISA plate, overnight at 4°C, washed 3 times with PBST, 5 min/time, and washed with PBST containing 10% calf serum. The washing solution was blocked overnight at 4°C, washed, and stored at -20°C for screening positive cloned cells.

4. Screening for positive clones

The established indirect ELISA method was used to detect the secretion of antibodies by hybridoma cells. The specific method is as follows: add the supernatant of hybridoma cells to the ELISA plate that has been pre-coated with the optimal coating concentration determined in step 3, 100 μL/well, and use the supernatant of SP2/0 cells as a negative control to immunize with mouse polyclonal antibody Serum was used as a positive control, water bathed at 37°C for 2 hours; washed with PBST for 3 times; added working concentration of horseradish peroxidase HRP-labeled goat anti-mouse IgG, 100 μL/well, water bathed at 37°C for 1.5 hours; after washing, added TMB for color development 10-15min, after the color development is terminated, the OD ₄₅₀ reading is determined by the microplate reader. The OD ₄₅₀ reading of the tested wells was more than 2 times higher than that of the negative control, and it was judged as positive. The screened positive clone was named 4G9.

5. Cloning of positive hybridoma cells

The screened positive cell clone 4G9 was subcloned 2-3 times by limiting dilution method and preserved.

Example 2: Preparation of bovine MCP-1 monoclonal antibody

1. Ascites Preparation

The method of inducing ascites in vivo was carried out according to the conventional method. 10-12-week-old healthy BALB/c mice were injected intraperitoneally with 0.3-0.5 mL/mice of liquid paraffin, and 7-10 days later, the hybridoma cells 4G9, 5×10 ⁵ diluted with PBS and cultured to log phase growth were intraperitoneally inoculated. seven days later, the ascites was collected, the precipitate was removed by centrifugation, the supernatant was collected, the antibody titer was determined by indirect ELISA, and the cells were aliquoted and stored at -70°C.

2. Antibody Purification

The prepared MAb 4G9 ascites was purified using Protein A affinity chromatography.

Example 3: Detection of Monoclonal Antibody Characteristics

1. Identification of mAb subclasses

According to the instructions of the monoclonal antibody subtype kit, the antigen-mediated ELISA method was used. Add 100 μL/well of cell culture supernatant to the coated ELISA plate, wash 3 times with PBST at 37°C for 1 h, 5 min each time; add 1:1000 diluted goat anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM subclass antibodies 50 μL/well, 37°C for 0.5 h, add two wells of each subclass to each monoclonal antibody, wash 3 times with PBST for 5 min each; add 1:5000 diluted rabbit anti-goat enzyme-labeled secondary antibody 50 μL/ Well, 37°C for 15min, washed 3 times with PBST; add 100μL/well of TMB color developing solution, 37°C dark for 10-15min, 2M H ₂ SO ₄ 50μL/well to stop the reaction, the color is obviously higher than other wells by visual observation The added subclass of antibody is the monoclonal antibody subclass.

The results showed that the monoclonal antibody MAb 4G9 subclass was IgA.

2. Determination of monoclonal antibody ascites titer

Dilute the detection antigen to a certain concentration with coating buffer, coat ELISA plate with 100 μL per well, overnight at 4°C; wash 3 times with PBST, add 200 μL of blocking solution to each well, overnight at 4°C; dilute the monoclonal antibody in ascites fluid, each Well 100 μL, the same fold dilution of SP2/0 ascites as a negative control, incubated at 37 °C for 2 h; washed 3 times with PBST, added the enzyme-labeled secondary antibody at the working concentration, 100 μL per well, incubated at 37 °C for 1.5 h; after washing with PBST, TMB The OD ₄₅₀ value was determined by an enzyme-linked detector, and the ascites titer of the monoclonal antibody was determined with P/N ≥ 2.1 as the criterion.

The results showed that the titer of mAb MAb 4G9 was 1:1024000. The titer of mAb MAb 4G9 is high, indicating that its use in the preparation of diagnostic reagents can produce high sensitivity.

3. Identification of mAb specificity

The specificity of mAbs was identified by indirect ELISA. The same concentrations of rGST-BoMCP-1, rHis-BoIL-2, rGST-BoIL-2, rHis-BoIL-4, rGST-BoIL-4, rHis-BoIP-10, rGST-BoIP-10 were coated overnight; 1 day, washed 3 times with PBST for 5 min each, patted the liquid as dry as possible, blocked with 2% bovine serum albumin BSA in phosphate-buffered saline for 2 h; washed 3 times with PBST for 5 min each, patted the liquid as dry as possible, and added each Hybridoma cell supernatant, 100 μL/well, incubated at 37°C for 2 h; washed 6 times with PBST for 5 min each time, patted the washing solution as dry as possible, and added HRP-goat anti-mouse IgG diluted to the working concentration in PBS with 1% BSA , 100 μL/well, incubated at 37°C for 1 h; washed 6 times with PBST, 5 min each time, patted the washing solution as dry as possible, added TMB chromogenic solution, 100 μL/well, incubated at 37°C for 5 min in the dark; added 2mol/LH ₂ SO ₄ The reaction was terminated, 50 μL/well, and the OD ₄₅₀ value was determined.

In the indirect ELISA test, the monoclonal antibody MAb 4G9 only reacted with rGST-BoMCP-1, but not with the other recombinant cytokines expressed in prokaryotic cells, indicating that the monoclonal antibody MAb 4G9 secreted by the hybridoma cell line of the 1 The antigenic protein has good reactivity, specificity and affinity.

4. Flow cytometry to identify the reactivity of mAbs with native antigens

Aseptically collect 5 mL of tail venous blood from healthy cows and add it to a blood collection tube containing heparin sodium, invert the anticoagulant upside down and mix it for several times; Slowly add the diluted bovine blood along the tube wall into a sterile centrifuge tube containing bovine lymphocyte separation solution at a ratio of 1:1 to form a clear interface, and centrifuge at 2000 rpm for 25 min at room temperature; the liquid in the tube is divided into four layers, and the plasma and lymphocytes There is a visible cloud layer of peripheral blood mononuclear cells between the separation fluids, which are lymphocytes. Use a sterile dropper to suck the lymphocyte layer into a clean centrifuge tube, add 1640 medium, mix the cells, centrifuge at 2000 rpm for 10 min at room temperature, repeat twice, discard the supernatant; discard the supernatant medium, add The cells were resuspended in complete 1640 medium. After the cells were counted, the cells were added to a 24-well plate and divided into two groups. One group was added with bacterial lipopolysaccharide LPS stimulator at a final concentration of 10 μg/mL, and the other group was not added with LPS stimulation. , placed in a 37°C 5% CO ₂ incubator for 24h. After stimulating and culturing for 4-6 hours, add BFA to block; the next day, centrifuge at 2000rpm for 10min to collect cells; wash cells with PBS containing 1% BSA, centrifuge at 2000rpm for 10min, discard the supernatant; add fixative and let stand at room temperature for 15min ; Wash cells with PBS containing 1% BSA; centrifuge at 2000 rpm for 10 min, discard the supernatant; dilute the monoclonal antibody ascites with membrane-breaking agent, add LPS-stimulated group and unstimulated group respectively, 2 μL/sample. Act at room temperature for 20 min; wash cells with PBS containing 1% BSA; centrifuge at 2000 rpm for 10 min, discard the supernatant; add FITC-labeled goat anti-mouse IgG diluted to the working concentration with PBS containing 1% BSA, act in the dark at room temperature for 20 min, and use the The cells were washed twice with PBS containing 1% BSA, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded; the cells were resuspended in 200 μL of PBS containing 1% BSA for FACS detection.

The results of flow cytometry showed that there was a certain difference between the LPS stimulated group and the unstimulated group, indicating that MAb4G9 monoclonal antibody could recognize bovine natural MCP-1, showing its application potential as a diagnostic reagent.

Example 4: Assembly of Western-Blot Kit

The assembly steps of the Western-blot kit are as follows:

1. Preparation of horseradish peroxidase-labeled bovine MCP-1 monoclonal antibody (named HRP-4G9):

Weigh 2 mg of horseradish peroxidase (HRP) in a 5 mL centrifuge tube and dissolve it in 0.5 mL of deionized water. At this time, the solution turns reddish brown and immediately save it from light; add 0.5 mL of freshly prepared 0.06mol/L sodium periodate. , the solution turns blue at this time, and protect from light at 4°C for 30min; add 160mmol/L ethylene glycol 0.5mL for 30min at room temperature; add purified monoclonal antibody 4G9 2mg and mix well, put the mixture into a dialysis bag at 2L 0.05mol/L Dialyze in carbonate buffer, overnight at 4 °C; transfer the dialysate to a centrifuge tube, add 0.2 mL of freshly prepared 5 mg/mL sodium borohydride, mix well, and act at 4 °C for 2 h; add an equal volume of saturated ammonium sulfate at 4 °C Act for 30min, centrifuge at 4000rpm/min for 20min, discard the supernatant, dissolve the precipitate with PBS and dialyze overnight at 4°C, add an equal volume of glycerol, add 0.1% ProClin300, filter with a 0.45μm filter, divide into 110μL/branch, and store at 2-8°C .

2. The horseradish peroxidase-labeled bovine MCP-1 monoclonal antibody MAb 4G9 (HRP-4G9) and the bacterial lipopolysaccharide LPS stimulator were packaged and assembled into a kit.

Further, the kit needs to be assembled in sequence: one or more of a blocking solution, a washing solution, and a substrate solution.

Example 5: Western-Blot detection of bovine recombinant MCP-1 protein

1. SDS-PAGE electrophoresis

The purified proteins rHis-BoMCP-1 and rGST-BoMCP-1 were added to protein loading buffer respectively, boiled at 100°C for 10 min, and subjected to SDS-PAGE electrophoresis.

2. Transfer

After electrophoresis, soak the gel in the transfer buffer for 5 minutes, and soak the NC membrane and filter paper at the same time. From top to bottom, the order is 2 pieces of filter paper - NC membrane - gel - 2 pieces of filter paper. Transfer to a semi-dry transfer machine, taking care to remove air bubbles. Transfer at 0.8 mA/cm ² for 2 h in a Bio-Rad semi-Dry transfer Cell system.

3. Detection

①Block with PBS containing 5% skimmed milk, and block overnight with shaking at room temperature;

② Wash the membrane with PBST, wash 4 times for 10 minutes each time, and dry the residual washing solution on the membrane as much as possible;

③Add HRP-4G9 diluted 1:1000 with PBS, shake at room temperature for 2h;

④ Wash the membrane with PBST for 4 times, 10 min each time, and dry the residual washing solution on the membrane as much as possible; after color development, the reaction was terminated with tap water, and the reaction result was photographed by a scanner.

The results are shown in Figure 1, A shows the detection result of recombinant His-BoMCP-1 protein, and B shows the detection result of recombinant GST-BoMCP-1 protein. The results showed that Western-Blot method could effectively detect bovine MCP-1 protein expressed in E. coli expression system, with good sensitivity and specificity.

Example 6: Western-Blot detection of bovine peripheral blood mononuclear cell samples

1. Preparation of Bovine Peripheral Blood Mononuclear Cells

① Aseptically take 5 mL of bovine blood to be tested and add it to a blood collection tube containing heparin sodium, invert and mix to obtain anticoagulation after blood collection;

② After diluting the anticoagulant and sterile PBS 1:1, slowly add the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation medium at a ratio of 1:1 to form a clear interface, at room temperature 2000rmp Centrifuge for 20-30min;

③ It can be seen that the peripheral blood mononuclear cells exist in the cloudy layer. Use a sterile dropper to suck the peripheral blood mononuclear cell layer into a clean centrifuge tube, add sterile PBS, mix the cells, and centrifuge at 2000rmp at 4°C 10min, repeated twice to obtain precipitated cells;

④ Discard the supernatant medium, add complete 1640 medium to resuspend the pelleted cells, take 10 μL of cell suspension, add 10 μL of phenol blue, mix well, add to a hemocytometer, count under a microscope, and use complete 1640 medium to suspend the cell suspension. Dilute to ¹ x 107 cells/mL.

2. Cell Incubation

Add the following reagents to a 24-well cell culture plate: 500 μL of culture medium to each control well, 500 μL of 10 μg/mL LPS to each positive well. Add 500 μL of diluted cell suspension to each well, place at 37°C, 5% CO ₂ incubator for 24-48 hours, collect cells as test samples;

3. SDS-PAGE electrophoresis

The cell lysate was added to protein loading buffer, boiled at 100 °C for 10 min, and subjected to SDS-PAGE electrophoresis.

4. Transfer

After electrophoresis, soak the gel in the transfer buffer for 5 minutes, and soak the NC membrane and filter paper at the same time. From top to bottom, the order is 2 pieces of filter paper - NC membrane - gel - 2 pieces of filter paper. Transfer to a semi-dry transfer machine, taking care to remove air bubbles. Transfer at 0.8 mA/cm ² for 2 h in a Bio-Rad semi-Dry transfer Cell system.

5. Detection

①Block with PBS containing 5% skimmed milk, and block overnight with shaking at room temperature;

② Wash the membrane with PBST, wash 4 times for 10 minutes each time, and dry the residual washing solution on the membrane as much as possible;

③Add HRP-4G9 diluted 1:1000 with PBS, shake at room temperature for 2h;

④ Wash the membrane with PBST for 4 times, 10 min each time, and dry the residual washing solution on the membrane as much as possible; after color development, the reaction was terminated with tap water, and the reaction result was photographed by a scanner.

The results showed that the Western-Blot detection kit could effectively detect the natural bovine MCP-1 protein secreted by bovine peripheral blood mononuclear cells, with good sensitivity and specificity.

Example 7: Assembly of the Direct Immunofluorescence (DFA) Detection Kit

The assembly steps of the DFA kit are as follows:

1. Preparation of fluorescein isothiocyanate-labeled bovine MCP-1 monoclonal antibody (named FITC-4G9):

The purified MAb 4G9 monoclonal antibody was labeled with standard fluorescein isothiocyanate labeling method. Dialyze the cross-linked monoclonal antibody MAb4G9 (concentration ≥ 1 mg/ml) against the cross-linking reaction solution at 4°C for three times to pH 9.0; dissolve freshly prepared FITC (concentration 1 mg/mL) in DMSO; press P:F (Protein: FITC) = 1 mg: 150 μg. Slowly add FITC to the antibody solution, shake gently while adding to mix it with the antibody evenly, and react at 4°C in the dark for 8 h; add 5 mol/L NH ₄ Cl to the end Concentration of 50 mmol/L, the reaction was terminated at 4°C for 2 h; the cross-linked product was dialyzed in PBS for more than four times until the dialysate was clear; the protein concentration and F/P ratio of the cross-linked product were identified; the protein cross-linked by FITC should be placed in Add 0.1% NaN ₃ and 1% BSA to phosphate buffer at pH 7.4, and store at 4°C in the dark.

2. Fluorescein isothiocyanate-labeled MCP-1 monoclonal antibody 4G9 (FITC-4G9) and bacterial lipopolysaccharide LPS stimulator were packaged and assembled into a kit respectively.

Further, one or more of a fixative and a washing solution are assembled into the kit as required.

Example 8: Direct immunofluorescence (DFA) detection of bovine recombinant MCP-1 protein

1. Transfection

①24h before transfection, digest and inoculate HEK293T cells in a 24-well plate, 2×10 ⁵ cells/well;

1.5μL至含Opti-MEM的1.5mL指形管中至总体积50μL；②Join

1.5 μL into a 1.5 mL finger tube with Opti-MEM to a total volume of 50 μL;

③ Add 1 μL each of recombinant plasmid pVAX1-GFP-BoMCP-1 and empty vector plasmid pVAX1, and 2 μL of P3000 ^TM to a 1.5 mL finger tube containing Opti-MEM to a total volume of 50 μL;

Opti-MEM 50μL，静置5min，分别加入细胞板中，置培养箱继续培养。④Add the containing

Opti-MEM 50 μL, let stand for 5 min, added to the cell plate respectively, and placed in an incubator to continue culturing.

2. Cytokine detection

①Aspirate the culture supernatant of HEK293T(pVAX1-GFP-BoMCP-1) and HEK293T(pVAX1) cells, wash twice with 500μL PBS, add 500μL -20℃ pre-cooled ice methanol and fix at -20℃ for 10min;

② Wash three times with 500 μL PBS, add MAb 4G9 diluted 1:1000, and incubate in a 37°C water bath for 2 hours;

③ Rinse three times with 500 μL PBS, add FITC-labeled goat anti-mouse IgG, dilute with PBS to a working concentration of 1:500, and place in a 37°C water bath for 1 h;

④ Wash three times with 500 μL PBS and observe with a fluorescent inverted microscope.

The results showed that the direct immunofluorescence (DFA) method could effectively detect bovine MCP-1 protein in eukaryotic expression system with good sensitivity and specificity.

Example 9: Direct Immunofluorescence (DFA) Detection of Bovine Peripheral Blood Mononuclear Cell Samples

1. Preparation of Bovine Peripheral Blood Mononuclear Cells

① Aseptically take 5 mL of bovine blood to be tested and add it to a blood collection tube containing heparin sodium, invert and mix to obtain anticoagulation after blood collection;

② After diluting the anticoagulant and sterile PBS 1:1, slowly add the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation medium at a ratio of 1:1 to form a clear interface, and centrifuge at 2000rmp at room temperature 20-30min;

③ It can be seen that the peripheral blood mononuclear cells exist in the cloudy layer. Use a sterile dropper to suck the peripheral blood mononuclear cell layer into a clean centrifuge tube, add sterile PBS, mix the cells, and centrifuge at 2000rmp at 4°C 10min, repeated twice to obtain precipitated cells;

④ Discard the supernatant medium, add complete 1640 medium to resuspend the pelleted cells, take 10 μL of cell suspension, add 10 μL of phenol blue, mix well, add to a hemocytometer, count under a microscope, and use complete 1640 medium to suspend the cell suspension. Dilute to ¹ x 107 cells/mL.

2. Cell Incubation

Add the following reagents to a 24-well cell culture plate: 500 μL of culture medium to each control well, 500 μL of 10 μg/mL LPS to each positive well. Add 500 μL of the diluted cell suspension to each well, place it in a 37°C, 5% _CO2 incubator for 24-48 hours, and collect the cells;

3. Cytokine detection

①Aspirate the culture supernatant of cells, wash twice with 500μL PBS, add 500μL -20℃ pre-cooled ice methanol and fix at -20℃ for 10min;

② Wash three times with 500 μL PBS, add MAb 4G9 diluted 1:1000, and incubate in a 37°C water bath for 2 hours;

③ Rinse three times with 500 μL PBS, add FITC-labeled goat anti-mouse IgG, dilute with PBS to a working concentration of 1:500, and place in a 37°C water bath for 1 h;

④ Wash three times with 500 μL PBS and observe with a fluorescent inverted microscope.

The results are shown in Figure 2. The results show that when detecting bovine peripheral blood mononuclear cell samples, the positive sample well (B) has obvious green fluorescence, while the control sample well (A) has no green fluorescence, which indicates that the kit can effectively detect LPS Stimulates cells secreting bovine MCP-1 in bovine peripheral blood with good sensitivity and specificity.

Example 10: Assembly of FCM Detection Kit

The assembly steps of the FCM detection kit are as follows:

1. Preparation of fluorescein isothiocyanate-labeled bovine MCP-1 monoclonal antibody (named FITC-4G9):

The purified MAb 4G9 monoclonal antibody was labeled with standard fluorescein isothiocyanate labeling method. Dialyze the cross-linked monoclonal antibody MAb4G9 (concentration>1mg/mL) against the cross-linking reaction solution at 4°C for three times to pH 9.0; dissolve freshly prepared FITC (concentration: 1mg/mL) in DMSO; press P:F (Protein: FITC) = 1 mg: 150 μg. Slowly add FITC to the antibody solution, shake gently while adding to mix it with the antibody evenly, and react at 4°C in the dark for 8 hours; add 5 mol/L NH ₄ Cl to The final concentration was 50 mmol/L, and the reaction was terminated at 4°C for 2 h; the cross-linked product was dialyzed in PBS for more than four times until the dialysate was clear; the protein concentration and F/P ratio of the cross-linked product were identified; the protein cross-linked by FITC should be placed Add 0.1% NaN ₃ and 1% BSA to phosphate buffer at pH 7.4, and store at 4°C in the dark.

2. Fluorescein isothiocyanate-labeled bovine MCP-1 monoclonal antibody and bacterial lipopolysaccharide LPS stimulator were packaged and assembled into a kit respectively.

Further, the kit needs to be assembled in sequence: one or more of the blocking agent Brefeldin, a fixative, a membrane breaking agent and a washing solution.

Example 11: FCM detection of bovine peripheral blood mononuclear cell samples

1. Preparation of Bovine Peripheral Blood Mononuclear Cells

① Aseptically take 5 mL of bovine blood to be tested and add it to a blood collection tube containing heparin sodium, invert and mix to obtain anticoagulation after blood collection;

② After diluting the anticoagulant and sterile PBS 1:1, slowly add the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation medium at a ratio of 1:1 to form a clear interface, at room temperature 2000rmp Centrifuge for 20-30min;

③ It can be seen that the peripheral blood mononuclear cells exist in the cloudy layer. Use a sterile dropper to suck the peripheral blood mononuclear cell layer into a clean centrifuge tube, add sterile PBS, mix the cells, and centrifuge at 2000rmp at 4°C 10min, repeated twice to obtain precipitated cells;

④ Discard the supernatant medium, add complete 1640 medium to resuspend the pelleted cells, take 10 μL of cell suspension, add 10 μL of phenol blue, mix well, add to a hemocytometer, count under a microscope, and use complete 1640 medium to suspend the cell suspension. Dilute to ¹ x 107 cells/mL.

2. Cell Incubation

①Add the following reagents to a 96-well cell culture plate: 50 μL of culture medium to each control well, 50 μL of 10 μg/mL LPS to each positive well. Add 50 μL of cell suspension to each well, and place the 96-well cell culture plate in a 37°C, 5% _CO2 incubator for 4-6 hours;

② Add the cytokine secretion blocking agent BFA, and culture in a 37°C, 5% CO ₂ incubator for 16 hours.

3. Cytokine detection:

① Collect the cultured cells the next day, wash the cells with PBS containing 1% BSA, centrifuge at 2000 rpm at 4°C for 10 min, and discard the supernatant;

②Add fixative and let stand at room temperature for 15min; wash cells with PBS containing 1% BSA; centrifuge at 4°C at 2000rpm for 10min, discard the supernatant, repeat twice;

③ Dilute the FITC-labeled mAb4G9 specific for bovine MCP-1 with a membrane-breaking agent to 0.5 μg/mL, let stand for 15 min at room temperature, wash the cells with PBS containing 1% BSA; centrifuge at 4°C at 2000 rpm for 10 min, discard the supernatant , repeated twice;

④ Resuspend the cells in 200 μL PBS, and perform FACS to detect the proportion of cells secreting bovine MCP-1.

The results are shown in Figure 3. The results show that when bovine peripheral blood mononuclear cell samples are detected, the proportion of cells secreting bovine MCP-1 in positive sample well B (0.345%) is higher than that in control sample wells (0.131%), which indicates that the kit is effective The detection of LPS-stimulated cells secreting bovine MCP-1 in bovine peripheral blood has good sensitivity and specificity.

The above are only preferred examples of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Without departing from the scope of the technical solution of the present invention, when the technical content disclosed above can be used to make some changes or modifications to equivalent embodiments with equivalent changes, but any content that does not depart from the technical solution of the present invention, according to the technical essence of the present invention Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solutions of the present invention.

Claims (10)

1. A hybridoma cell strain secreting bovine MCP-1 monoclonal antibody is characterized in that the hybridoma cell strain is a hybridoma cell strain 4G9 or a subcultured cell strain thereof, and the preservation number of the hybridoma cell strain 4G9 is CCTCC NO: C2017281.

2. A bovine MCP-1 monoclonal antibody MAb4G9 secreted by the hybridoma cell line secreting bovine MCP-1 monoclonal antibody according to claim 1.

3. A Western-Blot detection kit for detecting bovine MCP-1, which comprises a detection antibody and a bacterial Lipopolysaccharide (LPS) stimulant, wherein the detection antibody is the bovine MCP-1 monoclonal antibody MAb4G9 labeled by horseradish peroxidase according to claim 2.

4. The Western-Blot assay kit of claim 3, further comprising one or more of the following reagents:

1) sealing liquid;

2) substrate solution; and

3) and (4) washing liquid.

5. A direct immunofluorescence DFA detection kit for detecting bovine MCP-1, comprising a detection antibody and a bacterial Lipopolysaccharide (LPS) stimulant, wherein the detection antibody is fluorescein isothiocyanate labeled bovine MCP-1 monoclonal antibody MAb4G9 according to claim 2.

6. The direct immunofluorescence DFA detection kit according to claim 5, wherein said kit further comprises one or more of the following reagents:

1) a fixative; and

2) and (4) washing liquid.

7. An FCM detection kit of bovine MCP-1, comprising a detection antibody and a bacterial Lipopolysaccharide (LPS) stimulator, wherein the detection antibody is fluorescein isothiocyanate labeled bovine MCP-1 monoclonal antibody MAb4G9 according to claim 2.

8. The FCM detection kit according to claim 7, further comprising one or more of the following reagents:

1) a blocker brefeldin;

2) fixative, membrane rupture agent; and

3) and (4) washing liquid.

9. Use of the hybridoma cell line according to claim 1 or the bovine MCP-1 monoclonal antibody MAb4G9 according to claim 2 in preparation of a detection reagent or a diagnostic reagent for the immune state of a bovine body.

10. Use of a test kit according to any one of claims 3, 5, 7 for the detection of bovine MCP-1 for non-diagnostic purposes including the detection of recombinantly expressed bovine MCP-1, the detection of ex vivo tissue, epitope identification studies, epidemiological studies.

Images