A hybridoma cell line secreting monoclonal antibody against yak IgG, the monoclonal antibody and its application

Abstract

The present invention provides a hybridoma cell line secreting monoclonal antibodies against yak IgG, its monoclonal antibody and applications, belonging to the field of biotechnology. The preservation number of this hybridoma cell line is CGMCC No. 46011. The present invention provides a hybridoma cell line (i.e., anti-yak IgG hybridoma cell line EF37) capable of stably and efficiently secreting monoclonal antibodies against yak IgG. This hybridoma cell line can efficiently and stably secrete monoclonal antibodies against yak IgG, thus making up for the situation that there is no diagnostic reagent specifically for monoclonal antibodies against yak IgG at home and abroad. The monoclonal antibody against yak IgG prepared in the present invention has a good reaction effect on yak IgG, and the antibody titer is above 1:10000. When the monoclonal antibody against yak IgG is applied to the detection of antibodies against yak brucellosis, both the sensitivity and specificity are above 95%. The monoclonal antibody against yak IgG can be used for the research and detection of yak-related biology and diseases, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a hybridoma cell line secreting monoclonal antibodies against yak IgG, its monoclonal antibody and applications thereof. Background Art

[0002] With the development of China's economy and the improvement of the awareness of food quality. As an economic animal for specific purposes, the value generated by yaks in industries such as the textile industry, food industry and tourism industry is increasing year by year. Compared with other domestic animals, the types of biological products such as antibodies, vaccines and diagnostic products for yaks are very scarce, which causes difficulties in the prevention and control of yak diseases (such as brucellosis (referred to as brucellosis)).

[0003] Immunoglobulin IgG is the most important immunoglobulin produced by the body, the main component of immunoglobulins, accounting for about 75% of the total immunoglobulin content in serum. IgG is the main antibody in the body, with functions of antiviral, neutralizing virus, antibacterial and immunomodulation. Changes in IgG often indicate changes in the occurrence and development of diseases.

[0004] IgG has obvious species specificity, that is, each animal has its own unique IgG. From a genetic relationship perspective, yaks are in an independent branch among domestic cattle. Research shows that the genetic distance between yaks and domestic cattle is relatively close, but there are obvious differences in their genomes. Yaks are a bovine species in the genus Bos, and the genus Bos includes 15 species such as the common cattle species (Bos taurus), zebu species (Bos indicus), yak species (Bos grunniens), etc. The genetic differences between yaks and common cattle are obvious in the development and application of serological diagnostic reagents. When an indirect ELISA kit used for detecting diseases in common domestic cattle is directly used for yak diagnosis, the reactivity is greatly reduced and it cannot be used. The most important reason is that the enzyme-labeled secondary antibody against common cattle IgG in the kit reacts weakly with IgG in yak serum. If it is replaced with an enzyme-labeled secondary antibody against yak IgG at an appropriate concentration, the ELISA reaction result is significantly improved.

[0005] Currently, there are no monoclonal antibodies against yak IgG and their products at home and abroad. The serological diagnostic methods for various yak diseases have been blank at home and abroad, seriously restricting the development of serological diagnosis of yak diseases and disease prevention and control technologies. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the purpose of the present invention is to provide a hybridoma cell line EF37 against yak IgG. This hybridoma cell line EF37 against yak IgG can efficiently and stably secrete monoclonal antibodies against yak IgG, and the monoclonal antibodies against yak IgG can be used for the detection of yak IgG and yak-related diseases.

[0007] The present invention provides a hybridoma cell line with a preservation number of CGMCC No. 46011.

[0008] The present invention also provides a monoclonal antibody against yak IgG, which is characterized in that it is secreted by the above-mentioned hybridoma cell line.

[0009] Furthermore, the monoclonal antibody against yak IgG belongs to the IgG1 subtype.

[0010] The present invention also provides a method for preparing the above-mentioned monoclonal antibody against yak IgG. The method includes the following steps: injecting the above-mentioned hybridoma cell line into the abdominal cavity of a mouse, raising it, extracting ascites, and purifying it to obtain the monoclonal antibody against yak IgG.

[0011] The present invention also provides an immunoassay kit, which contains the above-mentioned monoclonal antibody against yak IgG.

[0012] Furthermore, the immunoassay kit is an enzyme-linked immunosorbent assay kit.

[0013] The present invention also provides the use of the above-mentioned monoclonal antibody against yak IgG and the above-mentioned immunoassay kit in the preparation of reagents for detecting yak IgG.

[0014] Furthermore, the reagent for detecting yak IgG is a reagent for detecting yak diseases.

[0015] Furthermore, the disease is brucellosis.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. By prokaryotic expression, purification of a partial sequence of the FC segment of yak IgG protein, immunizing mice, preparing monoclonal antibodies, and screening, the present invention obtained a hybridoma cell line (i.e., hybridoma cell line EF37 against yak IgG) that can stably and efficiently secrete monoclonal antibodies against yak IgG. This hybridoma cell line EF37 against yak IgG can efficiently and stably secrete monoclonal antibodies against yak IgG, making up for the lack of specific monoclonal antibodies against yak IgG at home and abroad.

[0018] 2. The monoclonal antibody against yak IgG prepared by the present invention has a good reaction effect on yak IgG, and the antibody titer is above 1:10000.

[0019] 3. The monoclonal antibody against yak IgG prepared by the present invention has no cross-reactivity with the immunoglobulins of other animals.

[0020] 4. When the monoclonal antibody against yak IgG prepared by the present invention is applied to the detection of yak brucellosis antibodies, the sensitivity and specificity of the established kit are both above 95%.

[0021] 5. The monoclonal antibody against yak IgG prepared by the present invention can be used for the research and detection of yak-related biology and diseases, and has broad application prospects.

[0022] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0023] The above content of the present invention will be further described in detail below through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0024] Figure 1 : SDS-PAGE electrophoresis pattern of yak IgG recombinant protein. In the figure, 1 is the marker, and 2 and 3 are two batches of purified yak IgG recombinant proteins.

[0025] Figure 2 : SDS-PAGE electrophoresis pattern of the purified monoclonal antibody. In the figure, 1 is the marker, and 2 is the purified monoclonal antibody.

[0026] Figure 3 : Distribution diagram of SP values detected by indirect ELISA of known positive and negative sera.

[0027] Figure 4 : ROC curve of the indirect ELISA method for yak brucellosis established based on HRP-labeled monoclonal antibody against yak IgG. Detailed Description of the Embodiments

[0028] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0029] Example 1 Preparation, Screening and Identification of Hybridoma Cells of Monoclonal Antibody Against Yak IgG

[0030] I. Expression and Purification of Yak IgG Recombinant Protein

[0031] (1) Design forward and reverse primers according to the gene sequence of yak IgG in NCBI (GenBank accession number: NM_001303570.1), and insert restriction enzyme site sequences into the forward and reverse primers. The primer sequences are FC-F: 5'-CTCGAGGCAGGGCCTGCT-3' (SEQ ID NO.1) and FC-R: 5'-GGATCCTCAGCATCTTTGGAAAG-3' (SEQ ID NO.2).

[0032] (2) Take yak spleen tissue, homogenize it and extract mRNA. After reverse transcription, use it as a template to amplify the target fragment, and the size of the target fragment is 759 bp.

[0033] (3) Perform reverse amplification on the pET-28a(+) empty vector using the PET28-F / R primers. The primer sequences are: PET28-F: 5'-GGCAGCAGCCATCATCATCATCATCACA-3' (SEQ ID NO.3) and PET28-R: 5'-CATGGTATATCTCCTTCTTAAAGTTAAACAAAATT-3' (SEQ ID NO.4).

[0034] (4) Use a homologous recombination cloning kit to ligate the target fragment and the vector by homologous recombination, and transform the product into BL21 competent cells.

[0035] (5) After culturing at 37°C for 16 - 18 hours, pick single colonies for identification.

[0036] (6) Inoculate positive clones into LB medium, culture them with shaking at 37°C, and add IPTG with a final concentration of 1 mM when the absorbance reaches 0.4 - 0.8 for induction for 3 - 6 hours.

[0037] (7) Centrifuge the culture medium to collect the bacteria. After washing twice with 0.01 M phosphate buffer, resuspend the precipitate with deionized water, freeze-thaw twice and then ultrasonically disrupt it.

[0038] (8) After centrifuging the ultrasonicated product, take the supernatant and purify it with a nickel column to obtain purified recombinant yak IgG protein. The SDS-PAGE electrophoresis results of the recombinant yak IgG protein are as Figure 1 shown.

[0039] II. Preparation of hybridoma cells secreting monoclonal antibodies against yak IgG

[0040] (1) Take the purified recombinant yak IgG protein, dilute it with physiological saline to 0.1 mg / ml, emulsify it with an equal volume of Freund's complete adjuvant, and immunize 8-week-old BALB / c mice by subcutaneous multi-point injection. Fourteen days after immunization, use an emulsion of Freund's incomplete adjuvant and recombinant protein to boost the immunization at the same dose, and boost the immunization continuously three times. The interval between booster immunizations is 7-10 days.

[0041] (2) Ten days after the last immunization, collect mouse serum from the tail vein for ELISA antibody detection. Select one mouse with an antibody level above 20,000 for the preparation of monoclonal antibody hybridoma cells.

[0042] (3) Sacrifice the mouse by bleeding, make a single-cell suspension from the spleen cells, and perform cell fusion with SP2 / 0 myeloma cells according to the standard procedure.

[0043] (4) Seven to ten days after fusion, select the wells containing single clusters of hybridoma cells to take the supernatant, and perform ELISA screening with an enzyme-linked immunosorbent assay plate coated with recombinant yak IgG protein to obtain strongly reactive positive hybridoma cell lines.

[0044] (5) For the selected hybridoma cells, perform two subclonings using the limiting dilution method, and perform ELISA screening with an enzyme-linked immunosorbent assay plate coated with recombinant yak IgG protein to obtain hybridoma cells with a high level of monoclonal antibody secretion. After expanded culture, freeze them in liquid nitrogen.

[0045] (6) Select the EF37 strain with the highest ELISA titer of monoclonal antibody secretion as the cell line for the preparation of monoclonal antibodies in this patent, named anti-yak IgG hybridoma cell line EF37.

[0046] The anti-yak IgG hybridoma cell line EF37 has been deposited in the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 1, 2024, and the deposit number is CGMCC No. 46011. The taxonomic name of this biological material is: anti-yak IgG hybridoma cells.

[0047] Example 2 Preparation, Purification and Identification of Anti-Yak IgG Monoclonal Antibody

[0048] I. Preparation of Ascites Containing Anti-Yak IgG Monoclonal Antibody

[0049] (1) Take BALB / c mice over 12 weeks old, and inject 0.5 mL of liquid paraffin intraperitoneally 7-10 days before ascites preparation to expand the abdomen.

[0050] (2) The anti-yak IgG hybridoma cell line EF37 that has been expanded and cultured for recovery is gently washed twice with serum-free DMEM, then pipetted evenly and counted. Take 50,000 - 100,000 cells per mouse and inject them intraperitoneally into the mice after laparotomy. Continue to raise the mice for 8 - 10 days. When the abdomen becomes swollen and pear-shaped, collect the ascites of the mice.

[0051] (3) Centrifuge the collected ascites at high speed at 4°C, 12,000 rpm for 15 minutes, collect the supernatant, and use an ELISA plate coated with yak IgG recombinant protein to measure the antibody titer. The antibody titer of the ascites should not be lower than 1:10,000.

[0052] (4) Aliquot the ascites and store it frozen at -65°C or below for later use.

[0053] II. Determination of the titer of monoclonal antibody

[0054] (1) Dilute the yak IgG recombinant protein to 1 μg / mL with carbonate buffer (pH 9.6). Take a 96-well ELISA plate and add 100 μl to each well, and incubate it overnight at 2 - 8°C for coating.

[0055] (2) Discard the coating solution, wash once with PBS (0.01 mol / L, pH 7.2), then add 100 μl of blocking solution (PBS (0.01 mol / L, pH 7.2) containing 5% BSA) to each well. After blocking at 37°C for 2 hours, discard the blocking solution and store it at 2 - 8°C for later use.

[0056] (3) Dilute the centrifuged ascites 1:1000 times with PBS (0.01 mol / L, pH 7.2), and then serially dilute it two-fold to 1:128,000 times.

[0057] (4) Take the ELISA plate prepared in (2), add the monoclonal antibody of each dilution, 100 μl per well, and incubate at 37°C for 30 minutes.

[0058] (5) Discard the reaction solution, add 350 μl of washing solution to each well, wash 3 times, and pat dry on absorbent paper.

[0059] (6) Dilute the HRP-labeled goat anti-mouse IgG 5000 times with PBS (0.01 mol / L, pH 7.2) containing 2% BSA, then add 100 μl to each well, and incubate at 37°C for 30 minutes.

[0060] (7) Repeat step (5).

[0061] (8) Add TMB chromogenic solution, 100 μl per well, and develop color at room temperature in the dark for 15 minutes. Then add 50 μl of stop solution (2 mol / L HCl), and measure the OD with an ELISA reader. 450nmValue. With OD 450nm The maximum dilution factor ≥ 0.2 was taken as the final titer of the monoclonal antibody.

[0062] III. Purification and Identification of Monoclonal Antibody

[0063] (1) After thawing the ascites stored in Step 1, centrifuge at 12,000 rpm for 15 minutes at 2 - 8°C, and take the supernatant.

[0064] (2) Add 2 volumes of acetate buffer to the supernatant, adjust the pH value to 4.5, and slowly add caprylic acid dropwise with stirring using a magnetic heating stirrer at room temperature. The addition amount is 33 μl / ml. After dropping, continue to stir for 30 minutes and let it stand at 4°C for 2 hours;

[0065] (3) Centrifuge at 12,000 rpm for 30 minutes, take the supernatant, filter it with filter paper, add 10×PBS buffer (0.01 mol / L, pH value 7.4) according to 10% volume, adjust the pH value to 7.4, add ammonium sulfate to a final concentration of 45% at 0.277 g / ml while stirring, continue to stir for 30 minutes after adding, let it stand overnight at 2 - 8°C, and centrifuge at 10,000 rpm for 20 minutes at 4°C to collect the precipitate.

[0066] (4) Dissolve the collected precipitate with PBS (0.01 mol / L, pH value 7.2) to restore the original volume, and dialyze at 2 - 8°C for 8 hours, with dialysis performed twice.

[0067] (5) After measuring the concentration of the protein solution after dialysis, identify the purification effect of the monoclonal antibody by SDS - PAGE. Figure 2 , after aliquoting, store it at -65°C or below, and the validity period is 72 months.

[0068] (6) Take the purified monoclonal antibody and detect it using an antibody subtype detection kit. The detection results are shown in Table 1, and the results indicate that the monoclonal antibody prepared from the anti - yak IgG hybridoma cell line EF37 is of the IgG1 subtype.

[0069] Table 1 Identification Results of Monoclonal Antibody Subtypes

[0070] Antibody subtype IgA IgG1 IgG2a Ig2b Ig3 IgM <![CDATA[Monoclonal antibody OD 450nm Mean value]]> 0.087 1.761 0.07032 0.0575 0.0832 0.137

[0071] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0072] Experimental Example 1 Identification of Reactivity of Anti - Yak IgG Monoclonal Antibody with Various Bovine Serum IgGs and Other Animal Serum IgGs

[0073] (1) Take yak serum, dairy cow serum, yellow cattle serum, Huaxi beef cattle serum, horse serum, and pig serum, add equal volumes of carbonate buffer respectively, coat 2 strips of an enzyme - linked immunosorbent assay (ELISA) plate with each, add 100 μl to each well, and place it at 2 - 8°C for overnight coating.

[0074] (2) Discard the coating solution. After washing once with PBS (0.01 mol / L, pH 7.2), add 100 μl of blocking solution [PBS (0.01 mol / L, pH 7.2) containing 5% BSA] to each well. After blocking at 37 °C for 2 hours, discard the blocking solution and store at 2 - 8 °C for later use.

[0075] (3) Dilute the prepared ascites with PBS (0.01 mol / L, pH 7.2) at a ratio of 1:20000, and then add 100 μl to each well of the enzyme - labeled plate coated with different types of bovine serum, horse serum, and porcine serum.

[0076] (4) Incubate at 37 ± 1 °C for 30 minutes. Then, take out the reaction plate, discard the reaction solution, add 350 μl of washing solution to each well, wash 3 times, and pat dry on absorbent paper.

[0077] (5) Dilute HRP - labeled goat anti - mouse IgG with PBS (0.01 mol / L, pH 7.2) at a ratio of 1:10000, add 100 μl to each well, incubate at 37 ± 1 °C for 30 minutes, discard the reaction solution, add 350 μl of washing solution to each well, wash 3 times, and pat dry on absorbent paper.

[0078] (6) Add the chromogenic solution, 100 μl per well, and develop color at room temperature in the dark for 15 minutes. Then add 50 μl of stop solution (2 mol / L hydrochloric acid) to each well to terminate the reaction. Measure the OD 450nm value with an enzyme - labeled instrument.

[0079] The results show that the anti - yak IgG monoclonal antibody prepared by the present invention has no reactivity to porcine serum and horse serum (OD450nm value < 0.2), has the strongest reactivity to yak serum, and has relatively weak reactivity to other bovine sera. The results are shown in Table 2.

[0080] Table 2 Reactivity results of anti - yak IgG monoclonal antibody to IgG in various bovine, horse, and porcine sera

[0081]

[0082]

[0083] Experimental Example 2 Method for using anti - yak IgG monoclonal antibody to detect yak brucellosis

[0084] I. Labeling of horseradish peroxidase (HRP)

[0085] This method is used for the HRP labeling of anti - yak IgG monoclonal antibody and the purification of the labeled product. The steps are as follows:

[0086] (1) Weigh 5 mg of HRP and dissolve it in 1 ml of distilled water. Add 0.2 ml of freshly prepared 0.1 mol / L sodium iodate solution thereto and stir gently in the dark at room temperature for 20 minutes.

[0087] (2) Put the above solution into a dialysis bag and dialyze it using sodium acetate buffer (0.001 mol / L, pH 4.4) overnight at 2 - 8°C.

[0088] (3) Immediately add 10 mg of anti - yak IgG monoclonal antibody to 1 ml of carbonate buffer (0.01 mol / L, pH 9.6) and stir gently in the dark at room temperature for 2 hours.

[0089] (4) Add 0.1 ml of freshly prepared NaBH4 solution (4 mg / ml), mix well, and keep at 2 - 8°C for 2 hours.

[0090] (5) Put the above solution into a dialysis bag and dialyze it with PBS (0.01 mol / L, pH 7.2) overnight at 2 - 8°C.

[0091] (6) Weigh 100 g of ammonium sulfate, add 90 ml of water, heat to dissolve, then let it cool at room temperature. After the ammonium sulfate crystals precipitate stably, adjust the pH value to 7.6 with ammonia water. Then, while stirring, add an equal volume of saturated ammonium sulfate drop - by - drop to the dialyzed horseradish peroxidase and antibody reaction solution, and let it stand at 2 - 8°C for 3 hours.

[0092] (7) Centrifuge at 8000 revolutions per minute for 30 minutes and discard the supernatant. Dissolve the precipitate in 1 ml of PBS (0.01 mol / L, pH 7.2). Finally, while shaking, add 0.5 ml of saturated ammonium sulfate drop - by - drop to make its final concentration reach 33%, let it stand at 2 - 8°C for 3 hours, centrifuge at 8000 revolutions per minute for 30 minutes, discard the supernatant, and dissolve the precipitate in 1 ml of PBS (0.01 mol / L, pH 7.2). Put the above solution into a dialysis bag and dialyze it with PBS (0.01 mol / L, pH 7.2) to remove ammonium ions (detected with Nessler's reagent), then centrifuge at 8000 revolutions per minute for 30 minutes to remove the precipitate. The supernatant is the enzyme - labeled antibody, which is aliquoted and stored below - 65°C with a validity period of 72 months.

[0093] II. Establishment of an indirect ELISA antibody detection method for yak brucellosis

[0094] (1) Determination of antigen coating concentration: The purified Brucella lipopolysaccharide (LPS) was diluted with coating buffer to 20 μg / ml, 10 μg / ml, 5 μg / ml, and 2.5 μg / ml for coating the enzyme-linked immunosorbent assay (ELISA) plates. ELISA was performed using positive and negative control sera for brucellosis. The test results are shown in Table 3. The dilution with a positive control absorbance value greater than 1.0 and the highest positive control / negative control (P / N value) was selected as the coating concentration of LPS.

[0095] Table 3 Comparison of the effects of different coating concentrations of Brucella LPS

[0096]

[0097]

[0098] The above test results showed that the best result was obtained at a coating concentration of 10 μg / ml, with a positive control absorbance value of about 1.7 and the highest P / N.

[0099] (2) Determination of the optimal working concentration of anti-yak IgG enzyme-labeled monoclonal antibody

[0100] The HRP-labeled anti-yak IgG monoclonal antibody was serially diluted starting from 1:5000 to 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000. Positive and negative sera were selected, and the dilution with the highest OD 450nm ratio was taken as the optimal working concentration. When the HRP-labeled anti-yak IgG monoclonal antibody was diluted 30000-fold, the OD450nm ratio of positive and negative sera was the highest. Therefore, it was determined that the best reaction occurred when the HRP-labeled anti-yak IgG monoclonal antibody was diluted 1:20000-fold.

[0101] (3) Determination of the cut-off value

[0102] 162 positive yak sera and 162 negative yak sera that were all positive in Rose Bengal plate agglutination test, tube agglutination test, and complement fixation test were collected. The OD 450nm values were measured according to the established indirect ELISA method. The SPSS 19.0 software was used to analyze the test results. A receiver operating characteristic (ROC) curve was plotted with sensitivity on the vertical axis and 1 - specificity on the horizontal axis. Each point on the curve represents the sensitivity and specificity corresponding to the cut-off value, as shown in Figure 4 . The cut-off point with the maximum Youden index was used as the critical point for positive and negative judgment, and the sensitivity and specificity of the kit were determined simultaneously. According to the SPSS statistical software, the maximum Youden index was 0.947, and the corresponding SP value was 0.297 ( Figure 3 ). Therefore, the cut-off value could be determined as 0.3, with a corresponding sensitivity of 97.8% and a specificity of 96.9%.

[0103] The above experimental results indicate that the anti-yak IgG monoclonal antibody secreted by the anti-yak IgG hybridoma cell line EF37 of the present invention can be used to prepare a kit for detecting yak brucellosis and for the detection of yak brucellosis.

[0104] In summary, during the implementation of the present invention, through the prokaryotic expression, purification of a partial sequence of the FC segment of yak IgG protein, immunization of mice, preparation of monoclonal antibodies, a hybridoma cell line that can stably and highly efficiently secrete anti-yak IgG monoclonal antibodies (i.e., anti-yak IgG hybridoma cell line EF37) was screened. The anti-yak IgG hybridoma cell line EF37 can highly efficiently and stably secrete monoclonal antibodies against yak IgG, and after identification, it is of the IgG1 subtype. The purified monoclonal antibody labeled with HRP is applied to the indirect ELISA antibody detection kit for yak brucellosis. This kit can effectively and accurately detect yak brucellosis, and its sensitivity and specificity are both above 95%.

[0105] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. A hybridoma cell line, characterized in that, Its preservation number is CGMCC No. 46011.

2. A monoclonal antibody against yak IgG, characterized in that, It is secreted by the hybridoma cell line described in claim 1.

3. A method for preparing the monoclonal antibody against yak IgG as claimed in claim 2, characterized in that, The method comprises the following steps: injecting the hybridoma cell line described in claim 1 into the abdominal cavity of a mouse, raising the mouse, extracting ascites, and purifying to obtain a monoclonal antibody against yak IgG.

4. An immunoassay kit, characterized in that, It contains the monoclonal antibody against yak IgG described in claim 2.

5. The immunoassay kit according to claim 4, wherein It is an enzyme-linked immunosorbent assay kit.

6. Use of the monoclonal antibody against yak IgG described in claim 2 in the preparation of a reagent for detecting yak IgG.

7. The use according to claim 6, characterized in that, The reagent for detecting yak IgG is a reagent for detecting yak diseases.

8. The use according to claim 7, characterized in that, The disease is brucellosis.

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