Monoclonal antibody with competitive effect for resisting brucella lipopolysaccharide and application of monoclonal antibody

Abstract

The invention relates to the technical field of biological detection, in particular to a Brucella lipopolysaccharide-resistant monoclonal antibody with a competitive effect and application of the Brucella lipopolysaccharide-resistant monoclonal antibody. The monoclonal antibody can be subjected to specific reaction with Brucella melitensis, Brucella bovis and Brucella suis, and is not subjected to specific reaction with Escherichia coli, salmonella and Yersinia enterocolitica. The positive serum of the Brucella antibody of cattle and sheep has an inhibiting effect on the combination of the monoclonal antibody and Brucella lipopolysaccharide, and can be used for establishing a Brucella antibody competitive ELISA (enzyme-linked immuno sorbent assay) detection method. The monoclonal antibody for resisting brucella lipopolysaccharide and the application of the monoclonal antibody provide solid material and technical support for the development of the detection technology of brucellosis in China.

Description

Technical Field

[0001] The present application relates to the technical field of biological detection, and in particular to a monoclonal antibody with competitive effect against Brucella lipopolysaccharide and its application. Background Art

[0002] Brucellosis (abbreviated as brucellosis) is a zoonotic infectious disease caused by Brucella bacteria, with fever and abortion as the main characteristics. It is prevalent in more than 170 countries and regions, posing a serious threat to the development of animal husbandry and the health of humans and animals. At present, Brucella can be divided into at least 12 species, among which sheep and cattle Brucella are the most virulent and account for the largest proportion of infections in human brucellosis cases. At present, the World Organization for Animal Health (WOAH) lists brucellosis as a notifiable animal disease, and my country lists it as a Class B legally reportable infectious disease, occupational disease, and a Class II animal disease managed in accordance with the "Animal Epidemic Prevention Law of the People's Republic of China".

[0003] Brucella lipopolysaccharide is an important virulence factor of Brucella, which consists of three parts: lipid A, core polysaccharide and O antigen. Studies have shown that Brucella lipopolysaccharide can induce a strong immune response in animals, is the most important effective component that stimulates the body to produce antibodies, and is a key factor in the serological diagnosis of smooth Brucella.

[0004] At present, animal brucellosis detection is mainly based on serological detection methods, such as tiger red plate agglutination test, tube agglutination test and enzyme-linked immunosorbent assay, etc. However, it is worth noting that since Brucella lipopolysaccharide and other Gram-negative bacteria LPS have the same antigenic epitopes, serological detection has certain false positive results. Therefore, it is particularly important to prepare specific monoclonal antibodies against Brucella lipopolysaccharide and establish detection methods based on specific monoclonal antibodies for brucellosis detection. Summary of the invention

[0005] The object of the present invention is to provide a monoclonal antibody against Brucella lipopolysaccharide with competitive effect and its application, so as to solve the problems existing in the above-mentioned prior art. The monoclonal antibody against Brucella lipopolysaccharide prepared by the present invention has the advantages of good specificity and strong affinity, and provides a solid foundation for the development of serological detection technology of Brucella in my country.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a monoclonal antibody 1B4 against Brucella lipopolysaccharide. The heavy chain variable region of the monoclonal antibody 1B4 includes an amino acid sequence CDR1 such as SEQ ID NO.5, an amino acid sequence CDR2 such as SEQ ID NO.6, and an amino acid sequence CDR3 such as SEQ ID NO.7. The light chain variable region of the monoclonal antibody 1B4 includes an amino acid sequence CDR1 such as SEQ ID NO.8, an amino acid sequence CDR2 such as SEQ ID NO.9, and an amino acid sequence CDR3 such as SEQ ID NO.10.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B4 is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of the monoclonal antibody 1B4 is shown in SEQ ID NO.4.

[0009] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a monoclonal antibody against Brucella lipopolysaccharide, wherein the nucleic acid sequence comprises CDR1, CDR2 and CDR3 of the heavy chain variable region, and CDR1, CDR2 and CDR3 of the light chain variable region, respectively, with amino acid sequences.

[0010] The present invention also includes a vector, which includes the nucleic acid molecule.

[0011] The present invention also provides a host cell, which comprises the nucleic acid molecule or the vector.

[0012] Furthermore, the monoclonal antibody 1B4 can produce specific immune reactions with Brucella melitensis, Brucella bovis and Brucella suis, but does not produce specific reactions with Escherichia coli, Salmonella and Yersinia enterocolitica.

[0013] Furthermore, the heavy chain of the monoclonal antibody 1B4 is IgG1, and the light chain is Kappa.

[0014] The present invention also provides an application of the monoclonal antibody in a Brucella antibody competitive ELISA detection method.

[0015] Furthermore, the Brucella antibody competition ELISA detection method specifically includes: diluting Brucella lipopolysaccharide to 0.1μg / ml, coating the ELISA plate at 100μl / well, and allowing to act at 4°C for 15 hours; discarding the solution in the wells and patting dry. Use 1% fish skin glue as a blocking solution, add 200μl to each well, and allow to act at 37°C for 2 hours; discarding the solution in the wells and patting dry. Add 1:5 diluted serum to be tested, 50μl per well, and immediately add HPR-monoclonal antibody 1B4, 50μl per well, and incubate at 37°C for 45 minutes. Shake off the liquid in the wells, wash the plate 5 times with washing solution, 300μl per well, and pat dry on absorbent paper for the last time; add substrate colorimetric solution, 100μl per well, and develop in the dark for 10 minutes; add stop solution, 50μl per well; use an enzyme reader to measure the OD of each well. 450nm value.

[0016] Further, the criteria for judging the Brucella competitive ELISA antibody detection method are:

[0017] When the inhibition rate (PI) of the serum sample to be tested is ≥50%, the Brucella antibody test result is judged to be positive.

[0018] When the inhibition rate (PI) of the serum sample to be tested is less than 50%, the Brucella antibody test result is judged to be negative.

[0019] The present invention discloses the following technical effects:

[0020] The immunogen used in the present invention is the whole inactivated Brucella melitensis (TZ strain), which makes the immunogen closer to the natural structure of the bacteria's own LPS and gives the antibody the advantage of better affinity.

[0021] The monoclonal antibody provided by the present invention can specifically react with Brucella melitensis, Brucella bovis and Brucella suis, and does not specifically react with positive serum of Escherichia coli, Salmonella and Yersinia enterocolitica, and has good specificity and affinity.

[0022] The monoclonal antibody provided by the present invention can effectively inhibit the binding of the monoclonal antibody to Brucella lipopolysaccharide in Brucella positive serum in competitive ELISA, indicating that the monoclonal antibody can be used to establish a Brucella competitive ELISA antibody detection method, providing a solid foundation for the development of Brucella serological detection technology in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Silver staining identification of Brucella lipopolysaccharide

[0024] Figure 2 Brucella lipopolysaccharide SDS-PAGE image

[0025] Figure 3This is a graph showing the competitive activity test of hybridoma cell supernatant

[0026] Figure 4 Figure 2 shows the results of the monoclonal antibody specificity test. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below through specific implementations, but the embodiments are only exemplary and do not limit the scope of the present invention in any way. Any simple modifications, changes and equivalent transformations made to the embodiments based on the technical essence of the present invention still fall within the scope of protection of the present invention.

[0028] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0029] Example 1 Extraction and Identification of Brucella Lipopolysaccharide

[0030] 1. Extraction of Brucella lipopolysaccharide

[0031] The inactivated Brucella melitensis (TZ strain) bacterial solution was centrifuged at 8000 r / min for 10 minutes, the bacterial cells were collected, and the bacterial weight was weighed. The following extraction process is based on a bacterial weight of 50.00 g. 50.00 g of bacterial cells were suspended in 170 mL of pure water and heated to 66°C; 190 mL of 90% (v / v) phenol solution at 66°C was added, and the mixture was shaken and mixed in a 66°C water bath for 30 minutes, and then centrifuged at 4°C and 10,000 r / min for 15 minutes; the lower phenol phase was aspirated with a long tube, and large pieces of bacterial fragments were removed by filtering with filter paper; 500 mL of saturated sodium acetate methanol solution precooled at 4°C was added to the filtrate, and the mixture was incubated at 4°C for 2 hours to precipitate LPS, and then centrifuged at 4°C and 10,000 r / min for 10 minutes, and the supernatant was discarded; the precipitate was resuspended in 80 mL of pure water, stirred at 4°C for 18 hours, and then centrifuged at 4°C and 10,000 r / min for 10 minutes, and the supernatant was collected and stored at 4°C; the precipitate was resuspended in 80 mL of pure water again, stirred at 4°C for 2 hours, and the supernatant was obtained by centrifugation according to the above method, and mixed with the above supernatant; 160 mL of the 160 mL of ... Add 8.00g trichloroacetic acid to the LPS extract, stir at room temperature for 15 minutes, centrifuge at 10000r / min for 15 minutes, discard the precipitate, and dialyze the supernatant with pure water; add proteinase K to the LPS extracted above to a final concentration of 15μg / mL, digest at 55℃ for 3 hours, and then digest at room temperature for 24 hours. Digest thoroughly with pure water, divide the dialyzed samples, place them in a freeze dryer for freeze drying, and weigh them. Dilute the freeze-dried LPS to 3mg / mL using 0.01mol / LPB buffer and store at -70℃ or below.

[0032] 2. Purity test of Brucella lipopolysaccharide

[0033] Take 40 μl of extracted Brucella lipopolysaccharide and place it in an EP tube, add 10 μl of 5×SDS-PAGE Loading Buffer, and place it in a 100°C water bath for 5 minutes for later use; place the prepared PAGE gel in the electrophoresis tank, and add about 700 ml of SDS-PAGE electrophoresis buffer; add 10 μl of sample and 5 μl of protein marker to the sample wells respectively, adjust the voltage to 80 V for electrophoresis, stop electrophoresis when bromophenol blue moves to the bottom of the gel, and stain the separation gel with Coomassie brilliant blue and silver nitrate respectively, the steps are as follows:

[0034] Coomassie Brilliant Blue Staining: Peel the separation gel into the staining solution (the staining solution must soak the gel surface), stain it on a shaker for 4 hours, put it into the destaining solution, and change the destaining solution every 30 minutes until clear protein bands can be observed. Put the completely destained gel into the gel imaging system and take pictures.

[0035] Silver nitrate staining: First, take 100 ml of fixative solution and oxidize and fix the protein gel at 22°C for 20 minutes, then wash with pure water 3 times, 5 minutes each time. Then add 50 ml of silver stain solution to stain the protein gel for 30 minutes, and rinse the gel with pure water once; finally, add color developing solution to develop the color, and observe at any time. After the bands appear, add stop solution to stop the color development, wash the gel with pure water, and take pictures with a digital camera.

[0036] The results showed that there were no obvious protein bands in the Coomassie Brilliant Blue staining test ( Figure 1 ), silver nitrate staining bands are evenly dispersed, showing polysaccharide-specific ladder-like bands ( Figure 2 ), indicating that the extracted Brucella lipopolysaccharide has a complete structure, high purity, and almost no foreign protein contamination.

[0037] 3. Test for the reactogenicity of Brucella lipopolysaccharide

[0038] Dilute the extracted LPS of Brucella melitensis (TZ strain) to 0.5μg / ml, coat the ELISA plate at 100μl / well, and incubate at 2-8℃ for 15 hours; discard the solution in the wells, pat dry, add 200μl blocking solution to each well, incubate at 37℃ for 2 hours; pat dry. Add 1:40 diluted Brucella positive serum and Brucella negative serum from cattle and sheep, 100μl per well. Incubate at 37℃ for 30 minutes. Shake off the liquid in the wells, wash the plate 5 times with washing solution, 300 μl per well, and pat dry on absorbent paper for the last time; add 1:40000 diluted HRP-rabbit anti-bovine IgG (HRP-rabbit anti-goat IgG), 100 μl per well, incubate at 37°C for 45 minutes; shake off the liquid in the wells, wash the plate 5 times with washing solution, 300 μl per well, and pat dry on absorbent paper for the last time; add substrate color development solution, 100 μl per well, develop color in the dark for 10 minutes; add stop solution, 50 μl per well; measure the OD of each well with an enzyme-labeled instrument450nm value.

[0039] The ELISA results showed that the extracted Brucella lipopolysaccharide could react with Brucella antibody-positive serum, indicating that it had good reactivity and could be used for subsequent hybridoma cell screening (Table 1).

[0040] Table 1 Reactogenicity test results

[0041]

[0042] Example 2 Screening of hybridoma cells secreting anti-Brucella lipopolysaccharide monoclonal antibodies

[0043] 1. Mouse immunization

[0044] Four 6-week-old female BALB / c mice were immunized with inactivated whole-cell Brucella melitensis (TZ strain) by subcutaneous injection of 1×10 whole-cell Brucella emulsified with equal volumes of Freund's complete adjuvant at multiple points in the first immunization. 9 CFU / mouse; immunization was conducted every 2 weeks thereafter, with whole bacteria and Freund's incomplete adjuvant mixed in equal volumes and emulsified, 1×10 9 CFU / mouse. 3 Two weeks after immunization, the antibody titer produced by the mice was detected by indirect ELISA method. If the antibody titer was lower than 1:12800, the immunization was continued. Mice with qualified antibody titers (not lower than 1:12800) were selected and injected intraperitoneally with 2×10 whole bacteria without adjuvant 4 days before fusion. 9 CFU / piece.

[0045] 2. Cell fusion

[0046] According to the conventional procedure, spleen cells were fused with myeloma cells SP2 / 0. The fused cells were seeded into 96-well plates containing feeder cells and cultured in a 37°C, 5% carbon dioxide incubator. When the fused cell colonies grew to 1 / 4 of the culture well and the culture medium turned slightly yellow, positive hybridoma cells were screened.

[0047] 3. Screening and subcloning of positive hybridoma cells

[0048] Use an ELISA plate coated with Brucella lipopolysaccharide to detect the supernatant of the hybridoma cells after fusion. Gently blow the positive hybridoma cells from the culture wells and count them. Dilute them in multiples in a 96-well plate, count them under a microscope, aspirate all the cells in the wells with a total cell count of about 100-200 into 10mL HT complete medium, and divide them into 96-well plates containing feeder cells. When the cell clones fill 1 / 3 to 1 / 2 of the culture wells, they can be detected. If the cell growth wells are positive, you can select cell wells with high antibody titers, monoclonal growth, and good morphology, and continue to clone them in the same way. If all three subclones are positive, they can be identified.

[0049] 4. Screening of hybridoma cells with competitive effects

[0050] Add 1:10 diluted positive serum of bovine Brucella and negative serum of bovine Brucella to the Brucella melitensis lipopolysaccharide coated plate (0.05 μg per well), 50 μl per well, and immediately add the screened positive hybridoma cell supernatant, 50 μl per well, gently shake the liquid in the well, and incubate at 37°C for 30 minutes. Discard the liquid in the well, wash 5 times, and pat dry; add 1:5000 diluted HRP-sheep anti-mouse IgG, 100 μl per well, and act at 37°C for 30 minutes. Discard the liquid in the well, wash 5 times, and pat dry; add substrate color development solution, 100 μl per well, and color for 10 minutes in the dark; add stop solution, 50 μl per well; use an enzyme reader to measure the OD of each well. 450nm The results of ELISA test showed that the Brucella antibody positive serum had an inhibitory effect on the binding of the antibody secreted by hybridoma cell 1B4 with Brucella lipopolysaccharide ( Figure 3 ).

[0051] Example 3 Verification of the properties of monoclonal antibody 1B4

[0052] 1. Monoclonal antibody subtype identification

[0053] The subtype of monoclonal antibody 1B4 was analyzed using the mouse monoclonal antibody subtype identification kit of Sigma. According to the instructions of the kit, the cell culture supernatant was identified. The results showed that the heavy chain of monoclonal antibody 1B4 was IgG1 and the light chain was Kappa (Table 2).

[0054] Table 2 Subtype identification

[0055]

[0056] 2. Specificity identification of monoclonal antibodies

[0057] The inactivated Brucella sarlingi, Brucella melitensis TZ strain, Brucella bovis A19 strain, Escherichia coli O157 strain, Yersinia enterocolitica and Salmonella SL1344 strain were ultrasonically disrupted, and the supernatant was collected as the sample to be tested. 40 μl of the sample to be tested was placed in an EP tube, 10 μl of 5×SDS-PAGE Loading Buffer was added, and the tube was bathed at 100°C for 5 minutes, followed by SDS-PAGE electrophoresis and transfer. After the transfer, the PVDF membrane was incubated with 5% skim milk TBST solution at room temperature for 2 hours, and then washed 4 times with TBST solution for 5 minutes each time; the purified monoclonal antibody 1B4 was diluted 10,000 times with TBST solution, and the PVDF membrane was placed in it, incubated at 37°C for 2 hours, and then washed 4 times with TBST solution for 5 minutes each time. HRP-goat anti-mouse IgG was diluted 20,000 times with TBST solution, and the PVDF membrane was placed in it and incubated at 37°C for 2 hours, then washed 4 times with PBST solution, each time for 5 minutes; finally, the luminescent signal was detected using a chemiluminescence imager.

[0058] The results showed that the prepared monoclonal antibody 1B4 could react specifically with Brucella suis S2, Brucella melitensis TZ and Brucella bovis A19, but had no specific reaction with Escherichia coli O157, Salmonella SL1344 and Yersinia enterocolitica positive serum ( Figure 4 ).

[0059] Example 4 PCR amplification and sequence determination of variable region genes of anti-Brucella lipopolysaccharide monoclonal antibody 1B4

[0060] The total RNA of hybridoma cells secreting anti-Brucella lipopolysaccharide monoclonal antibody 1B4 was extracted by Trizol lysis method, and then cDNA was synthesized by reverse transcription. 11 pairs of degenerate primers were designed for monoclonal antibody 1B4 light chain variable region gene amplification, and 12 pairs of degenerate primers were designed for monoclonal antibody 1B4 heavy chain variable region gene amplification. After amplification, agarose gel electrophoresis was performed, and the heavy chain and light chain variable region gene fragments of monoclonal antibody 1B4 were inserted into pMD 18T blunt-end vector, transformed into Escherichia coli, and positive clones were selected for sequencing.

[0061] Table 3 Primer sequences for amplifying light chain variable regions

[0062]

[0063] Table 4 Primer sequences for amplifying the heavy chain variable region

[0064]

[0065] The sequencing results showed that the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1B4 against Brucella lipopolysaccharide was shown as SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B4 against Brucella lipopolysaccharide was shown as SEQ ID NO.2; the nucleotide sequence of the light chain variable region of the monoclonal antibody 1B4 against Brucella lipopolysaccharide was shown as SEQ ID NO.3; and the amino acid sequence of the light chain variable region of the monoclonal antibody 1B4 against Brucella lipopolysaccharide was shown as SEQ ID NO.4.

[0066] The sequences of the heavy chain and light chain variable regions of the monoclonal antibody 1B4 against Brucella lipopolysaccharide obtained by sequencing were analyzed, and the amino acid sequences of the three CDR regions of the heavy chain variable region were shown in SEQ ID NO.5 to SEQ ID NO.7, respectively. The amino acid sequences of the three CDR regions of the light chain variable region were shown in SEQ ID NO.8 and SEQ ID NO.10, respectively.

[0067] Example 5 Establishment of Brucella competitive ELISA antibody detection method

[0068] Brucella lipopolysaccharide was diluted to 0.1 μg / ml, and coated on the ELISA plate at 100 μl / well, and allowed to react at 4°C for 15 hours; the solution in the wells was discarded and patted dry. 1% fish skin glue was used as a blocking solution, 200 μl was added to each well, and allowed to react at 37°C for 2 hours; the solution in the wells was discarded and patted dry. 1:5 diluted serum to be tested was added, 50 μl per well, and then HPR-monoclonal antibody 1B4 was immediately added, 50 μl per well, and incubated at 37°C for 45 minutes. The liquid in the wells was shaken off, and the plate was washed 5 times with washing solution, 300 μl per well, and the last time it was patted dry on absorbent paper; 100 μl of substrate colorimetric solution was added to each well, and color was developed in the dark for 10 minutes; 50 μl of stop solution was added to each well; the OD of each well was measured using an ELISA reader. 450nm value.

[0069] The criteria for Brucella competitive ELISA antibody detection method are:

[0070] When the inhibition rate (PI) of the serum sample to be tested is ≥50%, the Brucella antibody detection result is judged to be positive.

[0071] When the inhibition rate (PI) of the serum sample to be tested is less than 50%, the Brucella antibody detection result is judged to be negative.

[0072] Example 6 Performance test of Brucella competitive ELISA antibody detection method

[0073] 1. Sensitivity test

[0074] Brucella national standard positive serum (1000IU / mL) was diluted to 80 times, 160 times, 320 times, ..., 2560 times with sample diluent, and the inhibition rate (PI) value of serum sample to be tested was calculated. The result showed that when Brucella national standard positive serum was 80 times, 160 times, 320 times, 640 times, 1280 times with Brucella negative serum, the competitive ELISA test result was positive; when diluted to 2560 times, the test result was negative (Table 5).

[0075] Table 5 Sensitivity test results

[0076]

[0077] 2. Specificity test

[0078] Use above-mentioned Brucella competition ELISA antibody detection method to detect bovine Escherichia coli positive serum, sheep Escherichia coli positive serum, bovine Salmonella positive serum, sheep Salmonella positive serum, bovine enterocolitica Yersinia positive serum, sheep enterocolitica Yersinia positive serum and bovine Mycobacterium positive serum respectively, to assess the specificity of competition ELISA antibody detection method of the present invention. The result shows that above-mentioned serum detection results are all negative (Table 6).

[0079] Table 6 Specificity test results

[0080]

[0081] 3. Compliance test

[0082] Use the above-mentioned Brucella competition ELISA detection method and the Brucella cELISA antibody detection kit of Wuhan Keqian Biological Co., Ltd. to detect 124 sheep sera and 99 bovine sera respectively, and compare the compliance rates of the two methods. The results show that for sheep serum, the total compliance rate of the Brucella competition ELISA detection method and the commercial Brucella cELISA antibody detection kit is 93.55% (Table 7); for bovine serum, the total compliance rate of the Brucella competition ELISA detection method and the commercial Brucella cELISA antibody detection kit is 90.55% (Table 8).

[0083] Table 7 Sheep serum compliance test results

[0084]

[0085] Table 8 Bovine serum compliance test results

[0086]

[0087] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly or indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A monoclonal antibody 1B4 against Brucella lipopolysaccharide, characterized in that: The heavy chain variable region of the monoclonal antibody 1B4 includes an amino acid sequence CDR1 such as SEQ ID NO.5, an amino acid sequence CDR2 such as SEQ ID NO.6, and an amino acid sequence CDR3 such as SEQ ID NO.

7. The light chain variable region of the monoclonal antibody 1B4 includes an amino acid sequence CDR1 such as SEQ ID NO.8, an amino acid sequence CDR2 such as SEQ ID NO.9, and an amino acid sequence CDR3 such as SEQ ID NO.

10.

2. The monoclonal antibody 1B4 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B4 is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of the monoclonal antibody 1B4 is shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody 1B4 according to claim 1.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 3 or the vector of claim 4.

6. The monoclonal antibody 1B4 according to claim 1, characterized in that The monoclonal antibody 1B4 can generate specific immune reactions with Brucella melitensis, Brucella bovis and Brucella suis, but does not generate specific reactions with Escherichia coli, Salmonella and Yersinia enterocolitica.

7. The monoclonal antibody 1B4 according to claim 1, characterized in that The heavy chain of the monoclonal antibody 1B4 is IgG1, and the light chain is Kappa.

8. Use of the monoclonal antibody against Brucella lipopolysaccharide according to claim 1 in preparing a Brucella competitive ELISA antibody detection method.

9. The use according to claim 8, wherein the Brucella competitive ELISA antibody detection method is as follows: dilute Brucella lipopolysaccharide to 0.1 μg / ml, coat the ELISA plate at 100 μl / well, and allow to act at 4°C for 15 hours; discard the solution in the wells and pat dry. Use 1% fish skin glue as a blocking solution, add 200 μl to each well, and allow to act at 37°C for 2 hours; discard the solution in the wells and pat dry. Add 1:5 diluted serum to be tested, 50 μl per well, and then immediately add HPR-monoclonal antibody 1B4, 50 μl per well, and incubate at 37°C for 45 minutes. Shake off the liquid in the wells, wash the plate 5 times with washing solution, 300 μl per well, and pat dry on absorbent paper for the last time; add substrate color developing solution, 100 μl per well, and develop for 10 minutes in the dark; add stop solution, 50 μl per well; measure the OD of each well with an enzyme marker. 450nm value.

10. The Brucella competitive ELISA antibody detection method according to claim 9, characterized in that: The criteria are: When the inhibition rate (PI) of the serum sample to be tested is ≥50%, the Brucella antibody test result is judged to be positive. When the inhibition rate (PI) of the serum sample to be tested is less than 50%, the Brucella antibody test result is judged to be negative.

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