Monoclonal antibodies specifically binding brucella lps and use in the quantitative detection of lps antibody levels

Abstract

The application provides a monoclonal antibody specifically binding to Brucella LPS, the monoclonal antibody having three light chain complementarity determining regions and three heavy chain complementarity determining regions; the amino acid sequences of the light chain complementarity determining regions are respectively the 24th to 40th amino acids in the sequence table SEQ ID NO. 1, the 56th to 62nd amino acids in the sequence table SEQ ID NO. 1 and the 95th to 102nd amino acids in the sequence table SEQ ID NO. 1; the amino acid sequences of the heavy chain complementarity determining regions are respectively the 31st to 37th amino acids in the sequence table SEQ ID NO. 3, the 52nd to 67th amino acids in the sequence table SEQ ID NO. 3 and the 100th to 113th amino acids in the sequence table SEQ ID NO. 3. The application further discloses a preparation method of the monoclonal antibody and application of the monoclonal antibody in quantitative detection of Brucella LPS antibody level.

Description

Technical Field

[0001] The invention belongs to the technical field of serological detection, and particularly relates to a monoclonal antibody specifically binding to Brucella LPS and its application in quantitative detection of LPS antibody levels. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by multiple species of Brucella that affects livestock, wildlife, and humans. Brucella infection in animals can cause severe economic losses to the livestock industry and threaten human public health.

[0003] Currently, vaccination remains the most effective and economical method for brucellosis prevention and control. Live attenuated vaccines used in my country, including A19, M5-90, and S2, as well as gene-deleted vaccines A19-ΔVirB12, M5-90Δbp26, and BA0711, have played a significant role in brucellosis prevention and control. The sheep Rev.1 vaccine, currently imported from abroad and suitable for sheep immunization, has been approved for widespread use.

[0004] Serological antibody detection techniques are primarily used to evaluate the immune efficacy of Brucella vaccines and diagnose infection in unvaccinated animals. Current serological testing methods include the rosebengal test (RBT), serum agglutination test (SAT), complement fixation test (CFT), indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and fluorescence polarization assay (FPA). SAT and CFT can detect antibody titers against whole Brucella bacteria, but they have poor sensitivity and a large interquartile range. Results are significantly influenced by operator judgment. RBT, SAT, and CFT use whole bacteria as antigens, iELISA uses whole bacteria or lipopolysaccharide (LPS) as antigens, and FPA uses LPS as antigen. However, Brucella LPS and Yersinia enterocolitica O:9 LPS share cross-epitopes, and this technique can produce false positives when detecting Yersinia enterocolitica O:9-positive serum. While cELISA uses LPS and its monoclonal antibody as the antigen and indicator antibody, respectively, it is specific but can only determine positive and negative results and cannot measure antibody titers. To overcome these technical limitations, there is an urgent need to develop a serological technique based on Brucella LPS monoclonal antibodies as indicator antibodies that can specifically detect Brucella LPS antibodies and quantitatively measure LPS antibody levels, in order to provide important technical support for Brucella epidemiological surveys and vaccine immunity evaluations. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides monoclonal antibodies that specifically bind to Brucella lipopolysaccharides (LPS), as well as the use of such monoclonal antibodies in the quantitative detection of LPS antibody levels. The present invention immunizes mice with Brucella LPS as an antigen, then fuses the mouse spleen cells with myeloma SP2 / 0 cells to screen for a hybridoma cell line, LPS-2A6, that secretes a specific monoclonal antibody. The present invention inoculates the LPS-2A6 cell line into the peritoneal cavity of mice, collects the mouse ascites, and purifies the antibodies, resulting in a purified monoclonal antibody that specifically binds to Brucella LPS. The antibody does not react with Yersinia enterocolitica O:9, Chlamydia, Salmonella, Escherichia coli O:157, or Vibrio cholerae O11 / O139, thus ensuring the specificity of the reaction.

[0006] The first object of the present invention is to provide a monoclonal antibody that specifically binds to Brucella LPS, wherein the monoclonal antibody has three light chain complementary determining regions and three heavy chain complementary determining regions;

[0007] The amino acid sequences of the light chain complementary determining regions are: amino acids 24 to 40 in SEQ ID NO.1 in the sequence listing, amino acids 56 to 62 in SEQ ID NO.1 in the sequence listing, and amino acids 95 to 102 in SEQ ID NO.1 in the sequence listing;

[0008] The amino acid sequences of the heavy chain complementary determining regions are: amino acids 31 to 37 in SEQ ID NO.3, amino acids 52 to 67 in SEQ ID NO.3, and amino acids 100 to 113 in SEQ ID NO.3.

[0009] Preferably, the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID No. 1; and the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 3.

[0010] The monoclonal antibody specifically binding to Brucella LPS of the present invention can specifically bind to Brucella LPS, and its binding ability to Brucella LPS can be blocked by Brucella positive serum.

[0011] A second object of the present invention is to provide coding genes for encoding the above-mentioned monoclonal antibodies that specifically bind Brucella LPS.

[0012] Preferably, the encoding gene includes a DNA sequence as shown in SEQ ID No.2, which is used to encode the light chain variable region of the monoclonal antibody that specifically binds to Brucella LPS; and includes a DNA sequence as shown in SEQ ID No.4, which is used to encode the heavy chain variable region of the monoclonal antibody that specifically binds to Brucella LPS.

[0013] The third object of the present invention is to provide a method for preparing a monoclonal antibody that specifically binds to Brucella LPS, which comprises inoculating BALB / c female mice with hybridoma cells, collecting and purifying mouse ascites, and obtaining the monoclonal antibody that specifically binds to Brucella LPS.

[0014] The fourth object of the present invention is to provide the use of the above-mentioned monoclonal antibody that specifically binds to Brucella LPS in the quantitative detection of Brucella LPS antibody levels.

[0015] Preferably, the quantitative detection of Brucella LPS antibody levels comprises preparing a blocking ELISA kit for detecting Brucella LPS antibodies.

[0016] The fifth object of the present invention is to provide a blocking ELISA kit for quantitatively detecting Brucella LPS antibodies, wherein the blocking ELISA kit comprises an enzyme labeling plate coated with Brucella LPS and the above-mentioned monoclonal antibody that specifically binds to Brucella LPS.

[0017] Preferably, the coating concentration of the Brucella LPS is 2 μg / mL; and / or

[0018] The dilution ratio of the monoclonal antibody was 1:6000.

[0019] The test kit provided by the present invention uses Brucella LPS as an antigen and the specific monoclonal antibody prepared as a detection indicator antibody to prepare a blocking ELISA kit for detecting Brucella LPS serum antibodies. The antibody level of Brucella LPS can be quantitatively detected, thus filling the gap in the current Brucella ELISA antibody detection kit related technology. The test kit of the present invention utilizes blocking ELISA technology, uses a mouse monoclonal antibody specifically bound to Brucella LPS as a detection indicator antibody, can detect serum antibodies from various animal sources, and avoids the species restriction (as shown in Table 1) of the sample source. In addition, the test kit has good repeatability and all has a compliance rate of more than 95% with the Brucella antibody detection kit currently on sale, and has a long shelf life. The technical requirements are relatively loose during the operation of the test kit, and the test kit can be widely promoted and used in production, and is applied to Brucella epidemiological surveys and vaccine immunity level monitoring.

[0020] The blocking ELISA kit of the present invention can quantify and evaluate the immune antibody level of the inactivated Brucella vaccine currently used in the sheep breeding industry, and can also perform serological investigation and diagnosis on sheep that have not been immunized with the Brucella vaccine.

[0021] ELISA diagnostic reagents for quantitatively detecting Brucella LPS serum antibodies have long been a much-needed product in the cattle and sheep farming industry. The blocking ELISA kit of the present invention can quantify and evaluate the immune antibody levels of the attenuated Brucella vaccine currently used in the sheep farming industry. It can also conduct serological surveys and diagnoses on sheep that have not been immunized with the Brucella vaccine, achieving accurate evaluation of vaccine immunity and wild-type virus infection. The present invention uses a mouse monoclonal antibody as a detection indicator antibody, making it easy to prepare and produce in large quantities. The kit of the present invention can achieve high-throughput detection and can be operated by relevant professionals according to the instructions, thus having universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 It is purified Brucella LPS.

[0024] Figure 2 Flow chart for the preparation of hybridoma cell line LPS-2A6 that secretes a monoclonal antibody that specifically binds to Brucella LPS.

[0025] Figure 3 This is the electrophoresis result of the purified monoclonal antibody that specifically binds to Brucella LPS.

[0026] Figure 4 The standard curve is used to quantify the concentration of Brucella LPS antibodies. DETAILED DESCRIPTION

[0027] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0028] Example 1 Preparation of Brucella lipopolysaccharide (LPS)

[0029] (1) Brucella GS-XQ-A1 (bovine species, Brucella abortus) was propagated in a biosafety level 3 laboratory (BLS-3). This strain was isolated and identified by our laboratory and is stored at the Biosafety and Management Platform of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. GS-XQ-A1 glycerol culture stored at -40°C was inoculated into 2000 mL of tryptone soy broth (TSB) at a ratio of 1:100 and cultured at 37°C and 180 rpm for 72 h. The cells were collected after centrifugation and weighed. (2) 5 g of Brucella was dissolved in 17 mL of sterile distilled water and inactivated in a water bath at 80°C for 3 h. (3) After the inactivated bacterial suspension was cooled to 66°C, 19 mL of 90% (v / v) phenol solution preheated to 66°C was added and stirred at 66°C for 15 min. (4) Centrifuge at 4°C and 10,000 g for 15 min, remove the lower phenol phase with a 10 mL pipette, add 50 mL of cold methanol containing 1% saturated sodium acetate, and precipitate at 4°C for 2 h. (5) Discard the supernatant, add 30 mL of distilled water to the precipitate, stir at 4°C for 18 h, centrifuge at 4°C and 10,000 g for 10 min, and collect the supernatant (v1). (6) Add 30 mL of distilled water to the precipitate, stir at 4°C for 2 h, centrifuge at 4°C and 10,000 g for 10 min, and collect the supernatant (v2). (7) Mix the supernatants v1 and v2 collected twice, add trichloroacetic acid (g / mL) to a final concentration of 5%, and stir at room temperature for 10 min. (8) Centrifuge the solution at 10,000 g for 10 min, collect the supernatant, put it into a dialysis bag, and dialyze it against distilled water for 48 h to obtain crude LPS. (9) Nuclease was added to the crude LPS at a final concentration of 15 μg / mL, and the mixture was digested at 37°C for 24 h. (10) Proteinase K was added at a final concentration of 15 μg / mL, and the mixture was digested at 55°C for 3 h, followed by digestion at room temperature for 24 h. (11) The mixture was dialyzed against deionized water to obtain purified Brucella LPS. Grayscale scanning analysis showed that the purity of the Brucella LPS prepared by the present invention was above 95%.

[0030] Figure 1 The following table shows purified Brucella LPS. M: protein marker relative molecular mass standard; 1-2: purified Brucella LPS.

[0031] Example 2 Obtaining hybridoma cell line LPS-2A6 that secretes monoclonal antibodies that specifically bind to Brucella LPS

[0032] Five 6-8 week old female BALB / c mice were selected and immunized with the purified Brucella LPS from Example 1 and Freund's complete adjuvant in a 1:1 volume ratio. Immunizations were repeated every two weeks at a 1:1 volume ratio of Brucella LPS to Freund's incomplete adjuvant. Four immunizations were performed, with an antigen dose of 50 μg per mouse per dose. Two weeks after the fourth immunization, mouse serum was obtained and titered by indirect ELISA using Brucella LPS as the antigen. Positive titers were determined by a P / N ratio of ≥ 2.1, and immunization was considered acceptable if the titer reached 1:50,000 or higher. Splenocytes from these mice were obtained and fused with SP2 / 0 myeloma cells at a 5:1 ratio. The fused cells underwent a half and full medium exchange on days 7 and 10 after the fusion, respectively. On the third day after the full medium exchange, hybridoma cells secreting anti-Brucella LPS antibodies were screened using an indirect ELISA. Positive wells screened in the first screening were then subjected to a full medium exchange and a second screening. The wells that were still positive in the second screening were expanded and frozen, and subcloning was performed continuously for 3-5 times until the positive rate was 100%. The positive cell lines were frozen in a liquid nitrogen tank to obtain a hybridoma cell line that secreted monoclonal antibodies that specifically bound to Brucella LPS. The hybridoma cell line was named LPS-2A6.

[0033] Figure 2 Flow chart for the preparation of hybridoma cell line LPS-2A6 that secretes a monoclonal antibody that specifically binds to Brucella LPS.

[0034] Example 3 Preparation of Monoclonal Antibodies Specifically Binding to Brucella LPS

[0035] The method for preparing a monoclonal antibody that specifically binds to Brucella LPS is as follows:

[0036] BALB / C mice were sensitized by intraperitoneal injection of 1 mL of autoclaved liquid paraffin. 7 days later, 1.0×10 6 The hybridoma cell line LPS-2A6 of Example 2, which is growing well, was obtained. Changes in the mouse abdominal cavity were observed daily. When the abdominal cavity was swollen to the point of difficulty in movement, ascites was collected (ascites from BALB / c female mice was obtained, centrifuged, and the middle layer of pale yellow ascites was collected). The resulting ascites was purified using a Protein G purification column and then subjected to SDS-PAGE for purity observation. Grayscale scanning analysis showed a purity of over 90%, and the concentration was determined to be 4.2 mg / mL. The obtained LPS monoclonal antibody was stored at -80°C for future use. A monoclonal antibody that specifically binds to Brucella LPS was obtained.

[0037] The IgG subtype of the monoclonal antibody that specifically binds to Brucella LPS was identified using a commercial kit. The secreted antibody was assayed for Ig subclass and identified as IgG1, kappa chain.

[0038] Figure 3 The electrophoresis results of the purified monoclonal antibody that specifically binds to Brucella LPS are shown in Figure 1. Here, M is the relative molecular mass standard of the protein marker; I is the purified monoclonal antibody against Brucella LPS.

[0039] Example 4 Determination of the light and heavy chain variable region sequences of monoclonal antibodies that specifically bind to Brucella LPS

[0040] Based on the hybridoma cell line LPS-2A6 obtained above, the hybridoma cells were cultured to 10 7 RNA was extracted from hybridoma cells and reverse transcribed to generate cDNA. PCR amplification of these cDNAs was performed using a mouse IgG VH VL primer library designed by Wuhan Jinkairui Bioengineering Co., Ltd. The PCR reaction was performed under the following conditions: 98°C for 5 minutes of initial denaturation; 94°C for 30 seconds of denaturation; 54°C for 30 seconds of annealing; 72°C for 2 minutes of extension; and 30 cycles of extension at 72°C for 6 minutes. The resulting PCR products were subjected to agarose gel electrophoresis and purified. After ligation with the pUC19 vector, they were transformed into competent Escherichia coli TOP10 cells and plated onto LB plates containing 100 μg / mL ampicillin. Several colonies were selected for verification by colony PCR. Transformants were then sequenced using the company's sequencing platform. The sequencing primers were M13-F: GTAAAACGACGGCCAG. Through these steps, the variable regions of the heavy and light chains of the monoclonal antibody IgG were amplified and sequenced, thereby obtaining the sequence information for the antibody light and heavy chains.

[0041] The amino acid sequence of the light chain variable region of the monoclonal antibody that specifically binds to Brucella LPS is (SEQ ID NO.1 in the sequence listing):

[0042] DIVMSQSPSSLAVSAGEKVTMSC KSSQSQFNSRTRKNYLA WYQQKPGQSPKLLIY GASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLAVYYC KQSGYNPT FGSGTKLEIKR.

[0043] The underlined portions are the amino acid sequences of the light chain complementary determining regions (i.e., hypervariable regions) CDR1, CDR2, and CDR3, respectively, i.e., amino acids 24 to 40 in the above sequence, amino acids 56 to 62 in SEQ ID NO. 1, and amino acids 95 to 102 in SEQ ID NO. 1.

[0044] The gene sequence encoding the light chain variable region of the monoclonal antibody that specifically binds to Brucella LPS is (SEQ ID NO.2 in the sequence listing):

[0045] GACATCGTGATGAGCCAGAGCCCTAGCAGCCTGGCCGTGAGCGCCGGCGAGAAGGTGACCATGAGCTGCAAGAGCAGCCAGAGCCAGTTCAACAGCCGTACCCGTAAGAACTACCTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCTAAGCTGCTGATCTACGGAGC CAGCACCCGTGAGAGCGGCGTGCCTGACCGTTTCATCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCAAGCAGAGCGGATACAACCCTACCTTCGGCAGCGGCACCAAGCTGGAGATCAAGCGT.

[0046] The amino acid sequence of the heavy chain variable region of the monoclonal antibody that specifically binds to Brucella LPS is (SEQ ID NO.3 in the sequence listing):

[0047] QVSLKESGPGILQPSQTLSLTCSFSGFSLS TSGESVG WIRQPSGKGLEWLT NTQQDD SKYYNAALKS RLTISKETSKNQVFLKIASVDTADTATYYCAR QVQDYGNYLYAQDY WG QGTSVTVSS.

[0048] The underlined portions are the amino acid sequences of the heavy chain complementary determining regions (i.e., hypervariable regions) CDR1, CDR2, and CDR3, respectively, i.e., amino acids 31 to 37 in SEQ ID NO. 3, amino acids 52 to 67 in SEQ ID NO. 3, and amino acids 100 to 113 in SEQ ID NO. 3.

[0049] The gene sequence encoding the heavy chain variable region of the monoclonal antibody that specifically binds to Brucella LPS is (SEQ ID NO.4 in the sequence listing):

[0050] CAGGTGAGCCTGAAGGAGTCAGGACCAGGCATCCTGCAGCCTTCACAGACCCTGAGCCTGACCTGCAGCTTCTCAGGCTTCAGCCTGAGCACCAGCGGCGAGAGCGTGGGCTGGATCCGTCAGCCTAGCGGCAAGGGCCTGGAGTGGCTGACCAACACCCAGCAGGACGACAGCAAGTACTACAAC GCCGCCCTGAAGAGCCGTCTGACCATCAGCAAGGAGACCAGCAAGAACCAGGTGTTCCTGAAGATCGCCAGCGTGGACACCGCCGACACCGCCACCTACTACTGCGCCCGTCAGGTGCAGGACTACGGCAACTACCTGTACGCCCAGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0051] Example 5 Establishment of Brucella LPS Blocking ELISA Antibody Detection Method

[0052] 1. Blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies

[0053] The blocking ELISA kit for quantitatively detecting Brucella LPS serum antibodies is prepared by taking Brucella LPS as an antigen and a monoclonal antibody specifically binding to Brucella LPS as a detection indicator antibody.

[0054] The blocking ELISA kit for quantitatively detecting Brucella LPS serum antibodies of the present invention comprises:

[0055] (1) Two ELISA plates (96 wells / plate) coated with Brucella LPS, stored at 4°C;

[0056] (2) 1 bottle of 25x PBST concentrate (60 mL / bottle);

[0057] (3) One tube of positive control serum (1 mL / tube), stored at 4°C;

[0058] (4) One tube of negative control serum (1 mL / tube), stored at 4°C;

[0059] (5) 1 bottle of HRP-labeled monoclonal antibody working solution (25 mL / bottle), stored at 4°C;

[0060] (6) 1 bottle of TMB substrate colorimetric solution (25 mL / bottle), stored at 4°C;

[0061] (7) 1 bottle of stop solution (15 mL / bottle), stored at 4°C;

[0062] (8) 2 sheets of sealing film;

[0063] (9) One copy of the instruction manual.

[0064] The kit should be stored at 4°C for 12 months and can detect 184 serum samples.

[0065] Among them, the Brucella LPS for coating was prepared according to the method of Example 1 (LPS prepared using other field-isolated Brucella bovis can also achieve the effect of this kit. After experiments, LPS prepared from at least 2 other Brucella bovis can achieve the effect of this kit).

[0066] Brucella LPS coating method is:

[0067] Thaw the purified Brucella LPS antigen from Example 1, frozen at -40°C, dilute to 2 μg / mL with pH 9.6 carbonate buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight. The next day, discard the antigen solution, wash, and block with 5% skim milk powder (100 μL / well) at 37°C for 2 h. Discard the blocking solution and spin dry. Air-bleach the plate in a laminar flow hood for 1 h. Finally, place the plate in a packaging bag and seal with plastic.

[0068] The monoclonal antibody was prepared according to the method of Example 3, and the monoclonal antibody was labeled with a commercial horseradish peroxidase (HRP) labeling kit to obtain an HRP-labeled monoclonal antibody HRP-mAb.

[0069] The preparation method of positive control serum is as follows:

[0070] Sheep approximately 6 months old were selected and immunized for the first time with an inactivated Brucella GS-XQ-A1 strain of the present invention mixed with Freund's complete adjuvant in a 1:1 volume ratio. Two weeks later, the sheep were immunized for the second time with an inactivated Brucella GS-XQ-A1 strain of the present invention mixed with Freund's incomplete adjuvant in a 1:1 volume ratio. Another two weeks later, the sheep were immunized for the third time with an inactivated Brucella GS-XQ-A1 strain of the present invention mixed with Freund's incomplete adjuvant in a 1:1 volume ratio. The immunizing antigen dose was 500 μg per sheep per dose. Two weeks after the third immunization, a small amount of blood was collected to separate serum, which was tested using a Brucella rose bengalensis agglutination test. If the titer reached +++ or above, a large amount of blood was collected from the carotid artery to separate serum, which was frozen at -20°C for future use.

[0071] The negative control serum is the serum of healthy sheep that has neither been immunized with Brucella vaccine nor infected with Brucella. It is tested with Brucella red bengal agglutination test and the result is negative.

[0072] The enzyme-labeled monoclonal antibody diluent (used for preparing HRP-labeled monoclonal antibody working solution), TMB colorimetric solution, stop solution, and 25-fold PBST concentrate in the blocking ELISA kit for detecting Brucella LPS serum antibodies of the present invention were all purchased from Lanzhou Veterinary Research Biology Co., Ltd. of the Chinese Academy of Agricultural Sciences.

[0073] 2. How to use the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies

[0074] The optimal coating concentration of Brucella LPS was determined to be 2 μg / mL, 100 μL / well by chessboard titration test; serum was diluted 2-fold, 100 μL / well, and incubated for 20 minutes; monoclonal antibody was diluted 1:6000, 100 μL / well, and incubated for 20 minutes. The substrate solution TMB was added at 100 μL / well, and incubated for 10 minutes; the stop solution was added at 50 μL / well, and the OD was immediately measured after addition. 450 value.

[0075] The method of using the blocking ELISA kit for quantitatively detecting Brucella LPS serum antibodies of the present invention is as follows:

[0076] 1. Remove the blocking ELISA kit from the 4°C refrigerator and equilibrate to room temperature. Dilute the 25x concentrated PBST wash buffer to 1x the working concentration with deionized water.

[0077] 2. Take out the enzyme-labeled plate coated with Brucella LPS, determine the required enzyme-labeled plate strips according to the number of sera to be tested, place the remaining strips in the packaging bag, and store at 4°C.

[0078] 3. Add the serum to be tested to the serum dilution plate, 75 μL / well; then add 1x washing solution, 75 μL / well, and mix well.

[0079] 3. Sample addition: Add the diluted serum sample to the ELISA plate at 100 μL / well; add the positive control serum and negative control serum to 2 wells each at 100 μL / well.

[0080] 4. Incubation: Seal the ELISA plate with a sealing film and incubate it in a 37°C incubator for 20 minutes.

[0081] 5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds, and then discard. Repeat this process 4 times and pat dry.

[0082] 6. Add HRP-labeled monoclonal antibody working solution at a dilution ratio of 1:6000, 100 μL / well.

[0083] 7. Incubation: Same steps as 4.

[0084] 8. Washing: Same steps as 5.

[0085] 9. Color development: Add TMB color development solution, 100 μL / well, and incubate at 37°C in the dark for 10 min.

[0086] 10. Stop: Add stop solution, 50 μL / well.

[0087] 11. Determination: Immediately measure the absorbance of each well at 450 nm using a microplate reader (OD 450 value).

[0088] 12. Result determination

[0089] ①Calculate the OD of the positive control serum, negative control serum and each serum to be tested on the same ELISA plate 450 The inhibition rate (PI) of the positive control serum and the serum to be tested was calculated according to the following formula: PI = (1-sample OD 450 Average value / negative control serum OD 450 average value)×100%.

[0090] ②PI of positive control serum>70%, OD of negative control serum 450 >1.5, the test is established, otherwise the test result is invalid.

[0091] ③ For serum samples tested, a PI ≥ 50% is considered positive; a PI < 50% is considered negative. For samples determined to be positive, the corresponding serum antibody titer (IU / mL) is calculated based on the PI value corresponding to the standard curve (see step 3 for the preparation of the standard curve). If the antibody concentration is greater than 12.5 IU / mL, that is, the PI value is greater than 87.83%, the sample should be diluted 5-10 times and retested until the PI value is between 1.11% and 87.83% to obtain the accurate antibody concentration.

[0092] 3. Standard curve of the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention

[0093] Test kit among the present invention can quantitatively detect Brucella LPS serum antibody, therefore need to formulate standard curve, specific as follows: Brucella standard positive control serum (230039-201401) is purchased from China Veterinary Drug Supervision Institute. Brucella standard positive control serum (1000IU / mL) is diluted to 50IU / mL, then serial dilution, concentration is followed successively by 50IU / mL, 25IU / mL, 12.5IU / mL, 6.25IU / mL, 3.125IU / mL, 1.563IU / mL, 0.781IU / mL, 0.391IU / mL, 0.195IU / mL. The serum of above-mentioned serial dilution is detected, obtains PI value. Take PI value as ordinate, take standard serum antibody concentration (IU / mL) as horizontal ordinate, adopt the method for four-parameter logic analysis to draw standard curve, as Figure 4 shown.

[0094] Figure 4 The standard curve is used to quantify the concentration of Brucella LPS antibodies.

[0095] Depend on Figure 4 It can be seen that when the antibody concentration of Brucella LPS in the serum is between 0.195 IU / mL (PI = 1.11%) and 12.5 IU / mL (PI = 87.83%), the antibody concentration and the PI value show a good linear relationship, and the antibody concentration can be obtained based on the PI value. When the antibody concentration is greater than 12.5 IU / mL, that is, the PI value is greater than 87.83%, the sample needs to be diluted 5 to 10 times and tested again until the PI value is between 1.11% and 87.83% to obtain the accurate antibody concentration. The equation for calculating the antibody concentration is as follows (X represents the antibody concentration, Y represents the PI):

[0096]

[0097] R 2 =0.9467

[0098] Example 6 Performance test of the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention

[0099] 1. Sensitivity test of the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention

[0100] Brucella standard positive serum was diluted according to 50 IU / mL, 25 IU / mL, 12.5 IU / mL, 6.25 IU / mL, 3.125 IU / mL, 1.563 IU / mL, 0.781 IU / mL, 0.391 IU / mL, and 0.195 IU / mL, and the kit of the present invention was used to detect the sample of each dilution gradient, and the minimum antibody titer that the kit can detect was tested. The results are shown in FIG. Figure 4 The results showed that the positive result could still be detected when the positive serum was diluted to 3.125 IU / mL, indicating that the kit has a high sensitivity to Brucella LPS serum antibodies.

[0101] The minimum serum antibody concentrations detected by the two commercial competitive ELISA antibody detection kits were 7.5 IU / mL and 5 IU / mL, respectively, indicating that the sensitivity of the blocking ELISA kit of the present invention is higher than that of the currently commercial competitive ELISA antibody detection kits.

[0102] 2. Specificity experiment of the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention

[0103] The kit of the present invention was used to detect positive sera of pathogens such as Yersinia enterocolitica O:9, Salmonella, Escherichia coli O:157 and Vibrio cholerae O11 / O139, and the results were all negative, indicating that the kit of the present invention has good specificity.

[0104] A commercial indirect ELISA antibody detection kit for Brucella was used to test positive sera for Yersinia enterocolitica O:9, Salmonella, Escherichia coli O:157, and Vibrio cholerae O11 / O139. The results showed that positive sera for Yersinia enterocolitica O:9 and Escherichia coli O:157 all tested positive, indicating that the blocking ELISA technology of the present invention has higher specificity than the indirect ELISA kit.

[0105] 3. Repeatability Experiment of Blocking ELISA Kit for Quantitative Detection of Brucella LPS Serum Antibodies of the Present Invention

[0106] Ten Brucella-positive sera and ten Brucella-negative sera were tested three times using a test kit from the same batch under the same conditions at different times, and the results were identical. Three batches of test kits were used to test the sera, and the results were identical. These results indicate that the test kit of the present invention has good repeatability.

[0107] 3. Shelf life of the blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention

[0108] The same batch of test kits was used to test the same 10 positive and 10 negative sera every two weeks. The results showed that the test results of the same serum samples were the same after the test kit was stored for 15 months, indicating that the shelf life of the test kit is at least 12 months.

[0109] 4. The blocking ELISA kit for quantitative detection of Brucella LPS serum antibodies of the present invention detects serum from multiple hosts

[0110] After testing the sera immunized with different existing Brucella vaccines and infected sera from different host sources, the results showed that all were positive (Table 1), indicating that this method is applicable to antibodies produced by immunization with all currently used Brucella vaccines, as well as serum antibodies from different host sources of Brucella infection, and has wide applicability.

[0111] Table 1 Applicability analysis of Brucella blocking ELISA antibody detection kit

[0112]

[0113]

[0114] V. Clinical Detection Experiment of the Blocking ELISA Kit for Quantitative Detection of Brucella LPS Serum Antibodies of the Present Invention

[0115] 284 clinical sera (including 130 bovine sera and 154 goat sera) were tested using the kit of the present invention. The serum samples were also tested using the Brucella red bengalensis agglutination test and a commercial competitive ELISA antibody detection kit. The results are shown in Table 2.

[0116] Table 2 The conformity rate of the kit of the present invention with the bengalensis agglutination test and competitive ELISA kit

[0117]

[0118] As shown in Table 2, the above 284 parts of field sera were detected respectively using the test kit of the present invention, the Brucella rose bengal agglutination test and the commercial competitive ELISA test kit. The positive coincidence rate of the blocking ELISA test kit and the rose bengal agglutination test for quantitatively detecting Brucella LPS serum antibodies of the present invention was 100%, the negative coincidence rate was 95.74%, and the total coincidence rate was 97.18%. The positive coincidence rate of the commercial competitive ELISA test kit was 100%, the negative coincidence rate was 97.83%, and the total coincidence rate was 98.59%, which has a higher coincidence rate. This shows that the test kit of the present invention can effectively detect Brucella LPS serum antibodies and is widely used in production.

[0119] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to Brucella LPS, characterized in that: The monoclonal antibody has three light chain complementary determining regions and three heavy chain complementary determining regions; The amino acid sequences of the light chain complementary determining regions are: amino acids 24 to 40 in SEQ ID NO.1 in the sequence listing, amino acids 56 to 62 in SEQ ID NO.1 in the sequence listing, and amino acids 95 to 102 in SEQ ID NO.1 in the sequence listing; The amino acid sequences of the heavy chain complementary determining regions are: amino acids 31 to 37 in SEQ ID NO.3, amino acids 52 to 67 in SEQ ID NO.3, and amino acids 100 to 113 in SEQ ID NO.

3.

2. The monoclonal antibody specifically binding to Brucella LPS according to claim 1, wherein: The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID No. 1; and the heavy chain variable region has the amino acid sequence shown in SEQ ID No.

3.

3. Encoding gene, characterized in that: Used to encode the monoclonal antibody that specifically binds to Brucella LPS as described in claim 1 or 2.

4. The coding gene according to claim 3, characterized in that: The coding gene includes a DNA sequence as shown in SEQ ID No.2, which is used to encode the light chain variable region of the monoclonal antibody that specifically binds to Brucella LPS; and includes a DNA sequence as shown in SEQ ID No.4, which is used to encode the heavy chain variable region of the monoclonal antibody that specifically binds to Brucella LPS.

5. Use of the monoclonal antibody that specifically binds to Brucella LPS according to claim 1 or 2, and the encoding gene according to claim 3 or 4 in preparing a blocking ELISA kit for detecting Brucella LPS antibodies.

6. A blocking ELISA kit for quantitative detection of Brucella LPS antibodies, characterized in that: The blocking ELISA kit comprises an enzyme labeling plate coated with Brucella LPS and the monoclonal antibody specifically binding to Brucella LPS according to claim 1 or 2.

7. The blocking ELISA kit according to claim 6, wherein: The coating concentration of the Brucella LPS is 2 μg / mL; and / or The dilution ratio of the monoclonal antibody was 1:6000.

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